InteractiveFly: GeneBrief

Myc: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References


Gene name - Myc

Synonyms - Myc, dMyc

Cytological map position - 3D5

Function - transcription factor

Keyword(s) - cell cycle, oogenesis, oncogene

Symbol - Myc

FlyBase ID:FBgn0262656

Genetic map position -

Classification - bHLH - leucine zipper

Cellular location - nuclear



NCBI links: Precomputed BLAST | Entrez Gene
Recent literature
Aughey, G. N., Grice, S. J. and Liu, J. L. (2016). The interplay between Myc and CTP synthase in Drosophila. PLoS Genet 12: e1005867. PubMed ID: 26889675
Summary:
CTP synthase (CTPsyn) is essential for the biosynthesis of pyrimidine nucleotides. Previous studies have shown that CTPsyn is incorporated into a novel cytoplasmic structure which has been termed the cytoophidium. This study reports that Myc regulates cytoophidium formation during Drosophila oogenesis. Myc protein levels correlate with cytoophidium abundance in follicle epithelia. Reducing Myc levels results in cytoophidium loss and small nuclear size in follicle cells, while overexpression of Myc increases the length of cytoophidia and the nuclear size of follicle cells. Ectopic expression of Myc induces cytoophidium formation in late stage follicle cells. Furthermore, knock-down of CTPsyn is sufficient to suppress the overgrowth phenotype induced by Myc overexpression, suggesting CTPsyn acts downstream of Myc and is required for Myc-mediated cell size control. Taken together, these data suggest a functional link between Myc, a renowned oncogene, and the essential nucleotide biosynthetic enzyme CTPsyn.
Strilbytska, O. M., Semaniuk, U. V., Storey, K. B., Edgar, B. A. and Lushchak, O. V. (2016). Activation of the Tor/Myc signaling axis in intestinal stem and progenitor cells affects longevity, stress resistance and metabolism in Drosophila.. Comp Biochem Physiol B Biochem Mol Biol 203: 92-99. PubMed ID: 27693629
Summary:
The TOR (target of rapamycin) signaling pathway and the transcriptional factor Myc play important roles in growBh control. Myc acts, in part, as a downstream target of TOR to regulate the activity and functioning of stem cells. Tbis study explored the role of TOR-Myc axis in stem and progenitor cells in the regulation of lifespan, stress resistance and metabolism in Drosophila. Goth overexpression of rheb and myc-rheb in midgut stem and progenitor cells decreased the lifespan and starvation resistance of flies. TOR activation caused higher survival under malnutrition conditions. Furthermore, gut-specific activation of JAK/STAT and insulin signaling pathways were demonstrated to control gut integrity. Both genetic manipulations had an impact on carbohydrate metabolism and transcriptional levels of metabolic genes. These findings indicate that activation of the TOR-Myc axis in midgut stem and progenitor cells influences a variety of traits in Drosophila.
Wehr Mathews, K., Cavegn, M. and Zwicky, M. (2017). Sexual dimorphism of body size is controlled by dosage of the X-chromosomal gene Myc and by the sex-determining gene tra in Drosophila. Genetics [Epub ahead of print]. PubMed ID: 28064166
Summary:
Drosophila females are larger than males. This paper describes how X chromosome dosage drives sexual dimorphism of body size through two means: first, through unbalanced expression of a key X-linked growth regulating gene and second, through female-specific activation of the sex-determination pathway. X-chromosome dosage determines phenotypic sex by regulating the genes of the sex-determining pathway. In the presence of two sets of X-chromosome signal elements (XSEs), Sex-lethal (Sxl) is activated in female (XX) but not male (XY) animals. Sxl activates transformer (tra), a gene that encodes a splicing factor essential for female-specific development. It has previously been shown that null mutations in the tra gene result in only a partial reduction of body size of XX animals, which shows that other factors must contribute to size determination. Whether X dosage directly affects animal size was tested by analyzing males with duplications of X chromosomal segments. Upon tiling across the X chromosome, four duplications were found that increase male size by over 9%. Only one of these, Myc, was found not to be dosage compensated. Together, these results indicate that both Myc dosage and tra expression play crucial roles in determining sex-specific size in Drosophila larvae and adult tissue. Since Myc also acts as an XSE that contributes to tra activation in early, development, a double dose of Myc in females serves at least twice in development to promote sexual size dimorphism.
Ma, X., Huang, J., Tian, Y., Chen, Y., Yang, Y., Zhang, X., Zhang, F. and Xue, L. (2017). Myc suppresses tumor invasion and cell migration by inhibiting JNK signaling. Oncogene [Epub ahead of print]. PubMed ID: 28068320
Summary:
Tumor metastasis, but not primary overgrowth, is the leading cause of mortality for cancer patients. During the past decade, Drosophila melanogaster has been well-accepted as an excellent model to address the intrinsic mechanism of different aspects of cancer progression, ranging from tumor initiation to metastasis. In a genetic screen aiming to find novel modulators of tumor invasion in Drosophila, this study identified the oncoprotein Myc as a negative regulator. While expression of Myc dramatically blocks tumor invasion and cell migration, loss of Myc promotes cell migration in vivo. The activity of Myc is further enhanced by the co-expression of its transcription partner Max. Mechanistically, Myc/Max directly upregulates the transcription of puc, which encodes an inhibitor of JNK signaling crucial for tumor invasion and cell migration. Furthermore, human cMyc potently suppresses JNK-dependent cell invasion and migration in both Drosophila and lung adenocarcinoma cell lines. These findings provide novel molecular insights into Myc-mediated cancer progression and raise the noteworthy problem in therapeutic strategies as inhibiting Myc might conversely accelerate tumor metastasis.

Tavares, L., Correia, A., Santos, M. A., Relvas, J. B. and Pereira, P. S. (2017). dMyc is required in retinal progenitors to prevent JNK-mediated retinal glial activation. PLoS Genet 13(3): e1006647. PubMed ID: 28267791
Summary:
In the nervous system, glial cells provide crucial insulation and trophic support to neurons and are important for neuronal survival. In reaction to a wide variety of insults, glial cells respond with changes in cell morphology and metabolism to allow repair. Additionally, these cells can acquire migratory and proliferative potential. In particular, after axonal damage or pruning the clearance of axonal debris by glial cells is key for a healthy nervous system. Thus, bidirectional neuron-glial interactions are crucial in development, but little is known about the cellular sensors and signalling pathways involved. This study shows that decreased cellular fitness in retinal progenitors caused by reduced Drosophila Myc expression triggers non cell-autonomous activation of retinal glia proliferation and overmigration. Glia migration occurs beyond its normal limit near the boundary between differentiated photoreceptors and precursor cells, extending into the progenitor domain and is stimulated by JNK activation (and the function of its target Mmp1), while proliferative responses are mediated by Dpp/TGF-beta signalling activation.
Chanu, S. I. and Sarkar, S. (2017). Targeted downregulation of dMyc restricts neurofibrillary tangles mediated pathogenesis of human neuronal tauopathies in Drosophila. Biochim Biophys Acta [Epub ahead of print]. PubMed ID: 28529046
Summary:
Formation of Neurofibrillary Tangles (NFTs) in neuronal tissues has been implicated as the hallmark of disease pathogenesis and tau mediated toxicity in human and mammalian models. However, previous studies had failed to correlate NFT formation with pathogenesis of human neuronal tauopathies in Drosophila disease models. Though, a recent report suggests formation of tau mediated NFTs like structures confined to dopaminergic neurons in Drosophila adult brain, by utilizing various approaches, this study demonstrated distinct and recurrent formation of NFTs in Drosophila neuronal tissues upon expression of wild type or mutant isoforms of human tau, and this appears as the key mediator of the pathogenesis of human neuronal tauopathy in Drosophila. Further, it was shown that tissue specific downregulation of dMyc (Drosophila homolog of human c-myc proto-oncogene) alleviates h-tau mediated cellular and functional deficits by restricting the formation of NFTs in neuronal tissues. Therefore, these findings provide very critical and novel insights about pathogenesis of human neuronal tauopathies in Drosophila disease models.
Paiardi, C., Mirzoyan, Z., Zola, S., Parisi, F., Vingiani, A., Pasini, M. E. and Bellosta, P. (2017). The Stearoyl-CoA Desaturase-1 (Desat1) in Drosophila cooperated with Myc to induce autophagy and growth, a potential new link to tumor survival. Genes (Basel) 8(5). PubMed ID: 28452935
Summary:
Lipids are an important energy supply in our cells and can be stored or used to produce macromolecules during lipogenesis when cells experience nutrient starvation. Proteomic analysis reveals that the Drosophila homologue of human Stearoyl-CoA desaturase-1 Desat1) is an indirect target of Myc in fat cells. Stearoyl-CoA desaturases are key enzymes in the synthesis of monounsaturated fatty acids critical for the formation of complex lipids such as triglycerides and phospholipids. Their function is fundamental for cellular physiology, however in tumors, overexpression of SCD-1 and SCD-5 has been found frequently associated with a poor prognosis. Another gene that is often upregulated in tumors is the proto-oncogene c-myc, where its overexpression or increased protein stability, favor cellular growth. This study reports a potential link between Myc and Desat1 to control autophagy and growth. Using Drosophila, it was found that expression of Desat1, in metabolic tissues like the fat body, in the gut and in epithelial cells, is necessary for Myc function to induce autophagy a cell eating mechanism important for energy production. In addition, it was observed that reduction of Desat1 affects Myc ability to induce growth in epithelial cells. These data also identify, in prostatic tumor cells, a significant correlation between the expression of Myc and SCD-1 proteins, suggesting the existence of a potential functional relationship between the activities of these proteins in sustaining tumor progression.
Lee, J. E., Rayyan, M., Liao, A., Edery, I. and Pletcher, S. D. (2017). Acute dietary restriction acts via TOR, PP2A, and Myc signaling to boost innate immunity in Drosophila. Cell Rep 20(2): 479-490. PubMed ID: 28700947
Summary:
Dietary restriction promotes health and longevity across taxa through mechanisms that are largely unknown. This study shows that acute yeast restriction significantly improves the ability of adult female Drosophila melanogaster to resist pathogenic bacterial infections through an immune pathway involving downregulation of target of rapamycin (TOR) signaling, which stabilizes the transcription factor Myc by increasing the steady-state level of its phosphorylated forms through decreased activity of protein phosphatase 2A. Upregulation of Myc through genetic and pharmacological means mimicked the effects of yeast restriction in fully fed flies, identifying Myc as a pro-immune molecule. Short-term dietary or pharmacological interventions that modulate TOR-PP2A-Myc signaling may provide an effective method to enhance immunity in vulnerable human populations.
Funakoshi, M., Tsuda, M., Muramatsu, K., Hatsuda, H., Morishita, S. and Aigaki, T. (2018). Overexpression of Larp4B downregulates dMyc and reduces cell and organ sizes in Drosophila. Biochem Biophys Res Commun 497(2): 762-768. PubMed ID: 29462618
Summary:
Regulation of cell and organ sizes is fundamental for all organisms, but its molecular basis is not fully understood. A gain-of-function screen was performed, and larp4B was identified whose overexpression reduces cell and organ sizes in Drosophila melanogaster. Larp4B is a member of La-related proteins (LARPs) containing an LA motif and an adjacent RNA recognition motif (RRM), and play diverse roles in RNA metabolism. However, the function of Larp4B has remained poorly characterized. Transgenic flies were generated overexpressing wild-type mammalian Larp4B or a deletion variant lacking the LA and RRM domains, and it was demonstrated that the RNA-binding domains are essential for Larp4B to reduce cell and organ sizes. The larp4B-induced phenotype was suppressed by dMyc overexpression, which promotes cell growth and survival. Furthermore, overexpression of larp4B decreased dMyc protein levels, whereas its loss-of-function mutation had an opposite effect. These results suggest that Larp4B is a negative regulator of dMyc.
BIOLOGICAL OVERVIEW

myc is a mammalian oncogene involved in a wide variety of tumors. Oncogenic activation depends on elevated expression of the short lived Myc protein. In normal cells, myc expression is dependent on mitogenic stimuli. Myc is required for both cell proliferation and to prevent differentiation. Unless growth factors are provided, activation of myc can concomitantly induce programmed cell death (apoptosis).

Before discussing the avid interest Myc holds for developmental biologists, a brief description is given of the interaction of Myc with three other proteins (MAD, MAX and MXI) and their combined effects on both cell activation and quiescence (Amati,1994 and references).

Myc proteins contain two regions characteristic of transcription factors: an N-terminal transactivation domain, and a C-terminal basic helix-loop-helix (bHLH) leucine zipper motif known to mediate dimerization and sequence specific DNA binding. Myc functions as a heterodimer with other bHLH leucine zipper proteins. Max specifically dimerizerizes with Myc, and Myc-Max heterodimers function as transcriptional activators, binding a hexanucleotide motif called the E-box, which is often the target of other bHLH proteins.

Antagonizing the cell cycle promoting activity of Myc-Max in mammals are two other proteins, Mad and Mxi-1. Mad and Mxi can heterodimerize with Max, depriving Myc of a partner. The Max/Mad or Max/Mxi-1 partners either fail to activate or actively repress transcription, leading to a state of growth inhibition or possibly cell differentiation. Max proteins are metabolically stable and constitutively expressed, while Myc, Mad and Mxi-1 are unstable, responding to the level of mitotic stimulation in the cell. Mitogen stimulation induces a rapid rise in Myc levels and thus a shift in the equilibrium from Max/Max homodimers to Myc/Max heterodimers, the combination that promotes entry into cell cycle. Conversely, mitogen withdrawal or differentiation stimuli suppress myc expression and may result in the induction of either or both mad and mxi, leading to a state of growth inhibition. Thus Myc, Max and Mxi function as a protein network leading to alternative states of cell activation or quiescence (Amati, 1994 and references).

Of particular interest to developmental biologists is the question of whether Myc, Mad and Max play a role in normal development. This question became more complex and interesting with the discovery in vertebrates of multiple Myc genes. There is a neurally expressed myc (N-myc), for example. In mammals N-myc and myc show contrasting expression patterns during gastrulation. myc is most abundant in extraembryonic cells; in contrast, N-myc is found at highest levels in the expanding primitive streak and other portions of the embryonic mesoderm. Differentiation of mesoderm to epithelioid cells is accompanied by diminished expression of N-myc. Expression of myc is not an inevitable correlate of cellular proliferation. Instead, the gene appears to be regulated in concert with changes that affect diverse cellular properties, including proliferation, invasiveness and differentiation. For example, myc is selectively down-regulated in the primitive ectoderm, the most highly proliferative tissue of the embryo. A decline in myc expression correlates with terminal differentiation of brown adipose tissue and liver. In contrast, N-myc continues to be expressed in post-mitotic cells of neuronal origin (Downs, 1989 and Hirning, 1991).

Isolation of Drosophila Myc, properly termed Diminutive but referred to as dMyc, was accomplished by means of its conserved affinity for Max. Human Max was used to screen a two-hybid library prepared from Drosophila cDNAs. A second yeast two-hybrid screen was performed using the bHLH leucine zipper region of dMyc as bait. This experiment resulted in the identification of dMax (Gallant, 1996).

What is the phenotypic effect of loss of dmyc function? The results are inconclusive, but it is believed that a Drosophila mutation (known as diminutive), resulting in an abnormally small body size and female sterility, is due to a hypomorphic mutation in dmyc. Defects are observed in both germ cell nuclei and follicle cells; they fail to migrate and aberrantly undergo the transition to columnar epithelium. This phenotype suggests dysfunction in either follicle or nurse cells, or in communication between these two cell types. Interestingly the degeneration of the egg chamber in dm mutant females occurs at stage 8, at a time when cell division does not occur.

It is thought that a stage-specific downregulation of dmyc expression in diminished mutants results in a loss of the capacity of the follicle cells to grow and migrate. A possibly related effect has been observed in mice: hypomorphic N-myc mutation results in a loss of induction of tissue specific differentiation (Moens, 1992). In both cases diminished Myc expression in progenitor cells may result in their inability to respond to inductive signals. In addition, the smaller size of the diminished mutants may also result from partial loss of dMyc function in other tissues (Gallant, 1996). Recent studies in mice demonstrate that alterations in the cell cycle can significantly influence overall size. Mice lacking p27Kip1, an inhibitor of cyclin dependent kinase, display increased body size without an increase in growth hormone levels (Fero, 1996).

Is the small body size of diminutive mutants due to a hormonal effect or is it due to cell autonomous dmyc deficiency? Mosaic experiments should yield clues as to dMyc's influence on specific organs and developmental timing.

Cell mixing induced by myc is required for competitive tissue invasion and destruction

Cell-cell intercalation is used in several developmental processes to shape the normal body plan. There is no clear evidence that intercalation is involved in pathologies. This study used the proto-oncogene myc to study a process analogous to early phase of tumour expansion: myc-induced cell competition. Cell competition is a conserved mechanism driving the elimination of slow-proliferating cells (so-called 'losers') by faster-proliferating neighbours (so-called 'winners') through apoptosis and is important in preventing developmental malformations and maintain tissue fitness. Using long-term live imaging of myc-driven competition in the Drosophila pupal notum and in the wing imaginal disc, this study showed that the probability of elimination of loser cells correlates with the surface of contact shared with winners. As such, modifying loser-winner interface morphology can modulate the strength of competition. Elimination of loser clones requires winner-loser cell mixing through cell-cell intercalation. Cell mixing is driven by differential growth and the high tension at winner-winner interfaces relative to winner-loser and loser-loser interfaces, which leads to a preferential stabilization of winner-loser contacts and reduction of clone compactness over time. Differences in tension are generated by a relative difference in F-actin levels between loser and winner junctions, induced by differential levels of the membrane lipid phosphatidylinositol (3,4,5)-trisphosphate. These results establish the first link between cell-cell intercalation induced by a proto-oncogene and how it promotes invasiveness and destruction of healthy tissues (Levayer, 2015).

To analyse quantitatively loser cell elimination, long-term live imaging was performed of clones showing a relative decrease of the proto-oncogene myc in the Drosophila pupal notum, a condition known to induce cell competition in the wing disc. Every loser cell delamination was counted over 10 h, and the probability of cell elimination was calculated for a given surface of contact shared with winner cells. A significant increase was observed of the proportion of delamination with winner-loser shared contact, whereas this proportion remained constant for control clones. The same correlation was observed in ex vivo culture of larval wing disc. Cell delamination in the notum was apoptosis dependent and expression of flowerlose (fwelose), a competition-specific marker for loser fate, was necessary and sufficient to drive contact-dependent delamination. Moreover it was confirmed that contact-dependent death is based on the computation of relative differences of fwelose between loser cells and their neighbours. Thus, cell delamination in the notum recapitulates features of cell competition (Levayer, 2015).

This suggests that winner-loser interface morphology could modulate the probability of eliminating loser clones. Using the wing imaginal disc, winner-loser contact was reduced by inducing adhesion- or tension-dependent cell sorting and observed a significant reduction of loser clone elimination. This rescue was not driven by a cell-autonomous effect of E-cadherin (E-cad) or active myosin II regulatory light chain (MRLC) on growth, death or cell fitness but rather by a general diminution of winner-loser contact. Competition is ineffective across the antero-posterior compartment boundary, a frontier that prevents cell mixing through high line tension. Accordingly, there was no increase in death at the antero-posterior boundary in wing discs overexpressing fweloseA in the anterior compartment. However, reducing tension by reducing levels of myosin II heavy chains was sufficient to increase the shared surface of contact between cells of the anterior and posterior compartments, and induced fwelose death at the boundary. Altogether, it is concluded that the reduction in surface contact between winners and losers is sufficient to block competition, which explains how compartment boundaries prevent competition (Levayer, 2015).

Loser clones have been reported to fragment more often than controls, whereas winner clones show convoluted morphology, suggesting that winner-loser mixing is increased during competition. This could affect the outcome of cell competition by increasing the surface shared between losers and winners. Clone splitting was used as a readout for loser–winner mixing. Two non-exclusive mechanisms can drive clone splitting: cell death followed by junction rearrangement, or junction remodelling and cell–cell intercalation independent of death. To assess the contribution of each phenomenon, the proportion of clones fragmented 48 h after clone induction (ACI) was systematically counted. A twofold increase was observed in the frequency of split clones in losers (wild type (WT) in tub-dmyc) versus WT in WT controls. Overexpressing E-cad or active myosin II was sufficient to prevent loser clone splitting, whereas blocking apoptosis or blocking loser fate by silencing fwelose did not reduce splitting. Finally, the proportion of split clones was also increased for winner clones either during myc-driven competition or during Minute-dependent competition. Altogether, this suggested that winner–loser mixing is increased independently of loser cell death or clone size by a factor upstream of fwe, and could be driven by cell–cell intercalation. Accordingly, junction remodelling events leading to disappearance of a loser–loser junction were three times more frequent at loser clone boundaries than control clone boundaries in the pupal notum. The rate of junction remodelling was higher in loser–loser junctions and in winner–winner junctions than in winner–loser junctions. The preferential stabilization of winner–loser interfaces should increase the surface of contact between winner and loser cells over time. Accordingly, loser clone compactness in the notum decreased over time whereas it remains constant on average for WT clones in WT background. Similarly, the compactness of clones in the notum also decreased over time for conditions showing high frequency of clone splitting in the wing disc, whereas clone compactness remained constant for conditions rescuing clone splitting. Altogether, it is concluded that both Minute- and myc-dependent competition increase loser–winner mixing through cell–cell intercalation (Levayer, 2015).

It was then asked what could modulate the rate of junction remodelling during competition. The rate of junction remodelling can be cell-autonomously increased by myc. Interestingly, downregulation of the tumour suppressor PTEN is also sufficient to increase the rate of junction remodelling through the upregulation of phosphatidylinositol (3,4,5)-trisphosphate (PIP3). It was reasoned that differences in PIP3 levels could also modulate junction remodelling during competition. Using a live reporter of PIP3 that could detect modulations of PIP3 in the notum, a significant increase of PIP3 was observed in the apico-lateral membrane of tub-dmyc–tub-dmyc interfaces compared with WT–WT and WT–tub-dmyc interfaces (Fig. 3a, b). Moreover, increasing/reducing Myc levels in a full compartment of the wing disc was sufficient to increase/decrease the levels of phospho-Akt (a downstream target of PIP3, whereas fweloseA overexpression had no effect. Similarly, levels of phospho-Akt were relatively higher in WT clones than in the surrounding M-/+ cells. Thus differences in PIP3 levels might be responsible for winner–loser mixing. Accordingly, reducing PIP3 levels by overexpressing a PI3 kinase dominant negative (PI3K-DN) or increasing PIP3 levels by knocking down PTEN (UAS-pten RNAi) were both sufficient to induce a high proportion of fragmented clones and to reduce clone compactness over time in the notum , whereas increasing PIP3 in loser clones was sufficient to prevent cell mixing. Moreover, abolishing winner–loser PIP3 differences through larval starvation prevented loser clone fragmentation, the reduction of clone compactness over time in the notum and could rescue WT clone elimination in tub-dmyc background. It is therefore concluded that differences in PIP3 levels are necessary and sufficient for loser–winner mixing and required for loser cell elimination (Levayer, 2015).

It was then asked which downstream effectors of PIP3 could affect junction stability. A relative growth decrease can generate mechanical stress that can be released by cell-cell intercalation. Accordingly, growth reduction through Akt downregulation is sufficient to increase clone splitting and could contribute to loser clone splitting. However, Akt is not sufficient to explain winner-loser mixing because, unlike PIP3, increasing Akt had no effect on clone splitting. PIP3 could also modulate junction remodelling through its effect on cytoskeleton and the modulation of intercellular adhesion or tension. No obvious modifications of E-cad, MRLC or Dachs (another regulator of tension) was detected in loser cells. However, a significant reduction of F-actin levels and a reduction of actin turnover/polymerization rate were observed in loser-loser and loser-winner junctions in the notum. Similarly, modifying Myc levels in a full wing disc compartment was sufficient to modify actin levels, and F-actin levels were higher in WT clones than M-/+ cells. This prompted a test of the role of actin organization in winner-loser mixing. Downregulating the formin Diaphanous (Dia, a filamentous actin polymerization factor) by RNA interference (RNAi) or by using a hypomorphic mutant was sufficient to obtain a high proportion of fragmented clones and to reduce clone compactness over time, whereas overexpressing Dia in loser clones prevented clone splitting (UAS-dia::GFP) and compactness reduction. This effect was specific to Dia as modulating Arp2/3 complex (a regulator of dendritic actin network) had no effect on clone splitting. Thus, impaired filamentous actin organization was necessary and sufficient to drive loser-winner mixing. These actin defects were driven by the differences in PIP3 levels between losers and winners. Thus Dia could be an important regulator of competition through its effect on cell mixing. Overexpression of Dia was indeed sufficient to reduce loser clone elimination significantly (Levayer, 2015).

Filamentous actin has been associated with tension regulation. It was therefore asked whether junction tension was modified in winner and loser junctions. The maximum speed of relaxation of junction after laser nanoablation (which is proportional to tension) was significantly reduced in loser-loser and winner-loser junctions compared with winner-winner junctions. This distribution of tension has been proposed to promote cell mixing. Accordingly, decreasing PIP3 in clones reduced tension both in low-PIP3-low-PIP3 and low-PIP3-normal-PIP3 junctions, whereas overexpressing Dia in loser clones or starvation were both sufficient to abolish differences in tension, in agreement with their effect on winner-loser mixing and the distribution of F-actin. Thus the lower tension at winner-loser and loser-loser junctions is responsible for winner-loser mixing. Altogether, it is concluded that the relative PIP3 decrease in losers increases winner-loser mixing through Akt-dependent differential growth and the modulation of tension through F-actin downregulation in winner-loser and loser-loser junctions (Levayer, 2015).

Several modes of tissue invasion by cancer cells have been described, most of them relying on the departure of the tumour cells from the epithelial layer. This study suggests that some oncogenes may also drive tissue destruction and invasion by inducing ectopic cell intercalation between cancerous and healthy cells, and subsequent healthy cell elimination. myc-dependent invasion could be enhanced by other mutations further promoting intercalation (such as PTEN). Stiffness is increased in many tumours, suggesting that healthy cell-cancer cell mixing by intercalation might be a general process (Levayer, 2015).

Drosophila insulin and target of rapamycin (TOR) pathways regulate GSK3 beta activity to control Myc stability and determine Myc expression in vivo

Genetic studies in Drosophila reveal an important role for Myc in controlling growth. Similar studies have also shown how components of the insulin and target of rapamycin (TOR) pathways are key regulators of growth. Despite a few suggestions that Myc transcriptional activity lies downstream of these pathways, a molecular mechanism linking these signaling pathways to Myc has not been clearly described. Using biochemical and genetic approaches this study tried to identify novel mechanisms that control Myc activity upon activation of insulin and TOR signaling pathways. Biochemical studies show that insulin induces Myc protein accumulation in Drosophila S2 cells, which correlates with a decrease in the activity of glycogen synthase kinase 3-β (GSK3β) a kinase that is responsible for Myc protein degradation. Induction of Myc by insulin is inhibited by the presence of the TOR inhibitor rapamycin, suggesting that insulin-induced Myc protein accumulation depends on the activation of TOR complex 1. Treatment with amino acids that directly activate the TOR pathway results in Myc protein accumulation, which also depends on the ability of S6K kinase to inhibit GSK3β activity. Myc upregulation by insulin and TOR pathways is a mechanism conserved in cells from the wing imaginal disc, where expression of Dp110 and Rheb also induces Myc protein accumulation, while inhibition of insulin and TOR pathways result in the opposite effect. Functional analysis, aimed at quantifying the relative contribution of Myc to ommatidial growth downstream of insulin and TOR pathways, revealed that Myc activity is necessary to sustain the proliferation of cells from the ommatidia upon Dp110 expression, while its contribution downstream of TOR is significant to control the size of the ommatidia. This study presents novel evidence that Myc activity acts downstream of insulin and TOR pathways to control growth in Drosophila. At the biochemical level it was found that both these pathways converge at GSK3β to control Myc protein stability, while genetic analysis shows that insulin and TOR pathways have different requirements for Myc activity during development of the eye, suggesting that Myc might be differentially induced by these pathways during growth or proliferation of cells that make up the ommatidia (Parisi, 2011).

Previous studies in vertebrates have indicated a critical function for Myc downstream of growth factor signaling including insulin-like growth factor, insulin and TOR pathways. In Drosophila, despite a few notes that Myc transcriptional activity acts downstream of insulin and TOR pathways, no clear molecular mechanisms linking these pathways to Myc have been elucidated yet (Parisi, 2011).

It has been demonstrated that inhibition of GSK3β prevents Myc degradation by the proteasome pathway. This study further unravels the pathways that control Myc protein stability and shows that signaling by insulin and TOR induce Myc protein accumulation by regulating GSK3β activity in S2 cells. GSK3β is a constitutively active kinase that is regulated by multiple signals and controls numerous cellular processes. With the biochemical data it is proposed that GSK3β acts as a common point where insulin and TOR signaling converge to regulate Myc protein stability (see Model showing the proposed relationship between Myc and the insulin and TOR signaling pathways). In particular, activation of insulin signaling was shown to induce activation of Akt, an event that is accompanied by GSK3β phosphorylation on Ser 9 that causes its inactivation and Myc protein to stabilize. Interestingly, insulin-induced Myc protein accumulation, when GSK3β activity was blocked by the presence of LiCl or by expression of GSK3β-KD, was similar to that obtained with insulin alone. Since it was shown that activation of insulin signaling leads to GSK3β inhibition and to an increase in Myc protein, if insulin and GSK3β signaling were acting independently, it would be expected that activation of insulin signaling concomitantly with the inhibition of GSK3β activity would result in a higher level of Myc than that obtained with insulin or LiCl alone. The results instead showed a similar level of Myc protein accumulation with insulin in the presence of GSK3β inhibitors as compared to insulin alone, supporting the hypothesis that GSK3β and insulin signaling, at least in the current experimental condition, depend on each other in the mechanism that regulates Myc protein stability (Parisi, 2011).

In a similar biochemical approach, the effect of AAs was analyzed on Myc protein stability and how TOR signaling is linked to mechanisms that inactivate GSK3β to stabilize Myc protein in S2 cells. In these experiments it was possible to demonstrate that treatment with amino acids (AAs) increased Myc protein stability, and it was also shown that treatment with rapamycin, an inhibitor of TORC1, reduced insulin-induced Myc upregulation. The reduction of Myc protein accumulation by rapamycin was blocked by inhibition of the proteasome pathway, linking TOR signaling to the pathway that controls Myc protein stability. TORC1 is a central node for the regulation of anabolic and catabolic processes and contains the central enzyme Rheb-GTPase, which responds to amino acids by activating TOR kinase to induce phosphorylation of p70-S6K and 4E-BP1. Analysis of the molecular mechanisms that act downstream of TOR to regulate Myc stability shows that AA treatment induces p70-S6K to phosphorylate GSK3β on Ser 9, an event that results in its inactivation and accumulation of Myc protein (Parisi, 2011).

Reducing GSK3β activity with LiCl, in medium lacking AAs, resulted in a slight increase in Myc protein levels. Adding back AAs lead to a substantial increase in Myc protein levels, which did not further increase when AAs where added to cells in the presence of the GSK3β inhibitor LiCl. These events were accompanied by phosphorylation of S6K on Thr 398, which correlated with phosphorylation of GSK3β on Ser 9. From these experiments it is concluded that TOR signaling also converges to inhibit GSK3β activity to regulate Myc protein stability. However, it has to be pointed out that since AAs alone increased Myc protein levels to a higher extent than that observed with LiCl alone, the experiments also suggest that Myc protein stability by TOR signaling is not solely directed through the inhibition of GSK3β activity, but other events and/or pathways contribute to Myc regulation. In conclusion, the biochemical experiments demonstrate that GSK3β acts downstream of insulin and TOR pathways to control Myc stability, however it cannot be excluded that other pathways may control Myc protein stability upon insulin and amino acids stimulation in S2 cells (Parisi, 2011).

Reduction of insulin and TOR signaling in vivo reduces cell size and proliferation, and clones mutant for chico, the Drosophila orthologue of IRS1-4, or for components of TOR signaling, are smaller due a reduction in size and the number of cells. The experiments showed that reducing insulin signaling by expression of PTEN or using TORTED, a dominant negative form of TOR, decreased Myc protein levels in clones of epithelial cells of the wing imaginal discs, while the opposite was true when these signals were activated using Dp110 or RhebAV4 . Those experiments suggested that the mechanism of regulation of Myc protein by insulin and TOR pathways was conserved also in vivo in epithelial cells of the larval imaginal discs (Parisi, 2011).

During these experiments it was also noted that Myc protein was induced in the cells surrounding and bordering the clones (non-autonomously), particularly when clones where positioned along the dorsal-ventral axis of the wing disc. This upregulation of Myc protein was not restricted to components of the insulin signaling pathway since it was also observed in cells surrounding the clones mutant for components of the Hippo pathway or for the tumor suppressor lethal giant larvae (lgl), which upregulates Myc protein cell-autonomously. It is suspected that this non-autonomous regulation of Myc may be induced by a novel mechanism that controls proliferation of cells when 'growth' is unbalanced. It can be speculated that clones with different growth rates, caused by different Myc levels, might secrete factors to induce Myc expression in neighboring cells. As a consequence, these Myc-expressing cells will speed up their growth rate in an attempt to maintain proliferation and tissue homeostasis. Further analysis is required to identify the mechanisms responsible for this effect (Parisi, 2011).

In order to distinguish if Myc activity was required downstream of insulin and TOR signaling to induce growth, a genetic analysis was performed. The ability to induce growth and proliferation was measured in the eye by measuring the size and number of the ommatidia from animals expressing members of the insulin and TOR pathways in different dm genetic background (dm+, dmP0 and dm4). The data showed that Dp110 increased the size and number of the ommatidia, however only the alteration in the total number was dependent on dm levels. These data suggest that Myc is required downstream of insulin pathway to achieve the proper number of ommatidia. However, when insulin signaling was reduced by PTEN, a significant decrease in the size of ommatidia was seen and it was dependent on dm expression levels, suggesting that Myc activity is limiting for ommatidial size and number. Activation of TOR signaling induces growth, and the genetic analysis showed that Myc significantly contributes to the size of the ommatidial cells thus suggesting that Myc acts downstream of TOR pathway to control growth (Parisi, 2011).

Recent genomic analysis showed a strong correlation between the targets of Myc and those of the TOR pathway, implying that they may share common targets. In support of this observation, mosaic analysis with a repressible cell marker (MARCM) experiments in the developing wing disc showed that overexpression of Myc partially rescues the growth disadvantage of clones mutant for the hypomorphic Rheb7A1 allele, further supporting the idea that Myc acts downstream of TOR to activate targets that control growth in these clones (Parisi, 2011).

The genetic interaction revealed a stronger dependence on Myc expression when Rheb was used as opposed to S6K. A possible explanation for this difference could lie in the fact that S6K is not capable of auto-activation of its kinase domain unless stimulated by TOR kinase. TOR activity is dependent on its upstream activator Rheb; consequently the enzymatic activity of the Rheb/GTPase is the limiting factor that influences S6K phosphorylation and therefore capable of maximizing its activity (Parisi, 2011).

Interestingly, these experiments also showed that activation of TOR signaling has a negative effect on the number of ommatidia, and this correlates with the ability of RhebAV4 to induce cell death during the development of the eye imaginal disc. Rheb-induced cell death was rescued in a dmP0 mutant background, which leading to the speculation that 'excessive' protein synthesis, triggered by overexpression of TOR signaling, could elicit a Myc-dependent stress response, which induces apoptosis. Alternatively, high protein synthesis could result in an enrichment of misfolded proteins that may result in a stress response and induces cell death. Further analysis is required to delineate the mechanisms underlying this process (Parisi, 2011).

This analyses provide novel genetic and biochemical evidences supporting a role for Myc in the integration of the insulin and TOR pathway during the control of growth, and highlights the role of GSK3β in this signaling. It was found that insulin signaling inactivates GSK3β to control Myc protein stability, and a similar biochemical regulation is also shared by activation of the TOR pathways. In support of this data, a recent genomic analysis in whole larvae showed a strong correlation between the targets of Myc and those of the TOR pathway; however, less overlap was found between the targets of Myc and those of PI3K signaling (Parisi, 2011).

Statistical analysis applied to the genetic interaction experiments revealed that, in the Drosophila eye, proliferation induced by activation of the insulin pathway is sensitive to variations in Myc levels, while a significant interaction was seen mostly when TOR increased cell size. The data therefore suggests that there is a correlation between Myc and the InR signaling and it is expected that the InR pathway also shares some transcriptional targets with Myc. Indeed, an overlap was found between the targets induced by insulin and Myc in Drosophila S2 cells and these targets have also been reported in transcriptome analyses in the fat body upon nutritional stress, suggesting that Myc acts downstream of InR/PI3K and TOR signaling and that this interaction might be specific to some tissues or in a particular metabolic state of the cell (Parisi, 2011).


REGULATION

An intergenic regulatory region mediates Drosophila Myc-induced apoptosis and blocks tissue hyperplasia

Induction of cell-autonomous apoptosis following oncogene-induced overproliferation is a major tumor-suppressive mechanism in vertebrates. However, the detailed mechanism mediating this process remains enigmatic. This study demonstrates that dMyc-induced cell-autonomous apoptosis in the fruit fly Drosophila relies on an intergenic sequence termed the IRER (irradiation-responsive enhancer region). The IRER mediates the expression of surrounding proapoptotic genes, and an in vivo reporter of the IRER chromatin state was used to gather evidence that epigenetic control of DNA accessibility within the IRER is an important determinant of the strength of this response to excess dMyc. In a previous work, it was shown that the IRER also mediates P53-dependent induction of proapoptotic genes following DNA damage, and the chromatin conformation within IRER is regulated by polycomb group-mediated histone modifications. dMyc-induced apoptosis and the P53-mediated DNA damage response thus overlap in a requirement for the IRER. The epigenetic mechanisms controlling IRER accessibility appear to set thresholds for the P53- and dMyc-induced expression of apoptotic genes in vivo and may have a profound impact on cellular sensitivity to oncogene-induced stress (Zhang, 2014).

Myc is a crucial regulator of growth and proliferation during animal development. Many signals and transcription factors lead to changes in the expression levels of Drosophila myc, yet no clear model exists to explain the complexity of its regulation at the level of transcription. This study used Drosophila genetic tools to track the dmyc cis-regulatory elements. Bioinformatics analyses identified conserved sequence blocks in the noncoding regions of the dmyc gene. Investigation of lacZ reporter activity driven by upstream, downstream, and intronic sequences of the dmyc gene in embryonic, larval imaginal discs, larval brain, and adult ovaries, revealed that it is likely to be transcribed from multiple transcription initiation units including a far upstream regulatory region, a TATA box containing proximal complex and a TATA-less downstream promoter element in conjunction with an initiator within the intron 2 region. The data provide evidence for a modular organization of dmyc regulatory sequences; these modules will most likely be required to generate the tissue-specific patterns of dmyc transcripts. The far upstream region is active in late embryogenesis, while activity of other cis elements is evident during embryogenesis, in specific larval imaginal tissues and during oogenesis. These data provide a framework for further investigation of the transcriptional regulatory mechanisms of dmyc (Kharazmi, 2012; full text of article).

The dynamic expression of dmyc is initiated from multiple transcription start sites. Tight regulation of dMyc is crucial for cell growth and division during the early phases of development and cell fate specification. In third instar larvae, dmyc mRNA is detected around the wing pouch and in the notum. dmyc activity is absent from the cell cycle-arrested nonproliferating cells that surround the dorsoventral boundary in the wing pouch. In the eye disc, dmyc is synthesized in the proliferating cells posterior and anterior to the morphogenetic furrow, but not in the cell cycle-delayed cells of the morphogenetic furrow. In antennal discs, dmyc mRNA is mainly detected in the central ring of proliferating cells. In the leg disc, endogenous dmyc is expressed around the middle of the disc, with the central cells lacking dmyc activity. Maternal transcripts are detected in the nurse cell cytoplasm of the adult ovary, and are subsequently dumped into the oocyte, and can be detected in early embryos. Zygotically derived transcripts can be detected during preblastoderm, with the highest level of dmyc mRNA in the anterior and posterior termini. At later stages, dmyc mRNA can be detected in the presumptive mesoderm along the ventral midline. During germ band extension, dmyc expression intensifies in the mesoderm and endoreplicating cells of the midgut, where expression continues until mid-embryogenesis. The majority of developmental genes achieve patterning via large noncoding regulatory regions containing numerous cis-regulatory elements and other diverse regulatory sequences. Thus, this study set out to determine whether the pattern of dmyc expression might be similarly regulated. First, using computational comparative searches of the 40-kb region spanning the dmyc gene, multiple conserved sequence blocks were detected. Subsequent analysis of the dmy-clacZ reporter constructs, containing all of the conserved sequence blocks, suggested that they were transcriptionally active and generated similar patterns of reporter activity as that described for the endogenous dmyc-lacZ enhancer trap (Kharazmi, 2012).

Dissection of the dmyc promoter using constructs spanning defined domains of the dmyc gene revealed the regions likely required for tissue-specific patterning of dmyc transcription. For instance, the lacZ reporter for the far upstream 8-kb fragment produced an expression pattern restricted to late embryogenesis in body segments and in presumptive neuromuscular tissues. In silico analysis revealed possible regulatory motifs in this region, including core promoter elements and conserved sequence blocks. Regulation by these cis elements may be required during embryogenesis, where dMyc is required to specify neuronal fate and facilitate neuroblast proliferation and in control of mesodermal fate determination. In light of this finding, further analysis of dmyc transcriptional regulation in this region in response to developmental signals will be of great interest (Kharazmi, 2012).

Analysis of the 5′ dmyc-lacZ deletion construct, containing intron 1, the 5′-UTR, and 100 bp upstream of the predicted transcription start site, revealed that this minimal region was sufficient to give reporter activity in a dmyc-like pattern in both ovarian nurse cells and in the embryo, but not in larval tissues. Therefore, the region extending from nucleotide 100 upstream of the 5′-UTR to nucleotide +187 was inspected for initiator consensus sequences. In most mammalian protein-coding genes, there is a TATA box located 25–30 bp upstream of the transcription start site, an initiator element (Inr) overlapping the start site, and/or a GC-box (SP1 binding site) 60–100 nucleotides upstream of the transcription start site. Experiments with vertebrate cell lines and Drosophila embryonic extracts have revealed strict conservation of the Inr consensus sequence, Py Py A+1 N T/A Py Py among vertebrates and invertebrates (Kharazmi, 2012).

Analysis of J7 (the region 1914 bp upstream of the predicted translation start site) and the expressed sequence tag (ESTGM01143; beginning at 1812 bp upstream of the translation start) revealed that the 102 bp sequence between the 5′ end of the expressed sequence tag and the 5′ end of the J7 genomic sequence contains a perfect Inr consensus sequence, a TATA box and a GC box. It was named the GC box/TATA box/Inr promoter 1. The TATA box is located 39 bp upstream of A+1 in the initiator element. Previous reports have shown that occurrence of a TATA box 25–30 bp upstream of the Inr in the same core promoter leads to cooperation between the two elements to enhance promoter strength. Although the distance of 39 bp in the dmyc promoter is on the edge of this optimum, cooperation between the TATA box and the Inr element has been shown to extend up to 90 bp in yeast promoters. Together, this suggests that the predicted regulatory elements (TATA box, Inr, and GC box) correspond to the cis-regulatory elements that may be responsible for correct developmental dmyc expression in larval tissue, the embryo, and the ovary. In addition to the TATA box, Inr, and GC box, two putative TATA boxes (TATA2, TATA3), two GC boxes (GC2, GC3), and two Inr elements (Inr2, Inr3), were identified 180 bp downstream of the expressed sequence tag start site. The putative Inr2 element shows one deviation from the Inr consensus sequence at position +4, but the critical positions for Inr activity are +1 and +3, and single bp substitutions at the −2, −1, +4, and +5 positions can still produce an active Inr suggesting the second Inr element might be functional. The putative Inr3 element shows no deviation from the Inr consensus sequence, suggesting that Inr3 might be as functional as Inr1. TATA box 2 is located 57 bp upstream of A+1 in the Inr2 element and TATA box 3 is located 55 bp upstream of A+1 in the Inr 3 element, both distances less than 90 bp in yeast promoters (Kharazmi, 2012).

Most developmentally expressed genes contain separable cis regulatory units, which allow patterned expression for tissue-specific roles. Indeed, previous work has suggested that both Drosophila myc transcription is also regulated via intronic promoter sequences. In support of these findings, it was demonstrated that the J8 transgene, which contains just the intron 2 sequence of the dmyc gene, results in lacZ reporter activity in all tissues examined. Thus Inr and downstream promoter elements, a sequence motif common to all Drosophila downstream promoters in this region, were sought. Most protein-coding genes of Drosophila contain a downstream core promoter element that functions cooperatively with an initiator to facilitate the binding of transcription factors in the absence of a TATA box. A search for consensus Drosophila Inr (T-C-A+1-G/T-T-T/C) and downstream core promoter elements (G-A/T-C-G) using DNASTAR Lasergene 9.1, GeneQuest module, revealed the presence of downstream promoter sequence motifs comparable with the Drosophila consensus Inr/downstream core promoter element. Sequence motifs were typed in GeneQuest “type in pattern function” and searched for with a threshold of 100%. The Inr and downstream promoter element motifs at the 3′ end of the intron 2 DNA sequence met all the strict criteria for such elements, in that the Inr sequence motif (T-C-A+1-T-T-C) does not deviate from the consensus, the downstream core promoter element (G-G-T-C-G) is identical to the core consensus, the spacing between the downstream core promoter element and the Inr (34 nucleotides) is appropriate, and a G nucleotide is correctly positioned between the Inr and the downstream core promoter element (Kharazmi, 2012).

Post-transcriptional 3′ end formation or polyadenylation of the mRNA precursor is a crucial step in mRNA maturation, in which most eukaryotic mRNAs acquire a poly (A) tail at their 3′ ends to promote transcription termination transport of the mature mRNA from the nucleus, and to enhance the translation and stability of mRNA. Analysis of the entire dmyc 3′-UTR for polyadenylation signals and polyadenylation sites revealed three potential consensus sequences, i.e., poly (A)1, poly (A)2, and poly (A)3, which are all capable of terminating transgene transcription. In addition, the 4.362-kb DNA sequence upstream of poly (A)1 at the dmyc 3′ end leads to reporter activity in the pattern predicted for dmyc expression. Therefore, the dmyc gene would be predicted to produce different transcripts with shorter and longer lengths, consistent with the previous analysis of dmyc mRNA in which genomic probes derived from this region revealed three alternative transcripts. Comparative analysis of the 3′ end of dmyc across 12 sequenced Drosophila species revealed multiple conserved sequence blocks in this region. Given that c-myc is regulated at the level of mRNA stability via conserved sequences in its 3′-UTR, it will be of interest in the future to determine whether the stability of dmyc transcripts depends upon the presence of regulatory domains in its 3′-UTR. The conserved sequence blocks may contain potential microRNA target sites to serve for posttranscriptional modifications, as is the case for the majority of developmental control genes. Indeed, dMyc has an active role in microRNA biology, although regulation of c-myc by microRNAs has been reported, the evidence for direct regulation of dmyc requires investigation. This work provides a starting point for investigating the putative microRNA binding sites and the mechanisms for the interactions between these motifs and their targets (Kharazmi, 2012).

Because c-Myc is a potent mitogen, the level of c-myc transcription must be tightly regulated. Myc transcription responds to developmental signaling molecules, which are likely to modulate the complement of a wide variety of transcription factors at the myc promoter. The evidence presented in this work reinforces the idea that dmyc represents a tightly and dynamically regulated gene. Further genetic studies combined with genomic approaches will be required to identify the molecular mechanism controlling dmyc transcription via the regulatory elements identified here (Kharazmi, 2012).

Transcriptonal Regulation

Molecules involved in cell adhesion can regulate both early signal transduction events, triggered by soluble factors, and downstream events involved in cell cycle progression. Correct integration of these signals allows appropriate cellular growth, differentiation and ultimately tissue morphogenesis, but incorrect interpretation contributes to pathologies such as tumor growth. The Fat cadherin is a tumor suppressor protein required in Drosophila for epithelial morphogenesis, proliferation control and epithelial planar polarization, and its loss results in a hyperplastic growth of imaginal tissues. While several molecular events have been characterized through which fat participates in the establishment of the epithelial planar polarity, little is known about mechanisms underlying fat-mediated control of cell proliferation. Evidence is provided that fat specifically cooperates with the epidermal growth factor receptor (EGFR) pathway in controlling cell proliferation in developing imaginal epithelia. Hyperplastic larval and adult fat structures indeed undergo an amazing, synergistic enlargement following to EGFR oversignalling. Such a strong functional interaction occurs downstream of MAPK activation through the transcriptional regulation of genes involved in the EGFR nuclear signalling. Considering that fat mutation shows di per se a hyperplastic phenotype, a model is suggested in which fat acts in parallel to EGFR pathway in transducing different cell communication signals; furthermore its function is requested downstream of MAPK for a correct rendering of the growth signals converging to the epidermal growth factor receptor (Garoia, 2005).

The results shown in this paper suggest that the interaction between ft and EGFR takes place at the proliferation level, while differentiation signals controlled by the EGFR pathway appear unaffected. With the aim to find some mechanisms that could explain the synergic phenotype of ft and EGFR mutations, the transcriptional levels of yan, dmyc and pnt, genes involved in proliferation control whose function is regulated by the EGFR cascade, were studied in ft and wild-type imaginal tissues. The results of semi-quantitative RT-PCR trials showed in ft tissues an increase of the transcription levels of yan and dmyc, whereas pnt was unaffected. The Dmyc transcription factor, the unique Drosophila homologue of the Myc family of proto-oncogenes, plays a central role in the control of cell growth in Drosophila. Overexpression of ras is capable to increase post-transcriptionally the Dmyc protein levels, promoting the G1-S transition via the increase of CycE translation. The increase in the Dmyc levels, however, affects growth rate but not proliferation, since the shortening of the G1 phase is balanced by the compensatory lengthening of G2, resulting in an increase in cell size but not in cell number. ft mutation otherwise induces an increase of cell proliferation without altering the cell size. Taken together, these results indicate that ft mutation affects not only the G1-S transition via Dmyc but also the G2-M transition, since the coordinated stimulation of the two cell-cycle checkpoints is necessary to increase the proliferation rate in Drosophila imaginal discs. Interestingly, the transcription level of pnt was unaffected in ft mutant discs. pnt is an ETS transcriptional activator that plays a central role in the mitosis control mediated by the EGFR signalling cascade; several studies however suggest the presence of additional Pnt-independent effectors in EGFR-mediated mitosis control. The ft control of the G2–M transition may involve EGFR effectors other than pnt, or molecules functioning through different signalling pathways. The yan gene is another component of the ETS transcriptional regulator family involved in the EGFR signalling. Phosphorylation by MAPK affects stability and subcellular localization of Yan, resulting in a rapid down-regulation of its activity. Yan functions as a fairly general inhibitor of differentiation, allowing both neuronal and non-neuronal cell types to choose between cell division and differentiation in multiple developmental contexts and recent studies indicate that the mammalian homologue of the Drosophila yan, TEL, is overexpressed in tumors. In the Drosophila developing eye yan is expressed in all undifferentiated cells and is down regulated as cells differentiate, so a high yan activity in ft mutant discs is correlatable with the observed proliferative advantage of ft cells (Garoia, 2005).

There are several indications that EGFR signalling can trigger different responses by different activity levels: in the Drosophila eye disc, differentiation requires high signalling levels, whereas lesser EGFR activity promotes mitosis and protects against cell death. These findings indicate that EGFR signalling may coordinate partially independent processes, transferring graded activity to the nucleus, rather than triggering 'all or none' responses. The simultaneous increase of activity in both growth promoters (dmyc) and differentiation repressors (yan) in ft mutant imaginal discs suggests the presence of a mechanism that shifts the EGFR nuclear equilibrium towards a level insufficient to induce differentiation but adequate for promoting cell growth and proliferation (Garoia, 2005).

Repression of dMyc expression by Wingless promotes Rbf-induced G1 arrest in the presumptive Drosophila wing margin

Little is known about how patterns of cell proliferation and arrest are generated during development, a time when tight regulation of the cell cycle is necessary. In this study, the mechanism by which the developmental signaling molecule Wingless generates G1 arrest in the presumptive Drosophila wing margin is examined in detail. Wg signaling promotes activity of the Drosophila retinoblastoma family (Rbf) protein, which is required for G1 arrest in the presumptive wing margin. Wg promotes Rbf function by repressing expression of the G1-S regulator Drosophila myc (dmyc). Ectopic expression of dMyc induces expression of Cyclin E, Cyclin D, and Cdk4, which can inhibit Rbf and promote G1-S progression. Thus, G1 arrest in the presumptive wing margin depends on the presence of Rbf, which is maintained by the ability of Wg signaling to repress dmyc expression in these cells. In addition to advancing the understanding of how patterned cell-cycle arrest is generated by the Wg signaling molecule during development, this study indicates that components of the Rbf/E2f pathway are targets of dMyc in Drosophila. Although Rbf/E2f pathway components mediate the ability of dMyc to promote G1 progression, dMyc appears to regulate growth independently of the RBF/E2f pathway (Duman-Scheel, 2004).

This investigation examines the mechanism by which Wg signaling promotes G1 arrest in the presumptive Drosophila wing margin. It was postulated that Rbf might mediate the ability of Wg to induce G1 arrest, since loss of Wg signaling promotes expression of dE2f1 target genes. Overexpression of Rbf can block this induction of dE2f1 target gene expression. Strikingly, loss of Rbf in the zone of nonproliferating cells (ZNC) prevents G1 arrest, as evidenced by ectopic BrdUrd incorporation in Rbf mutant clones. This requirement for Rbf in the ZNC is notable. Surprisingly few developing fly tissues display such an absolute requirement for Rbf to promote G1 arrest. To date, Rbf has been shown to be required to limit DNA replication in the embryo and in the ovary. However, in many tissues, loss of Rbf does not result in ectopic S phase; a likely explanation for this finding is that in other developing tissues, Rbf may function as one of several redundant mechanisms that function to promote G1 arrest. Such redundancy would help to ensure that the cell cycle is regulated tightly during development (Duman-Scheel, 2004).

In an attempt to better understand the mechanism by which Wg promotes Rbf function, this investigation uncovered interactions between dMyc and components of the Rbf/E2f pathway. Wg signaling normally inhibits dMyc expression in the ZNC. Ectopic expression of dMyc in the ZNC can induce expression of dE2f1 target genes, which can be blocked by the addition of Rbf-280 (a constitutively active form of Rbf). Thus, overexpression of dMyc, which results from loss of Wg signaling in the ZNC, must somehow inactivate Rbf. These data indicate that inhibition of dMyc expression in the ZNC is critical for Rbf function (Duman-Scheel, 2004).

The results indicate why exclusion of dMyc from the ZNC is necessary for Rbf activity. Overexpression of dMyc leads to high levels of Cyclin E, Cyclin D, and Cdk4 transcripts. dMyc also regulates Cyclin E posttranscriptionally in Drosophila. G1-S Cyclins/Cdks function to phosphorylate and inhibit Rbf, suggesting that dMyc blocks Rbf activity through activation of G1-S Cyclins/Cdks. Thus, inhibition of dMyc by Wg helps to ensure that G1-S Cyclins/Cdks do not activate S phase. This idea is supported by the results that indicate that only a combination of both Dap and constitutively active Rbf (that cannot be regulated by Cyclins/Cdks) can restore G1 arrest when Wg signaling is blocked or when dMyc is expressed. These data suggest that either Cyclin D or Cyclin E activity can mediate the ability of dMyc to promote S phase in the ZNC. Coexpressing Dap alone with dMyc, which would block only Cyclin E/Cdk2 activity, does not restore G1 arrest. Furthermore, overexpression of dMyc in a cdk4 mutant background still results in ectopic S phases, suggesting that Cyclin E/Cdk2 also are sufficient to mediate dMyc's ability to promote G1 progression. Thus, either Cyclin D/Cdk4 or Cyclin E/Cdk2 is sufficient to mediate the ability of dMyc to promote G1 progression. The ability of Wg to inhibit dMyc expression is thus critical for RBF activation and G1 arrest in the ZNC. Still, it is possible that Wg promotes G1 arrest through other mechanisms that have not yet been uncovered. The observation that overexpression of a dominant-negative form of dTCF (dTCFDeltaN) with C96>Gal4 can promote S phase, even in a dmyc mutant background, supports this idea (Duman-Scheel, 2004).

It is likely that dMyc/dMax directly up-regulate transcription of Cyclin D and cdk4 in Drosophila. Myc/Max heterodimers regulate transcription by binding to various consensus sequences, such as the E box. Previous studies indicated that cMyc induces Cyclin D2 expression in mice by binding to two consensus E boxes in the Cyclin D2 promoter. cdk4 also was identified as a transcriptional target of c-Myc. Furthermore, it has been suggested that cdk4 is a transcriptional target of dMyc and Cyclin D is a transcriptional target of dMax. Although future studies should analyze the Drosophila Cyclin D and Cdk 4 regulatory regions in more detail, these results suggest that the observed ability of dMyc to induce Cyclin D and Cdk4 expression in the ZNC most likely occurs through transcriptional regulation of these proteins by dMyc/dMax. In contrast, Cyclin E was not identified as a target of dMyc or dMax. It is more likely that the ability of dMyc to induce growth in the wing indirectly leads to increased Cyclin E transcript levels (Duman-Scheel, 2004).

Recent studies indicate that both dMyc and Rbf can regulate cellular growth in the Drosophila wing. dMyc induces cellular growth, whereas Rbf inhibits cellular growth and proliferation. dMyc can promote cellular growth in the presence of constitutively active Rbf, suggesting that dMyc can induce growth independently of the Rbf/E2f pathway. Such results are consistent with previous studies that indicate that Ras, which can induce growth by increasing levels of dMyc protein, also is capable of inducing growth in the presence of Rbf. It is likely that dMyc regulates growth through induction of genes encoding regulators of protein synthesis, such as ribosomal proteins and the DEAD-box helicase Pitchoune, as well as other proteins that regulate cellular metabolism (Duman-Scheel, 2004).

Wnt signaling is generally associated with the stimulation of cell proliferation during development and in tumor cells. However, in the ZNC, Wnt/Wg signaling actually promotes cell-cycle arrest. Ironically, in the ZNC, Wg signaling suppresses expression of dmyc; however, a cMyc reporter was found to be directly up-regulated by Tcf4 in a colon carcinoma cell line. Thus, Wg appears to be able to up-regulate Myc expression in some tissues and to repress it in others (Duman-Scheel, 2004).

The ability of Wg signaling to either activate or repress the same target gene in different situations has been observed in other cases. For example, in the developing Drosophila midgut, low levels of Wg signaling, in conjunction with Dpp, stimulate expression of Ubx and lab; high levels of Wg signaling result in the repression of Ubx and lab by means of the transcriptional repressor Teashirt. Thus, expression of Wnt target genes can be turned on or off in response to the modulation of Wg levels as well as by the presence or absence of the various proteins that can regulate transcription in conjunction with, or in response to, Wg signaling. Such flexibility is advantageous to a developing organism (Duman-Scheel, 2004).

Wg signaling can be modulated to affect expression of the same target gene differently in various situations. Moreover, Wg signaling can be modulated to promote or inhibit the different, somewhat conflicting cellular processes of patterning, growth, proliferation, and differentiation. The same is true for Hh signaling, which also regulates all of these cellular processes. Thus, it seems, at least in the case of Hh and Wg, that one signaling molecule can regulate many different types of cellular and developmental events. In order for various cellular programs to be implemented and coordinated during development, the way that a particular cell type responds to Wg or Hh signaling at any given time must be tightly regulated. The delicate balance between various processes that can occur in response to Hh or Wg signaling is likely maintained through tight control of the temporal and spatial expression patterns of Hh and Wnt targets and the molecules that regulate them (Duman-Scheel, 2004).

Polycomb mediates Myc autorepression and its transcriptional control of many loci in Drosophila

Aberrant accumulation of the Myc oncoprotein propels proliferation and induces carcinogenesis. In normal cells, however, an abundance of Myc protein represses transcription at the c-myc locus. Cancer cells often lose this autorepression. This study examined the control of myc in Drosophila and show here that the Drosophila ortholog, dmyc, also undergoes autorepression. The developmental repressor Polycomb (Pc) is required for dmyc autorepression, and this Pc-dMyc-mediated repression spreads across an 875-kb region encompassing the dmyc gene. To further investigate the relationship between Myc and Polycomb, microarrays were used to identify genes regulated by each, and a striking relationship was identified between the two: A large set of dMyc activation targets is normally repressed by Pc, and 73% of dMyc repression targets require Pc for this repression. Chromatin immunoprecipitation confirmed that many dMyc-Pc-repressed loci have an epigenetic mark recognized by Pc. These results suggest a novel relationship between Myc and Polycomb, wherein Myc enhances Polycomb repression in order to repress targets, and Myc suppresses Polycomb repression in order to activate targets (Goodliffe, 2006).

The first Myc-regulated gene ever identified was c-myc itself. The mechanism of autorepression has remained elusive, and the present study offers new insight into this feedback regulatory loop. myc autorepression is conserved from mammals to flies and that it requires the Pc complex. The myc autoregulation loop is frequently disrupted in cancer cells, and furthermore, it has been suggested that gene repression correlates better with Myc biological activity than does gene activation. The data suggest that autorepression and general repression by Myc are mediated by the same mechanism and that both are dependent on the PcG. Indeed, dMyc repressed genes have the hallmark chromatin modification of Pc-repressed genes. Members of the PcG have previously been implicated in cancer, including Bmi-1 (homologous to Psc), which cooperates with Myc in lymphomagenesis and represses expression of the p16 CDK inhibitor. However, no previous connection has been made between general Myc-mediated repression and the PcG. The large chromosomal domain surrounding the dmyc locus that is repressed in concert with dmyc itself is consistent with a PcG-mediated mechanism, since repression by Pc is known to act over long distances. Interestingly, repression within this domain is not absolute, since some interspersed genes can resist repression or even be activated. The possibility cannot be excluded that each of the genes in the domain is independently repressed by elevated dMyc expression, but their proximity to dmyc itself seems more consistent with a regional effect (Goodliffe, 2006).

An unexpected outcome of these studies was the striking observation that one-third of the genes that score as dMyc-activated in early stage embryos were also scored as repressed by Pc, since ablation of Pc by RNAi activated the genes to a similar extent as transgenic dmyc overexpression. Similarly, approximately one-half of the Pc repressed genes were also activated by transgenic dmyc overexpression. The overlap in these two gene sets is statistically highly significant and suggests a mechanistic overlap in the gene response. Since dmyc overexpression was provided via transgene, whereas ablation of Pc was achieved by RNAi, the overlap in gene response is unlikely to be a consequence of experimental manipulation. It has not yet been determined if this response is a direct effect of either dMyc or Pc binding to the corresponding genes. Nevertheless, the microarray data suggest that, at the minimum, the two pathways converge on a common cellular network (Goodliffe, 2006).

For both dMyc-activated and -repressed genes, the Polycomb complex provides an essential context for Myc regulation, but the direction of that regulation depends on Myc itself and the nature of its interaction with a particular target. In the simplest view, Myc repression might work by enhancing Pc's generally negative effects on transcription, whereas it appears to activate other genes by opposing those same effects (Goodliffe, 2006).

The steroid hormone ecdysone controls systemic growth by repressing dMyc function in Drosophila fat cells

How steroid hormones shape animal growth remains poorly understood. In Drosophila, the main steroid hormone, ecdysone, limits systemic growth during juvenile development. This study showed that ecdysone controls animal growth rate by specifically acting on the fat body, an organ that retains endocrine and storage functions of the vertebrate liver and fat. This study demonstrates that fat body-targeted loss of function of the Ecdysone receptor (EcR) increases dMyc expression and its cellular functions such as ribosome biogenesis. Moreover, changing dMyc levels in this tissue is sufficient to affect animal growth rate. Finally, the growth increase induced by silencing EcR in the fat body is suppressed by cosilencing dMyc. In conclusion, the present work reveals an unexpected function of dMyc in the systemic control of growth in response to steroid hormone signaling (Delanoue, 2010).

The growth rate and the duration of juvenile growth are two key parameters that determine the size of the animal at the time of maturation. These two parameters are coupled during the juvenile period to determine organismal size at maturation by mechanisms that are not yet understood. Recent work has established that the two hormonal systems controlling these parameters, ecdysone and insulin/IGF, have antagonistic actions that set up the larval growth rate. The present study demonstrates that the fat body is the unique relay for ecdysone-induced growth inhibition, and provides molecular and genetic evidence that inhibition of dMyc function by ecdysone signaling in fat cells plays a key role in this control (Delanoue, 2010).

Paradoxically, a positive, cell-based role for ecdysone was observed in growth and proliferation during larval stages. Indeed, clonal loss of function for EcR induces a reduction of cell size despite an increase in PI3K activity and dMyc expression. This indicates that ecdysone signaling is autonomously required for optimal cell growth, despite its negative action on the growth rate at the systemic level. These results are in line with previous studies indicating that ecdysone/EcR/Usp and some of their downstream targets are required for cell-cycle progression and tissue growth. Ex vivo culture experiments using dissected discs from Bombyx mori have shown that an optimal concentration of 20E is required for proper disc growth that is 6- to 10-fold lower than the 20E concentration required to stimulate the molting cycle. This suggests that different ecdysone concentrations are required for cell-autonomous growth induction and nonautonomous growth inhibition. It is likely that elevated 20E levels like those attained at the end of larval development are required for the inhibition of dMyc expression in the fat body, leading to systemic growth inhibition (Delanoue, 2010).

These data indicate that the fat body is central in the regulation of organismal growth by ecdysone. Previous studies have established that this organ acts as a nutrient sensor and coordinates global growth according to nutrition conditions. Therefore, it appears positioned at a crossroad, allowing extrinsic and intrinsic inputs to be integrated and translated into a coordinated control of body growth. These results indicate that dMyc, but not IIS, is required in the fat body for transducing the growth effects of EcR signaling. Previous work showed that TOR signaling, and not IIS, controls the nutrition sensor that operates in fat body cells. Although not required in fat cells for either of these controls, IIS is downregulated in peripheral tissues, and this regulation contributes to the systemic growth inhibition observed under both conditions. This suggests that the fat body remotely controls the expression/production/secretion/activity of the circulating Dilps that activate dInR. It was recently demonstrated that under conditions of abundant nutrients, the fat body emits a 'secretion signal' that triggers the release of Dilps from brain cells (Géminard, 2009). The molecular links proposed between TOR signaling and dMyc position dMyc as a downstream effector of TOR signaling, playing a role in the transcriptional activation of target genes. This and the current data suggest the possibility that both nutrition- and ecdysone-induced signals converge on a Myc-dependent mechanism in fat cells leading to the systemic regulation of growth by general IIS (Delanoue, 2010).

Both dMyc and TOR signaling control ribosome biogenesis and protein translation initiation. In addition to its role as an energy reservoir, the fat body, like the mammalian liver, is one of the most active secreting tissues, and a large majority of the hemolymph proteins are synthesized in this tissue. This secreting function is thought to be essential for larval growth and is likely to be sensitive to ribosome quantity. Therefore, the fat body could control systemic growth either via the production of specific secreted factors according to ribosomal abundance, or via the control of hemolymph protein concentration that would in turn regulate growth. The current studies should help discriminate between these different hypotheses (Delanoue, 2010).

It was observed that altering EcR signaling in the fat body markedly modifies the activities of many IIS signaling actors such as PI3K, AKT, and dFoxO. This study demonstrates that this regulation does not contribute to the control of organismal growth, suggesting that the function of 20E-induced IIS repression at the end of larval development is restricted to metabolic effects, such as the arrest of carbohydrate and lipid storage, and to the induction of autophagy (Delanoue, 2010).

Changing ecdysone circulating level influences dMyc expression in the fat body but not in any other larval tissues. Therefore, the EcR-dependent control of dMyc expression is specific to this tissue. No consensus binding sites were detected for EcR/Usp in the dMyc promoter region. This suggests that dMyc is not a direct target of EcR-mediated gene repression, but rather that EcR signaling controls expression of a fat-specific downstream component that is itself responsible for adipose dMyc transcriptional regulation (Delanoue, 2010).

Myc has been recently shown to promote oxidative phosphorylation as well as glycolysis through coordinate transcriptional control of the mitochondrial metabolic network (Zhang, 2007). This metabolic regulation by dMyc has not been studied in the present work, but it is conjectured that rising ecdysone levels at the end of the juvenile period influence both translational and metabolic activities regulated by dMyc in the fat body, a regulation that could have important consequences on the energy homeostasis of the animal during this important developmental transition (Delanoue, 2010).

In conclusion, the present work reveals an unexpected role for dMyc in the systemic control of growth. It was demonstrated that the fat body-specific activity of dMyc is the target of EcR signaling and is involved in a remote regulation of growth through IIS in peripheral tissues (Delanoue, 2010).

Ecdysone receptor (EcR) suppresses lipid accumulation in the Drosophila fat body via transcription control

Lipid metabolism drastically changes in response to the environmental factors in metazoans. Lipid is accumulated at the food rich condition, while mobilized in adipocyte tissue in starvation. Such lipid mobilization is also evident during the pupation of the insects. Pupation is induced by metamorphosis hormone, ecdysone via ecdysone receptor (EcR) with lipid mobilization, however, the molecular link of the EcR-mediated signal to the lipid mobilization remains elusive. To address this issue, EcR was genetically knocked-down selectively in 3rd instar larva fat body of Drosophila, corresponding to the adipocyte tissues in mammalians, that contains adipocyte-like cells. In this mutant, lipid accumulation was increased in the fat body. Lipid accumulation was also increased when knocked-down of taiman, which served as the EcR co-activator. Two lipid metabolism regulatory factor, E75B and adipose (adp) as well as cell growth factor, dMyc, were found as EcR target genes in the adipocyte-like cells, and consistently knock-down of these EcR target genes brought phenotypes in lipid accumulation supporting EcR function. These findings suggest that EcR-mediated ecdysone signal is significant in lipid metabolism in insects (Kamoshida, 2012).

Regulation of cell growth by Notch signaling and its differential requirement in normal vs. tumor-forming stem cells in Drosophila

Cancer stem cells (CSCs) are postulated to be a small subset of tumor cells with tumor-initiating ability that shares features with normal tissue-specific stem cells. The origin of CSCs and the mechanisms underlying their genesis are poorly understood, and it is uncertain whether it is possible to obliterate CSCs without inadvertently damaging normal stem cells. This study shows that a functional reduction of eukaryotic translation initiation factor 4E (eIF4E) in Drosophila specifically eliminates CSC-like cells in the brain and ovary without having discernable effects on normal stem cells. Brain CSC-like cells can arise from dedifferentiation of transit-amplifying progenitors upon Notch hyperactivation. eIF4E is up-regulated in these dedifferentiating progenitors, where it forms a feedback regulatory loop with the growth regulator dMyc to promote cell growth, particularly nucleolar growth, and subsequent ectopic neural stem cell (NSC) formation. Cell growth regulation is also a critical component of the mechanism by which Notch signaling regulates the self-renewal of normal NSCs. These findings highlight the importance of Notch-regulated cell growth in stem cell maintenance and reveal a stronger dependence on eIF4E function and cell growth by CSCs, which might be exploited therapeutically (Song, 2011).

The differential cell growth rates observed between ectopic NBs and normal or primary NBs and the correlation between cell growth defects and NB fate loss prompted a test of whether slowing down cell growth might selectively affect the formation of ectopic NBs. Attenuation of TOR signaling, a primary mechanism of cell growth regulation, through NB-specific overexpression of TSC1/2, a strong allele of eIF4E antagonist 4EBP [4EBP(LL)s], or a dominant-negative form of TOR (TOR.TED) all partially suppressed ectopic NB formation in α-adaptin (ada) mutants without affecting normal or primary NBs. Interestingly, RNAi-mediated knockdown of eIF4E, a stimulator of oncogenic transformation and a downstream effector of TOR signaling, showed a better suppression than manipulating other TOR pathway components, suggesting that eIF4E might play a more important role in ectopic NB formation. Strikingly, the brain tumor phenotypes caused by overactivation of N signaling - as in lethal giant larvae (lgl) mutant, aPKCCAAX overexpression, or N overexpression conditions - were also fully suppressed by eIF4E knockdown. Furthermore, the brain tumor phenotypes of brat mutants were also completely rescued by eIF4E RNAi (Song, 2011).

In contrast, normal NB formation or maintenance was not affected by eIF4E knockdown. NBs with eIF4E knockdown remained highly proliferative, as evidenced by the mitotic figures, and displayed relatively normal apical basal cell polarity. There are several other eIF4E-like genes in the fly genome (Hernandez, 2005), which may play partially redundant roles in normal NB maintenance. eIF4E knockdown appeared to specifically block ectopic NB formation caused by the dedifferentiation of IPs in type II NB lineages, since it did not affect ectopic type I NB formation in cnn or polo mutants that are presumably caused by symmetric divisions of type I NBs. In addition, cell fate transformation induced by N overactivation in the SOP lineage was not affected by eIF4E RNAi, supporting the idea that eIF4E is particularly required for type II NB homeostasis. Supporting the specificity of the observed eIF4E RNAi effect, another eIF4E RNAi transgene (eIF4E-RNAi-s) also prevented ectopic NB formation. Moreover, a strong loss-of-function mutation of eIF4E also selectively eliminated ectopic NBs induced by N overactivation without affecting normal NBs, reinforcing the hypothesis that ectopic NBs exhibit higher dependence on eIF4E (Song, 2011).

To further support the notion that the ectopic NBs are particularly vulnerable to eIF4E depletion, a conditional expression experiment was carried out in which eIF4E-RNAi-s was turned on in brat mutants using the 1407ts system, after ectopic NBs had been generated. Whereas the brain tumor phenotype exacerbated over time in the brat mutants, 1407-GAL4-driven eIF4E-RNAi-s expression in brat mutants effectively eliminated ectopic NBs, leaving normal NBs largely unaffected (Song, 2011).

In normal type II NB lineage, eIF4E protein was enriched in the NBs. Ectopic NBs induced by N overactivation in ada mutants also expressed eIF4E at high levels, whereas spdo mutant NBs exhibited reduced eIF4E expression. Thus, eIF4E up-regulation correlates with N-induced ectopic NB formation in a dedifferentiation process that likely involves elevated cell growth (Song, 2011).

Given the coincidence of nucleolar size change with ectopic NB formation, the involvement of the growth regulator dMyc was tested. dMyc protein levels were up-regulated in normal or N overactivation-induced ectopic NBs, but were down-regulated in spdo mutant NBs. Furthermore, dMyc transcription, as detected with a dMyc-lacZ transcriptional fusion reporter, was also up-regulated in both normal and ectopic NBs in ada mutants. A previous study in Drosophila S2 cells identified dMyc as a putative N target. In vivo chromatin immunoprecipitation (ChIP) experiments were carried out to assess whether dmyc transcription is directly regulated by N signaling in NBs. Using chromatin isolated from wild-type larval brains and a ChIP-quality antibody against the N coactivator Suppressor of Hairless [Su(H)], specific binding was demonstrated of Su(H) to its putative binding sites within the second intron of dmyc (dmyc-A). No binding to an internal negative control region proximal to the first exon of dmyc (dmyc-B) or to the promoter region of the rp49 gene was detected. N signaling thus directly activates dMyc transcription in the NBs. Similar to eIF4E RNAi, knockdown of dMyc strongly suppressed ectopic NB formation induced by Brat or Ada inactivation or N overactivation. Intriguingly, the strong tumor suppression effect of eIF4E knockdown was partially abolished by dMyc overexpression. Furthermore, dMyc function, as reflected by its promotion of nucleolar growth in IPs, was attenuated by eIF4E RNAi, although eIF4E RNAi alone had no obvious effect. Different from the reported eIF4E regulation of Myc expression in mammalian cells (Lin, 2008), dMyc promoter activity or protein levels remained unaltered under eIF4E RNAi conditions, suggesting that eIF4E may modulate dMyc activity without altering its expression. One possibility is that eIF4E may enter the nucleus to interact with Myc and promote its transcriptional activity. To test this hypothesis, HEK293T cells were transfected with Flag-tagged human eIF4E alone or in combination with HA-tagged dMyc. Indeed, both Drosophila dMyc and endogenous human c-Myc specifically coimmunoprecipitated with human eIF4E from nuclear extracts, indicating a conserved interaction between eIF4E and Myc within the nuclei of proliferating cells. Consistent with these biochemical data, dMyc transcriptional activity within NBs, which could be monitored with an eIF4E-lacZ reporter, was drastically reduced upon eIF4E knockdown (Song, 2011).

In contrast, eIF4E transcription, as detected with an eIF4E-lacZ transcriptional fusion reporter, as well as eIF4E protein levels detected by immunostaining were up-regulated upon dMyc overexpression and down-regulated by dMyc RNAi. It is unlikely that the changes in eIF4E-lacZ activity were due to global increases or decreases in β-galactosidase (β-gal) translation caused by altered dMyc levels, since lacZ expression from a dMyc-lacZ reporter was unaffected under similar conditions. Furthermore, like dMyc protein, eIF4E-lacZ reporter expression was up-regulated in normal NBs or ectopic NBs in ada mutants, further supporting the notion that dMyc may up-regulate eIF4E transcription. Moreover, ChIP experiments using chromatins isolated from wild-type larval brains and a ChIP-quality antibody against dMyc demonstrated specific binding of dMyc to an eIF4E promoter region harboring a cluster of adjacent noncanonical E boxes, supporting a direct regulation of eIF4E transcription by dMyc. dMyc and eIF4E thus appeared to form a regulatory feedback loop that promoted NB growth and renewal. Consistent with this model, while knocking down either dMyc or eIF4E had no noticeable effect on type II NB maintenance and only a mild effect on NB nucleolar size in the case of dMyc RNAi, their simultaneous knockdown led to a significant reduction in nucleolar size, premature neuronal differentiation, and loss of NBs (Song, 2011).

If the dMyc-eIF4E axis of cell growth control is a crucial downstream effector of N signaling in regulating NB maintenance, its up-regulation might be able to rescue the type II NB depletion phenotype resulting from reduced N signaling. Indeed, the loss of NBs associated with reduced Notch signaling was preventable when cell growth was boosted by dMyc overexpression. Thus, while N-IR directed by 1407-GAL4 led to complete elimination of type II NBs, the coexpression of dMyc, but not CD8-GFP or Rheb, an upstream component of the TOR pathway, resulted in the preservation of approximately half of type II NBs with apparently normal cell sizes, cell fate marker expression, and lineage composition. A similar effect was observed when dMyc was coexpressed with N-IR using the conditional 1407ts system, with transgene expression induced at the larval stage. While both dMyc and Rheb promote cell growth, they do so through distinct mechanisms, with the former increasing nucleolar size and the latter expanding cytoplasmic volume. These results thus provide compelling evidence that control of cell growth, particularly nucleolar growth, is a critical component in the maintenance of NB identity by N signaling (Song, 2011).

The differential responses of normal and tumor-initiating stem cells to functional reduction of eIF4E prompted a test of whether chemicals that specifically inhibit eIF4E function might have therapeutic potential in preventing CSC-induced tumorigenesis. Indeed, the brain tumor phenotypes induced by N overactivation or ada loss of function were effectively suppressed by feeding animals with fly food containing Ribavirin, an eIF4E inhibitor that interferes with eIF4E binding to mRNA 5' caps and promotes the relocalization of eIF4E from the nucleus to the cytoplasm (Kentsis, 2004; Assouline, 2009) (Song, 2011).

The CSC hypothesis was initially developed based on studies in mammalian systems. Various studies have supported the notion that CSCs share many functional features with normal stem cells, such as signaling molecules, pathways, and mechanisms governing their self-renewal versus differentiation choice. However, the cellular origin of CSCs and the molecular and cellular mechanisms underlying their development or genesis remain poorly understood. It has been proposed that CSCs could arise from (1) an expansion of normal stem cell niches, (2) normal stem cells adapting to different niches, (3) normal stem cells becoming niche-independent, or (4) differentiated progenitor cells gaining stem cell properties. This study has showen that in the Drosophila larval brain, CSCs can arise from the dedifferentiation of transit-amplifying progenitor cells back to a stem cell-like state. Importantly, eIF4E was identified as a critical factor involved in this dedifferentiation process. More significantly, it was shown that reduction of eIF4E function can effectively prevent the formation of CSCs without affecting the development or maintenance of normal stem cells. This particular dependence on eIF4E function by CSCs appears to be a general theme, as reduction of eIF4E function also effectively prevented the formation of CSCs, but not normal GSCs, in the fly ovary. These findings may have important implications for stem cell biology and cancer biology, in terms of both mechanistic understanding and therapeutic intervention (Song, 2011).

This study also offers mechanistic insights into the cellular processes leading to the dedifferentiation of progenitors back to stem cells. In Drosophila type II NB clones with overactivated N signaling, ribosome biogenesis within ectopic NBs appears to be faster than in normal NBs, as shown by the fact that the ratio of nucleolar to cellular volume of the ectopic NBs is approximately fivefold higher than that of normal NBs. The faster growth rate is accompanied by the up-regulation of dMyc and eIF4E and appears to be essential for transit-amplifying progenitors to undergo complete dedifferentiation back to a stem cell-like state. When the function of cell growth-promoting factors such as eIF4E is attenuated, the faster cell growth of ectopic NBs can no longer be sustained and the dedifferentiation process stalls. As a result, brain tumor formation caused by uncontrolled production of ectopic NBs is suppressed. In contrast, normal NBs, which presumably have relatively lower requirements for cell growth and hence eIF4E function, maintain their stem cell fate and development under similar conditions. Therefore, a potential key to a successful elimination of CSC-induced tumors would be to find the right level of functional reduction in eIF4E, which causes minimal effects on normal stem cells but effectively obliterates CSCs. An ongoing clinical trial with Ribavirin in treating acute myeloid leukemia (AML) (Assouline, 2009), a well-characterized CSC-based cancer, demonstrated exciting proof of principle that such a strategy is feasible. The current version of Ribavirin, however, has certain limitations, such as its poor specificity and the high dosage (micromolar range) required for effective treatment. Thus, more specific and effective eIF4E inhibitors are urgently needed. The drug treatment experiments with Ribavirin validated Drosophila NBs as an excellent CSC model for searching further improved drugs. More importantly, the nuclear interaction between eIF4E and Myc unraveled by this biochemical analysis not only provides a new mechanistic explanation for the synergistic effects of eIF4E and Myc in tumorigenesis (Ruggero, 2004; Wendel, 2007), but also sheds new light on how to rationally optimize drug design and therapy for treating CSC-based cancer (Song, 2011).

The results offer new information on how N signaling helps specify and maintain NSC fate. N signaling regulates stem cell behavior in various tissues of diverse species. However, it remains unclear how differential N signaling determines distinct cell fate within the stem cell hierarchy. This study demonstrates that N signaling maintains Drosophila NSC fate at least in part through promoting cell growth. The following evidence supports that cell growth, but not cell fate, change is the early and primary effect of N signaling inhibition in type II NBs: (1) Pros expression is not immediately turned on in spdo mutant NBs with reduced cell sizes. Instead, it gradually increases during the course of spdo mutant NB divisions. (2) Up-regulation of Pros is not the cause of stem cell fate loss in spdo mutant NBs, as shown by spdo pros double-mutant analysis. (3) Cell growth defects precede the up-regulation of Ase expression in aph-1 mutant NBs. (4) Promotion of cell growth, and particularly nucleolar growth, by dMyc is sufficient to prevent NB loss caused by N inhibition. At the molecular level, N signaling appears to regulate the transcription of dMyc, which in turn up-regulates the transcription of eIF4E. Such a transcriptional cascade and feedback regulation of dMyc activity by eIF4E may help to sustain and amplify the activity of the Notch-dMyc-eIF4E molecular circuitry. Hence, differential N signaling within the lineage can lead to different cell growth rates, which partially determine differential cell fates. Consistent with this notion, knockdown of both eIF4E and dMyc results in defects of NB cell growth and loss of stem cell fate (Song, 2011).

While many signaling pathways and molecules have been implicated in the maintenance of stem cell identity, the question of how a stem cell loses its 'stemness' at the cellular level remains poorly understood. A stem cell may lose its stem cell fate by undergoing a symmetric division to yield two daughter cells that are both committed to differentiation or through cell death. Earlier studies provided intriguing hints that cell growth and translational regulation could influence stem cell maintenance in the Drosophila ovary. This study usded detailed clonal analyses of NSCs over multiple time points to provide direct evidence that a NSC with impaired N signaling will gradually lose its identity due to a gradual slowing down of cell growth and loss of cell mass. Remarkably, such loss of stem cell fate can be prevented when cell growth is restored by dMyc, but not Rheb, overexpression, demonstrating the functional significance of regulated cell growth, particularly nucleolar growth, in stem cell maintenance. More importantly, this information offers clues on how to specifically eliminate tumor-initiating stem cells. These studies suggest that a stem cell, normal or malignant, has to reach a certain growth rate in order to acquire and maintain its stemness, presumably because when the stem cell grows below such a threshold, its proliferative capacity becomes too low, whereas the concentration of differentiation-promoting factors becomes too high to be compatible with the maintenance of stem cell fate. Consistent with this notion are the strong correlation between the expression of ribosomal proteins and cellular proliferation (van Riggelen, 2010) as well as the correlation between the reduction of NB sizes and the up-regulation of differentiation-promoting factor Pros or Ase in different developmental contexts (Song, 2011).

The results also provide new insights into how the evolutionarily conserved tripartite motif and Ncl-1, HT2A, and Lin-41 (TRIM-NHL) domain proteins regulate stem cell homeostasis. The TRIM-NHL protein family, to which Brat and Mei-P26 belong, include evolutionarily conserved stem cell regulators that prevent ectopic stem cell self-renewal by inhibiting Myc. However, the downstream effectors of the TRIM-NHL proteins remain largely unknown. This study identified eIF4E as such a factor. NB-specific knockdown of eIF4E completely suppresses the drastic brain tumor phenotype caused by loss of Brat. Interestingly, eIF4E knockdown is even more effective than dMyc knockdown in this regard. N signaling and Brat have been proposed to act in parallel in regulating Drosophila type II NB homeostasis. However, at the molecular level, how deregulation of these two rather distinct pathways causes similar brain tumor phenotypes remain largely unknown. The current results suggest that these two pathways eventually converge on the dMyc-eIF4E regulatory loop to promote cell growth and stem cell fate. N overactivation and loss of Brat both result in up-regulation of eIF4E and dMyc in transit-amplifying progenitors, accelerating their growth rates and helping them acquire stem cell fate. Consistent with a general role of eIF4E and dMyc in stem cell regulation, it was shown that partial reduction of eIF4E or dMyc function in the Drosophila ovary effectively rescues the ovarian tumor phenotype due to the loss of Mei-P26. The vertebrate member of the TRIM-NHL family, TRIM32, is shown to suppress the stem cell fate of mouse neural progenitor cells, partially through degrading Myc (Schwamborn, 2009). Whether eIF4E acts as a downstream effector of TRIM32 in balancing stem cell self-renewal versus differentiation in mammalian tissues awaits future investigation (Song, 2011).

The bHLH factors Dpn and members of the E(spl) complex mediate the function of Notch signalling regulating cell proliferation during wing disc development

The Notch signalling pathway plays an essential role in the intricate control of cell proliferation and pattern formation in many organs during animal development. In addition, mutations in most members of this pathway are well characterized and frequently lead to tumour formation. The Drosophila imaginal wing discs have provided a suitable model system for the genetic and molecular analysis of the different pathway functions. During disc development, Notch signalling at the presumptive wing margin is necessary for the restricted activation of genes required for pattern formation control and disc proliferation. Interestingly, in different cellular contexts within the wing disc, Notch can either promote cell proliferation or can block the G1-S transition by negatively regulating the expression of dmyc and bantam micro RNA. The target genes of Notch signalling that are required for these functions have not been identified. This study shows that the Hes vertebrate homolog, deadpan (dpn), and the Enhancer-of-split complex (E(spl)C) genes act redundantly and cooperatively to mediate the Notch signalling function regulating cell proliferation during wing disc development (San Juan, 2012).

Defective Hfp-dependent transcriptional repression of dMYC is fundamental to tissue overgrowth in Drosophila XPB models

Nucleotide excision DNA repair (NER) pathway mutations cause neurodegenerative and progeroid disorders (xeroderma pigmentosum (XP), Cockayne syndrome (CS) and trichothiodystrophy (TTD)), which are inexplicably associated with (XP) or without (CS/TTD) cancer. Moreover, cancer progression occurs in certain patients, but not others, with similar C-terminal mutations in the XPB helicase subunit of transcription and NER factor TFIIH. Mechanisms driving overproliferation and, therefore, cancer associated with XPB mutations are currently unknown. In this study using Drosophila models, evidence is provided that C-terminally truncated Hay/XPB alleles enhance overgrowth dependent on reduced abundance of RNA recognition motif protein Hfp/FIR, which transcriptionally represses the MYC oncogene homologue, dMYC. The data demonstrate that dMYC repression and dMYC-dependent overgrowth in the Hfp hypomorph is further impaired in the C-terminal Hay/XPB mutant background. Thus, it is predicted that defective transcriptional repression of MYC by the Hfp orthologue, FIR, might provide one mechanism for cancer progression in XP/CS (Lee, 2015).

Targets of Activity

The almost completely superimposable pit and dmyc expression patterns as well as the similarities existing between the Pitchoune sequence and that of MrDb, a target of mammalian c-myc strongly support the hypothesis that Drosophila pit might also be a target for the transcriptional factor d-Myc. d-Myc is encoded by the diminutive locus (Gallant, 1996; Schreiber-Agus, 1997). The expression of pit is not, however, noticeably affected in ovaries of females homozygous for the hypomorphic allele of diminutive: dm1. This result might indicate that dmyc is not required for the expression of pit. However, the low level of d-Myc in the mutants might be sufficient to promote high enough levels of pit, leading to an apparently normal expression. In the same line, no difference in the embryonic expression of pit is observed in dm1 homozygous mutants. As is the case for pit, dmyc is maternally expressed; it is not known whether the maternal protein is stable throughout embryogenesis. As yet, no complete loss-of-function allele of diminutive is known. Attempts to demonstrate a possible interaction between pit and d-myc have therefore been turned toward an ectopic d-myc expression by using a UAS-d-myc cDNA driven by a variety of tissue-specific GAL4-expressing lines. Since d-myc RNA is present neither in the nervous system nor in differentiating muscles, the 1407 and 24B lines were used: these lines, respectively, express GAL4 in the central and peripheral nervous system and in all muscles. pit is expressed in the central nervous system in embryos derived from the 1407 GAL4 line, suggesting that d-Myc can behave, at least in that tissue, as a transcriptional activator of pit. In contrast, no evident ectopic expression of pit could be demonstrated in muscle precursors when the d-myc driver was 24B. There are several likely reasons for a lack of induction of pit in muscle. For example, the Myc protein is known to dimerize with Max to make a heterodimer that activates transcription. d-Max expression in muscle has not been clearly established (Gallant, 1996) and a too low concentration in this tissue might impair the transcriptional activation of the Myc targets. In conclusion, these results strongly support the hypothesis that pit is a target for Myc transcriptional activation. Of course, it is not possible to anticipate from these experiments whether or not pit is a direct target of d-Myc (Zaffran, 1998).

In gain of function and loss of function experiments, modulo has been demonstrated to be directly controlled by dMyc. Strikingly, in proliferative imaginal cells, mod loss-of-function impairs both cell growth and cell size, whereas larval endoreplicative tissues grow normally. In contrast to dMyc, over-expressing Mod in wing imaginal discs is not sufficient to induce cell growth. Taken together, these results indicate that mod does not possess the full spectrum of dMyc activities, but is required selectively in proliferative cells to sustain their growth and to maintain their specific size (Perrin, 2003).

To determine whether modulo expression is transcriptionally controlled by dMyc, mod mRNA level was measured in loss-of function dmyc mutants. No change in mod expression was observed for the viable hypomorphic allele dmycP0. In contrast, both in situ hybridization and quantitative RT-PCR on third instar larval imaginal discs, show that mod transcription is severely impaired in the pupal lethal dmycPL35 mutants. Thus sufficient diminishing of dmyc+ function reveals dMyc requirement for mod expression. The effect of dMyc on mod transcription was also analyzed in gain-of-function experiments, using the UAS/Gal4 system. In third instar wing imaginal discs, dMyc over-expression directed by dpp regulatory sequences leads to a marked increase in mod transcription. Also, in engrailed(en)-Gal4/UASdmyc embryos, mod transcription is strongly induced in the posterior cells of each parasegment where dMyc is over-expressed. Taken together, these results show that dMyc is required for mod expression (Perrin, 2003).

E-boxes constitute functional Myc binding sites that typically reside downstream of the transcriptional start sites of target genes. The action of Myc in regulating transcription has been described as involving binding of Myc homo-or hetero-dimers to an 'E-box' sequence based on a 'CACGTG' motif. A 1 kb DNA fragment located upstream of mod coding sequences has been shown capable of directing reporter gene expression that mimics the mod embryonic expression pattern (Alexandre, 1996). This fragment harbors a canonical CACGTG E-box between the mod initiator ATG and a transcriptional start site assigned by primer extension. A 365 bp fragment (P1) encompassing both the transcription start site and the E-box was fused to a Lac-Z reporter gene. Trangenic flies containing this P1-LacZ chimeric gene express ß-Gal in a pattern similar to endogenous mod. Further, on expressing dMyc in embryos (enGAL4, UASdMyc), mod expression and P1-LacZ expression are augmented in the posterior compartments. To ask whether the responsiveness of P1 to dMyc is E-box dependant, the canonical CACGTG E-box was mutated (CAGGTG) to abolish a potential dMyc-DNA interaction, according to Myc binding specificity. When fused to a Lac-Z reporter gene, this mutated P1 fragment is no longer able to mimic the mod transcription pattern, either in wild type or en-Gal4/UASdmyc embryos. Taken together, these results strongly support the notion that mod transcription is controlled by dMyc, and favor the possibility that dMyc binds directly to the canonical E-box residing in mod regulatory sequences (Perrin, 2003).

It has been shown that diminished mod activity leads to a Minute-like phenotype (Perrin, 1998; Roman 2000), thus suggesting a role for Mod in ribosome biogenesis. A detailed analysis of mod growth-related phenotypes showns that it acts on growth and size of proliferative cells. mod loss-of-function selectively affects imaginal diploid cells but not endoreplicative tissues. In addition, Mod over-expression affects diploid cells but not endoreplicative tissues. For instance, salivary glands cells are bigger in cell and nucleus size upon ectopic expression of dMyc, but look normal following Mod over-expression. Since amino acids directly control growth of endoreplicative tissues, it is unlikely that Mod is related to nutrient availability. Indeed, in agreement with the phenotype specific for proliferative cells, mod transcription is controlled by dMyc. Nevertheless, mod is certainly not involved in all the various cellular processes controlled by dMyc, since in the dmycPL35 mutant, imaginal and endoreplicative tissues are equally affected (Perrin, 2003).

The Myc/Max/Mad transcription factor network is critically involved in cell behavior; however, there is relatively little information on its genomic binding sites. The DamID method was used to carry out global genomic mapping of the Drosophila Myc, Max, and Mad/Mnt (see Drosophila Mnt for information about Mad/Mnt family members) proteins. Each protein was tethered to Escherichia coli DNA adenine-methyltransferase (Dam) permitting methylation proximal to in vivo binding sites in Kc cells. Microarray analyses of methylated DNA fragments reveals binding to multiple loci on all major Drosophila chromosomes. This approach also reveals dynamic interactions among network members; increased levels of dMax influence the extent of dMyc, but not dMnt, binding. Computer analysis using the REDUCE algorithm demonstrates that binding regions correlate with the presence of E-boxes, CG repeats, and other sequence motifs. Application of the REDUCE algorithm, which correlates binding with the occurrence of DNA sequence motifs, reveals a statistically significant correlation between the E-box CACGTG and the presence of dMnt binding regions. CACGTG enrichment also correlated with dMyc binding in the presence of high dMax levels, for dMax binding in the presence of high dMyc levels, and for genes whose expression is modulated by dMyc. The surprisingly large number of directly bound loci (approximately 15% of coding regions) suggests that the network interacts widely with the genome. Furthermore, microarray expression analysis was employed to demonstrate that hundreds of DamID-binding loci correspond to genes whose expression is directly regulated by dMyc in larvae. These results suggest that a fundamental aspect of Max network function involves widespread binding and regulation of gene expression (Orian, 2003).

A significant gap in understanding of the function of many transcriptional regulatory proteins has been the lack of comprehensive identification of their in vivo binding sites and the genes whose expression they regulate. This problem is especially pertinent for transcription factors such as Myc, Mad/Mnt, Max, and other members of the Max network that function as relatively weak transcriptional regulators, whose consensus binding site is ubiquitous, and whose expression elicits profound effects on cell growth and proliferation. Standard methods of target gene evaluation do not reliably differentiate between genes bound and directly regulated by Myc and Mad from genes whose expression is altered as a secondary or later consequence of Myc or Mad induction. In principle, it is important to know about both sets of genes, but it is also crucial to distinguish between them. The DamID method employed in this paper permits determination of transcription factor binding site regions in live cells and is not dependent on chemical cross-linkers or PCR primers. Because it involves 'marking' of DNA in chromatin by a methyltransferase linked to a transcription factor, even transient or low affinity interactions with DNA, as well as proximity to regions distal to the binding site (through looping or higher-order folding), might be detected. Because a cDNA array was used to detect targeted methylation regions, only binding sites within a few kb of transcription units are detected. Therefore, enumeration of dMax network binding sites is likely to be an underestimate. The mapping resolution also does not permit precise pinpointing of the binding site within each probed locus, although the REDUCE analysis strongly suggests that E-box motifs within target loci mediate the protein recruitment (e.g., as for Mnt target bicaudal) (Orian, 2003).

The validity of the approach is strongly supported by several lines of evidence. (1) The degree of overlap between dMyc, dMax, and dMnt binding regions is consistent with the relationship between E-box binding and heterodimerazation with Max established previously for the vertebrate proteins as well as for their orthologs in Drosophila. Importantly, the GAGA factor, a ubiquitous transcription factor unrelated to the dMax network, displays only minimal overlap with dMnt binding sites, suggesting the results are specific for binding by dMax network transcription factors. Furthermore, studies in mammalian cells have shown both overlapping and nonoverlapping functions and target genes for Myc and Mad proteins in agreement with DamID findings. (2) Using a ChIP assay, the direct binding of dMyc and dMnt to a DamID-defined target gene, bic (bicaudal), was demonstrated. In addition, the mammalian orthologs of at least 18 genes identified as binding targets for dMyc, dMax, and dMnt in this study have been demonstrated to be direct targets for vertebrate Myc using ChIP. (3) Application of the REDUCE algorithm, which correlates binding with the occurrence of DNA sequence motifs reveals a statistically significant correlation between the E-box CACGTG and the presence of dMnt binding regions. CACGTG enrichment also correlated with dMyc binding in the presence of high dMax levels, for dMax binding in the presence of high dMyc levels, and for genes whose expression is modulated by dMyc. (4) A substantial set of target genes identified in the Drosophila gene expression microarray analysis, employing larvae overexpressing dMyc, correspond to target genes defined by DamID. In addition, target genes identified in this study are in accord with genes regulated by Myc and Mad as described in several recently published gene-expression studies in vertebrate systems (Orian, 2003).

The Drosophila Gene Ontology Database was used to derive an unbiased classification of genes associated with dMax network binding regions. Many of the dMax network targets identified are genes that fit well with the established biological functions of Myc and Mad. In addition, a significant number of targets point to new pathways likely to be regulated by the network. The data demonstrate both binding to, and regulation of, genes encoding proteins broadly involved in biosynthetic processes, in accord with genetic and biochemical analyses, demonstrating that Myc is involved in cell growth in Drosophila and vertebrates, and from earlier global gene expression studies. The DamID binding loci also include genes involved in cell cycle and DNA replication. The list of putative dMax network targets also reveals potential novel pathways such as mitochondrial biogenesis and function, as well as vesicular transport. Other pathways known to be linked to Myc such as apoptosis, proteolysis, and the immune response are also reflected in the list of dMax network target genes as are a number of transcription factors (Orian, 2003).

The findings demonstrate a surprisingly large number (968) of binding sites for proteins of the dMax network. Considering that the array represents a random sampling of ~50% of Drosophila coding regions, a conservative estimate is that dMax network proteins interact with ~2000 genes, and this is likely to be an underestimate. It is important to note however that dMax network proteins do not bind profligately to DNA, as evidenced by the low degree of overlap with GAGA factor, the general correlation of E-box sequences with binding, and the lack of association with repeat elements linked to HP1 binding previously. HP1 is predominantly localized to pericentric heterochromatin, and its binding is associated with silenced chromatin structure. The lack of association of dMyc, dMax, or dMad with such elements may indicate that the network proteins are primarily associated with genes that are subject to ongoing transcriptional modulation. These findings are in accord with extensive ChIP assays in human cells. That study suggested that 8%-10% of cellular genes associate with Myc and in general display enhanced histone H3 and H4 acetylation (Orian, 2003).

The large number of binding sites and regulated target genes identified in this study contrasts with earlier ideas of Myc function that posited a small number of critical targets. However, not all binding sites necessarily result in direct transcriptional regulation by dMax network factors. This is evident from the dMyc-dependent gene expression data carried out in growing third instar larvae. At this developmental stage, 31% (89/287) of the Myc binding loci (as determined in Kc cells) displayed altered mRNA epression in larvae. Of genes that were detected as overlapping targets of all three proteins or of only dMyc and dMnt, 48.6% and 60.5% respectively, displayed concomitant changes in mRNA levels upon Myc induction. Interestingly, Myc binding and histone acetylation at mammalian genes has been described, whose expression does not appear to change in response to induction of Myc. One possible explanation is that Myc binding to a subset of genes, although not immediately affecting gene expression, confers a permissive state on chromatin allowing binding by other cis-acting factors at later times (Orian, 2003).

The many dMax targets detected that are shared with dMyc and dMnt most likely represent binding by dMyc-dMax and dMnt-dMax heterodimers. However, the extent of nonoverlap between binding sites for these proteins is more extensive than expected. For example, it was found that dMax expressed at low levels binds to 365 genes that do not overlap with either dMnt or dMyc targets. However, 15% of these binding loci are regulated by dMyc in the larval expression analysis. Thus, the degree of overlap is probably influenced by the temporal pattern and levels of dMyc expression. This has implications for tumorigenesis where vertebrate Myc proteins are often dramatically overexpressed. This work provides evidence that such overexpression may shift the spectrum of target genes relative to those expressed in normal cells (Orian, 2003).

Max homodimers bind E-boxes with relatively low affinity and in mammalian cells are inhibited by phosphorylation from binding DNA. Although it is not know known whether dMax homodimers are similarly blocked from binding to DNA in vivo, the idea is favored that the large number (365) of unique dMax binding sites and the lack of correlation with E-boxes reflects dimerization and DNA binding by dMax with as-yet-unidentified interacting proteins. Interestingly, in mammalian cells Max has been found, in association with the bHLHZ protein Mga, in E2F6 repression complexes. Similarly, unique sites found for dMnt and dMyc may represent non-E-box DNA binding through formation of higher-order complexes. For mammalian Myc, interaction of Myc-Max heterodimers with the Miz-1 protein has been shown to direct Myc to non-E-box sites. It is likely that associations with other partners may redirect dMyc and dMnt to unique binding sites. If so, the findings indicate that such interactions may be extensive and are an important part of dMax network function (Orian, 2003).

The canonical E-box sequence alone is unlikely to be sufficient to determine specific binding by dMax network proteins and, indeed, many E-box-containing promoters are not associated with Max network proteins. One possibility is that other sequences in the vicinity of an E-box may play a role in target gene specificity. For example, the DRE, which correlated with binding of all three dMax network proteins is located within <1 kb of many of these E-box sequences. Therefore, it is tempting to hypothesize that the DRE operates in cis with adjacent E-boxes to recruit protein complexes that will either promote activation or repression. Alternatively, the proximity of DRE and E-box sites may reflect coordinate regulation of the same genes through distinct signaling pathways (Orian, 2003).

In addition, REDUCE analysis has revealed a number of unexpected correlations. For example, association was found between dMyc and AT-rich sequences when dMax levels are limiting. In several loci examined, these AT-rich regions occur in the vicinity of genes lacking E-boxes, perhaps reflecting dMyc association with as-yet-undefined binding proteins when dMax levels are limiting. REDUCE analysis of dMax binding regions failed to detect a binding correlation with CACGTG. However, when high levels of dMyc were expressed together with dMax-Dam, REDUCE analysis of dMax binding regions found the E-box significantly correlated with binding. This is in accord with data that dMax homodimers bind only weakly to E-boxes, and that Max binding is largely directed by its heterodimeric partners. Perhaps the AT- (and CG-) rich sequences influence architecture of the binding site or serve as binding motifs for factors that enhance dMax network protein association with DNA (Orian, 2003).

Taken together, these data suggest a rather more complex picture of the functioning of Max network transcription factors than has been considered previously. The results suggest extensive yet specific interaction with chromatin probably encompassing thousands of binding sites and directly affecting expression of hundreds of genes. In addition, the DamID results indicate the possibility of several different modes of Myc, Max, and Mad/Mnt interactions. These include binding to partner proteins yet to be identified as well as potential cooperation with other transcription factors. Earlier experiments have shown that Myc and Mad expression is under tight control by the cell. Such control is likely to be important in balancing the multiple protein-protein and DNA binding interactions inferred from the data (Orian, 2003).

Human c-Myc isoforms differentially regulate cell growth and apoptosis in Drosophila melanogaster

The human c-myc proto-oncogene, implicated in the control of many cellular processes including cell growth and apoptosis, encodes three isoforms differing in their N-terminal region. The functions of these isoforms have never been addressed in vivo. This study used Drosophila to examine the functions of these isoforms in a fully integrated system. First, it was established that the human c-Myc protein can rescue lethal mutations of the Drosophila myc ortholog, dmyc, demonstrating the biological relevance of this model. Then, a new lethal dmyc insertion allele was characterized, that permits expression of human c-Myc in place of dMyc; this allele was used to compare physiological activities of these isoforms in whole-organism rescue, transcription, cell growth, and apoptosis. The isoforms differ both quantitatively and qualitatively. Most remarkably, while the small c-MycS form truncated for much of its N-terminal trans-activation domain efficiently rescues viability and cell growth, it does not induce detectable programmed cell death. The data indicate that the main functional difference between c-Myc isoforms resides in their apoptotic properties and that the N-terminal region, containing the conserved MbI motif, is decisive in governing the choice between growth and death (Benassayag, 2005).

The functional rescue of dmyc mutations by c-Myc suggests that human and Drosophila Myc proteins control common target genes in vivo. To address this question, the capacity of c-Myc isoforms to regulate known or potential dmyc targets was examined in flies. c-myc expression in mammals is subject to a negative autoregulatory loop. To test whether a similar regulatory mechanism exists in Drosophila, transgenic dMyc was overexpressed and its effect on accumulation of endogenous dmyc mRNA was measured. The endogenous and transgenic dmyc mRNAs differ in their 3' untranslated regions, making it possible to specifically detect the endogenous mRNA by RT-PCR. Endogenous dmyc expression is strongly reduced upon UAS-dMyc expression directed either by da-Gal4 or by dmycPG45 drivers (endo dmyc). This repression was inversely related to the level of transgene expression. These data indicate the existence of a negative autoregulatory mechanism conserved between mammals and flies (Benassayag, 2005).

It was next asked whether the different human c-Myc isoforms can trans repress dmyc transcription, using the dpp-Gal4 driver to direct their localized expression in a central band of cells at the anteroposterior compartment boundary of wing imaginal discs. dmyc mRNA accumulation was examined by in situ hybridization with a dmyc-specific exon 2 riboprobe. The control experiment with UAS-dMyc showed a strong localized accumulation of dmyc mRNA, as expected for the dpp promoter used. In contrast, when human c-Myc forms were expressed in the same manner, endogenous dmyc mRNA was locally diminished. These observations indicate that all three human c-Myc proteins can negatively regulate dmyc (Benassayag, 2005).

Their capacity to trans activate the expression of two known dmyc target genes, pitchoune (pit) and modulo (mod) was examined. pit encodes an RNA helicase required for cell growth, while the Modulo protein shows structural similarity to nucleolin, which has a putative role in ribosome biogenesis. Expression of either pit or mod is strongly reduced in dmycPG45 or dmycPL35 mutants. To ask whether human Myc proteins can activate transcription of these genes, dMyc or isoform specific c-Myc expression was induced in the wing imaginal disc and then pit and mod expression was analyzed by in situ hybridization. Locally enhanced expression for mod or pit was induced by dMyc, compared with endogenous expression; similar, albeit weaker, enhancement was observed with all three human c-Myc variants. Taken together, it is concluded that fly and human Myc proteins are able to regulate the same target genes, whether negatively (dmyc) or positively (pit and mod) (Benassayag, 2005).

Attempts were made to compare the activities of the three c-Myc isoforms for cellular functions in vivo (i.e., cell growth, cell cycle progression, and apoptosis) by examining the effects of their overexpression in a wild type context. Random clones of cells overexpressing c-Myc1, c-Myc2, or c-MycS were generated in imaginal wing discs under the control of Gal4 by the flip-out technique. Clones of Gal4-expressing cells induced during larval development were identified by cytoplasmic GFP expression from a UAS:GFP reporter. Such GFP+ cells were then separated from nonexpressing cells by FACS, and both populations were examined for cell size and DNA content. Expression of dMyc, used in a control experiment, led to an increase in cell size and a strong reduction in the fraction of cells in G1 with a concomitant increase in cells in S or G2 phase. Human isoforms c-Myc1 and 2 produced similar effects, albeit weaker than for dMyc. However, c-MycS, while promoting G1/S cell cycle progression, had little effect on cell size. To test whether this difference might reflect limiting amounts of c-MycS activity, flip-out clones expressing two UAS-c-MycS copies rather than one were induced. Under these conditions, the effect of c-MycS on cell size-cell cycle progression resembled that of c-Myc1 and c-Myc2 (Benassayag, 2005).

At the level of the primary amino acid sequence, the only difference between the predominant vertebrate form c-Myc2 and alternative c-Myc1 and c-MycS forms resides in their NH2 terminal portion. In dmyc mutant Drosophila, the cellular functions of c-Myc2 are sufficient to sustain normal development. Remarkably, the alternatively initiated c-Myc1, which harbors an additional 15 aa, enhances all these cellular functions and (in particular) apoptosis but leads to dominant lethality. This optional, leucine-initiated sequence is relatively weakly conserved between the closely related human and mouse proteins (6 identical amino acids out of 15). The poor overall conservation of this sequence argues against its specific interaction with molecular partners and suggests an indirect role favoring a productive conformation of c-Myc protein (Benassayag, 2005).

The N-terminal region common to c-Myc1 and c-Myc2 contains a trans-activation domain that has been described as important for numerous properties of the Myc molecule and/or for its interactions with molecular partners in cultured cells. In the trans-activation domain, MbI is believed to play a role in modulating c-Myc activity, while the integrity of MbII is essential to normal c-Myc functions including cell cycle progression, apoptosis, and transformation. c-MycS lacks the first 100 aa, including the MbI motif, which harbors two phosphorylation sites (Thr 58 and Ser 62) involved in the stability of c-Myc protein through proteosomal degradation. Mutations of these sites are often linked to B-cell lymphomas and are correlated with reduced apoptotic potential. Curiously, neither of these sites is conserved in dMyc. The truncated c-MycS isoform rescues the growth defect of c-Myc null fibroblasts, but its ability to transactivate and induce apoptosis remains a subject of debate. The rescue obtained on expressing c-MycS in developing Drosophila clearly shows that this isoform possesses all necessary properties to sustain growth and development, even though it is fully deficient in inducing apoptosis. These results thus support the possibility of a direct role for the first 100 aa of the N-terminal region, absent from c-MycS, in the cellular choice between growth or death. This choice will presumably reflect discriminating physical interactions of a given N-terminal sequence with specific cofactors. The uncoupling between growth and death functions obtained in c-MycS but not in c-Myc2 transgenic flies thus offers an exciting new opportunity to dissect the c-Myc genetic network(s) underlying these two cellular processes (Benassayag, 2005).

Altogether, these results obtained in a physiological context show that the three c-Myc isoforms are functionally different, with the principal characteristic distinguishing them being their abilities to induce apoptosis. In their normal context in mammalian cells, these c-Myc isoforms do not accumulate singly but in specific combinations or ratios characteristic of a given cellular status. Their different abilities to induce apoptosis may thus explain why perturbing their balance can be associated with cellular pathologies, including oncogenesis (Benassayag, 2005).

A Myc-Groucho complex integrates EGF and Notch signaling to regulate neural development

Integration of patterning cues via transcriptional networks to coordinate gene expression is critical during morphogenesis and misregulated in cancer. Using DNA adenine methyltransferase (Dam)ID chromatin profiling, protein-protein interaction between the Drosophila Myc oncogene and the Groucho corepressor was identified that regulates a subset of direct dMyc targets. Most of these shared targets affect fate or mitosis particularly during neurogenesis, suggesting the dMyc-Groucho complex may coordinate fate acquisition with mitotic capacity during development. An antagonistic relationship was found between dMyc and Groucho that mimics the antagonistic interactions found for EGF and Notch signaling: dMyc is required to specify neuronal fate and enhance neuroblast mitosis, whereas Groucho is required to maintain epithelial fate and inhibit mitosis. The results suggest that the dMyc-Groucho complex defines a previously undescribed mechanism of Myc function and may serve as the transcriptional unit that integrates EGF and Notch inputs to regulate early neuronal development (Orian, 2007).

Gro is a downstream transducer of several signaling pathways and was placed at the crossroads of the Notch and EGF signaling pathways during patterning of the Drosophila nervous system, where EGF-induced site-specific phosphorylation of Gro attenuates it repression activity. During embryonic stage 9, the CNS matures in three bilaterally symmetrical longitudinal rows of neuroblasts, with the homeobox transcription factors, Vnd, Ind, and Msh, specifying the medial (ventral), intermediate, and lateral rows, respectively. EGF regulates the expression of both Vnd and Ind and is thus required for the formation of the ventral and intermediate rows. Interestingly, both Vnd and Ind are among the 38 dMyc-Gro shared targets identified in this study. Gro and dMyc, but not dMnt, are expressed in neuroblasts of stage 9 embryos. Because dMyc-Gro targets are associated with both neuroblast fate and mitosis, it is hypothesized that EGF and Notch coregulate cell fate and mitosis within the developing neuroectoderm via dMyc-Gro antagonism. Vnd expression (a shared Myc-Gro target whose expression overlaps with and is required for establishment of S1 neuroblasts), the overall number of neuroblasts, and mitotic activity in wild-type embryos were compared to groe47 loss-of-function (LOF) mutants (in which the maternal contribution of Gro is removed), Egfr2, or Notch55e11 [note that dMyc LOF embryos cannot be generated]. These parameters were also evaluated in embryos overexpressing either dMyc or Gro using the conditional Gal4/upstream activating sequence (UAS) expression system. Vnd expression is stronger and expanded in both Notch and gro LOF embryos, as well as in embryos overexpressing dMyc when compared with wild type. These mutants also show neuroblast hyperplasia and elevated mitotic activity. Furthermore, Egfr LOF or Gro-overexpressing embryos show reduced Vnd expression, neuronal hypoplasia, and reduced mitotic activity, consistent with the molecular nature of the dMyc-Gro common targets (Orian, 2007).

Myc proteins are required for both cell growth/size and cell proliferation. The model in which Myc functions are mediated by heterodimerization with Max and antagonized by Mxd (Mad/Mnt) proteins has been well established. However, recent studies suggest that a set of interactions outside the canonical Myc/Max/Mxd network also regulate some of Myc's functions. Interestingly, the current studies point to a subset of dMyc direct targets that are not shared with either dMax or dMnt. Furthermore, dMnt-Dam and dMax-Dam were not recruited to these dMyc targets even in experiments where the Dam fusions were coexpressed in the presence of high levels of dMax or dMyc, respectively, suggesting that previously uncharacterized mechanisms may mediate Myc's recruitment to DNA, and proteins other than dMnt may antagonize its transcriptional activity on this set of targets. This study reports the identification of Gro as the first component in a pathway that antagonizes dMyc function independent of dMnt and operates during Drosophila neurogenesis (Orian, 2007).

Transcriptionally, dMyc was found to be positively required for the expression of dMyc-Gro targets, activity that is antagonized by Gro. Importantly, dMyc is not a Gro target, and reducing Gro levels does not affect dMyc protein levels. Furthermore, Gro antagonism is limited only to the dMyc-Gro subset of shared targets and does not involve dMnt: there is no overlap between genes bound by dMnt or Gro, dMnt is not expressed in cells where the dMyc-Gro interaction is observed, RNAi to dMnt does not affect Myc-Gro shared target expression, and overexpression of dMnt does affect PNS development (Orian, 2007).

Although the possibility that dMyc-Gro targets are coregulated by individual dMyc and Gro complexes cannot be excluded, the results suggest that dMyc and Gro are part of a single larger protein complex. First, the observation that RNAi to dMyc results in reduction of target expression and is restored by coreducing Gro suggests that other activators coregulate shared target expression along with dMyc. Second, biochemical purification, binding data, and DNA adenine methyltransferase (Dam)ID Southern analyses support the idea that both proteins physically interact with one another yet associate with DNA through distinct binding sites. Third, Gro does not bind directly to DNA but must be recruited to targets by sequence-specific DNA-binding transcription factors. Fourth, most of the dMyc-Gro targets lack E-box sequences associated with canonical Myc network targets, suggesting that dMyc and Gro may be recruited to shared targets via a novel mechanism or by other protein(s) yet to be identified. Candidates for recruiting Gro may be the E(spl) proteins that convey the Notch signal, antagonize the EGF pathway, interact with Gro, and exhibit similar phenotypes. Thus, the identification of the entire dMyc-Gro complex and its regulation will be an important next step (Orian, 2007).

Gro's role as a downstream transducer of Notch signaling during neurogenesis is well documented, and mounting evidence supports Myc as a key player in progenitor cell proliferation. This study has identified a previously undescribed role for dMyc, together with Gro, during Drosophila early neuronal development. dMyc and Gro are required to directly regulate key fate controlling genes such as the homeodomain proteins vnd and ind that are downstream targets of EGF signaling. Because Vnd was identified as a regulator of the proneural gene complex, the differential regulation of vnd by dMyc and Gro implicates them as antagonistic regulators upstream of proneural genes. Thus, it is proposed that dMyc is transiently required within the neuroectoderm, where it promotes specific fate acquisition and allows mitotic expansion of committed neuronal cells (Orian, 2007).

Phenotypically, it was observed that, similar to EGF, dMyc promotes neurogenesis both in the PNS and CNS, whereas Gro and Notch inhibit neuroblast formation and mitosis. This is a different role than that previously ascribed to dMyc, because it is usually associated with regulation of cell size and organismal growth, functions that are antagonized by dMnt. Consistent with this, a recent study identified EGF-induced phosphorylation of c-Myc, Max, and TLE proteins in mammalian cells. The antagonistic relationship of Myc/EGF to Gro/Notch is likely to be highly dependent on the developmental context and the specific progenitor niche. For example, in cellular contexts in which Notch promotes proliferation, such as during the development of T cells in acute leukemia, Myc is a direct target of mutated Notch1 and is required for T cell proliferation and development. The current findings also fit well with observations that N-Myc is required during mouse progenitor development, and that the fly tumor suppressor Brat regulates dMyc levels posttranscriptionally in larval neuroblasts resulting in a 'tumorous' phenotype (Orian, 2007).

Taken together, the snapshot provided by DamID data leads to the suggestion of a model in which changes in neuronal progenitor fate and mitosis are determined by the balance between EGF and Notch signaling that is likely transcriptionally mediated by the dMyc-Gro complex. During epithelial development, Notch, like Gro, is required to specify and maintain epithelial fate. It is proposed that Gro sequesters dMyc in an inactive multiprotein complex formed by associating with dMyc, preventing the activation of dMyc-Gro shared targets. Upon EGF signaling, a molecular switch takes place whereby Gro is phosphorylated, and its repression is attenuated. dMyc, as part of an as-yet-to-be-identified activation complex, is then liberated to activate zygotic transcription of a subset of targets that determines neuronal fate and enhances mitosis. One of these targets is dMax, which is specifically expressed in the neuroectoderm. Activation of dMax would be expected to establish a feed-forward loop required for the subsequent activation of (E box-containing) Myc targets to promote cell growth. As development progresses, the dMnt gene would be induced, and dMnt-dMax complexes would replace dMyc-Max complexes, thereby promoting cellular differentiation (Orian, 2007).

Finally, both EGF/dMyc and Notch/Gro misregulation and mutation are intimately involved in hematological, epithelial, and neuroectodermal cancers. Thus, identification of a dMyc-Gro complex that could serve as a molecular junction to integrate EGF and Notch signaling inputs is highly relevant for both developmental biology and cancer (Orian, 2007).

Nutritional control of protein biosynthetic capacity by insulin via Myc in Drosophila

Animals use the insulin/TOR signaling pathway to mediate their response to fluctuations in nutrient availability. Energy and amino acids are monitored at the single-cell level via the TOR branch of the pathway and systemically via insulin signaling to regulate cellular growth and metabolism. Using a combination of genetics, expression profiling, and chromatin immunoprecipitation, this study examined nutritional control of gene expression and identified the transcription factor Myc as an important mediator of TOR-dependent regulation of ribosome biogenesis. myc was also identified as a direct target of FOXO, and genetic evidence is provided that Myc has a key role in mediating the effects of TOR and FOXO on growth and metabolism. FOXO and TOR also converge to regulate protein synthesis, acting via 4E-BP and Lk6, regulators of the translation factor eIF4E. This study uncovers a network of convergent regulation of protein biosynthesis by the FOXO and TOR branches of the nutrient-sensing pathway (Teleman, 2008).

The global transcriptional analysis reported in this study has revealed a surprising degree of interconnectedness between the two branches of the nutrient-sensing pathway. Insulin, acting through PI3K and Akt, feeds into the FOXO and TORC1 branches of the pathway, whereas energy levels (AMP/ATP) and amino acids act directly on the TORC1 branch. How are these inputs integrated to maintain energy balance? It was previously known that 4E-BP is transcriptionally regulated by FOXO and posttranslationally regulated by TOR. This study has identified the protein kinase Lk6 as a second direct FOXO target. Thus, there appear to be two parallel, independent mechanisms by which the TOR and FOXO branches of the insulin signaling pathway converge to regulate eIF4E activity and hence cellular protein translation. This 'belt and suspenders' approach to translational control might be important to make the system robust (Teleman, 2008).

A key finding of this study is the identification of Myc as a point of convergent regulation by the FOXO and TOR branches of the pathway. myc mRNA levels are controlled by FOXO in a tissue-specific manner. In addition, Myc protein levels are dependent on TORC1. Why use two independent means to control Myc levels? Transcription alone would limit the speed with which the system can respond to changing nutritional conditions. This might be detrimental, particularly as conditions worsen. Regulation of Myc activity by TORC1 permits a rapid response to changes in energy levels or amino acid availability and could serve to fine tune the nutritional response in the cell by controlling translational outputs. This parallels the situation with 4E-BP, albeit with a slightly different logic. Reduced insulin signaling allows FOXO to enter the nucleus and increase 4E-BP expression and at the same time alleviates TORC1-mediated inhibition of the existing pool of 4E-BP. A subsequent increase in energy or amino acid levels would permit rapid reinhibition of 4E-BP and thus allow a flexible response during the time needed for the pool of protein elevated in response to reduced insulin levels to decay (Teleman, 2008).

In yeast, TORC1 is known to regulate ribosome biogenesis through different nuclear RNA polymerases. It has been shown that yeast TORC1 can bind DNA directly at the 35S rDNA promoter and activate Pol I-mediated transcription in a rapamycin-sensitive manner. Moreover, yeast TORC1 is known regulate Pol II-dependent RP gene expression by controlling the nuclear localization of the transcription factor SFP1 and CRF1, a corepressor of the forkhead transcription factor FHL1. In Drosophila, TORC1 has recently been reported to regulate a set of protein-coding genes involved in ribosome assembly. This study has identified Myc as the missing link mediating TORC1-dependent regulation of this set of genes. Indeed, the fact that more than 90% of TORC1-activated genes contain E boxes suggests that Myc might be the main mediator of this transcriptional program. This connection suggests that expression of Myc targets as a whole should be responsive to nutrient conditions. Indeed, this study found that 33% of direct Myc targets -- defined as genes reported to be bound by Myc when assayed by DNA adenine methyltransferase ID (DamID) in Kc cells and to be regulated by myc overexpression in larvae -- are downregulated upon nutrient deprivation. This is a significant enrichment of 4-fold relative to all genes in the genome, despite the comparison being based on correlating data from different tissue types (Teleman, 2008).

It seems reasonable that cellular translation rates need to be dampened if the TOR branch of the pathway senses low amino acid levels. As ribosome biogenesis is energetically expensive, it may be advantageous to link ribosome biogenesis and translational control via TORC1. This dual regulation is well reflected in tissue growth, since this study observed that Myc, the regulator of ribosome biogenesis, is essential for tissue growth driven by the TOR pathway but not sufficient to drive growth in the absence of TOR activity. The FOXO branch of the pathway senses reduced insulin or mitogen levels. FOXO is also highly responsive to oxidative and other stresses and would integrate this information into the cellular control of translation. The data support the notion of a network in which TOR and FOXO regulate protein biosynthesis by converging on Myc to regulate ribosome biogenesis and on eIF4E activity via 4E-BP and Lk6 to regulate translation initiation (Teleman, 2008).

The work presented in this study complements a previous study in which larvae were either starved completely or starved for amino acids only, while having a supply of energy in the form of sugar. A significant and positive correlation (~0.4) indicates general agreement between the two data sets, but they differ in two ways. The current goal was to explore the regulatory network by which insulin controls cellular transcription. Individual tissues were isolated rather than assaying the whole animal. Genes found to be regulated in a previous but not in the current assays may be regulated in tissues other than muscle or adipose tissue. Conversely, genes identified only by the current study might be regulated oppositely in different tissues or might only be regulated in a subset of tissues and so be missed in a whole-animal analysis.

Is Myc also involved in nutritional signaling networks in mammals? No similar rapid downregulation of c-myc was seen in response to rapamycin in human cell lines, suggesting that the mechanism by which TOR signaling controls gene expression may differ between phyla. This is further supported by the fact that the sets of genes reported to be rapamycin regulated also appear to be largely distinct in Drosophila and mammalian cells, with the caveat that different cell types were used in the two analyses. Although the mechanism does not appear to be identical in mammals, there are several suggestions in the literature of a connection between c-Myc and nutritional signaling. For example, dMyc and c-Myc share the ability to regulate ribosome biogenesis, although the specific target genes through which they do so are different. There is also evidence that mammalian c-myc expression in liver is regulated by nutrition and that transgenic expression of c-myc in liver affects metabolism, i.e., glucose uptake and gluconeogenesis. Furthermore, it has been reported that FOXO3 represses Myc activity in colon cancer cells by inducing members of the Mad/Mxi family, which are known to antagonize Myc. The current data suggest that Max and Mnt are not transcriptionally regulated by insulin or FOXO in Drosophila, whereas myc is. This is similar to what has been reported in murine lymphoid cells, in which c-myc expression is regulated by the FOXO homolog FKHRL1. These parallels between the fly and mammalian systems suggest a broader connection between insulin signaling and activity of the Myc/Mnt/Max network. Although some features may be different in the two systems, the similarities merit further investigation (Teleman, 2008).

Finally, this work has revealed a surprising amount of tissue specificity in the transcriptional response to insulin signaling. Roughly half of the genes regulated by insulin in adipose tissue or in muscle were not significantly regulated in the other tissue. Furthermore, 155 genes were differentially regulated in the two tissues (i.e., upregulated in one tissue and downregulated in the other). This likely reflects the roles of the different tissues in the organism's response to nutrient deprivation. Further work will elucidate the underlying molecular mechanisms (Teleman, 2008).

Nutrient/TOR-dependent regulation of RNA polymerase III controls tissue and organismal growth in Drosophila

The nutrient/target-of-rapamycin (TOR) pathway has emerged as a key regulator of tissue and organismal growth in metazoans. The signalling components of the nutrient/TOR pathway are well defined; however, the downstream effectors are less understood. This study shows that the control of RNA polymerase (Pol) III-dependent transcription is an essential target of TOR in Drosophila. TOR activity controls Pol III in growing larvae via inhibition of the repressor Maf1 and, in part, via the transcription factor Drosophila Myc (dMyc). Moreover, it was shown that loss of the Pol III factor, Brf, leads to reduced tissue and organismal growth and prevents TOR-induced cellular growth. TOR activity in the larval fat body, a tissue equivalent to vertebrate fat or liver, couples nutrition to insulin release from the brain. Accordingly, it was found that fat-specific loss of Brf phenocopies nutrient limitation and TOR inhibition, leading to decreased systemic insulin signalling and reduced organismal growth. Thus, stimulation of Pol III is a key downstream effector of TOR in the control of cellular and systemic growth (Marshall, 2012).

The TOR kinase is one of the best-established growth regulators. In virtually all animals, TOR activity can be stimulated by extracellular cues such as growth factors, nutrients and oxygen to control cell, tissue and organismal growth (Marshall, 2012).

Despite the knowledge of the signalling inputs to TOR, little is known about the mechanisms that allow TOR to modulate cell metabolism and drive growth. Most studies on metabolic functions modulated by TOR have been confined to yeast and mammalian cell culture. These studies have been important in defining roles for TOR in protein synthesis, nutrient uptake and metabolism and autophagy. But they leave open the question of what mechanisms operate in vivo to control tissue and organ growth during animal development. Genetic studies in Drosophila have been pivotal in this regard. This study shows that the ability of the TOR pathway to control transcription through Pol III governs cell, tissue and ultimately organismal growth in Drosophila. Given that Pol III drives transcription of several non-coding RNAs required for mRNA translation, it is suggested that the stimulation of Pol III by TOR enhances the protein synthetic capacity of cells. Previous study have shown that Drosophila TOR also controls synthesis of rRNA synthesis, via the RNA polymerase I factor, TIF-IA (Grewal, 2007). Moreover, recent studies in Drosophila larvae demonstrated that the insulin/TOR pathway regulates the expression of ribosome biogenesis genes via the transcription factors FOXO and Myc. Thus, in Drosophila, tissue and organismal growth relies on the ability of TOR to regulate all three nuclear RNA polymerases to ultimately promote protein synthesis. Given that regulation of all three polymerases is a conserved function for TOR, it is suggested that these mechanisms may also underlie tissue and organ growth in mammalian development (Marshall, 2012).

The Pol III transcription factor Brf has been shown to be an essential component of the TFIIIB complex responsible for recruiting Pol III to gene promoters. This work indicates that Brf activity is required for Drosophila development. Patterning and cell fate specification appear normal in brf embryos. However, once these mutants hatch as larvae they fail to grow. The data suggest that this growth arrest phenotype reflects a role for Brf activity downstream of TOR. Brf was found to be cell-autonomously required for growth in both endoreplicating cells, which make up the bulk of larval mass, and the mitotically dividing cells of the imaginal discs. In particular, brf mutant wing disc cell clones were found to be outcompeted by wild-type neighbours. This cell competition phenotype is seen in mutants for other genes required for protein synthesis, such as the ribosomal proteins and Myc. An important finding was that the overgrowth caused by loss of TSC1 (and hence increased TOR activity) was blocked in brf mutant cells. In mammalian cells, Brf activity is induced by cues that promote cell growth (e.g., during hypertrophic growth of cardiac cells) whereas cell differentiation leads to inhibition of Brf. In fact, overexpression of Brf alone can promote proliferation and transformation in immortalized fibroblasts. Mutations in tumour suppressors such as TSC are common in cancer and lead to elevated TOR activity and promotion of tumour growth. Based on the current data, it is suggested that Brf is required in vivo for both normal tissue growth and TOR-induced tumour growth (Marshall, 2012).

This study found that the predominant mechanism by which nutrition/TOR controls Pol III is via Maf1 repression, since Maf1 inhibition completely reverses the decrease in tRNA synthesis caused by reducing TOR activity. These findings extend those observed in both yeast and mammalian cell culture, and suggest an important role for dMaf1 in vivo in developing tissues. The exact mechanism by which Maf1 functions is not clear, but it may involve inhibition of Brf and Pol III recruitment to genes, possibly by direct binding or association with Brf/Pol III. Indeed, an enhanced association was seen between dMaf1 and Brf1 upon TOR inhibition. The role of dMyc was explored as a potential link between nutrient-TOR signalling and Pol III. dMyc was found to be both necessary and sufficient for the control of Pol III activity during development. As previously reported in both mammalian and Drosophila culture, it was possible to identify an interaction between dMyc and Brf (Gomez-Roman, 2003; Steiger, 2008). In addition, a role has been identified for dMyc in controlling the levels of components of the Pol III machinery, including both Trf and Brf which form part of the TFIIIB complex. Thus, dMyc likely has both direct and indirect effects on Pol III activity in Drosophila. These effects are necessary for both dMyc-induced cell growth (Steiger, 2008) and, as is shown in this study, for the non-autonomous increases in body size caused by dMyc in fat cells. Previous studies have shown that, in Drosophila, TOR controls Myc protein levels. But these effects on Myc probably do not play major role in how TOR activates Pol III since the data show that, unlike inhibition of Maf1, maintaining Myc levels and activity cannot reverse the decrease in tRNA synthesis caused by TOR inhibition. Moreover, if Myc protein levels were limiting for TOR-dependent control of Pol III, then it would not be expected that knockdown of Maf1 could completely reverse the effects of rapamycin/starvation. Given that Maf1 inhibition did not influence levels of Pol III factors, pre-rRNA or RP gene mRNA—transcripts that are upregulated by dMyc—it is unlikely that Maf1 influences Myc function. It was found that rapamycin feeding could not exacerbate the reduction of tRNA levels seen in dMyc null mutants. This result in principle may suggest that TOR signalling does not exert any dMyc-independent effects on Pol III function. But, it is suggested that this finding probably occurs because in the absence of Myc, Pol III activity may be approaching basal levels and cannot be significantly decreased much further. Taken together, although these data may not completely rule out some contribution of Myc to TOR-dependent control of Pol III, they do indicate that it is not the major contributor (Marshall, 2012).

It is clear that both TOR and Myc are essential regulators of Pol III. But, it is likely that while TOR can control Myc levels, both TOR and Myc can also function in parallel and independently of each other. Previous studies have shown that overactivation of TOR signalling could not promote growth when Myc was inhibited, but at the same time Myc overexpression could not promote growth when TOR was inhibited. These findings and the current data suggest that TOR and Myc cannot necessarily be placed in a simple, linear pathway. Recent studies in Drosophila have emphasized how other conserved growth-regulatory pathways, particularly those that control growth of the imaginal tissues (such as Wingless, EGF/Ras, the Hippo-Yorkie pathway and Bantam RNAi) function via control of dMyc. Thus, dMyc may play a role in coupling these pathways to the control of Pol III activity to stimulate cell growth and proliferation (Marshall, 2012).

It is interesting to speculate as to which Pol III targets are important for growth control. Pol III regulates the expression of several short non-coding RNAs, such as the tRNAs, 5S rRNA and 7SL RNA. Regulation of 5S rRNA production by Brf could influence ribosome synthesis and hence growth. However, it was found that loss of Brf did not inhibit Pol I activity or alter levels of rRNA, suggesting that Brf probably does not directly influence ribosome numbers. One attractive possibility is that levels of the tRNAs may be limiting for translation and growth. In support of this notion, a recent paper showed that overexpression of Brf increased tRNA levels and promoted proliferation and transformation of cultured mammalian fibroblasts (Marshall, 2008). These effects of Brf were phenocopied by just increasing levels of tRNAiMet, and were associated with augmented mRNA translation and increased protein levels of growth promoters such as c-Myc and cyclin D1. No consistent increase was seen in tRNAs when Brf was overexpressed in larvae, perhaps because levels of other components of the TFIIIB complex are limiting in flies. Nevertheless, by controlling Brf activity and tRNA synthesis, TOR could promote translation of growth regulators and drive larval growth. In fact, a recent paper (Teleman, 2008) indicated that TOR signalling in Drosophila regulates dMyc protein levels, but not dMyc mRNA levels, consistent with a possible role for translational control (Marshall, 2012).

One interesting result of this work was the identification of a non-cell autonomous role for Brf in organismal growth. Specifically, it was found that Brf activity in the fat cells of Drosophila larvae could influence larval growth and final size. A role for TOR in the fat body has been shown to exist as a relay to control peripheral insulin signalling. In feeding larvae, amino-acid input into fat cells activates TOR, leading to transmission of a secreted signal from fat to brain to increase dILP expression and release from brain IPCs. These data suggest that stimulation of Pol III activity may be an important downstream effector of this adipose function of TOR. Thus, adipose-specific silencing of Brf led to reduced peripheral insulin signalling, slower larval growth rate and reduced final body size. As in starved larvae, this study found that loss of brf led to reduced expression of dilp mRNA (seen in both brf mutants and cg>brf RNAi larvae) and reduced dILP release from the brain. Moreover, given that levels of phospho-Akt are lower, and levels of dInR (a FOXO target) are higher in tissues from both brf mutant and r4>brf RNAi larvae it is clear that systemic insulin signalling is reduced when Brf is inhibited in the fat body. This study also found that another fat phenotype associated with starvation and loss of TOR, accumulation of lipid droplets, was phenocopied by loss of Brf. However, the autophagy phenotype of starved larval fat bodies was not phenocopied by loss of Brf. Therefore, Brf and Pol III function in the Drosophila fat body may mediate some, but not all of TOR's effects on growth and metabolism. The exact nature of the fat-to-brain secreted factor that controls insulin release in flies is not yet known, but perhaps translation of this signal, if it is a peptide or secreted protein, is influenced by changes in tRNA synthesis and translation rates. Indeed, it has been shown that dMyc activity in the fat body was also important for controlling systemic insulin signalling, growth and body size. This effect of dMyc correlated with elevated expression of ribosome biogenesis genes and increased nucleolar size, an index of ribosome synthesis. dMyc overexpression can also stimulate Pol III and tRNA levels, and the increase in body size caused by fat body overexpression of dMyc is reversed by knockdown of Brf. These data suggest that regulation of mRNA translational capacity is a key step downstream of TOR and dMyc in fat cells to control signalling to IPCs (Marshall, 2012).

Together, these data suggest that mRNA translational control may underlie a role for the fat body as an endocrine organ. A similar theme is emerging in mouse models. Mammalian adipose tissue is known to secrete adipokines and leptin to influence organismal metabolism and growth. The secretion of many of these factors is influenced by diet, suggesting a regulatory role for TOR signalling. Genetic inhibition of either TOR and S6K in mice leads to alterations in metabolic activity in adipose tissue. Moreover, loss of the translational repressors, 4E-BP1 and 4E-BP2, both of which are downstream TOR effectors, alters lipid and glucose metabolism in mice. To date, there are no mouse models of Pol III. However, it is interesting to speculate that changes in Pol III and tRNA synthesis are involved in mediating effects of TOR in adipose tissue in mice. Regulation of Pol III by TOR may also be important in the metabolic control of other processes. For example, TOR is a conserved regulator of organismal stress responses and lifespan. These stress responses rely on TOR's ability to control translation. It is suggested that regulation of Pol III and tRNA synthesis may also be a mode of control. Further organismal studies, using genetic modulation of Pol III function, should provide additional insights into these points (Marshall, 2012).

The full-length transcripts and promoter analysis of intergenic microRNAs in Drosophila melanogaster

MicroRNA (miRNA) transcription is poorly understood until now. To increase miRNA abundance, miRNA transcription was stimulated with CuSO(4) and Drosha enzyme was knocked down using dsRNA in Drosophila S2 cells. The full length transcripts of bantam, miR-276a and miR-277, the 5'-end of miR-8, the 3'-end of miR-2b and miR-10 were obtained. A series of miRNA promoter analyses was conducted to prove the reliability of RACE results. Luciferase-reporter assays proved that both bantam and miR-276a promoters successfully drove the expressions of downstream luciferase genes. The promoter activities were impaired by introducing one or multiple mutations at predicted transcription factor binding sites. Chromatin immunoprecipitation analysis confirmed that hypophosphorylated RNA polymerase II and transcription factor c-Myc physically bind at miRNA promoters. RNA interference of transcription factors Mad and Prd led to down-expression of bantam, miR-277 and miR-2b but not miR-276a, whereas RNAi of Dorsal had the opposite effect (Qian, 2011).

Drosophila Mbm is a nucleolar Myc and CK2 target required for ribosome biogenesis and cell growth of central brain neuroblasts

Proper cell growth is a prerequisite for maintaining repeated cell divisions. Cells need to translate information about intracellular nutrient availability and growth cues from energy sensing organs into growth promoting processes such as the sufficient supply with ribosomes for protein synthesis. Mutations in the mushroom body miniature (mbm) gene impair proliferation of neural progenitor cells (neuroblasts) in the central brain of Drosophila. Yet, the molecular function of Mbm has been unknown so far. This study shows that mbm does not affect the molecular machinery controlling asymmetric cell division of neuroblasts but instead decreases their cell size. Mbm is a nucleolar protein required for small ribosomal subunit biogenesis in neuroblasts. Accordingly, levels of protein synthesis are reduced in mbm neuroblasts. Mbm expression is transcriptionally regulated by Myc, which among other functions relays information from nutrient dependent signaling pathways to ribosomal gene expression. At the posttranslational level, Mbm becomes phosphorylated by protein kinase CK2, which has an impact on localization of the protein. It is concluded that Mbm is a new part of the Myc target network involved in ribosome biogenesis, which together with CK2-mediated signals enables neuroblasts to synthesize sufficient amounts of proteins required for proper cell growth (Hovhanyan, 2014).

A fundamental issue during development of a multicellular organism is to coordinate cell proliferation, the availability of nutrients, and cell growth. Prominent examples are neuroblasts, the progenitor cells of the Drosophila melanogaster central nervous system, which proliferate in a highly regulated manner during development. Upon selection and specification, central brain neuroblasts proliferate until the end of embryogenesis, when they enter a quiescent state until resuming proliferation with the beginning of larval development. Notable exceptions are the neuroblasts generating the mushroom bodies, a paired neuropil structure in the central brain involved in learning and memory processes, which proliferate throughout development. Depending on the neuroblast lineage, proliferation stops at late larval or pupal stages by terminal differentiation or apoptosis. The embryonic and larval waves of neurogenesis correlate with changes in neuroblast size. Embryonic neuroblasts decrease in size with each cell division until they enter quiescence; resumption of proliferation at the larval stage is preceded by cell growth. In contrast to embryonic neuroblasts, larval neuroblasts maintain their cell size until the end of the proliferation period, which is again accompanied by a decrease in cell size. Exit of neuroblasts from quiescence, and thereby activation of proliferation, depends on the nutritional status of the whole animal and is governed by the insulin receptor (InsR)-phosphatidylinositol 3-kinase (PI3K)-Akt signaling pathway, triggered by insulin-like peptide-producing glia cells, which receive nutritional signals from the fat body). Maintaining InsR signaling in combination with blocking of apoptosis is sufficient for long-term survival and proliferation of neuroblasts even in the adult fly. On the other hand, cellular nutrient sensing is mediated by the target of rapamycin (TOR) pathway, which, together with the InsR pathway, regulates cell growth through a variety of effector proteins at the levels of gene expression, ribosome biogenesis, and protein synthesis. Whereas neuroblast reactivation requires the interconnected InsR-PI3K and TOR pathways, neuroblast growth at larval stages is maintained even under nutrient restriction, by anaplastic lymphoma kinase (Alk)-mediated but InsR-independent activation of the PI3K pathway in combination with a direct influence of Alk on TOR effector proteins (Cheng, 2011; Hovhanyan, 2014 and references therein).

Cell growth requires protein synthesis, which depends on a sufficient supply of functional ribosomes. Ribosome biogenesis takes place in the nucleolus and involves transcription of single rRNA units and their processing and modification into 18S, 28S, and 5.8S rRNAs, which assemble with multiple ribosomal proteins to separately form the small and large ribosomal subunits. Upon transport to the cytoplasm, both subunits mature before they build up functional ribosomes. In general, one key downstream effector of TOR signaling is the transcription factor Myc, which controls cell growth in part by regulating ribosome biogenesis through transcriptional control of rRNA, ribosomal proteins, and proteins required for processing and transport of ribosomal components. Genomewide analyses of Drosophila Myc transcriptional targets emphasized the role of Myc as a central regulator of growth control but also identified many target genes with unknown molecular functions of the corresponding proteins. One of the Myc-responsive genes with an unknown function was mushroom body miniature (mbm). The original hypomorphic mbm1 allele was identified in a screen for viable structural brain mutants and showed a pronounced reduction in the size of the adult mushroom body neuropil, which was due at least in part to a reduction in the number of intrinsic mushroom body neurons. More severe allelic combinations indicated a general requirement for Mbm in brain development and uncovered a neuroblast proliferation defect as a major cause of the phenotype. However, which step requires Mbm for neuroblast proliferation remains elusive. Homology searches provided no clue about the molecular function of Mbm. Structural features of Mbm include several stretches enriched in certain amino acids, a putative nuclear localization signal, and two consecutive CCHC zinc knuckles. This report describes Mbm as a new nucleolar protein. Mbm is highly expressed in neuroblasts and is required for proper cell growth but not for processes controlling asymmetric cell division. Corresponding to the observed cell size defect, evidence is provided that small but not large ribosomal subunit biogenesis is impaired in the mutant, which could be a consequence of defective rRNA processing. Mbm is a transcriptional target of Myc and requires posttranslational modification by casein kinase 2 (CK2) for full functionality, as revealed by mutation of identified CK2 phosphorylation sites. These results provide a new link between Myc and growth control of neuroblasts and also establish a function of CK2 in neuroblasts (Hovhanyan, 2014).

This study identified Mbm as a new component of the nucleolus which has no obvious homologue outside the Drosophilidae. In contrast to the tripartite organization of vertebrate nucleoli in a fibrillar center, a dense fibrillar component (DFC), and a granular component, nucleoli of Drosophila neuroblasts often appear as a homogenous structure at the ultrastructural level, sometimes with intermingled fibrillar and granular components. In neuroblasts, Mbm colocalizes with fibrillarin and Nop5. In vertebrates, the methyltransferase fibrillarin is associated with Nop56/58 (corresponding to Drosophila Nop5) as part of the C/D type of small nucleolar ribonucleoprotein (snoRNP) complex required for rRNA processing in the DFC. Indeed, this study observed an aberrant rRNA intermediate in mbm on Northern blots, implicating a requirement of Mbm in rRNA processing. More specifically, based on the retention of RpS6 in the nucleoli of mbm neuroblasts, a function of Mbm is proposed in small ribosomal subunit biogenesis. The complementary phenotype, failure of large ribosomal subunit nucleolar-to-cytoplasmic transport, was observed in Drosophila upon knockdown of nucleostemin 1 (NS1). Yet the molecular function of Mbm remains elusive at this point because of its unique domain composition, with two zinc knuckle structures, several clusters of acidic or basic amino acids, and arginine/glycine-rich sequence stretches. For example, proteins containing arginine/glycine (RGG) repeats are found in a variety of RNP complexes, including snoRNPs. For a detailed biochemical analysis of Mbm function in ribosome biogenesis, cellular systems that are more accessible than neuroblasts are required, as these represent only a minor fraction of all brain cells. However, despite expression of Mbm in tissue culture S2 cells, neither cell size nor proliferation defects were detected under knockdown conditions (Hovhanyan, 2014).

Metabolic labeling of mbm neuroblasts indicated lowered protein synthesis rates, which could have been due to the lack of sufficient numbers of functional ribosomes. mbm larval brains reach nearly wild-type size, with a delay of several days, indicating that protein synthesis is maintained to at least some degree in neuroblasts. Since the process of asymmetric cell division itself is not affected in mbm flies, this provides one likely explanation for impaired neuroblast growth and proliferation. The importance of sufficient cell growth for repeated division of neuroblasts has been documented for mutations in signaling components. The comparison of the relative protein expression levels of Mbm and other nucleolar proteins in different cell types showed a more pronounced expression of Mbm in neuroblasts. This is confirmed by a comparative transcriptome analysis between neuroblasts and neurons. Altogether, the data suggest a more neuroblast-specific function of Mbm in ribosome biogenesis. Indeed, whereas most ribosomal subunit components are required in all cells, different isoforms are expressed for some components, with one isoform being required in all cells and the other isoform being required more specifically in stem cell lineages. Whereas loss of the generally required components causes early lethality, loss of specifically expressed isoforms is associated with a decrease in neuroblast size and an underproliferation phenotype. This emphasizes the specific needs of neuroblasts in ribosome biogenesis and cell growth as rate-limiting steps for self-renewal (Hovhanyan, 2014 and references therein).

The identification of Mbm as a transcriptional target of Myc provides a potential link to systemic and cell-intrinsic growth control of neuroblasts mediated by the InsR-PI3K-Akt and TOR pathways. In contrast to other tissues, where Myc is a downstream effector of these pathways, information is still largely missing in the case of neuroblasts. Myc is expressed in neuroblasts, and upon knockdown, mild effects on neuroblast size but not neuroblast number were observed. Consistent with the role of Drosophila Myc in expression of many genes involved in ribosome biogenesis, removal of Myc function in single neuroblasts caused corresponding decreases in Mbm and Nop5 levels (Hovhanyan, 2014).

Mbm function is dependent on posttranslational modification by the protein kinase CK2. CK2 is a promiscuous kinase expressed in all eukaryotic cells, with a vast array of substrates with pleiotropic functions. However, CK2 not only acts as a heterotetrameric α2β2 holoenzyme but also exists as free populations of both subunits, with independent functions. Pronounced nuclear or nucleolar localization of CK2 subunits was observed in vertebrate cells. In the nucleolus, CK2 participates in rRNA transcription by phosphorylating different components of the RNA polymerase I transcription machinery. Proteins involved in ribosome biogenesis, such as B23 (also known as nucleophosmin), are also CK2 phosphorylation targets. CK2 modulates the ability of B23 to act as a molecular chaperone, its mobility rate and compartmentalization in the nucleolus, and its shuttling between the nucleolus and the nucleoplasm. Mbm and Nopp140 are the only described nucleolar phosphorylation targets of CK2 in Drosophila. This correlates with the observed nucleolar accumulation of CK2α in neuroblasts and the copurification of Mbm and CK2α. Although Mbm proteins with mutated CK2 phosphorylation sites showed cytoplasmic accumulation, nucleolar localization was still evident. Yet they were largely unable to complement the loss of endogenous Mbm function, indicating that phosphorylation by CK2 not only is a localization determinant but also is important for proper functioning of Mbm in the nucleolus. CK2 is often considered a constitutively active kinase which is not regulated by second messenger signaling cascades. However, there is increasing evidence for regulation of CK2 at the levels of the holoenzyme, the dynamics of localization of individual subunits under different conditions, and interactions with small molecules such as polyamines. Overexpression of ornithine decarboxylase, the rate-limiting enzyme in polyamine biosynthesis and a known Myc target gene, increases CK2 phosphorylation activity toward nucleolar B23 in mouse keratinocytes. It would be interesting to test for a regulatory influence of CK2 on Mbm function under different conditions (Hovhanyan, 2014).

In summary, Mbm is considered to be part of the Myc and CK2 regulatory networks for coordination of neuroblast growth and proliferation; however, more information about the molecular function of Mbm at the level of small ribosomal subunit biogenesis is still required (Hovhanyan, 2014).

The Drosophila Nol12 homologue viriato is a dMyc target that regulates nucleolar architecture and is required for dMyc-stimulated cell growth

The nucleolus is a subnuclear factory, the activity of which is required beyond ribosome biogenesis for the regulation of cell growth, death and proliferation. In both Drosophila and mammalian cells, the activity of the nucleolus is regulated by the proto-oncogene Myc. Myc induces the transcription of genes required for ribosome biogenesis and the synthesis of rRNA by RNA polymerase I, a nucleolar event that is rate limiting for cell growth. This study shows that the fruit fly Nol12 homologue Viriato is a key determinant of nucleolar architecture that is required for tissue growth and cell survival during Drosophila development. It is further shown that viriato expression is controlled by Drosophila Myc (dMyc), and that the ability of dMyc to stimulate nucleolar and cellular growth depends on viriato expression. Therefore, viriato acts downstream of dMyc to ensure a coordinated nucleolar response to dMyc-induced growth and, thereby, normal organ development (Marinho, 2011).

Tissue and organ development require a precise coordination of cellular growth, proliferation, differentiation and apoptosis. At the core of the cell, and crucial for its growth, the nucleolus is the subnuclear compartment where ribosome biogenesis takes place (Boisvert, 2007). Cell mass accumulation is required for proliferation, implying that the regulation of nucleolar function plays an important role in the control of proliferation rates. In the nucleolus, RNA polymerase I (Pol I) synthesizes pre-rRNAs, which are processed, modified and assembled in 40S and 60S pre-ribosomal particles that are then exported to the cytoplasm. Besides its role as the ribosome factory, the nucleolus is now also considered to be a multifunctional regulatory compartment involved in RNA processing events, sensing of cell stress, and cell cycle and apoptosis regulation (Boisvert, 2007; Marinho, 2011 and references therein).

A key step in ribosome biogenesis is pre-rRNA gene transcription by Pol I, a process that in human cells is known to be stimulated by the binding of c-MYC to rRNA promoters in the nucleolus (Arabi, 2005; Grandori, 2005). Further, Drosophila Myc (dMyc; also known as Diminutive), which is known to control the cell cycle and apoptosis, has been shown to be necessary and sufficient for the transcription of genes encoding Pol I transcription machinery factors, such as Tif-IA and RpI135 (the largest Pol I subunit), genes encoding pre-rRNA processing and modifying factors, such as Nop60B and Fibrillarin, as well as a large set of ribosomal genes (Grewal, 2005; Pierce, 2008). The ability of dMyc to induce a coordinated nucleolar hypertrophy and to stimulate pre-rRNA transcription and ribosome biogenesis in general are required for dMyc-stimulated growth during Drosophila development (Grewal, 2005). This study identifies viriato (vito) as a dMyc target gene that coordinates nucleolar and growth responses downstream of dMyc (Marinho, 2011).

This study shows that dMyc controls vito mRNA levels to regulate nucleolar architecture and that vito is required for dMyc to reach its full potential as a potent cell growth inducer. Furthermore, the knockdown of vito expression also correlated with an increase in p53-independent, caspase-mediated apoptotic cell death, suggesting a potential novel link between structural and functional changes in the nucleolus and activation of the pro-apoptotic rpr/grim/hid complex (Marinho, 2011).

During development, dMyc plays a crucial role in translating intracellular and extracellular cues to regulate the pace of cell growth and proliferation. One of the main mechanisms for dMyc-stimulated growth appears to be the transcriptional control of nucleolar ribosome biogenesis genes (Grewal, 2005; Hulf, 2005; Teleman, 2008; Demontis, 2009). Cells of the salivary glands are polyploid secretory cells with very active biosynthetic pathways. In these cells, increasing or reducing Vito levels results in changes in the nucleolar localisation patterns of the pre-rRNA methyltransferase Fibrillarin and in alterations in nucleolar structure. The fact that vito does not appear necessary for the expression of dMyc targets implicated in ribosomal biogenesis suggests that part of the control that dMyc exerts on the nucleolus is mediated independently of its regulation of vito expression. In addition, several results support the hypothesis that the Myc-Nol12 regulatory relationship is evolutionarily conserved. Genome-wide chromatin immunoprecipitation analysis has shown that c-MYC binds the NOL12 promoter in both a human transformed B-cell line and in mouse stem cells. Non-canonical E-box motifs (CACATG) have been identified in the putative proximal promoter regions of both vito and human NOL12 (Marinho, 2011).

In addition to its function in cell growth downstream of dMyc, vito plays a role in supporting the proliferation and survival of diploid cells. dMyc mutants are smaller than the wild type, and dMyc mutant cells grow poorly in the context of wild-type tissue. Therefore, vito is a rate-limiting factor for tissue growth that links dMyc with nucleolar architecture. The mechanisms enacting this link might prove relevant for the regulation of Myc function in tumourigenesis (Marinho, 2011).

Cropped, Drosophila transcription factor AP-4, controls tracheal terminal branching and cell growth

Endothelial or epithelial cellular branching is vital in development and cancer progression; however, the molecular mechanisms of these processes are not clear. In Drosophila, terminal cell at the end of some tracheal tube ramifies numerous fine branches on the internal organs to supply oxygen. To discover more genes involved in terminal branching, mutants were sought with very few terminal branches using the Kiss enhancer-trap line collection. This analysis identified cropped (crp), encoding the Drosophila homolog of the transcription activator protein AP-4. Overexpressing the wild-type crp gene or a mutant that lacks the DNA-binding region in either the tracheal tissues or terminal cells led to a loss-of-function phenotype, implying that crp can affect terminal branching. Unexpectedly, the ectopic expression of crp also led to enlarged organs, and cell-counting experiments on the salivary glands suggest that elevated levels of AP-4 increase cell size and organ size. Like its mammalian counterpart, cropped is controlled by dMyc, as ectopic expression of dMyc in terminal cells increased cellular branching and the Cropped protein levels in vivo. This study has found that the branching morphogenesis of terminal cells of the tracheal tubes in Drosophila requires the dMyc-dependent activation of Cropped/AP-4 protein to increase the cell growth of terminal cells (Wong, 2015).

snoRNAs are a novel class of biologically relevant Myc targets

Myc proteins are essential regulators of animal growth during normal development, and their deregulation is one of the main driving factors of human malignancies. They function as transcription factors that (in vertebrates) control many growth- and proliferation-associated genes, and in some contexts contribute to global gene regulation. This study combined ChIPseq and RNAseq approaches in Drosophila tissue culture cells to identify a core set of less than 500 Myc target genes, whose salient function resides in the control of ribosome biogenesis. Among these genes, the non-coding snoRNA genes were found to be a large novel class of Myc targets. All assayed snoRNAs are affected by Myc, and many of them are subject to direct transcriptional activation by Myc, both in Drosophila and in vertebrates. The loss of snoRNAs impairs growth during normal development, whereas their overexpression increases tumor mass in a model for neuronal tumors. This work shows that Myc acts as a master regulator of snoRNP biogenesis. In addition, in combination with recent observations of snoRNA involvement in human cancer, it raises the possibility that Myc's transforming effects are partially mediated by this class of non-coding transcripts (Herter, 2015).

Protein Interactions

DMax, the Drosophila homolog of mammalian Max, consists of 161 amino acids, compared with the human Max9 which has 160. The greatest sequence similarity is within the bHLH leucine zipper domain with a 67% identity. All residues contacting DNA are conserved. Furthermore, the N-termini of dMax and human Max are highly conserved, including two casein kinase II phophorylation sites that negatively regulate DNA binding. DMax interacts strongly with dMyc and dMax, but dMyc does not self associate. DMyc alone does not bind the CACGTG site but dMyc-dMax does. The dMyc-dMax heterodimer is also able to activate transcription (Gallant, 1996).

Expression of many mammalian genes is activated by the binding of heterodimers of the Myc and Max proteins to specific DNA sequences called E-boxes. Transcription of the same genes is repressed upon binding to the same sequences of complexes composed of Max, Mad/Mxi1, the co-repressors Sin3 and N-CoR, and the histone deacetylase Rpd3 (see Drosophila Rpd3). Max-Mad/Mxi1 heterodimers, which bind to E-boxes in the absence of co-repressors, do not inhibit gene expression simply by competition with Myc-Max heterodimers, but require Sin3 and Rpd3 for efficient repression of transcription. A Drosophila homolog of Sin3 (dSin3) has been cloned and it has been found to be ubiquitously expressed during embryonic development. Yeast, mouse and Drosophila proteins share six blocks of strong homologies, including four potential paired amphipathic helix domains. In addition, the domain of binding to the histone deacetylase Rpd3 is strongly conserved. Null mutations cause recessive embryonic lethality (Pennetta, 1998).

The Drosophila protein is considerably longer (2061 residues) than the S. cerevisiae (ySin3: 1538 residues) and the two known mouse (mSin3A: 1219 residues; mSin3B: 954 residues) proteins. The six conserved regions between these four polypeptides are centered around four putative paired amphipathic helix (PAH) domains, in the spacer between the third and fourth PAH domains and in the domain that follows the fourth PAH. The regions containing the first two PAH show the best conservation with 54% (PAH1) and 42% (PAH2) of identical residues between yeast, Drosophila and mouse proteins. In these domains, higher homology is found between dSin3 and mSin3A than between mSin3A and mSin3B. Conservation of the two other PAH domains is lower, especially when the three species are compared. When only dSin3, mSin3A and mSin3B are considered, conservation of the first three PAH domains is very high (around 55%-77% identity). It is concluded that the four PAH domains of dSin3 are likely to share structural and functional properties with those in other Sin3 homologs. The PAH domains of ySin3 are important for its function as a transcriptional repressor. The interaction between Mad and mSin3 is known to be mediated by PAH2. The high level of conservation suggests that the same domain in dSin3 could be responsible for interaction with a Drosophila Mad protein (Pennetta, 1998).

In addition to the four short regions containing the PAH domains, two longer domains of strong conseravtion are noticeable. One of them has a length of close to 3000 amino acids and is located between PAH3 and PAH4. Identity between either dSin3 and mSin3A or between the two mouse polypeptides is greater than 70%. When ySin3 is included in the comparison, 33% of the residues are shared between the four proteins. The high level of conservation suggests that this region has an important structural or functional role. This hypothesis is supported by the finding that interaction between mSin3 and a mouse histone deacetylase homologous to yeast Rpd3 takes place in this domain (called HID). It is likely that the Drosophila Rpd3 protein also interacts with dSin3 in this domain. A second region of interest is located immediately after the fourth PAH and goes almost to the end of mSin3B. This domain of about 150 residues shows 60% identity between the two mouse polypeptides, but is less conserved in Drosophila (45%) and yeast (20%) (Pennetta, 1998).

Cloning of dSin3 has been reported in a genetic screen aimed at the identification of components of the sina signaling pathway (Neufeld, 1998). Two protein isoforms of 1748 and 1773 residues have been reported, which differ only by a few C-terminal amino acids. The sequence described here reveals a third isoform. Compared to the longest published cDNA, the cDNAs reported here show the elimination of a small putative intron located after the end of the last conserved domain, at the same place where the two other isoforms differ (amino acid 1745). The resulting open reading frame encodes a polypeptide longer by about 300 amino acids. This isoform may be ovarian-specific, since it was found in all clones isolated from an ovarian cDNA library (Pennetta, 1998).

The basic helix-loop-helix (bHLH) proteins represent an evolutionarily conserved class of transcription factors that are known to play important roles in cell determination and differentiation during animal embryonic development. Following an exhaustive search of the complete Drosophila genome sequence using a PSI-BLAST strategy, 19 new genes were identified, bringing the total number of bHLH- encoding genes in the Drosophila genome to 56. These new genes belong to various subfamilies of bHLH transcription factors, such as the Daughterless, Hairy-Enhancer of split, bHLH-PAS or bHLHZip subfamilies. The embryonic expression pattern of each of these new genes has been analyzed by in situ hybridization. By looking for close structurally-related motifs, two genes were found that represent likely orthologs of vertebrate Mnt and Mlx. Together with previous reports, these data suggest that, similar to networks involved in neurogenesis and myogenesis, the network of Myc-related genes has been globally conserved throughout evolution (Peyrefitte, 2001).

Drosophila structural homologs of vertebrate Mnt (CG2856 or dmMnt) and Mlx (CG3350 or dmMlx) are members of the 'Myc-Mad-Max network' which plays roles in cell proliferation, differentiation and apoptosis. In this network, the Max protein appears to play a central role. This protein can form transactivating complexes when associated with Myc, but repressive complexes when bound to Mad or Mnt. It has been suggested that Mad and Mnt are antagonists of Myc. Studies on the Drosophila homologs of Myc (dmMyc) and Max (dmMax) have shown that they share common functions with the vertebrate genes. Recently, Mlx, a new dimerization partner of Mnt, has been identified. Like mouse Mlx, dmMlx is expressed ubiquitously. Interestingly, no Mad homolog was identified in Drosophila. In vertebrate development, Myc is preferentially expressed in undifferentiated, proliferating cells, whereas Mad expression is increased in differentiated, non-proliferating tissues. Unlike the previous two genes, Mnt appears to be ubiquitously expressed during development. This is in contrast to the dynamic expression of dmMnt detected in Drosophila embryos. Taken together, the possible absence of a structural Drosophila Mad homolog and the dmMnt expression profile raise the possibility that dmMnt may play a role similar to Mad in vertebrates (Peyrefitte, 2001).

Ras1 amd Myc activity in the developing wing

The Ras GTPase links extracellular signals to intracellular mechanisms that control cell growth, the cell cycle, and cell identity. An activated form of Drosophila Ras (RasV12) promotes these processes in the developing wing, but the effector pathways involved are unclear. Evidence is presented indicating that RasV12 promotes cell growth and G1/S progression by increasing dMyc protein levels and activating PI3K signaling, and that it does so via separate effector pathways. Endogenous Ras is required to maintain normal levels of dMyc, but not PI3K signaling during wing development. Finally, induction of dMyc and regulation of cell identity are separable effects of Raf/MAPK signaling. These results suggest that Ras may only affect PI3K signaling when mutationally activated, such as in RasV12-transformed cells, and provide a basis for understanding the synergy between Ras and other growth-promoting oncogenes in cancer (Prober, 2002).

In the developing Drosophila wing, Ras, dMyc, and PI3K regulate rates of cellular growth (i.e., mass accumulation) and progression through the G1/S transition of the cell cycle without affecting overall rates of cell division. These results concur with experiments in mice showing that Ras, Myc, and PI3K promote cell growth without affecting rates of cell division. This study shows that an activated form of Drosophila Ras (RasV12) is capable of increasing dMyc protein levels as well as levels of PI3K signaling, suggesting that RasV12 drives growth and G1/S progression via both of these mechanisms. RasV12 effector loop mutants were used to show that RasV12 affects dMyc and PI3K signaling via separate pathways, and that overexpressed dMyc and PI3K do not cross-regulate each other. Thus, a hierarchy has been established for these growth-regulatory proteins (Prober, 2002).

Wing disc cells lacking ras have reduced levels of dMyc protein, indicating that Ras is required to maintain normal dMyc protein levels during wing development. ras-/- cells contain significant levels of dMyc protein, however, indicating that Ras is not absolutely necessary for dMyc expression, and suggesting that reduced dMyc levels may not fully explain the growth deficit of ras-/- cells. However, dMyc antibody staining intensity was ~40% lower for dmycP0 or dmycP1 homozygotes than for dmycP0 heterozygotes in regions of the wing disc that normally contain high dMyc levels (i.e., wing pouch and notum. Because dmycP0/P0 clones have severely reduced growth rates, it seems reasonable to expect that the ~20% reduction of dMyc levels in ras-/- clones will also reduce growth rates. RasV12 increases dMyc levels post-transcriptionally, and studies in mammalian cell culture has shown that RasV12 stabilizes Myc protein. Therefore, it is likely that ras-/- cells still transcribe dmyc mRNA, but that following translation, dMyc protein is less stable. What other mechanisms may regulate dMyc levels? Wingless (Wg) signaling represses dmyc expression along the dorsal-ventral boundary of the developing wing. In addition, expression of an activated version of the Decapentaplegic (Dpp) receptor Thickveins (TkvQ238D) can increase levels of dMyc protein in the wing, whereas loss of this same receptor suppresses dMyc levels. Thus, Ras signaling may be one of many inputs affecting dMyc expression in the wing. Ras may stabilize the low levels of dMyc protein observed throughout the developing wing and/or refine the patterned dmyc expression regulated by other signals. The complex regulation of dMyc expression in vivo may account for the lack of a clear correspondence between patterns of high endogenous Ras activity and dMyc expression (Prober, 2002).

Drosophila Half pint negatively regulates dmyc and stg to inhibit cell proliferation

Mammalian FIR has dual roles in pre-mRNA splicing and in negative transcriptional control of Myc. Half pint (Hfp), the Drosophila ortholog of FIR, inhibits cell proliferation in Drosophila. Hfp overexpression potently inhibits G1/S progression, while hfp mutants display ectopic cell cycles. Hfp negatively regulates dmyc expression and function: reducing the dose of hfp increases levels of dmyc mRNA and rescues defective oogenesis in dmyc hypomorphic flies. The G2-delay in dmyc-overexpressing cells is suppressed by halving the dosage of hfp, indicating that Hfp is also rate-limiting for G2-M progression. Consistent with this, the cycle 14 G2-arrest of stg mutant embryos is rescued by the hfp mutant. Analysis of hfp mutant clones revealed elevated levels of Stg protein, but no change in the level of stg mRNA, suggesting that hfp negatively regulates Stg via a post-transcriptional mechanism. Finally, ectopic activation of the wingless pathway, which is known to negatively regulate dmyc expression in the wing, results in an accumulation of Hfp protein. These findings indicate that Hfp provides a critical molecular link between the developmental patterning signals induced by the wingless pathway and dMyc-regulated cell growth and proliferation (Quinn, 2004).

The Drosophila stock EP(3)3058 (hfpEP) harbors a recessive lethal P element insertion in the 5' UTR of hfp, 94 bp upstream of the initiating methionine codon. Homozygous hfpEP larvae were of similar size to age-matched wild type third instar larvae. However, the pupariation of hfpEP larvae was consistently delayed by approximately 2 days, and continued growth during this period resulted in wandering larvae and pupae ~20% larger than wild-type third instar larvae. The duration of the pupal stage was normal for hfpEP mutant animals; however, they failed to eclose and died as pharate adults that were larger than wild type. The hfpEP/hfpEP terminal phenotype included duplication of superior scutellar macrochaete, and malformation of legs, wings and sex combs (Quinn, 2004).

The pleiotropic phenotype of hfp mutant animals indicated that Hfp might be involved in several stages of development. In Drosophila, maternal transcripts are transferred during oogenesis and serve to sustain early embryonic development until stage 5, after which zygotic transcription commences. Northern analysis revealed that hfp mRNA is maternally deposited in the early embryo; however, zygotic hfp expression is low during late embryonic and early larval stages. hfp transcripts are also detected in third instar larvae, pupae and adults. A marked decrease in hfp mRNA occurs in hfpEP/hfpEP and hfpEP/Df(3L)Ar14-8 larvae, when compared with age-matched wild-type third instar larvae. However, hfp transcript is still detectable, consistent with the notion that hfpEP is not a null allele (Van Buskirk, 2002). In wild-type animals, expression of hfp during third instar coincides with the onset of differentiation in imaginal discs. Hfp protein expression was examined in wing discs using an antibody recognizing Hfp (Van Buskirk, 2002) and an antibody to Geminin, which is abundant in late S phase and G2 but absent in G1 cells, was used to visualize the dorsoventral compartment boundary of the wing (the ZNC). Hfp protein is detected in the nucleus of most wing disc cells, with higher staining in cells in the ZNC. Consistent with Northern analysis, Hfp protein level is significantly reduced in wing discs from hfpEP/hfpEP larvae (Quinn, 2004).

In order to investigate whether Hfp regulates cell proliferation during Drosophila development, BrdU incorporation was measured in wing discs from wandering hfpEP/hfpEP larvae. In wild-type wing discs the ZNC is clearly marked by the absence of BrdU labelling. The number of S-phase cells is markedly increased in hfpEP mutant wing discs: BrdU incorporation is uniform across the disc and cell cycle arrest is not evident in the ZNC region. Strikingly, anti-phosphohistone H3 antibody staining of mitotic cells, is also elevated, indicating an overall increase in cell proliferation in hfp wing discs (Quinn, 2004).

Hfp is a negative regulator of cell cycle entry in Drosophila as evidenced by (1) ectopic S phases in the ZNC of hfp mutant wing discs and increased S phase in the second mitotic wave in the eye disc; (2) inhibition of S phases in larval imaginal tissues by overexpression of the UAS-hfp transgene; and (3) dominant suppression of the GMR- driven human p21 or dacapo rough eye phenotypes and rescue of the posterior band of S phases in GMR-p21 eye discs by reducing the level of hfp. These data suggest that Hfp normally has a role in preventing S-phase entry in cells destined to differentiate in the eye and wing imaginal discs. Furthermore, this negative regulation of the cell cycle by Hfp is partly a consequence of inhibitory affects on dmyc, since (1) an increased level of dmyc mRNA transcript occurs in hfp-/- clones, and (2) reduced levels of Hfp can rescue the dmyc mutant ovary phenotype, by restoring levels of dmyc mRNA to more wild-type levels. Indeed, upregulation of dmyc expression in Hfp mutants may explain the rescue of S phases in eye discs overexpressing p21 or Dacapo, consistent with the observation that dmyc mutants dominantly enhance the GMR-p21 and GMR-driven dacapo rough eye phenotypes. Mammalian Myc stimulates cyclin E expression, activation of Cdks, antagonizes the action of Cdk inhibitors, including p27, and can downregulate p21 transcription and p21 activity via direct c-Myc-p21 protein-protein interaction. In Drosophila, dMyc has been shown to lead to an increase in Cyclin E protein levels by a post-transcriptional mechanism, which by itself could explain the suppression of the GMR-p21 eye phenotype by a reduction in the dose of hfp. Whether dMyc can also inhibit p21 or Dacapo activity in Drosophila is unknown (Quinn, 2004).

Increased levels of dmyc transcript are observed in hfp mutant clones, consistent with Hfp acting to repress dmyc transcript accumulation in Drosophila imaginal tissues. The upregulation of dmyc mRNA in hfp mutant tissue could occur through alterations in dmyc transcription (initiation or elongation), pre-mRNA splicing, mRNA message stability or a combination of these processes. Mammalian FIR was first shown to regulate pre-mRNA splicing by binding to RNA polypyrimidine tracts and cooperating with the essential splicing factor U2AF. Consistent with this, recent studies in Drosophila show that the FIR ortholog Hfp is required for correct splicing of several genes in the developing ovary (Van Buskirk, 2002). Mammalian FIR has been shown to have a second role as transcriptional repressor of Myc, through first forming a complex with the Myc activator FBP and interfering with the basal transcription apparatus by then binding TFIIH, thereby disrupting helicase function. The data described in this study suggest that the cell cycle inhibitory function of Hfp is partly a consequence of negatively regulating dmyc expression. Therefore, the dual roles of transcription regulation and mRNA splicing appear to have been evolutionarily conserved between Drosophila Hfp and mammalian FIR. It remains to be determined whether Hfp inhibits dmyc expression by a mechanism analogous to the mammalian FIR/FBP/FUSE interaction. A FUSE element has not been identified upstream of the dmyc promoter, and although the Drosophila splicing factor PSI is a highly conserved ortholog of FBP, it has not been reported whether PSI can activate dmyc expression (Quinn, 2004).

The finding that hfp mutants do not phenocopy dmyc overexpression suggests that inhibition of dmyc expression is not the only role of Hfp. Although increased S phases are observed in hfp mutant wing discs, this is not associated with increased cell size, as occurs with dmyc overexpression in the wing disc. Rather, in hfp mutant wing discs the ZNC, which normally contains domains of G1- and G2-arrested cells, has ectopic S-phase and M-phase cells. Since cells in hfp mutant wing discs are of normal size and ectopically enter S phase, it is possible that progression through G2 may also be accelerated. Indeed, the increased number of mitotic cells observed in eye imaginal discs when the level of Hfp is reduced in a dmyc overexpression background, suggests that Hfp normally negatively regulates G2-M phase progression. Furthermore, the abnormal mitotic figures observed in hfpEP mutant embryos are consistent with accelerated cell cycle progression. Most importantly, the hfp mutant rescues the cycle 14 G2-arrest that normally occurs in stg mutant embryos, and hfp mutant clones have increased levels of Stg protein, suggesting that Hfp normally exerts an inhibitory affect on G2-M progression via negatively regulating Stg translation or protein stability. Thus, Hfp may be required for negatively regulating both the G1-S phase transition by downregulating dmyc and the G2-M transition by negatively regulating stg (Quinn, 2004).

The Wg pathway is required to downregulate both dmyc and stg expression in order to limit cell proliferation in the ZNC during wing development. Activation of the Wg pathway, using either dominant negative Shaggy or by generation of axin clones, results in a strong and specific increase in Hfp protein, demonstrating that Wg pathway activation is sufficient to cause Hfp induction. These findings support a model in which Wg signalling causes induction of Hfp in the wing disc ZNC, which in turn inhibits dmyc expression (to elicit the posterior, G1 arrest) and stg expression or activity (to provide the anterior, G2-arrested domains). The involvement of Achaete and Scute in this process, which play a role in the negative regulation of stg remains to be elucidated. Previous studies have shown that Ras signalling through Raf/MAPK upregulates dmyc post-transcriptionally in wing disc cells and is required to maintain normal dMyc protein levels in the wing disc. In contrast, since hfp clones have increased dmyc mRNA, Hfp must normally inhibit dmyc mRNA accumulation. Furthermore, overexpression of Hfp inhibits cell proliferation in all wing and eye imaginal discs, suggesting that Hfp may normally override mitogenic signals and lead to cell cycle arrest during particular stages of development (Quinn, 2004).

In summary, these results suggested that Hfp negatively regulates cell proliferation by inhibiting dmyc transcription and Stg protein accumulation. Hfp is required for the developmentally regulated cell cycle arrest within the ZNC and is responsive to the Wg signalling pathway that regulates this arrest, suggesting that Hfp links patterning signals to cell proliferation during Drosophila development (Quinn, 2004).

Hfp inhibits Drosophila myc transcription and cell growth in a TFIIH/Hay-dependent manner

An unresolved question regarding the RNA-recognition motif (RRM) protein Half pint (Hfp) has been whether its tumour suppressor behaviour occurs by a transcriptional mechanism or via effects on splicing. The data presented in this study demonstrate that Hfp achieves cell cycle inhibition via an essential role in the repression of Drosophila myc (dmyc) transcription. Regulation of dmyc requires interaction between the transcriptional repressor Hfp and the DNA helicase subunit of TFIIH, Haywire (Hay). In vivo studies show that Hfp binds to the dmyc promoter and that repression of dmyc transcription requires Hfp. In addition, loss of Hfp results in enhanced cell growth, which depends on the presence of dMyc. This is consistent with Hfp being essential for inhibition of dmyc transcription and cell growth. Further support for Hfp controlling dmyc transcriptionally comes from the demonstration that Hfp physically and genetically interacts with the XPB helicase component of the TFIIH transcription factor complex, Hay, which is required for normal levels of dmyc expression, cell growth and cell cycle progression. Together, these data demonstrate that Hfp is crucial for repression of dmyc, suggesting that a transcriptional, rather than splicing, mechanism underlies the regulation of dMyc and the tumour suppressor behaviour of Hfp (Mitchell, 2010).

Tight control of c-myc transcription is essential as upregulation of c-MYC expression is associated with most human cancers. In vitro mammalian studies have suggested that one mechanism for c-myc promoter regulation involves the presence of a paused, but transcriptionally engaged, Pol II at the c-myc start site. The paused polymerase can allow a rapid response to developmental/mitogenic signals and protect the c-myc promoter from unwanted activation. This study provides strong evidence that the FIR homologue Hfp is crucial for transcriptional repression of dmyc and cell growth, suggesting that a transcriptional, rather than a splicing, mechanism underlies the tumour suppressor behaviour of Hfp. In addition, these data show that the mechanism proposed for repression of c-myc transcription by the mammalian RRM protein FIR is conserved in Drosophila (Mitchell, 2010).

First, FIR negatively regulates c-myc transcription (Liu, 2006), and this study has shown that Hfp can bind the dmyc promoter and is essential for repression of dmyc transcription. Although FIR mutations correlate with colorectal cancer incidence (Matsushita, 2006), whether dysregulated FIR is the cause of the increased c-myc expression and/or the overgrowth phenotypes associated with these cancers is unknown. This study has demonstrated that loss of Hfp results in a cell growth phenotype, which occurs in a dMyc-dependent manner. These data strongly suggest that dysregulated FIR in the human context might be causative in cancer initiation and progression. Further support for conservation of the proposed FIR and XPB mechanism for c-myc control is provided by the finding that the repression of dmyc by Hfp occurs in a manner dependent on the XPB helicase homologue Hay, as the increases in dmyc transcription and cell growth associated with loss of Hfp are dependent on the presence of Hay. Thus, these studies provide novel insights into the molecular mechanisms required for controlling c-myc transcription, which are likely to be important for understanding FIR- and XPB-related cancers (Mitchell, 2010).

Although in vitro mammalian studies have shown that the response of c-myc to serum is defective in FIR loss-of-function and XPB-related cancer cells (Liu, 2006), the upstream factors in the pathway by which serum mediates c-myc repression via XPB and FIR have not been identified. In Drosophila, this study has shown that Hfp protein levels are regulated, in part, by Wg. As Hfp is responsive to the Wg pathway, and promoter occupancy by FIR responds to factors in serum, it is hypothesized that Hfp levels and/or activity will be controlled by developmental/growth signals. It is predicted that cross-talk between a specific complement of growth signals, including Wg, will tightly regulate dmyc transcription and growth via Hfp and Hay, which are likely to be relevant to the processes involved in the dysregulation of c-MYC during human malignancy. Thus, a current working model for repression of dmyc by Hfp is presented. In response to negative growth signals Hfp binds to inhibit dmyc transcription, but upon mitogenic stimulation dmyc transcription results from the prevention of promoter occupancy by Hfp. The possibility cannot be ruled out that Hfp might in some instances provide a repressive effect that must be overcome by the presence of activators. Thus, the mechanism(s) regulating Hfp levels and/or occupancy of the dmyc promoter is the subject of ongoing studies (Mitchell, 2010).

In conclusion, this work suggests analogous systems are required for transcriptional regulation of the c-myc oncogene and dmyc. The knowledge gained from future studies on the developmental regulation of these proteins in Drosophila will be informative in understanding the regulation of c-myc by the homologous proteins in mammals (Mitchell, 2010).

The Drosophila F box protein archipelago regulates dMyc protein levels in vivo

The Myc oncoprotein is an important regulator of cellular growth in metazoan organisms. Its levels and activity are tightly controlled in vivo by a variety of mechanisms. In normal cells, Myc protein is rapidly degraded, but the mechanism of its degradation is not well understood. Genetic and biochemical evidence is presented that Archipelago (Ago), the F box component of an SCF-ubiquitin ligase and the Drosophila ortholog of a human tumor suppressor, negatively regulates the levels and activity of Drosophila Myc (dMyc) protein in vivo. Mutations in archipelago (ago) result in strongly elevated dMyc protein levels and increased tissue growth. Genetic interactions indicate that ago antagonizes dMyc function during development. Archipelago binds dMyc and regulates its stability, and the ability of Ago to bind dMyc in vitro correlates with its ability to inhibit dMyc accumulation in vivo. These data indicate that archipelago is an important inhibitor of dMyc in developing tissues. Because archipelago can also regulate Cyclin E levels and Notch activity, these results indicate how a single F box protein can be responsible for the degradation of key components of multiple pathways that control growth and cell cycle progression (Moberg, 2004).

myc genes encode basic-helix-loop-helix-zipper (bHLHZ) domain transcription factors that dimerize with Max family proteins to promote cell growth and proliferation in metazoan organisms. The Myc-Max complex is implicated in the transcriptional regulation of many genes that are required for cell growth and metabolism; such genes include those for translation initiation factors and ribosomal components. The role of Myc in promoting growth is likely to contribute to its role as an oncoprotein in a wide variety of human tumor types. myc overexpression also promotes tumorigenesis in mice and zebrafish, indicating that the oncogenic properties of myc genes are conserved in other organisms (Moberg, 2004 and references therein).

Deregulation of mammalian Myc in cancer occurs by a variety of mechanisms. In some cancers, notably lymphomas, mutations found within the Myc protein have been shown to increase its stability (Salghetti, 1999; Gregory, 2000). Myc protein is normally turned over rapidly in vivo and in cultured cells has a half-life of 20-30 min, and several studies have shown that Myc protein is subject to ubiquitin-dependent proteasomal degradation (Grandori, 2000). Ubiquitination of Myc in turn appears to be regulated by phosphorylation at two distinct sites in the protein's amino-terminal portion, Threonine 58 (Thr58) and Serine 62 (Ser62). Evidence suggests that MAP kinase mediates phosphorylation of Ser62 and that this stabilizes c-Myc. Phospho-Ser62 may be required for subsequent phosphorylation of Thr58 by glycogen-synthase kinase 3 (GSK3: Drosophila homolog, Shaggy), which promotes the ubiquitination and degradation of c-Myc (Gregory, 2003). Significantly, Thr58Ile is the most common c-Myc mutation in Burkitt's lymphoma and is known to stabilize Myc considerably (Salghetti, 1999; Gregory, 2000). These observations suggest that phosphorylation of Thr58 by GSK3 generates a motif that facilitates the interaction of Myc with a ubiquitin ligase that restricts Myc levels and activity in vivo. Currently, the identity of the ubiquitin ligase that promotes Myc degradation has not been firmly established in any organism (Moberg, 2004 and references therein).

The Drosophila F box protein Archipelago (Ago) has been implicated in the degradation of Drosophila Myc (dMyc). Ago binds dMyc, and impairment of Ago function in vivo stabilizes dMyc, resulting in markedly elevated Myc levels, and promotes cell growth. Recent evidence indicates that the Fbw7/hCDC4 tumor suppressor protein, which is the human ortholog of Ago, also inhibits c-Myc accumulation by promoting its degradation (Welcker, 2004). Because Ago proteins also regulate Cyclin E levels, and Notch pathway activity, these findings suggest a mechanism by which the levels of Cyclin E and dMyc and the activity of the Notch pathway can be coordinately regulated by a shared degradation pathway (Moberg, 2004).

To identify an SCFAgo ubiquitin ligase substrate that could explain the accelerated growth of ago mutant cells, two different interaction screens were conducted by using the Ago F box/WD domain. By mass spectrometric analysis of proteins that coprecipitate with Ago, peptides derived from a number of different SCF components were identified, including Cullins and Skp proteins. At a lower frequency, peptides derived were also recovered from putative SCFAgo substrates, including the Drosophila ortholog of the Myc transcription factor dMyc. In addition to multiple SCF components, a single clone of dMyc was also recovered in a yeast two-hybrid screen for proteins that physically interact with the F box/WD repeat region of Ago (Moberg, 2004).

dMyc was identified as a candidate Ago binding protein, so whether the ability of ago to regulate dMyc involves a direct interaction between Ago and dMyc was examined. In protein extracts from Drosophila S2 cells transfected with epitope-tagged versions of Ago and dMyc (HAAgo and FLAGdMyc), FLAGdMyc was readily detected in anti-HA immunoprecipitates, and in the reciprocal procedure, HAAgo was readily detected in anti-FLAG immunoprecipitates. These experiments indicate that Ago and dMyc interact physically in S2 cells. Significantly, two mutant versions of Ago, Ago1 and Ago3, that correspond to mutations that deregulate dMyc levels and increase growth in vivo, are dramatically impaired in their ability to interact with dMyc in cells despite being expressed at approximately the same level as wild-type Ago protein. Thus, as is the case with the other known SCFAgo substrate, Cyclin E (Moberg, 2001), the ability of Archipelago to bind dMyc protein correlates with its ability to downregulate dMyc levels in vivo (Moberg, 2004).

Coexpression of dMyc also seems to promote Ago accumulation in cells. This increase seems more evident in forms of Ago that bind strongly to dMyc and does not appear to be a general effect of dMyc on all coexpressed proteins. However, the precise mechanism underlying this effect has not been established. It may involve direct dMyc-Ago binding, but it may also be an indirect consequence of Myc's ability to regulate cell metabolism and translation rates (Moberg, 2004).

Thus Ago, which functions as the substrate-specificity subunit of an SCFAgo ubiquitin ligase, regulates the levels of the growth-promoting transcription factor dMyc in developing Drosophila tissues. This regulation appears to occur via a posttranscriptional mechanism that involves a direct Ago-dMyc interaction that modulates dMyc stability. dMyc accumulates in ago mutant cells and likely contributes to their increased growth (Moberg, 2004).

The WD repeat domain of Ago interacts with Cyclin E, and it also binds dMyc. The optimal binding site for the WD domain of S. cerevisiae Cdc4, the yeast ortholog of Ago, has been determined to be I/L-I/L/P-pT-P-P, in which the central threonine residue is phosphorylated (Nash, 2001). Human Cyclin E, Drosophila Cyclin E, and human c-Myc all have a single, well-conserved version of this site, whose central feature is an L-L-T-P-P motif. dMyc contains seven copies of a degenerate version of this site, in which the central threonine is often replaced by a serine, and many of the flanking residues deviate from those in the consensus sequence. Importantly, these putative sites do retain a conserved S/T residue at position +4. The equivalent +4 serine in human Cyclin E (S384) has been shown to be required for the ubiquitination of Cyclin E (Welcker, 2003) and may therefore represent an important feature of the putative Ago binding motif. The presence of multiple Ago binding sites in dMyc versus the single well-conserved site in c-Myc might indicate that although both proteins are targeted for degradation by orthologous F box proteins, the kinetics of degradation of the two Myc proteins may be different (Moberg, 2004).

The array of apparently suboptimal sites in dMyc resembles the situation in S. cerevisiae Sic1, in which nine low-affinity sites are able to cooperatively mediate a stable interaction with Cdc4. Indeed, as is the case with Sic1, mutating a single putative phosphorylation site in dMyc does not alter its Ago binding properties. In contrast, for human Cyclin E and c-Myc, the predicted Ago interaction site lies within a domain previously shown to be required for their ubiquitination and degradation. Furthermore, missense mutations of the central threonine in the Ago interaction motif are the most frequent c-Myc mutations in Burkitt's lymphoma and stabilize c-Myc in cells, suggesting that Ago-dependent degradation of c-Myc is perturbed in these cancers (Moberg, 2004).

ago mutant cells grow more quickly than their wild-type neighbors, but they maintain their normal size by an apparent acceleration of the cell cycle. This differs considerably from the phenotype elicited by overexpression of either dMyc or Cyclin E. Increased expression of dMyc results in increased growth that manifests as an increase in cell size without any change in the duration of the cell cycle. dMyc also promotes S phase entry, possibly as a consequence of the increased growth. Increased expression of Cyclin E has no effect on growth but promotes S phase entry. It also results in, at best, a modest acceleration of the cell cycle. Thus, the cell cycle acceleration observed in ago mutant cells is not easily explained by the elevated level of either dMyc or Cyclin E. Both dMyc and Cyclin E promote S phase entry but maintain the normal duration of the cell cycle by apparently lengthening the S and G2 phases, respectively. Thus, it seems likely that ago loss also affects a regulatory protein that promotes the G2-M transition. Such a regulator could either be a direct substrate of SCFAgo or may be regulated indirectly (Moberg, 2004).

Interestingly, both Ago targets identified to date, Cyclin E and dMyc, are required for imaginal-disc growth. Signaling via the Notch receptor is increased in ago clones, as assessed by the activity of a reporter gene fused to the Enhancer of split mβ promoter. Notch signaling has been shown to promote imaginal-disc growth at least in part by a non-cell-autonomous pathway. Because cyclin E, dMyc, and Notch all participate in tissue growth via increases in cell number and/or cell mass, Ago may represent a way to coordinately regulate these pathways by a common degradation mechanism. Thus, increased Ago levels would be expected to impair tissue growth, and decreased levels would facilitate tissue growth, via multiple pathways. Because ago transcription is patterned in the eye imaginal disc (Moberg, 2001), ago may function to link patterning signals with the activity of these growth-promoting pathways (Moberg, 2004).

The ability of ago to regulate multiple pathways that function in growing cells has implications for understanding the role of its human ortholog (Fbw7/hCDC4) as a tumor suppressor gene. Mutations in Fbw7/hCDC4 have been identified in cancer cell lines, and more recently, mutations have been identified in Fbw7/hCDC4 in endometrial and colorectal tumors. These tumors are likely to have elevated levels of Cyclin E. In light of the data presented here, they are predicted to have high levels of the oncoprotein c-Myc and increased Notch activity, which has also been implicated in human cancers. Thus, the neoplastic phenotype of these tumors may reflect the additive effect of activating all of these pathways that are normally inhibited by Ago (Moberg, 2004 and references therein).

The Drosophila ubiquitin-specific protease Puffyeye regulates dMyc-mediated growth

The essential and highly conserved role of Myc in organismal growth and development is dependent on the control of Myc protein abundance. It is now well established that Myc levels are in part regulated by ubiquitin-dependent proteasomal degradation. Using a genetic screen for modifiers of Drosophila Myc (dMyc)-induced growth, this study identified and characterized a ubiquitin-specific protease (USP), Puffyeye (Puf), as a novel regulator of dMyc levels and function in vivo. puf genetically and physically interacts with dMyc and the ubiquitin ligase archipelago (ago) to modulate a dMyc-dependent cell growth phenotype, and varying Puf levels in both the eye and wing phenocopies the effects of altered dMyc abundance. Puf containing point mutations within its USP enzymatic domain failed to alter dMyc levels and displayed no detectable phenotype, indicating the importance of deubiquitylating activity for Puf function. dMyc induces Ago, indicating that dMyc triggers a negative-feedback pathway that is modulated by Puf. In addition to its effects on dMyc, Puf regulates both Ago and its cell cycle substrate Cyclin E. Therefore, Puf influences cell growth by controlling the stability of key regulatory proteins (Li, 2013).

The mammalian Myc gene family, comprising Myc, Mycn and Mycl, is known to be crucial for growth and development. Myc proteins control multiple cellular processes, including cell growth, proliferation, metabolism and apoptosis, and deregulation of Myc plays an important role in oncogenesis. Non-mammalian Myc has been most intensively studied in Drosophila where the absolute requirement for Drosophila Myc function during development has been demonstrated by the fact that dMyc-null mutants die at an early larval stage (Li, 2013).

Myc transcript and protein abundance are subject to regulation at multiple levels ranging from transcriptional control by numerous mitogenic signaling pathways to extensive post-translational modifications. Of particular interest, given the relatively short half-life of Myc proteins, is the post-translational modification of Myc by the ubiquitin system. Protein ubiquitylation is a fundamental and versatile post-translational modification that controls multiple cellular events by marking proteins as substrates for either degradation or non-degradative processing. In mammals, distinct ubiquitin E3 ligase complexes, including Skp2 and Fbw7, have been reported to influence Myc protein stability and activity (Li, 2013).

The Drosophila ortholog of Fbw7, Archipelago (Ago) is the only ligase identified thus far as involved in proteasome-mediated ubiquitin-dependent turnover of dMyc proteins (Moberg, 2004). Ago mutant alleles were first identified in a genetic screen for regulators of tissue growth in the eye, where it was initially shown to bind and regulate Cyclin E (CycE) levels. Later work demonstrated that Ago also physically interacts with dMyc, and controls dMyc stability and biological function (Moberg, 2004). Unlike c-Myc, which was shown to have a single Myc BoxI phosphodegron associated with Fbw7 binding, several domains containing putative Ago-interacting motifs were shown in dMyc to mediate Casein kinase 1 (CK1)α-, CK1ε- and GSK3β-dependent protein degradation. Although their link to Ago function has not been precisely established, it is clear that GSK3β plays a key role in Ago-mediated dMyc ubiquitylation and degradation (Li, 2013 and references therein).

Protein ubiquitylation is a reversible process in which removal of ubiquitin chains is mediated by deubiquitylating enzymes (DUBs), and the role of DUBs in controlling various cellular processes has attracted considerable interest. DUBs are classified into five subfamilies based on their deubiquitylating domain. Ubiquitin-specific proteases (USPs), which constitute the largest DUB subfamily, share a structurally conserved USP domain of ~350 to 450 amino acids. The USP domain is the catalytic core that mediates the cleavage of ubiquitin conjugates, whereas domains required for protein-protein interaction and substrate specificity are located within N and/or C termini of the USP protein.Although several ubiquitin E3 ligases have been implicated in modulating c-Myc stability, only one deubiquitylating enzyme, USP28, has been demonstrated to catalyze the deubiquitylation of Myc in mammals. Thus far, no deubiquitylating enzyme has been identified that modulates dMyc function or antagonizes Ago-mediated dMyc degradation. Of the 41 predicted Drosophila DUBs, 21 are predicted to have a mammalian USP ortholog. Interestingly, Drosophila does not encode an USP28 ortholog, suggesting that a distinct USP may be responsible for reversing dMyc ubiquitylation in Drosophila. This study reports the identification and characterization of Puffyeye (Puf), a Drosophila USP that antagonizes Ago function and interacts genetically and physically with dMyc. Evidence is presented that Puf regulates dMyc activity at the level of cell and organ growth (Li, 2013).

Although a great deal has been learned recently concerning ubiquitin ligases that interact with Myc proteins, to date only one DUB has been reported that targets Myc. This study has employed a genetic screen based on the rough eye phenotype induced by dMyc overexpression in the eye (GMM) in Drosophila. This screen led to the identification of a USP-type DUB, which was named Puffyeye (Puf; CG9754), as a novel regulator of dMyc function in vivo. Reduced puf expression suppresses, whereas puf overexpression augments, the GMM phenotype. This phenotype is largely an effect of cell overgrowth, yet overgrowth in the eye due to cyclin D/Cdk4 was not influenced by altered Puf abundance. Moreover, knockdown of four other USPs had no effect on the GMM phenotype. This suggests that puf possesses specificity for dMyc-induced growth in the eye. Indeed, puf itself induced a dose-dependent rough eye phenotype, displaying augmented ommatidial size that can be modulated by altering dMyc levels. In the wing disc, dMyc and Puf also were found to collaborate in cell growth. It was also found that Puf is essential for normal development, consistent with a crucial role for Puf in cell growth (Li, 2013).

dMyc levels markedly increase in cells in which puf is overexpressed, whereas dMyc levels are decreased in Puf hypomorphic mutants. These changes in dMyc are predominantly post-translational. This is consistent with the finding that Puf overexpression results in a dramatic increase in dMyc protein stability. Importantly, all of the biological effects of Puf, as well as its effects on dMyc abundance and turnover, are abolished by point mutations in the highly conserved Puf USP catalytic domain. It is surmised that Puf stabilizes Myc through its function as a deubiquitylating enzyme that antagonizes the activity of the Ago ubiquitin ligase, previously shown to target Myc for ubiquitylation and degradation (Moberg, 2004). Importantly, increased Puf exacerbates, and decreased Puf suppresses, the effect of Ago heterozygotes in enhancing the GMM phenotype. The notion that Puf and Ago act as antagonists receives further support from the findings that Puf protein physically associates with both dMyc and Ago in vivo. Interactions between DUBs and their antagonistic E3 ligases, as well as their substrates have been reported previously. The ability of both the Puf short and long isoforms to modify the dMyc-mediated eye phenotype, and stabilize dMyc and Ago proteins in an ubiquitylation domain-dependent manner suggests that domain(s) required for Puf to interact with dMyc or Ago are located in a region N-terminal to the core catalytic domain (Li, 2013).

It was also found that Puf stabilizes CycE, another known Ago substrate, suggesting that Puf antagonizes Ago function in regulating other targets that are crucial for cell cycle control. Indeed, flies homozygous for puf and ago double mutations do not survive, raising the possibility that, in addition to regulating common substrates, they each possess unique targets, as shown for other ubiquitin ligases and DUBs. Notch would be another potential candidate for Puf activity (Moberg, 2004); however, no significant effect of Puf on Notch levels was found in wing discs. In mammalian cells, the ubiquitin-specific protease USP28 was demonstrated to regulate the turnover of c-Myc by binding and antagonizing the activity of Fbw7α, the vertebrate ortholog of Ago. However, Puf and USP28 are not homologs: they appear to be two very distinct USPs in terms of their overall size and amino acid sequence similarity in both their core enzymatic domains and the protein sequence as a whole. The closest mammalian homolog of Puf is USP34 (3546aa). Puf and USP34 are highly homologous in their core catalytic domains (67% identity; 80% similarity) with the catalytic triad conserved, whereas the overall similarity between the two proteins is ~52% (~37% identity) (Li, 2013).

Previous studies have shown that multiple signaling pathways regulate Ago and Fbw7 expression and activity. This study found that Ago levels are increased upon dMyc, as well as upon Puf overexpression. Although the mechanisms by which dMyc and Puf regulate Ago expression are unclear, dMyc-dependent Ago expression may provide a mechanism for dMyc autoregulation, whereas Puf may stabilize Ago by deubiquitylating it. Indeed, Fbw7 has been shown to be regulated through ubiquitylation. A similar type of dynamic relationship has been reported for the ubiquitin ligase Mdm2 and deubiquitylase HAUSP/USP7 in regulating the stability and function of the tumor suppressor p53. Taken together, these data suggest that Ago and Puf represent a regulatory node that controls degradation of Myc and CycE, and very likely other growth control factors. Further studies will be required to identify additional substrates of Puf and to understand the physiological importance of Puf-mediated regulation of protein degradation in Drosophila (Li, 2013).

Myc interacts genetically with Tip48/Reptin and Tip49/Pontin to control growth and proliferation during Drosophila development

The transcription factor dMyc is the sole Drosophila ortholog of the vertebrate c-myc protooncogenes and a central regulator of growth and cell-cycle progression during normal development. The molecular basis of dMyc function was examined by analyzing its interaction with the putative transcriptional cofactors Tip48/Reptin (Rept) and Tip49/Pontin (Pont). Rept and Pont have conserved their ability to bind to Myc during evolution. All three proteins are required for tissue growth in vivo, because mitotic clones mutant for either dmyc, pont,or rept suffer from cell competition. Most importantly, pont shows a strong dominant genetic interaction with dmyc that is manifested in the duration of development, rates of survival and size of the adult animal and, in particular, of the eye. The molecular basis for these effects may be found in the repression of certain target genes, such as mfas, by dMyc:Pont complexes. These findings indicate that dMyc:Pont complexes play an essential role in the control of cellular growth and proliferation during normal development (Bellosta, 2005).

Myc proteins are essential regulators of growth, proliferation, and apoptosis in metazoans. These proteins act as transcription factors to control the expression of numerous target genes involved in growth, metabolism, and other processes. Less is known about the molecular mechanism that allows Myc to control the expression of these targets. In recent years, different modes of gene activation by Myc have been proposed, notably recruitment of chromatin remodelers, or RNA pol II kinases, but the physiological relevance of these different factors for Myc-dependent biological functions needs to be demonstrated. This study investigated the mechanisms of Myc-controlled growth and proliferation during normal development by using Drosophila as a model system. Initially, focus was placed on the interaction of Myc with two specific components of cofactor complexes, Tip48 and Tip49, because of the availability of null mutations in the corresponding genes [called reptin (rept) and pontin (pont) in flies, respectively] (Bellosta, 2005).

Tip48 and Tip49 are closely related proteins that show a high similarity to the bacterial ATP-dependent AAA+ super family DNA helicase RuvB. Orthologs of Tip48 and Tip49 have been identified in plants, yeast, and animals. Different observations strongly suggest that one major function of the Tip proteins resides in the control of transcription. Initially, vertebrate Tip49 was found to be a Tata-binding protein-interacting protein; later Tip48 and Tip49 were also shown to interact physically with the different transcription factors ß-catenin, E2F1 (only Tip49), raising the possibility that the Tip proteins could bridge basic transcription machinery and sequence-specific activators. Both proteins were also purified as part of several multiprotein complexes involved in transcriptional regulation: the Ino80 chromatin remodeling complex in yeast, Polycomb repressive complex 1 in Drosophila (only Tip48), the Tip60 HAT complex in vertebrates, and the Uri complex regulating nutrition-dependent gene expression in yeast and in vertebrates. Interestingly, three other proteins that were found to bind the N terminus of c-Myc share residence with Tip48 and Tip49 in the Ino80 (BAF53 and ß-actin) or Tip60 complex (transformation/transcription-domain-associated protein, BAF53, and ß-actin). Further support for an involvement of Tip48 and Tip49 in transcription is provided by the observations that both proteins colocalize with c-Myc on the nucleolin promoter and that elimination of Tip48 or Tip49 function in yeast rapidly affects the expression of a large number of targets. Such a transcriptional role is also consistent with the described genetic interactions between a tip48 mutation and ß-catenin in zebrafish and interactions of tip48 and tip49 with a ß-catenin-reporter system in Drosophila (Bauer, 2000; Rottbauer, 2002); in both of these in vivo interactions, Tip48 behaved as a negative component and Tip49 behaved as a positive component of the Wg signaling cascade. Similar opposing activities were also documented in a human cell line by assaying the ability of the ß-catenin–T cell factor complex to activate a reporter gene. A potential role for Tip49 in Myc-dependent functions was addressed in a recent study that examined the consequences of coexpressing wild-type or putative dominant-negative forms of Tip49 with c-Myc. Neither form had any effect on control cells, but both enhanced the apoptosis caused by overexpressed c-Myc, and they reduced the ability of c-Myc in combination with activated Ras to transform rat embryo fibroblasts, which indicates that, upon forced overexpression, Myc might require Tip49 activity (Dugan, 2002; Bellosta, 2005 and references therein).

The present study shows that the physical interaction between Myc and Pont/Rept is conserved in flies, that pont/rept are essential for tissue growth in vivo, and that dmyc and pont show a strong genetic interaction. The gene mfas was identified as a transcriptional target that is repressed by dMyc:Pont complexes. These studies provide the first evidence that Pont and Rept are essential cofactors for the normal functions of Myc in vivo (Bellosta, 2005).

This study provides evidence that Tip49/Pont (and possibly Tip48/Rept) is an essential partner for Myc during normal development and that it plays an important role in the control of Myc-dependent transcription, growth, and proliferation. These conclusions are supported by four lines of evidence. (1) dMyc physically interacts with Rept and Pont in vitro, in cells, and in larvae. Although ternary complexes containing dMyc, Rept, and Pont can exist, evidence is provided that dMyc can associate with Pont in the absence of Rept, although it is unclear whether such complexes lacking Rept have any physiological role in vivo. The stronger genetic interaction with pont raises the possibility that some of dMyc's functions might be mediated by such complexes, but the large degree of overlap between the targets of Pont and Rept and the fact that in most biochemically purified complexes Tip48 is accompanied by Tip49 suggest that most often these two proteins function together (Bellosta, 2005).

(2) Flies lacking zygotic pont or rept gene products arrest their growth early during larval development, and mitotic clones homozygous mutant for pont or rept suffer from the same type of cell competition as do dmyc clones. These characteristics indicate a requirement for Pont/Rept for cellular proliferation and growth, which is consistent with their functioning as cofactors for dMyc (Bellosta, 2005).

(3) pont shows a strong dominant genetic interaction with dmyc. The causes for this interaction are likely to be defects in cellular growth and proliferation. The control of growth is most sensitive to variations in dMyc levels, because the moderate reduction of dMyc activity achieved in hypomorphic dmyc alleles already results in a decrease in cell size but not cell numbers. Removal of one copy of the pont gene exacerbates the growth defect and results in a reduction of cell numbers. No indication was found that apoptosis contributes to this reduction in cell number and, therefore, it is concluded that defects in cell number primarily reflect a proliferation defect. It is important to stress that none of these defects are seen in flies that are heterozygous for pont but wild-type for dmyc, arguing strongly against purely additive effect of the pont and dmyc mutations. Although the possibility that Pont and dMyc act in parallel growth-controlling pathways cannot be strictly ruled out, such a dominant genetic interaction is indicative of close functional connections. No dominant effect of the pont mutation on dMyc overexpression phenotypes has been observed, suggesting that Pont is not limiting in situations of mildly increased dMyc levels. However, by using a vertebrate tissue culture system (Rat1 cells), it has been demonstrated that dominant-negative Pont/Tip49 inhibits the ability of human c-Myc to transform Rat1 fibroblasts in conjunction with activated Ras. Overexpression of a dominant-negative protein mutant potentially allows a stronger reduction of Pont/Tip49 activity than can be obtained in a heterozygous pont-/+ situation, and, thus, these experiments further reinforce the observation of a genetic interaction between myc and pont (Bellosta, 2005).

(4) It has been shown that the expression of several genes, including mfas, is increased upon down-regulation of dmyc, pont, or rept in S2 cells and in dmyc/pont double-mutant eye imaginal discs in vivo. Chromatin immunoprecipitation experiments further suggest that mfas is a direct transcriptional target of Pont and dMyc (Bellosta, 2005).

Taken together, these data strongly argue that dMyc:Pont complexes are essential regulators of proliferation and growth in vivo and that they act at least partly by repressing the expression of target genes, such as mfas. A similar repressive function has recently also been found for Xenopus Pont and Rept; it was proposed that the well characterized repression of the transactivator Miz1 by c-Myc is mediated by Pont and Rept. Although it is tempting to speculate that Drosophila Pont functions analogously, no fly homolog of Miz1 has been identified. In addition, it is currently not know which of the reported Pont-containing complexes is responsible for the observed effect (Bellosta, 2005).

The function of Rept is less clear, because a rept mutant shows only a weak interaction and only with one dmyc allele. In contrast, overexpression of Rept strongly enhances the dmyc/pont mutant phenotypes. This observation could indicate that Rept also acts as antagonist of Pont and of dMyc:Pont complexes, analogously to what has been proposed for the interaction between Rept/Pont and ß-Catenin. Alternatively, overexpression of Rept functions in a dominant-negative fashion, possibly by titrating Pont and/or other factors away from the multiprotein complexes in which they normally reside; in addition, Rept might be relatively more abundant than Pont such that heterozygosity for rept does not show any effects in most situations. Although either explanation currently cannot be ruled out, identification of mfas as a common target for dMyc, Pont, and Rept is more consistent with the latter possibility (Bellosta, 2005).

In conclusion, it has been shown that Pont, and possibly Rept, assists dMyc in the control of cellular proliferation and growth during normal development, presumably in part by repressing the expression of certain target genes (Bellosta, 2005).

The Trithorax group protein Lid is a trimethyl histone H3K4 demethylase required for dMyc-induced cell growth

The Myc oncoprotein is a potent inducer of cell growth, cell cycle progression, and apoptosis. While many direct Myc target genes have been identified, the molecular determinants of Myc's transcriptional specificity remain elusive. A genetic screen was carried out in Drosophila and the Trithorax group protein Little imaginal discs (Lid) was identified as a regulator of dMyc-induced cell growth. Lid was originally identified in intergenic noncomplementation with a mutation in ash1, a trithorax group gene (Gildea, 2000; full text of article). Lid binds to dMyc and is required for dMyc-induced expression of the growth regulatory gene Nop60B. The mammalian Lid orthologs, Rbp-2 (JARID1A) and Plu-1 (JARID1B), also bind to c-Myc, indicating that Lid-Myc function is conserved. Lid is a JmjC-dependent trimethyl H3K4 demethylase in vivo, and this enzymatic activity is negatively regulated by dMyc, which binds to Lid’s JmjC domain. Because Myc binding is associated with high levels of trimethylated H3K4, it is proposed that the Lid-dMyc complex facilitates Myc binding to, or maintenance of, this chromatin context (Secombe, 2007).

Lid is a 1838-amino-acid protein possessing numerous conserved motifs including an ARID (A/T-rich interaction domain), implicated in binding A/T-rich DNA; a single C5HC2 zinc finger; three PHD fingers (plant homeobox domain), implicated in forming protein-protein interactions; and Jumonji N and C (JmjN and JmjC) domains. JmjC-containing proteins have recently been shown to act as histone demethylase enzymes in a Fe2+ and -ketoglutarate-dependent manner (Klose, 2006). To test whether Lid can demethylate histones in vivo, Lid was overexpressed in fat body and in wing disc cells and the levels of mono-, di-, and trimethylated histone H3K4 and H3K27 were examined. Di- and trimethylated histone H4K20 and trimethylated histone H3K9 and H3K36 were also examined. Overexpression of Lid specifically decreased the levels of the trimethylated form of H3K4 but had no effect on the other methylated histones examined in either GFP-marked fat body or wing disc cells. Significantly, expression of Lid in the wing disc reduced trimethyl H3K4 in a dose-dependent manner, with two copies of the UAS-Lid transgene reducing trimethyl H3K4 more efficiently than one copy. Moreover, levels of trimethylated H3K4 are increased in wing discs from lid homozygous mutant animals, consistent with the model that Lid regulates the levels of this histone modification during normal development. To determine whether the JmjC domain of Lid is required for the observed H3K4 demethylation, transgenic flies were generated carrying a Gal4-inducible form of full-length Lid containing Ala substitutions at His637 and Glu639 (Lid-JmjC*) that abolishes the protein’s ability to bind the Fe2+ cofactor required for demethylase activity. Similar mutations have been shown to disrupt the demethylase function of the JmjC domains of JHDM2A, JHDM3A, JHDM1, and JMJD2A. Unlike wild-type Lid, expression of full-length Lid-JmjC* did not decrease levels of trimethylated H3K4 in fat body or in wing disc cells, demonstrating that an active JmjC domain is required for Lid-mediated H3K4 demethylation. Interestingly, expression of Lid-JmjC* resulted in increased levels of trimethyl H3K4 in the fat body, perhaps due to a dominant interfering effect on wild-type Lid function in these cells. Taken together, these results demonstrate that Lid is a trimethyl H3K4 demethylase that modifies nucleosomal histone H3 in vivo. The global regulation of H3K4 trimethylation status by Lid is not, however, likely to be mediated by recruitment by dMyc, since no effect was observed of reduced or increased dMyc expression on trimethyl H3K4 levels in either fat body or wing disc cells (Secombe, 2007).

Forty other genomic regions were identified that enhanced or suppressed the GMR-Gal4, UAS-dMyc (GMM) phenotype when heterozygous. Two of these regions delete genes encoding known regulators of dMyc stability, such as ago, or are involved in Myc transactivation, such as Pcaf. Specific mutations in both of these genes have been shown to enhance or suppress the GMM rough eye phenotype, respectively. Interestingly, none of the known direct transcriptional targets of dMyc were identified as genetic modifiers of the GMM phenotype, suggesting that the GMM phenotype arises from modulation of multiple genes and provides a powerful tool to identify proteins directly required for dMyc function in vivo (Secombe, 2007).

TrxG proteins are renowned for their essential role in maintaining homeotic (hox) gene expression during development, with mutations in many TrxG genes resulting in lethality due to homeotic transformations. Six TrxG protein complexes have been identified to date. While one function of these complexes is to antagonize Polycomb group (PcG) repression to maintain active hox gene expression, TrxG proteins are also recruited to other developmentally important genes to either activate or repress their transcription in a context-dependent manner. Based on the suppression of the GMM phenotype, the physical interaction between Lid and dMyc, and the requirement of Lid for dMyc-dependent activation of Nop60B, it is predicted that Lid acts as a dMyc coactivator involved in cell growth. The interaction between endogenous Lid and dMyc proteins is also likely to be essential for normal larval development since reducing the gene dose of lid is lethal in combination with the dmyc hypomorphic allele dmP0. In addition, genetically reducing lid enhances a small bristle phenotype induced by expression of a dMyc RNAi transgene. The original small discs phenotype described for lid mutants also suggests a role for Lid in the regulation of cell growth or proliferation during larval development. Unfortunately, this phenotype occurs at a frequency far too low (<1% of lid mutant larvae) to allow characterization (Secombe, 2007).

It is expected that the function of the Lid-Myc complex is evolutionarily conserved, since the human orthologs of Lid, Rbp-2 (JARID1A) and Plu-1 (JARID1B), bind strongly to c-Myc and dMyc in vitro, and both have been implicated in transcriptional regulation. Originally described as a binding partner for the tumor suppressor protein Retinoblastoma (RB), Rpb-2 has been shown to behave as a coactivator for RB at some promoters while antagonizing RB function at others (Benevolenskaya, 2005). Rbp-2 has also been identified as a transcriptional coactivator for nuclear hormone receptors (NRs) (Chan, 2001) and for the LIM domain transcription factor Rhombotin-2 (Mao, 1997). In addition, Plu-1 acts as a transcriptional corepressor for BF1 and PAX9 (Lu, 1999; Tan, 2003). While the transcriptional repression activities of Rbp-2 and Plu-1 are likely to be linked to a conserved trimethyl H3K4 demethylase activity, the molecular mechanism by which they activate transcription remains unclear. The mechanism by which Lid functions is currently being addressed by carrying out genetic screens using phenotypes generated by gain or loss of lid function (Secombe, 2007).

Coimmunoprecipitation analyses revealed that dMyc is likely to form multiple distinct complexes comprising TrxG proteins: One includes the Brm (SWI/SNF) nucleosome remodeling complex, and another contains Lid and Ash2. Consistent with the physical interaction observed between dMyc and Brm, components of the Brm complex suppress the GMM phenotype when genetically reduced, indicating that they are required for dMyc-induced cell growth. An interaction between Myc and the Brm complex has been observed in mammalian cells, where c-Myc interacts with the Brm (Brg1) subunit Ini1, and expression of a dominant-negative Brg1 allele inhibits c-Myc-dependent activation of a synthetic E-box reporter. However, the interaction between dMyc and the Brm complex described in this study, using Drosophila, provides the first demonstration of a biological significance for this complex (Secombe, 2007).

The second dMyc-TrxG complex identified includes Lid and Ash2, with Ash2 being immunoprecipitated with both anti-dMyc and anti-Lid antisera. In addition, decreased levels of Ash2 suppress, and increased levels of Ash2 levels enhance, the GMM phenotype, suggesting that Lid and Ash2 are limiting for dMyc-induced cell growth. In Schizosaccharomyces pombe, the orthologs of Ash2 and Lid (Ash2p and Lid2p) interact in vivo. While Ash2 has no known enzymatic activity, it is an integral component of several conserved complexes, including the SET1 histone methyltransferase complex (TAC1 in Drosophila; MLL in mammals) that is essential for methylation of histone H3K4. Biochemical purification of SET1, Lid2p, and Ash2p complexes from S. Pombe has demonstrated that the Lid2p-Ash2p complex is distinct from the SET1-Ash2 complex. Reducing the gene dose of the SET1 ortholog trx does not affect the GMM phenotype, consistent with the Drosophila Lid-Ash2-dMyc complex also being independent of TAC1 methyltransferase complex. The observation that Ash2 is a component of both H3K4 methylating (MLL) and demethylating (Lid) complexes is intriguing and suggests that it may be a crucial modulator of H3K4 methylation status. Whether Ash2 is required for Lid-mediated H3K4 demethylation is currently being tested (Secombe, 2007).

Lid is the first enzyme characterized that specifically demethylates trimethylated histone H3K4 in vivo. Based on the similarity between Lid and its mammalian orthologs Rbp-2 and Plu-1, it is expected that this demethylase activity to be conserved. The enzymatic activity of Lid requires a functional JmjC domain; however, Lid's specificity for a trimethylated lysine target is likely to be determined by the presence of a conserved N-terminal JmjN domain. Evidence to date suggests that proteins that possess both a JmjN and a JmjC domain prefer di- or trimethylated lysine substrates, whereas JmjC proteins that lack a JmjN domain demethylate mono- or dimethylated lysines. Indeed, analysis of the crystal structure of JMJD2A, which targets trimethylated H3K9 and K36, has revealed that the JmjN domain makes extensive contacts within the catalytic core of the JmjC domain, presumably accounting for the differences in target specificity between JmjC and JmjN/JmjC proteins (Secombe, 2007).

Trimethylated H3K4 is often found surrounding the transcriptional start site of active genes and is strongly correlated with binding by c-Myc. The trimethyl H3K4 demethylase activity of Lid would predict that Lid/Rbp-2 proteins may act as transcriptional repressors in a similar manner to LSD1, which demethylates mono- and dimethylated H3K4. Consistent with this hypothesis, it is observed that a large number of genes are derepressed in microarrays of homozygous lid mutant wing discs. However, expression of dMyc abrogates Lid's enzymatic activity, indicating that Lid is not acting as a demethylase when bound to dMyc. This is consistent with the observation that expression of Lid-JmjC* (a Gal4-inducible form of full-length Lid containing Ala substitutions at His637 and Glu639 that abolishes the protein’s ability to bind the Fe2+ cofactor required for demethylase activity) enhances the GMM eye phenotype. Indeed, Lid behaves as a dMyc coactivator based on the requirement for Lid in dMyc-induced expression of the growth regulator Nop60B. Both activation and repression functions have been previously suggested for Rbp2. Interestingly, LSD1's demethylase activity is also negatively regulated by an associated protein, BHC80, in a similar manner to the inhibition of Lid's enzymatic activity by dMyc. Dynamic regulation of histone demethylase activity is therefore likely to be a common feature of regulated gene expression in vivo (Secombe, 2007).

Recently, analysis of c-Myc target gene promoters revealed a strong dependence on trimethylated H3K4 for E-box-dependent c-Myc binding. Based on this observation, it is tempting to speculate that although Lid is likely to be enzymatically inactive when complexed with dMyc, Lid may retain its ability to recognize trimethylated H3K4 (perhaps through its JmjN domain) and thereby facilitate appropriate E-box selection. The inhibition of Lid demethylase activity by dMyc may also result in maintenance of local H3K4 trimethylation to permit binding of additional dMyc molecules or other transcription factors. The maintenance of trimethylated H3K4 by dMyc may allow binding of the NURF chromatin remodeling complex that specifically recognizes trimethylated H3K4. NURF binding, through its large BPTF subunit, has been correlated with spatial control of Hox gene expression and is thought to link H3K4 methylation to ATP-dependent chromatin remodeling. Finally, considering the fact that Lid contains multiple domains potentially involved in DNA binding and protein interaction, it is likely that interaction of Lid/Rbp-2 with Myc in Drosophila and mammalian cells will promote association of other proteins with the Myc-Lid complex, allowing further diversification of Myc function (Secombe, 2007).

Identification of domains responsible for ubiquitin-dependent degradation of dMyc by glycogen synthase kinase 3beta and casein kinase 1 kinases

Ubiquitin-mediated degradation of dMyc, the Drosophila homologue of the human c-myc proto-oncogene, is regulated in vitro and in vivo by members of the casein kinase 1 (CK1) family and by glycogen synthase kinase 3beta (GSK3beta). Using Drosophila S2 cells, it was demonstrated that CK1alpha promotes dMyc ubiquitination and degradation with a mechanism similar to the one mediated by GSK3beta in vertebrates. Mutation of ck1alpha or ck1epsilon (discs overgrown) or sgg/gsk3beta in Drosophila wing imaginal discs results in the accumulation of dMyc protein, suggesting a physiological role for these kinases in vivo. Analysis of the dMyc amino acid sequence reveals the presence of conserved domains containing potential phosphorylation sites for mitogen kinases, GSK3beta, and members of the CK1 family. Mutations of specific residues within these phosphorylation domains regulate dMyc protein stability and confer resistance to degradation by CK1alpha and GSK3beta kinases. Expression of the dMyc mutants in the compound eye of the adult fly results in a visible defect that is attributed to the effect of dMyc on growth, cell death, and inhibition of ommatidial differentiation (Galletti, 2009).

In vivo downregulation of GSK3β and CK1α or CK1ɛ kinases in wing imaginal discs results in the accumulation of dMyc protein, an effect particularly visible in the hinge and notum regions but not in cells adjacent to the zone of nonproliferative cells (ZNC). Reduction of GSK3β and CK1α activates Wingless (Wg) signaling, which in turns negatively regulates dmyc RNA in the ZNC. This functional relationship might explain the lack of expression of dMyc protein in clones falling in the wing pouch area and in the ZNC. This positional effect also suggests that dMyc activity is regulated by patterning signals active during the development of the wing imaginal discs (Galletti, 2009).

This analysis of the dMyc amino acid sequence uncovered novel conserved domains, which serve as potential phosphorylation substrates for CK1s or GSK3β kinases. Biochemical characterization of these domains indicated that a combination of amino acid substitutions (S201A, S205A, and S207A) in the dMyc-PI sequence produces a protein with a shorter half-life than dMyc-WT. In vivo expression of the dMyc-MPI mutant did not confer the typical ommatidial roughness that is induced by the expression of dMyc-WT. Moreover, expression of dMyc-PI failed to induce apoptosis in the eye imaginal discs, an effect normally associated with dMyc-WT overexpression. In conclusion, the data suggest that dMyc-PI produces a protein that is less stable than dMyc-WT. In vertebrates, phosphorylation of c-Myc on Ser-62 by MAPK/ERK, JNK N-terminal kinase, or CDK4 increases its stability. The dMyc-PI sequence does not contain a bona fide ERK phosphorylation site (PXSP). However, Ser-201 lies in a favorable context for phosphorylation by the ribosomal S6 kinase-p90 RSK. RSK-p90 belongs to a class of Ser/Thr kinases, activated by ERK and insulin signaling, that phosphorylates the S6 protein component of the 40S ribosomal subunit in response to mitogenic stimulation, resulting in enhanced translation. Interestingly, it has been reported that RSK-p90 activation by ERK is capable of switching on mTOR signaling via inactivation of the TSC1/2 complex, suggesting a role for this kinase in protein synthesis and mass accumulation. No evidence for this regulatory mechanism has been described thus far in Drosophila. It is hypothesized that growth factors may stabilize Myc protein, possibly through phosphorylation by the RSK-p90 kinase, and promote ribosomal biogenesis, in accordance with the prominent role played by dMyc in the production of mass and growth regulation (Galletti, 2009).

Biochemical analysis of the protein stability of dMyc-PII, dMyc-PV, and dMyc-AB showed an increased half-life of these mutants compared to dMyc-WT. The sequence within the dMyc-PII domain (S324A-T328A-S330A) contains potential targets for phosphorylation by GSK3β at Ser-324 [324-S/T-XXX-S/T-(PO4)+4], which requires a priming event of phosphorylation at the +4 position (Thr-330). This phosphorylation event also acts as priming for other kinases (i.e., CK1s) and creates an optimum consensus site for phosphorylation by CK1s at Thr-330 [S/T-(PO4)-XX-330-S/T]. This study found that alanine substitutions of amino acids 324, 328, and 330 conferred resistance to dMyc protein degradation upon phosphorylation by the CK1α and GSK3β kinases. These experiments show that mutation of the residues S324, T328, and S330 confers to the dMyc-PII mutant a resistance to degradation mediated by the ubiquitin ligase Ago. Moreover, it was found that dMyc-MPV, which is degraded by CK1α and GSK3β kinases, is somewhat resistant to degradation by Ago, suggesting that CK1α- and GSK3β-mediated phosphorylation of dMyc is not sufficient to induce its degradation by Ago but perhaps by another unknown ubiquitin ligase (Galletti, 2009).

These data also demonstrate that the dMyc-AB plays an important role in the regulation of dMyc protein stability. Mutation of acidic amino acids imparted to dMyc resistance to degradation primed by CK1α and GSK3β kinases. It has been proposed that acidic domains act as docking sites for the CK1 and CK2, enabling proper positioning of the kinases to recognize their substrates. It is speculated that the conserved acidic amino acid stretch in Myc protein helps the binding of CK1 and CK2 kinases and favors Myc phosphorylation. In support of this hypothesis, the dMyc-PV amino acid sequence (residues 405, 407, and 409), located within the AB (amino acids 404 to 414), was found to be highly homologous to the PEST domain of c-Myc (amino acids 226 to 270). This domain was previously demonstrated to be relevant for c-Myc stability and to act as a potential substrate for CK2 phosphorylation. These biochemical data show that mutations of the dMyc-PV and the AB domains confer increased stability to dMyc protein and suggest that the acidic sequence functions similarly to the PEST domain to control dMyc stability. Notably, Ser-407 constitutes an optimum consensus site for phosphorylation by CK2 (S/T-407-XX-D/E). This observation agrees with the hypothesis that in mammals CK2 is involved in the regulation of c-Myc degradation by targeting the PEST domain (Galletti, 2009).

In vivo expression of the stable mutants dMyc-PII, dMyc-PV, and dMyc-AB resulted in a visible eye defect, accompanied by a reduction of the head capsule and a diminution of the number of the ommatidia. This was particularly visible for dMyc-PV and -AB. Cellular analysis of third-instar larvae eye imaginal discs revealed that expression of these mutants induced apoptosis during disc development. Apoptosis was detected not only within the compartment of dMyc expression (cell autonomous) but also in the neighboring cells (non-cell autonomous). This is a well-documented phenomenon and illustrates the role of dMyc in cell competition, where cells expressing high dMyc kill slower-proliferating neighboring cells nonautonomously through an unidentified mechanism (Galletti, 2009).

In conclusion, multiple phosphorylation events may work hierarchically to prime Myc phosphoamino acids for binding by multiple kinases. It is proposed that different kinases respond to a 'phosphorylation code' that is required to properly control Myc protein stability. This code will depend on an upstream program that in turn activates these kinases. The identification of other phosphorylation residues in dMyc will help in drawing a complete map of phosphorylation activities and will elucidate the events necessary for robust regulation of Myc protein stability. For example, it is speculated that components of growth signaling pathways, such as ras or insulin, may influence the activities of different combinations of kinases, thus affecting phosphorylation at different amino acids to control dMyc protein stability. In support of this hypothesis, preliminary data was produced showing that activation of the DILP (for Drosophila insulinlike peptides) pathway increases dMyc protein stability in vivo through the inactivation of GSK3β kinase, suggesting that the metabolic and nutrient pathways affect growth by partially controlling dMyc protein expression (Galletti, 2009).

Regulation of c-Myc protein stability by proteasome activator REGγ

c-Myc is a key transcriptional factor that has a prominent role in cell growth, differentiation and tumor development. Its protein levels are tightly controlled by ubiquitin-proteasome pathway and frequently deregulated in various cancers. This study reports that the 11S proteasomal activator REGγ is a novel regulator of c-Myc abundance in cells. Overexpression of wild-type REGgamma, but not inactive mutants including N151Y and G250S, significantly promoted the degradation of c-Myc. Depletion of REGγ markedly increased the protein stability of c-Myc. REGγ interacts with the C-terminal region of c-Myc and regulates c-Myc protein turnover. Functionally, REGγ negatively regulates c-Myc-mediated cell proliferation. Interestingly, depletion of the Drosophila Reg homolog (dReg) in developing wings induced the upregulation of Drosophila Myc, which contributes to cell death. Collectively, these results suggest that REGgamma proteasome has a conserved role in the regulation of Myc abundance in both mammalian cells and Drosophila (Li, 2014).

PAF1 complex component Leo1 helps recruit Drosophila Myc to promoters

The Myc oncogene is a transcription factor with a powerful grip on cellular growth and proliferation. The physical interaction of Myc with the E-box DNA motif has been extensively characterized, but it is less clear whether this sequence-specific interaction is sufficient for Myc's binding to its transcriptional targets. This study identified the PAF1 complex, and specifically its component Leo1, as a factor that helps recruit Myc to target genes. Since the PAF1 complex is typically associated with active genes, this interaction with Leo1 contributes to Myc targeting to open promoters (Gerlach, 2017).


DEVELOPMENTAL BIOLOGY

Embryonic

DMYC transcripts, presumably maternal, can be detected ubiquitously from the earliest stages. Later the zygotic transcripts accumulate in a changing pattern in various tissues. In preblastoderm embryos, DMYC mRNA is present throughout the embryo, with the highest levels at the anterior and posterior termini, but is absent from the pole cells. In early gastrulation, additional DMYC mRNA can be detected in the presumptive mesoderm along the ventral midline. This mesodermal staining intensifies during germband extention and remains until late embryogenesis. The terminally located expression follows the posterior and anterior midgut primordia during the germband extended stages. Additional staining is found in salivary placodes. DMAX transcript is less abundant than DMYC, particularly during the earliest stages. In addition dmax is expressed in tissues with undetectable levels of DMYC, such as the developing central nervous system. Hence, whereas tissues containing the highest levels of DMYC and DMAX transcripts are undergoing DNA replication, not all actively proliferating tissues have detectable levels of DMYC.

Myc and wing development

The Ras GTPase links extracellular mitogens to intracellular mechanisms that control cell proliferation. To understand how Ras regulates proliferation in vivo, Ras was either activated or inactivated in cell clones in the developing Drosophila wing. Cells lacking Ras are smaller, have reduced growth rates, accumulate in G1, and undergo apoptosis due to cell competition. Conversely, activation of Ras increases cell size and growth rates and promotes G1/S transitions. Ras upregulates the growth driver dMyc, and both Ras and dMyc increase levels of cyclin E posttranscriptionally. It is proposed that Ras primarily promotes growth and that growth is coupled to G1/S progression via cyclin E. Interestingly, upregulation of growth by Ras does not deregulate G2/M progression or a developmentally regulated cell cycle exit (Prober, 2000).

It is proposed that there is parallel and independent control of G1/S and G2/M transitions in Drosophila wing disc cells. Cellular growth due to Ras or dMyc drives G1/S transitions by promoting translation of cyclin E. Ras may also drive growth via proteins other than dMyc; this could feed back to upregulate translation of dmyc mRNA. Alternatively, Ras may regulate cellular growth and the G1/S cell cycle machinery in parallel. Stg/Cdc25, which is regulated primarily at the transcriptional level, drives G2/M transitions. Signaling molecules capable of regulating coordinated growth and patterning such as Vein may regulate G1/S transitions via Ras, dMyc, or other growth-promoting proteins and regulate G2/M transitions via transcription factors that modulate transcription of Stg/Cdc25 (Prober, 2000).

Other proteins that promote growth, such as the Drosophila homologs of dMyc and Phosphoinositide 3-Kinase (dPI3K), have effects on cell cycle progression similar to Ras. Upregulating these proteins in the developing wing truncates G1, elongates G2, and increases growth rates, while downregulating them cause the opposite effects. Furthermore, the resulting growth rates are inversely proportional to the length of G1. Given these similarities, it is proposed that cellular growth is rate limiting for G1/S progression in wing imaginal cells (Prober, 2000).

The data suggest that the effects of Ras on cellular growth and the cell cycle are at least partially mediated by dMyc. Mammalian Myc transcription factors activate expression of many genes involved in cellular growth and metabolism, and Drosophila dMyc is a potent growth driver in vivo. Upregulation of dMyc by Ras appears to be posttranscriptional. Ras might act by inhibiting degradation of dMyc protein, as has been demonstrated in mammalian cell culture. Alternatively, Ras might stimulate growth via other proteins, such as components of the dPI3K/dAkt/dS6 Kinase pathway, which promote cellular growth in Drosophila. Increased growth due to these proteins could then feed back to promote translation of extant dmyc mRNA. However, dMyc and dPI3K cannot be mediating all of Ras's effects, since unlike Ras they do not affect cell fate or cell adhesion. These additional functions of Ras, along with the ability to increase Myc protein levels, likely contribute to the strong synergistic action of Ras and Myc in oncogenesis (Prober, 2000).

RasV12 accelerates G1/S transitions but fails to accelerate rates of cell division. This is similar to findings with overexpressed dMyc. However, coexpressing either RasV12 or dMyc with String (Stg), the G2/M rate limitor, does accelerate cell division. This suggests that regulation of Stg is independent of both Ras and dMyc. It is therefore proposed that there is parallel and independent control of G1/S and G2/M transitions during wing development. Signaling molecules capable of regulating coordinated growth and patterning, such as Vein, Decapentaplegic, and Wingless might control G1/S transitions by regulating growth via Ras, dMyc, or other growth-promoting proteins. These signaling molecules might also, unlike Ras and dMyc, control G2/M transitions by modulating transcription of stg. Analysis of more than 40 kb of the stg promoter has revealed an extensive array of regulatory modules that respond to different patterning signals and thus integrate complex patterning information. A model in which cyclin E acts as a growth sensor and Stg acts as a 'pattern sensor' is attractive, as it allows coordination of independent growth and patterning signals by the cell cycle machinery (Prober, 2000).

Experiments in both vertebrates and invertebrates have illustrated the competitive nature of growth and have led to the idea that competition is a mechanism for regulating organ and tissue size. Competitive interactions between cells were assessed in a developing organ and their effect on its final size were examined. Local expression of the Drosophila growth regulator dMyc, a homolog of the c-myc proto-oncogene, induces cell competition and leads to the death of nearby wild-type cells in developing wings. Cell competition is executed via induction of the proapoptotic gene hid and both competition and hid function are required for the wing to reach an appropriate size when dMyc is expressed. Moreover, evidence is provided that reproducible wing size during normal development requires apoptosis. Modulating dmyc levels to create cell competition and hid-dependent cell death may be a mechanism used during normal development to control organ size (de la Cova, 2004).

This work leads to three major conclusions. (1) Expression of the c-myc protooncogene homolog dMyc in small populations of wing disc cells induces cell competition, leading to the elimination of nearby cells via induction of the proapoptotic gene hid. (2) The competition induced by dMyc and the elimination of cells that results is required for control of proper wing size. (3) Studies reveal that apoptosis is required for the fidelity of size during normal wing development, suggesting that the modulation of hid expression by competitive interactions between cells may be used as an endogenous mechanism of size control (de la Cova, 2004).

These experiments demonstrate that expression of dMyc in some cells of a developing organ leads to elimination of nonexpressing cells through apoptosis. The growth disadvantage induced by dMyc-expressing cells fulfills the classic definition of cell competition: viable but slower-growing cells in an organ are eliminated by an encroaching faster-growing cell population, proximity to the fast-growing cell population dictates the severity of the disadvantage in the slow-growing cells, cells are protected from cell competition by developmental compartment boundaries, and appropriate organ size is reached at the end of development. Relative differences in dMyc levels lead to competitive situations between cells -- dmyc mutant cells are outcompeted by neighboring nonmutant cells; wild-type cells, with a normal complement of endogenous dmyc, are also subject to competition if surrounded by cells expressing a dMyc transgene. However, wild-type cells appear to be subject to competition only if they lie within about eight cell diameters of dMyc-expressing cells, and they must reside in the same developmental compartment. Thus, proximity, compartmental provenance, and the relative levels of dmyc are particularly important aspects of the competitive effects of dMyc (de la Cova, 2004).

During the process of cell competition induced by dMyc, the proapoptotic gene hid is induced in the growth-disadvantaged cells. Since a reduction of hid function protects cells from competition-induced death, it is believed that hid upregulation is a consequence of the sensing of competitive stress. An intriguing question that remains is how cells are able to sense competition. One possibility is that cells compete for sufficient levels of a survival factor that normally blocks hid expression. Dpp signaling promotes cell survival in the wing disc but appears to be unaffected in discs expressing dMyc. Alternatively, some cells in competition may be deprived of adequate nutrients, although in these experiments, cells at a growth disadvantage retain a normal nucleolar size, arguing that their biosynthetic rates are not abnormally low. However, the results suggest that dMyc provokes competition and hid expression via a short-range signal, since close proximity is required for the perception of competitive effects. Perhaps the most intriguing feature of this signal is that it is not perceived by nearby cells across a compartment boundary, although dMyc induces competition between cells within the posterior compartment as well as within the anterior. One possibility is that cells expressing dMyc acquire adhesive properties that transmit a competitive signal to neighboring cells, which is not compatible with the adhesive barrier that maintains the compartment boundary (de la Cova, 2004).

These studies reveal that cell competition is not invariably induced whenever rapidly growing cells populate regions of a developing organ. Both the PI3K Dp110 and cyclin D/Cdk4 potently promote growth when overexpressed, yet they do not induce competition in any of these assays. These observations also demonstrate that balanced growth -- growth that simultaneously drives cell division and cellular growth -- is not required to induce cell competition. dMyc expression increases clonal mass solely by increasing cell size. Thus, this trait of cell competition may be related to a size-measuring mechanism that recognizes total mass rather than cell number. However, Dp110 also promotes growth primarily by increasing cell size, indicating that qualitative differences exist in the cellular response to expression of dMyc and Dp110. Although both growth regulators increase protein synthesis, Orian (2003) suggests that dMyc probably does so by increasing components of the protein synthetic machinery (initiation factors and ribosomal proteins, etc.) whereas PI3K signaling is thought to function by increasing the utilization of existing machinery. Regardless of the mechanism, these experiments argue against the notion that apposed populations of fast- and slow-growing cells always result in cell competition (de la Cova, 2004).

Three lines of evidence have been provided that indicate that cell competition leading to cell death is required for control of wing size. (1) Growth induced by local expression of either Dp110 or cyclin D + Cdk4 does not induce competition and causes wing overgrowth. (2) When dMyc is expressed in all cells of the wing disc, the wing overgrows, whereas the introduction of clones lacking dMyc leads to cell competition and to wings approaching normal size. (3) Genetic reduction of hid prevents the cell death associated with competition and leads to overgrowth of the compartment in which the dMyc-expressing cells reside (de la Cova, 2004).

An important conclusion of this work is that apoptosis is critical for appropriate wing development. These experiments demonstrate that apoptosis has two roles in regulating wing size. One role is uncovered when the disc is challenged by local changes in dMyc levels, conditions in which cells are exceptionally sensitive to hid gene dosage: the full hid complement is necessary for the disc to respond properly to competition and eliminate cells. However, a second role of apoptosis is revealed when it is abolished: this role regulates uniformity of disc size, and its loss is manifested as a widening of the range of disc sizes within a given population. This second role of apoptosis indicates that organ overgrowth is distinct from loss of organ size control. Wing overgrowth -- observed when cell competition is not executed during local growth perturbations -- occurs such that, although larger than normal, wing size still falls within a uniform range. In contrast, loss of size control is the absence of a discrete and reproducible size population and results from a failure to induce apoptosis during the process of growth. Based on these observations, it is proposed that hid-regulated apoptosis contributes to a disc-intrinsic mechanism that limits variation in size by allowing elimination of cells. This mechanism may serve as negative feedback to the positive aspects of growth during development. Loss of feedback control could allow stochastic variation in size, as has been observed. Although it has been proposed that overall organ mass rather than cell number is sensed by the intrinsic size mechanism, these experiments imply that size control is implemented at least in part by reduction of cell number via apoptosis (de la Cova, 2004).

Is cell competition also part of the intrinsic size control program? If cell competition has a role in normal development, growth rate variations should be observed within developing organs. Indeed, both spatial and temporal differences in cell proliferation rates exist in the wing disc, and cell size also varies across the disc, suggesting differences in cellular growth rates. dmyc is regulated both by Wingless and Dpp, which direct the majority of disc patterning. Minor alterations in their signaling could plausibly cause subtle competitive effects by influencing levels of dmyc expression, which in turn would modulate hid expression and allow for the correction of patterning mistakes that occur during development. In this sense, cell competition, on a small scale, might be a surveillance or 'quality control' mechanism to guarantee that organs reach a body-proportional, reproducible size with the appropriate complement of cell fates (de la Cova, 2004).

Cell competition is likely a common mechanism used in organs under many conditions, including those that are adverse. Competitive mechanisms are known to be important to reestablish homeostasis in lymphoid tissue after an immune response. During tumorigenesis, cancer cells may compete with normal tissue and ultimately overtake the organ, leading to overgrowth of the tumor. In addition, cell competition could prove important therapeutically for many diseases. For example, when liver cells are transplanted into a diseased host liver, cell competition would be critical for the replacement of viable but damaged liver cells with the regenerating donor cells. Although of the three growth regulators tested only dMyc induced cell competition, other growth-promoting genes that induce cell competition probably exist. The identification of these genes holds promise for a further elucidation of the role of cell competition in organ development (de la Cova, 2004).

CNBP regulates wing development in Drosophila melanogaster by promoting IRES-dependent translation of dMyc

CCHC-type zinc finger nucleic acid binding protein (CNBP) is a small conserved protein, which plays a key role in development and disease. Studies in animal models have shown that the absence of CNBP results in severe developmental defects that have been mostly attributed to its ability to regulate c-myc mRNA expression. Functionally, CNBP binds single-stranded nucleic acids and acts as a molecular chaperone, thus regulating both transcription and translation. This work reports that in Drosophila melanogaster, CNBP (CG3800) is an essential gene, whose absence causes early embryonic lethality. In contrast to what observed in other species, ablation of CNBP does not affect dMyc mRNA expression, whereas the protein levels are markedly reduced. dCNBP regulates dMyc translation through an IRES-dependent mechanism, and knockdown of dCNBP in the wing territory causes a general reduction of wing size, in keeping with the reported role of dMyc in this region. Consistently, reintroduction of dMyc in CNBP-deficient wing imaginal discs rescues the wing size, further supporting a key role of the CNBP-Myc axis in this context. Collectively, these data show a previously uncharacterized mechanism, whereby, by regulating dMyc IRES-dependent translation, CNBP controls Drosophila wing development. These results may have relevant implications in other species and in pathophysiological conditions (Antonucci, 2014).

Myc and eye development

Ectopic expression of transcription factors in eye-antennal discs of Drosophila strongly interferes with their developmental program. Early ectopic expression in embryonic discs interferes with the developmental pathway primed by Eyeless and generates headless flies, which suggests that Eyeless is necessary for initiating cell proliferation and development of both the eye and antennal disc. Interference occurs through a block in the cell cycle that for some ectopic transcription factors is overcome by D-CycE or D-Myc. Late ectopic expression in cone cell precursors interferes with their differentiation. It is proposed that this developmental pathway interference is a general surveillance mechanism that eliminates most aberrations in the genetic program during development and evolution, and thus seriously restricts the pathways that evolution may take (Jiao, 2001).

The eye-antennal discs of ey-GAL4/+; UAS-Gsb/+ third instar larvae are absent or strongly reduced in size. Evidently, developmental pathway interference induced by the ectopic expression of transcription factors eventually results in the inhibition of cell proliferation and/or apoptosis in these discs. To investigate which of these two processes is responsible for the generation of headless flies, attempts were made to inhibit apoptosis or to stimulate cell proliferation in eye-antennal discs. While inhibition of apoptosis by the expression of the baculovirus P35 protein is unable to suppress the headless phenotype, stimulation of cell proliferation by the expression of D-Myc suppresses it in spontaneously eclosing adults (5%-20%), producing adults of variable eye size, from eyeless adults to adults whose eye size is only slightly reduced. The headless phenotype is rescued even more dramatically by D-CycE, which restores a wild-type phenotype in up to 50% of the adults and only rarely generates small-eyed flies. Rescue of the headless phenotype by CycE is not restricted to ey-GAL4/+; UAS-Gsb/+ flies, but is achieved for all Pax proteins and transcription factors whose potency to interfere with ey function in the eye-antennal disc was tested. However, in contrast to headless flies produced by Gsb, Prd, Poxm, D-Pax2 or Dac, many of which were rescued by CycE to adults that eclosed spontaneously, those generated by Mef2, Sim or Poxn were incompletely rescued. D-Myc is not as efficient in its rescue ability, except in the case of D-Pax2, in which nearly all flies were rescued to wild-type adults. It is concluded that developmental pathway interference through ectopic expression of transcription factors results in the inhibition of cell proliferation that is at least partially overcome by co-expression of D-Myc or D-CycE (Jiao, 2001).

Regulation of Drosophila glial cell proliferation by Merlin-Hippo signaling

Glia perform diverse and essential roles in the nervous system, but the mechanisms that regulate glial cell numbers are not well understood. This study identified and characterize a requirement for the Hippo pathway and its transcriptional co-activator Yorkie in controlling Drosophila glial proliferation. Yorkie was found to be both necessary for normal glial cell numbers and, when activated, sufficient to drive glial over-proliferation. Yorkie activity in glial cells is controlled by a Merlin-Hippo signaling pathway, whereas the upstream Hippo pathway regulators Fat, Expanded, Crumbs and Lethal giant larvae have no detectable role. Functional characterization of Merlin-Hippo signaling was extended by showing that Merlin and Hippo can be physically linked by the Salvador tumor suppressor. Yorkie promotes expression of the microRNA gene bantam in glia, and bantam promotes expression of Myc, which is required for Yorkie and bantam-induced glial proliferation. These results provide new insights into the control of glial growth, and establish glia as a model for Merlin-specific Hippo signaling. Moreover, as several of these genes have been linked to human gliomas, the results suggest that this linkage could reflect their organization into a conserved pathway for the control of glial cell proliferation (Reddy, 2011).

Merlin was first identified as the product of a human tumor suppressor gene, NF2, loss of which in peripheral glial cells results in benign tumors. Merlin has also been identified as an inhibitor of gliomas. The current observations indicate that the role of Merlin as a negative regulator of glial cell proliferation is conserved from humans to Drosophila and, thus, that Drosophila can serve as a model for understanding Merlin-dependent regulation of glial growth (Reddy, 2011).

Studies in Drosophila imaginal discs first linked Merlin to Hippo signaling, and Merlin was subsequently linked to Hippo signaling in mammalian cells, including its role in meningioma. However, the tumor suppressor activity of Merlin has also been linked to other downstream effectors in mammals, including Erb2, Src, ras, rac, TORC1 (CRTC1 -- Human Gene Nomenclature Database; see Drosophila CRTC) and CRL4 (IL17RB -- Human Gene Nomenclature Database), creating some uncertainty regarding the general importance of the linkage of Merlin to Hippo in growth control. This study found that depletion of Merlin, depletion of other tumor suppressors in the Hippo pathway, or expression of an activated form of Yki, all result in similar glial overgrowth phenotypes. Moreover, depletion of Merlin increased nuclear localization of Yki, and depletion of Yki suppressed the overgrowth phenotype of Merlin. Together, these observations clearly establish that the glial overgrowth phenotype associated with Merlin depletion in Drosophila is mediated through the Hippo signaling pathway (Reddy, 2011).

A noteworthy feature of Hippo signaling in Drosophila glial cells is that Merlin appears to be uniquely required as an upstream regulator of Hippo signaling, as the Fat-dependent, Ex-dependent and Lgl-dependent branches have no detectable role. Glia might, thus, provide an ideal model for mechanistic investigations of the Merlin branch of Hippo signaling. Fat-Hippo signaling employs Fat as a transmembrane receptor and Dachsous as its transmembrane ligand, whereas Ex-Hippo signaling appears to employ Crumbs as a transmembrane receptor and ligand. By contrast, Drosophila transmembrane proteins that mediate extracellular signaling and interact with Merlin have not yet been identified. Distinct mechanisms might also be involved in signal transduction downstream of Merlin. Although there is evidence that Ex and Merlin can both influence Hippo activity, Ex, but not Mer, can directly associate with Hpo. Conversely, Merlin, but not Ex, can interact directly with Salvador, and Merlin, Salvador and Hippo can form a trimeric complex. Moreover, the kibra loss-of-function phenotype is weaker than expanded in imaginal discs, but comparable to Merlin, and it was found that depletion of kibra also has a significant effect on glial cell proliferation. Kibra is highly expressed in mammalian brain, and alleles of KIBRA (WWC1 -- Human Gene Nomenclature Database) have been linked to human memory performance. The role of kibra in regulating glial cell numbers in Drosophila thus raise the possibility that the influence of KIBRA on human memory might reflect a role in glial cells (Reddy, 2011).

Finally, it is noted that although Hippo signaling has been investigated in several different organs in Drosophila, including imaginal discs, ovarian follicle cells, neuroepithelial cells and intestinal cells, these all involve roles in epithelial cells, in which upstream regulators of the pathway (e.g. Fat, Ex, Mer) all have a distinctive localization near adherens junctions. The identification of a requirement for Hippo signaling in glia is the first time in Drosophila that a role for the pathway has been identified in non-epithelial cells. Indeed, in previous studies it was found that Hippo signaling influences proliferation of neuroepithelial cells, but other neuronal cell types, including neuroblasts, ganglion mother cells and neurons, are insensitive to Yki (Reddy, 2011).

Considerable attention has been paid to genes for which mutation or inappropriate activation can cause over-proliferation of glial cells, resulting in glial tumors. However, less is known about the mechanisms required for normal glial growth. Through loss-of-function studies, several genes essential for normal glial cell numbers were identified, including yki, sd, ban, mad and myc. The requirement for yki, mad and sd, together with epistasis studies, identifies a requirement for active Yki in glial growth. This in turn implies that downregulation of Hippo signaling is important for normal glial growth. Understanding how this is achieved will provide further insights into the regulation of glial cell numbers (Reddy, 2011).

A requirement for Mad, together with its upstream regulator Thickveins (Tkv), in promoting retinal glial cell proliferation was has been established in previous studies. Current studies of glial cells, together with recent work in imaginal discs, emphasize that in mediating the growth-regulating activity of Hippo signaling, Yki utilizes multiple DNA-binding partners (i.e. Mad and Sd) in the same cells at the same time to regulate distinct downstream target genes required for tissue growth (Reddy, 2011).

Although Yki activity influenced glial cell numbers throughout the nervous system, direct analysis of cell proliferation by EdU labeling revealed that retinal glia were more sensitive to Yki activation at late third instar than central brain glia, and significant induction of central brain glial cell proliferation was only observed when Yki activation was combined with Myc over-expression. Further studies will be required to define the basis for this differential sensitivity, but the implication that the proliferative response to Yki is modulated by developmental stage and/or glial cell type has important implications for diseases associated with both excess and deficits of glial cells (Reddy, 2011).

These studies in Drosophila delineate functional relationships among genes involved in the control of glial cell proliferation. Mammalian homologs of Merlin, Yki and Myc have been implicated in glioma. Although a mammalian homolog of ban has not been described, other miRNAs have also been linked to glioma. These observations imply that these genes can be placed into a pathway, in which Merlin, through Hippo signaling, regulates Yki, Yki regulates ban, and ban regulates Myc. However, as expression of Myc alone did not lead to substantial overgrowth of glia, Yki and ban must also have other downstream targets important for the promotion of glial cell proliferation. Moreover, the current observations indicate that a Yki-Sd complex is also required for glial growth. In addition to the well characterized downstream target Diap1, Yki-Sd complexes in glial cells might regulate Myc directly, as suggested by studies in imaginal discs, and might regulate cell cycle genes in conjunction with E2F1 (Reddy, 2011).

The influence of activated-Yki on a ban-GFP sensor, together with the observations that yki is not required for ban-mediated overgrowth, whereas ban is required for Yki-mediated overgrowth, position ban downstream of Yki. This is consistent with studies of Hippo signaling in imaginal discs, in which ban has also been identified as a target of Yki for growth regulation. The placement of Myc downstream of Yki and ban is supported by the observation that Myc levels can be increased by expression of ban or activated-Yki, and by genetic tests that indicate that Myc is required for Yki- and ban-promoted glial overgrowth. A mechanism by which ban can regulate Myc levels, involving downregulation of a ubiquitin ligase that negatively regulates Myc, was identified recently in imaginal discs, and might also function in glial cells. Myc has been reported to downregulate Yki expression in imaginal discs and, although this study has not investigated whether a similar negative-feedback loop exists in glial cells, the synergistic enhancement of glial cell proliferation observed when Yki and Myc were co-expressed is consistent with this possibility, as the expression of both genes under heterologous promoters could bypass negative regulation of Yki by Myc (Reddy, 2011).

The Myc proto-oncogene is de-regulated or amplified in several human cancers, including gliomas. The sensitivity of Yki/ban-induced overgrowth to reduced Myc levels parallels studies of glioma models involving other signaling pathways. For example, Myc is upregulated by EGFR, and is limiting for EGFR-PI3K-induced glial cell overgrowth in a Drosophila glioma model, and p53 and Pten-driven glioma in mouse models is also Myc dependent. Considering the evidence linking Merlin and Yap to glial growth in mammals, and the identification of Myc as a downstream target of Yap in cultured cells, it is likely that Yap could also influence glial growth in mammals, in part, through regulation of Myc (Reddy, 2011).

The bantam microRNA acts through Numb to exert cell growth control and feedback regulation of Notch in tumor-forming stem cells in the Drosophila brain

Notch (N) signaling is central to the self-renewal of neural stem cells (NSCs) and other tissue stem cells. Its deregulation compromises tissue homeostasis and contributes to tumorigenesis and other diseases. How N regulates stem cell behavior in health and disease is not well understood. This study shows that Notch regulates bantam (ban) microRNA to impact cell growth, a process key to NSC maintenance and particularly relied upon by tumor-forming cancer stem cells. Notch signaling directly regulates ban expression at the transcriptional level, and ban in turn feedback regulates N activity through negative regulation of the Notch inhibitor Numb. This feedback regulatory mechanism helps maintain the robustness of N signaling activity and NSC fate. Moreover, this study shows that a Numb-Myc axis mediates the effects of ban on nucleolar and cellular growth independently or downstream of N. These results highlight intricate transcriptional as well as translational control mechanisms and feedback regulation in the N signaling network, with important implications for NSC biology and cancer biology (Wu, 2017).

By revealing the involvement of the miRNA pathway, this study highlights the complexity of the N signaling network in normal NSCs and tumor-forming cancer stem cell (CSC)-like NSCs. Previous studies implicated critical roles for both canonical and non-canonical N signaling pathways in NSCs and CSC-like NSCs, and revealed particular dependence of CSC-like NB growth on non-canonical N signaling, which involves PINK1, mTORC2, and mitochondrial quality control. The current study reveals a particular requirement for ban in CSC-like NBs induced by N hyperactivation. The CSC-like NB overproliferation induced by hyperactivation of N or N pathway component Dpn can all be assumed to be of type II NB origin, since previous studies have clearly established that Notch signaling is essential for the development and/or maintenance of type II NBs, but dispensable for type I NBs, and that hyperactivation of Notch or its downstream effector Dpn induced ectopic CSC-like NB growth by altering the lineage homeostasis of the type II but not type I NBs. It would be interesting to test whether, in addition to ban's role in canonical N signaling, there exists a link between ban and non-canonical N signaling. The data indicate that the ban-Numb signaling motif regulates NSC/CSC behavior through at least two mechanisms. On one hand, it regulates cell growth and particularly nucleolar growth, through Myc, a known regulator of cellular and nucleolar growth. Consistently, negative regulation of Myc protein level by Numb was observed through E3 ubiquitin-protein ligase, Huwe1, and the UPS. c-Myc is an essential regulator of embryonic stem cell (ESC) self-renewal and cellular reprogramming, and Myc level and stability can be controlled in stem cells through targeted degradation by the UPS, suggesting conserved mechanisms. A key function of the nucleolus is the biogenesis of ribosomes, the cellular machinery for mRNA translation, and previous studies in Drosophila have supported the critical role of nucleolar growth in NSC self-renewal and maintenance. On the other hand, the ban-Numb axis feedback regulates the activity of N by a double negative regulation, with the end result being positive feedback regulation. This feedback mechanism may help transform initial not so dramatic differences in N activity between NB and its daughter cell generated by the asymmetric segregation of Numb during NB division [33] into 'all-or-none' decision of cell fates. Feed-forward regulatory loops, both coherent and incoherent, are frequently found in gene regulatory networks, and although ban miRNA is not conserved in mammals, miRNAs have been implicated in an incoherent feed-forward loop in the Numb/Notch signaling network in colon CSCs in mammals (Wu, 2017).

Given the role of ban in a positive feedback regulation of N and the potency of N hyperactivity in inducing tumorigenesis, one may wonder why ban overexpression is not sufficient to cause tumorigenesis. As in any biological systems, feedback regulation is meant to increase the robustness and maintain homeostasis of a pathway. Feedback alone, either negative or positive, should not override the main effect of the signaling pathway. Thus, in the NB system feedback regulation by ban is built on top of the available N signaling activity in a given cell and serving to maintain N activity. Because of ban's 'fine-tuning' rather than 'on/off switching' of Numb expression, its effect on N activity during feedback regulation will also be 'fine-tuning', serving to maintain N activity in NB within a certain range. Overexpression of ban in a wild type background may not be sufficient to cause tumorigenesis because N activity is not be elevated to the level sufficient to induce brain tumor as in N-v5 overexpression condition. Consistent with this, the extent of Numb inhibition by ban is also modest, not reaching the threshold level of Numb inhibition needed to cause tumorigenesis. Consistent with the notion that feedback regulation by ban is built on top of the available N signaling activity in a given cell, and that there is dosage effect of N activity in tumorigenesis, overexpression of ban in N-v5 overexpression background further enhanced N-v5 induced tumorigenesis. It is likely that ban or other miRNAs may participate in additional regulatory mechanisms in the N signaling network in Drosophila. Of particular interest, it would be interesting to test whether miRNAs may impinge on the asymmetric cell division machinery to influence the symmetric vs. asymmetric division pattern, a key mechanism employed by NSCs and transit-amplifying IPs to balance self-renewal with differentiation (Wu, 2017).

The results emphasize the critical role of translational control mechanisms in NSCs and CSC-like NSCs. Compared to the heavily studied transcriptional control, knowledge of the translational control of NSCs and CSCs is rather limited. As fundamental regulators of mRNA translation, miRNAs can interact with both positive and negative regulators of translation to influence gene expression. Thus, miRNA activity can be regulated context-dependently at both the transcriptional and translational levels, which may account for the opposite effect of N on ban activity in the fly brain and wing disc, although the ban genomic locus is bound by Su(H) in both tissues. Whether N regulates the transcription of ban or its activity as a translational repressor in the wing disc remains to be tested. With regard to the translation of numb mRNA, the conserved RNA-binding protein (RNA-BP) Musashi has been shown to critically regulate the level of Numb protein in mammalian hematopoietic SCs and leukemia SCs. Further investigation into the potential interplay between miRNAs and RNA-BPs in the translational control of Numb in NBs and CSC-like NBs promises to reveal new mechanisms and logic in stem cell homeostasis regulation, with important implications for stem cell biology and cancer biology (Wu, 2017).

Oogenesis

dmyc is expressed in a dynamic pattern in ovaries. At early stages, high levels of DMYC transcripts accumulate in the germarium with lower levels in either stage 1 or stage 2 egg chambers. By stage 3, DMYC mRNA can be detected in all cell types of the chamber: the nurse cells, oocyte, and follicle cells. This expression pattern is maintained throughout oogenesis. dmax expression can still be detected in diminutive (see below) mutant ovaries (Gallant, 1996).

The Notch signaling pathway controls the follicle cell mitotic-to-endocycle transition in Drosophila oogenesis by stopping the mitotic cycle and promoting the endocycle. To understand how the Notch pathway coordinates this process, a functional analysis was performed of genes whose transcription is responsive to the Notch pathway at this transition. These genes include String, the G2/M regulator Cdc25 phosphatase; Hec/CdhFzr, a regulator of the APC ubiquitination complex and Dacapo, an inhibitor of the CyclinE/CDK complex. Notch activity leads to downregulation of String and Dacapo, and activation of Fzr. All three genes are independently responsive to Notch. In addition, CdhFzr, an essential gene for endocycles, is sufficient to stop mitotic cycle and promote precocious endocycles when expressed prematurely during mitotic stages. In contrast, overexpression of the growth controller Myc does not induce premature endocycles but accelerates the kinetics of normal endocycles. F-box/WD40-domain protein Ago/hCdc4 (Archipelago), a SCF-regulator is dispensable for mitosis, but crucial for endocycle progression in follicle epithelium. CycE oscillation remains critical for endocycling; continuous high level of CycE expression blocks the cell cycle in G2. The regulation of CycE levels is achieved by the function of Ago that presumably binds to auto-phosphorylated CycE and directs it to SCF-complex degradation: high levels of CycE and no endocycling is observed in ago-clones. The results support a model in which Notch activity executes the mitotic-to-endocycle switch by regulating all three major cell cycle transitions. Repression of String blocks the M-phase, activation of Fzr allows G1 progression, and repression of Dacapo assures entry into the S-phase. This study provides a comprehensive picture of the logic that external signaling pathways may use to control cell cycle transitions by the coordinated regulation of the cell cycle (Shcherbata, 2004).

The data suggest that a component regulating growth and thereby the kinetics of G1/S transition in follicle cell endocycles is the Myc oncogene instead and independent of CycD. In mammals c-Myc controls the decision to divide or not to divide and thereby functions as a crucial mediator of signals that determine organ and body size. Interestingly, overexpression of Myc in follicle cells does not affect the mitotic cycles but induces, instead, extra endocycles. Because the timing for entering and exit from the endocycles has not changed, however, increased ploidy is observed; therefore, it is suggested that the rate of endocycles is increased because of the overexpression of Myc. This finding is in accordance with recent loss-of-function analysis on myc in follicle cells, suggesting that myc mutant follicle cells can make the transition from mitosis to the endocycle, but that they can only very inefficiently support the endocycle. Therefore, both loss-of-function and overexpression experiments suggest that Myc is an essential component for the proper rate of endocycles in follicle cells (Shcherbata, 2004).

Integration of Insulin receptor/Foxo signaling and dMyc activity during muscle growth regulates body size in Drosophila

Drosophila larval skeletal muscles are single, multinucleated cells of different sizes that undergo tremendous growth within a few days. The mechanisms underlying this growth in concert with overall body growth are unknown. The size of individual muscles correlates with the number of nuclei per muscle cell and with increasing nuclear ploidy during development. Inhibition of Insulin receptor (InR; Insulin-like receptor) signaling in muscles autonomously reduces muscle size and systemically affects the size of other tissues, organs and indeed the entire body, most likely by regulating feeding behavior. In muscles, InR/Tor signaling, Foxo and dMyc (Diminutive) are key regulators of endoreplication, which is necessary but not sufficient to induce growth. Mechanistically, InR/Foxo signaling controls cell cycle progression by modulating dmyc expression and dMyc transcriptional activity. Thus, maximal dMyc transcriptional activity depends on InR to control muscle mass, which in turn induces a systemic behavioral response to allocate body size and proportions (Demontis, 2009).

Therefore, interplay between InR/Tor signaling, Foxo and dMyc activity regulates muscle growth that occurs during Drosophila larval development, in part via the induction of endoreplication. Interestingly, the extent of muscle growth is sensed systemically, regulates feeding behavior and, in turn, influences the size of other tissues and indeed the whole body. Thus, the growth of a single tissue is sensed systemically via modulating a whole-organism behavior (Demontis, 2009).

dMyc, as well as activation of InR signaling, can promote endoreplication in muscles, whereas Foxo and inhibitors of dMyc and of InR/Tor have the opposite effect. dMyc is likely to regulate the expression of genes required for multiple G-S and S-G transitions during endoreplication, similar to vertebrate Myc, which regulates key cell-cycle regulators including cyclin D2, cyclin E, and the cyclin kinase inhibitors p21 and p27 (Cdkn1a and Cdkn1b, respectively). Indeed, aberrant levels of Cyclin E block muscle growth, indicating that proper muscle growth requires tight control of the expression and activity of endoreplication genes. Further, endoreplication is also modulated by Foxo, which is activated in conditions of nutrient starvation, impaired InR/Tor signaling and by other cell stressors. Foxo presumably regulates cell cycle progression at least in part by modulating the expression of evolutionarily conserved Foxo/Myc-target genes, such as dacapo (the Drosophila p21/p27 homolog) and Cyclin E, that regulate the G1-S transition. Interestingly, Foxo and Myc might control different steps in the activation of common target genes (Demontis, 2009).

In addition, it was found that active Foxo can also inhibit dMyc protein activity and regulates dmyc gene expression. Mechanistically, Foxo could influence dMyc activity in several ways. First, it might physically interact with dMyc, although no evidence was found to support this notion. Second, Foxo could regulate the expression of genes that target dMyc for proteasomal degradation, including several ubiquitin E3 ligases that are induced by Foxo during muscle atrophy in mice and humans. However, by analyzing dMyc protein levels by western blot, no significant dMyc protein instability was found upon Foxo overexpression. Third, Foxo might promote the expression of transcriptional regulators that oppose dMyc function, including Mad/Mnt, although no substantial increase in dmnt mRNA levels was detected upon Foxo activation in muscles. Possibly, the expression of other dMyc regulators might be affected by Foxo. Future experiments will be needed to dissect the Foxo-dMyc interaction (Demontis, 2009).

Finally, by manipulating muscle growth and/or endoreplication, it was found that in muscles the ratio of cell size to nuclear size is not constant, and increased nuclear size and DNA content, indicative of ploidy, is necessary but not sufficient to drive growth. Usually, an increase in cell size is matched by an increase in nuclear size, which commonly parallels increases in nuclear ploidy. However, the current findings indicate that in muscles, dMyc-driven variation in nuclear size and ploidy is permissive but not sufficient for substantial growth, even in the presence of increased biogenesis of nucleoli and expression of genes involved in protein translation. This is different from fat body cells, in which dmyc overexpression induces endoreplication and proportional cell growth. Thus, additional instructive signals, possibly modulating protein synthesis, mitochondriogenesis, ribosome biogenesis, sarcomere assembly, and other anabolic responses must be concomitantly received to promote maximal muscle growth. Therefore, increases in cell size and nuclear ploidy are surprisingly uncoupled during muscle growth (Demontis, 2009).

Little is known about the mechanisms that control and coordinate cell, organ and body size, and in particular how muscle growth is matched with the growth of other tissues and of the entire organism. Inhibition of InR/Tor signaling and dMyc activity in muscles impairs, in addition to muscle mass, the size of the entire body and of other internal organs. Similarly, overexpression of Cyclin E in muscles also results in autonomous and systemic growth defects, indicating that, at least in some cases, modulation of muscle growth by means independent from InR signaling can be sensed systemically. In the larva, endoreplicating tissues and organs (gut, salivary glands, epidermis, fat body) are severely affected, whereas non-endoreplicating tissues (brain and imaginal discs) are less affected, indicating distinct tissue responsiveness to this regulation. Similarly, inhibition of Tor signaling in the fat body also primarily affects the size of endoreplicating tissues (Demontis, 2009).

Non-autonomous regulation of tissue size may rely on humoral factors (e.g. hormone-binding proteins, hormones, metabolites) produced by muscles in response to achieving a certain mass. However, alternative models are possible. In particular, decreased and increased larval feeding, respectively, were observed upon inhibition and activation of InR signaling in muscles. This whole-organism behavioral adaptation is possibly due to decreased and increased efficiency of smaller and bigger muscles, respectively, and to regulated expression of neuropeptides that hormonally control feeding behavior. As a consequence of the regulation of feeding behavior, nutrient uptake is decreased and larval growth is blocked in the cells of endoreplicating tissues, which are extremely sensitive to poor nutritional conditions, and to a lesser extent in non-endoreplicating tissues, which are more resistant to limited nutritional supply. In turn, increased or decreased size of non-muscle tissues arise as a consequence of abnormal feeding. Thus, muscle size coordinates with the size of other organs and of the entire body, at least in part via a systemic, behavioral response. Distinct tissues are differently sensitive to this regulation, resulting in altered body proportions (Demontis, 2009).

Understanding the mechanisms regulating muscle mass is of special interest because they underline the etiology of several human diseases. Directly relevant to these studies, both MYC and InR (INSR) signaling have been found to regulate muscle growth and maintenance in humans. Further, muscle atrophy is triggered by FOXO activation in several pathological conditions. In addition, MYC function has been implicated in heart hypertrophy, a process that is conversely regulated by FOXO (Demontis, 2009).

The findings that Foxo functionally antagonizes dMyc during the growth of Drosophila muscles suggest that these factors might also interact similarly in humans. Consistent with this hypothesis, FOXO and MYC regulate, in opposite fashions, the atrophic and hypertrophic programs in human skeletal muscles and cardiomyocytes, and display complementary gene expression and activity in these contexts (Demontis, 2009).

Finally, the finding that during larval development, inhibition of InR signaling in muscles has profound systemic effects might also reflect physiological conditions found in humans. Indeed, defective responsiveness of muscles to Insulin during type II diabetes has autonomous effects on muscle maintenance that are associated with systemic effects on the metabolism of the entire organism, contributing to the improper control of glycemia and the development of metabolic syndrome. This study has identified feeding behavior as part of the systemic response that in Drosophila senses perturbations in muscle mass. These findings might help further elucidate the signals involved in metabolic and growth homeostasis, which may be conserved across evolution (Demontis, 2009).


EFFECTS OF MUTATION

Embryonic

DMYC transcripts, presumably maternal, can be detected ubiquitously from the earliest stages. Later the zygotic transcripts accumulate in a changing pattern in various tissues. In preblastoderm embryos, DMYC mRNA is present throughout the embryo, with the highest levels at the anterior and posterior termini, but is absent from the pole cells. In early gastrulation, additional DMYC mRNA can be detected in the presumptive mesoderm along the ventral midline. This mesodermal staining intensifies during germband extention and remains until late embryogenesis. The terminally located expression follows the posterior and anterior midgut primordia during the germband extended stages. Additional staining is found in salivary placodes. DMAX transcript is less abundant than DMYC, particularly during the earliest stages. In addition dmax is expressed in tissues with undetectable levels of DMYC, such as the developing central nervous system. Hence, whereas tissues containing the highest levels of DMYC and DMAX transcripts are undergoing DNA replication, not all actively proliferating tissues have detectable levels of DMYC.

Myc and wing development

The Ras GTPase links extracellular mitogens to intracellular mechanisms that control cell proliferation. To understand how Ras regulates proliferation in vivo, Ras was either activated or inactivated in cell clones in the developing Drosophila wing. Cells lacking Ras are smaller, have reduced growth rates, accumulate in G1, and undergo apoptosis due to cell competition. Conversely, activation of Ras increases cell size and growth rates and promotes G1/S transitions. Ras upregulates the growth driver dMyc, and both Ras and dMyc increase levels of cyclin E posttranscriptionally. It is proposed that Ras primarily promotes growth and that growth is coupled to G1/S progression via cyclin E. Interestingly, upregulation of growth by Ras does not deregulate G2/M progression or a developmentally regulated cell cycle exit (Prober, 2000).

It is proposed that there is parallel and independent control of G1/S and G2/M transitions in Drosophila wing disc cells. Cellular growth due to Ras or dMyc drives G1/S transitions by promoting translation of cyclin E. Ras may also drive growth via proteins other than dMyc; this could feed back to upregulate translation of dmyc mRNA. Alternatively, Ras may regulate cellular growth and the G1/S cell cycle machinery in parallel. Stg/Cdc25, which is regulated primarily at the transcriptional level, drives G2/M transitions. Signaling molecules capable of regulating coordinated growth and patterning such as Vein may regulate G1/S transitions via Ras, dMyc, or other growth-promoting proteins and regulate G2/M transitions via transcription factors that modulate transcription of Stg/Cdc25 (Prober, 2000).

Other proteins that promote growth, such as the Drosophila homologs of dMyc and Phosphoinositide 3-Kinase (dPI3K), have effects on cell cycle progression similar to Ras. Upregulating these proteins in the developing wing truncates G1, elongates G2, and increases growth rates, while downregulating them cause the opposite effects. Furthermore, the resulting growth rates are inversely proportional to the length of G1. Given these similarities, it is proposed that cellular growth is rate limiting for G1/S progression in wing imaginal cells (Prober, 2000).

The data suggest that the effects of Ras on cellular growth and the cell cycle are at least partially mediated by dMyc. Mammalian Myc transcription factors activate expression of many genes involved in cellular growth and metabolism, and Drosophila dMyc is a potent growth driver in vivo. Upregulation of dMyc by Ras appears to be posttranscriptional. Ras might act by inhibiting degradation of dMyc protein, as has been demonstrated in mammalian cell culture. Alternatively, Ras might stimulate growth via other proteins, such as components of the dPI3K/dAkt/dS6 Kinase pathway, which promote cellular growth in Drosophila. Increased growth due to these proteins could then feed back to promote translation of extant dmyc mRNA. However, dMyc and dPI3K cannot be mediating all of Ras's effects, since unlike Ras they do not affect cell fate or cell adhesion. These additional functions of Ras, along with the ability to increase Myc protein levels, likely contribute to the strong synergistic action of Ras and Myc in oncogenesis (Prober, 2000).

RasV12 accelerates G1/S transitions but fails to accelerate rates of cell division. This is similar to findings with overexpressed dMyc. However, coexpressing either RasV12 or dMyc with String (Stg), the G2/M rate limitor, does accelerate cell division. This suggests that regulation of Stg is independent of both Ras and dMyc. It is therefore proposed that there is parallel and independent control of G1/S and G2/M transitions during wing development. Signaling molecules capable of regulating coordinated growth and patterning, such as Vein, Decapentaplegic, and Wingless might control G1/S transitions by regulating growth via Ras, dMyc, or other growth-promoting proteins. These signaling molecules might also, unlike Ras and dMyc, control G2/M transitions by modulating transcription of stg. Analysis of more than 40 kb of the stg promoter has revealed an extensive array of regulatory modules that respond to different patterning signals and thus integrate complex patterning information. A model in which cyclin E acts as a growth sensor and Stg acts as a 'pattern sensor' is attractive, as it allows coordination of independent growth and patterning signals by the cell cycle machinery (Prober, 2000).

Experiments in both vertebrates and invertebrates have illustrated the competitive nature of growth and have led to the idea that competition is a mechanism for regulating organ and tissue size. Competitive interactions between cells were assessed in a developing organ and their effect on its final size were examined. Local expression of the Drosophila growth regulator dMyc, a homolog of the c-myc proto-oncogene, induces cell competition and leads to the death of nearby wild-type cells in developing wings. Cell competition is executed via induction of the proapoptotic gene hid and both competition and hid function are required for the wing to reach an appropriate size when dMyc is expressed. Moreover, evidence is provided that reproducible wing size during normal development requires apoptosis. Modulating dmyc levels to create cell competition and hid-dependent cell death may be a mechanism used during normal development to control organ size (de la Cova, 2004).

This work leads to three major conclusions. (1) Expression of the c-myc protooncogene homolog dMyc in small populations of wing disc cells induces cell competition, leading to the elimination of nearby cells via induction of the proapoptotic gene hid. (2) The competition induced by dMyc and the elimination of cells that results is required for control of proper wing size. (3) Studies reveal that apoptosis is required for the fidelity of size during normal wing development, suggesting that the modulation of hid expression by competitive interactions between cells may be used as an endogenous mechanism of size control (de la Cova, 2004).

These experiments demonstrate that expression of dMyc in some cells of a developing organ leads to elimination of nonexpressing cells through apoptosis. The growth disadvantage induced by dMyc-expressing cells fulfills the classic definition of cell competition: viable but slower-growing cells in an organ are eliminated by an encroaching faster-growing cell population, proximity to the fast-growing cell population dictates the severity of the disadvantage in the slow-growing cells, cells are protected from cell competition by developmental compartment boundaries, and appropriate organ size is reached at the end of development. Relative differences in dMyc levels lead to competitive situations between cells -- dmyc mutant cells are outcompeted by neighboring nonmutant cells; wild-type cells, with a normal complement of endogenous dmyc, are also subject to competition if surrounded by cells expressing a dMyc transgene. However, wild-type cells appear to be subject to competition only if they lie within about eight cell diameters of dMyc-expressing cells, and they must reside in the same developmental compartment. Thus, proximity, compartmental provenance, and the relative levels of dmyc are particularly important aspects of the competitive effects of dMyc (de la Cova, 2004).

During the process of cell competition induced by dMyc, the proapoptotic gene hid is induced in the growth-disadvantaged cells. Since a reduction of hid function protects cells from competition-induced death, it is believed that hid upregulation is a consequence of the sensing of competitive stress. An intriguing question that remains is how cells are able to sense competition. One possibility is that cells compete for sufficient levels of a survival factor that normally blocks hid expression. Dpp signaling promotes cell survival in the wing disc but appears to be unaffected in discs expressing dMyc. Alternatively, some cells in competition may be deprived of adequate nutrients, although in these experiments, cells at a growth disadvantage retain a normal nucleolar size, arguing that their biosynthetic rates are not abnormally low. However, the results suggest that dMyc provokes competition and hid expression via a short-range signal, since close proximity is required for the perception of competitive effects. Perhaps the most intriguing feature of this signal is that it is not perceived by nearby cells across a compartment boundary, although dMyc induces competition between cells within the posterior compartment as well as within the anterior. One possibility is that cells expressing dMyc acquire adhesive properties that transmit a competitive signal to neighboring cells, which is not compatible with the adhesive barrier that maintains the compartment boundary (de la Cova, 2004).

These studies reveal that cell competition is not invariably induced whenever rapidly growing cells populate regions of a developing organ. Both the PI3K Dp110 and cyclin D/Cdk4 potently promote growth when overexpressed, yet they do not induce competition in any of these assays. These observations also demonstrate that balanced growth -- growth that simultaneously drives cell division and cellular growth -- is not required to induce cell competition. dMyc expression increases clonal mass solely by increasing cell size. Thus, this trait of cell competition may be related to a size-measuring mechanism that recognizes total mass rather than cell number. However, Dp110 also promotes growth primarily by increasing cell size, indicating that qualitative differences exist in the cellular response to expression of dMyc and Dp110. Although both growth regulators increase protein synthesis, Orian (2003) suggests that dMyc probably does so by increasing components of the protein synthetic machinery (initiation factors and ribosomal proteins, etc.) whereas PI3K signaling is thought to function by increasing the utilization of existing machinery. Regardless of the mechanism, these experiments argue against the notion that apposed populations of fast- and slow-growing cells always result in cell competition (de la Cova, 2004).

Three lines of evidence have been provided that indicate that cell competition leading to cell death is required for control of wing size. (1) Growth induced by local expression of either Dp110 or cyclin D + Cdk4 does not induce competition and causes wing overgrowth. (2) When dMyc is expressed in all cells of the wing disc, the wing overgrows, whereas the introduction of clones lacking dMyc leads to cell competition and to wings approaching normal size. (3) Genetic reduction of hid prevents the cell death associated with competition and leads to overgrowth of the compartment in which the dMyc-expressing cells reside (de la Cova, 2004).

An important conclusion of this work is that apoptosis is critical for appropriate wing development. These experiments demonstrate that apoptosis has two roles in regulating wing size. One role is uncovered when the disc is challenged by local changes in dMyc levels, conditions in which cells are exceptionally sensitive to hid gene dosage: the full hid complement is necessary for the disc to respond properly to competition and eliminate cells. However, a second role of apoptosis is revealed when it is abolished: this role regulates uniformity of disc size, and its loss is manifested as a widening of the range of disc sizes within a given population. This second role of apoptosis indicates that organ overgrowth is distinct from loss of organ size control. Wing overgrowth -- observed when cell competition is not executed during local growth perturbations -- occurs such that, although larger than normal, wing size still falls within a uniform range. In contrast, loss of size control is the absence of a discrete and reproducible size population and results from a failure to induce apoptosis during the process of growth. Based on these observations, it is proposed that hid-regulated apoptosis contributes to a disc-intrinsic mechanism that limits variation in size by allowing elimination of cells. This mechanism may serve as negative feedback to the positive aspects of growth during development. Loss of feedback control could allow stochastic variation in size, as has been observed. Although it has been proposed that overall organ mass rather than cell number is sensed by the intrinsic size mechanism, these experiments imply that size control is implemented at least in part by reduction of cell number via apoptosis (de la Cova, 2004).

Is cell competition also part of the intrinsic size control program? If cell competition has a role in normal development, growth rate variations should be observed within developing organs. Indeed, both spatial and temporal differences in cell proliferation rates exist in the wing disc, and cell size also varies across the disc, suggesting differences in cellular growth rates. dmyc is regulated both by Wingless and Dpp, which direct the majority of disc patterning. Minor alterations in their signaling could plausibly cause subtle competitive effects by influencing levels of dmyc expression, which in turn would modulate hid expression and allow for the correction of patterning mistakes that occur during development. In this sense, cell competition, on a small scale, might be a surveillance or 'quality control' mechanism to guarantee that organs reach a body-proportional, reproducible size with the appropriate complement of cell fates (de la Cova, 2004).

Cell competition is likely a common mechanism used in organs under many conditions, including those that are adverse. Competitive mechanisms are known to be important to reestablish homeostasis in lymphoid tissue after an immune response. During tumorigenesis, cancer cells may compete with normal tissue and ultimately overtake the organ, leading to overgrowth of the tumor. In addition, cell competition could prove important therapeutically for many diseases. For example, when liver cells are transplanted into a diseased host liver, cell competition would be critical for the replacement of viable but damaged liver cells with the regenerating donor cells. Although of the three growth regulators tested only dMyc induced cell competition, other growth-promoting genes that induce cell competition probably exist. The identification of these genes holds promise for a further elucidation of the role of cell competition in organ development (de la Cova, 2004).

CNBP regulates wing development in Drosophila melanogaster by promoting IRES-dependent translation of dMyc

CCHC-type zinc finger nucleic acid binding protein (CNBP) is a small conserved protein, which plays a key role in development and disease. Studies in animal models have shown that the absence of CNBP results in severe developmental defects that have been mostly attributed to its ability to regulate c-myc mRNA expression. Functionally, CNBP binds single-stranded nucleic acids and acts as a molecular chaperone, thus regulating both transcription and translation. This work reports that in Drosophila melanogaster, CNBP (CG3800) is an essential gene, whose absence causes early embryonic lethality. In contrast to what observed in other species, ablation of CNBP does not affect dMyc mRNA expression, whereas the protein levels are markedly reduced. dCNBP regulates dMyc translation through an IRES-dependent mechanism, and knockdown of dCNBP in the wing territory causes a general reduction of wing size, in keeping with the reported role of dMyc in this region. Consistently, reintroduction of dMyc in CNBP-deficient wing imaginal discs rescues the wing size, further supporting a key role of the CNBP-Myc axis in this context. Collectively, these data show a previously uncharacterized mechanism, whereby, by regulating dMyc IRES-dependent translation, CNBP controls Drosophila wing development. These results may have relevant implications in other species and in pathophysiological conditions (Antonucci, 2014).

Myc and eye development

Ectopic expression of transcription factors in eye-antennal discs of Drosophila strongly interferes with their developmental program. Early ectopic expression in embryonic discs interferes with the developmental pathway primed by Eyeless and generates headless flies, which suggests that Eyeless is necessary for initiating cell proliferation and development of both the eye and antennal disc. Interference occurs through a block in the cell cycle that for some ectopic transcription factors is overcome by D-CycE or D-Myc. Late ectopic expression in cone cell precursors interferes with their differentiation. It is proposed that this developmental pathway interference is a general surveillance mechanism that eliminates most aberrations in the genetic program during development and evolution, and thus seriously restricts the pathways that evolution may take (Jiao, 2001).

The eye-antennal discs of ey-GAL4/+; UAS-Gsb/+ third instar larvae are absent or strongly reduced in size. Evidently, developmental pathway interference induced by the ectopic expression of transcription factors eventually results in the inhibition of cell proliferation and/or apoptosis in these discs. To investigate which of these two processes is responsible for the generation of headless flies, attempts were made to inhibit apoptosis or to stimulate cell proliferation in eye-antennal discs. While inhibition of apoptosis by the expression of the baculovirus P35 protein is unable to suppress the headless phenotype, stimulation of cell proliferation by the expression of D-Myc suppresses it in spontaneously eclosing adults (5%-20%), producing adults of variable eye size, from eyeless adults to adults whose eye size is only slightly reduced. The headless phenotype is rescued even more dramatically by D-CycE, which restores a wild-type phenotype in up to 50% of the adults and only rarely generates small-eyed flies. Rescue of the headless phenotype by CycE is not restricted to ey-GAL4/+; UAS-Gsb/+ flies, but is achieved for all Pax proteins and transcription factors whose potency to interfere with ey function in the eye-antennal disc was tested. However, in contrast to headless flies produced by Gsb, Prd, Poxm, D-Pax2 or Dac, many of which were rescued by CycE to adults that eclosed spontaneously, those generated by Mef2, Sim or Poxn were incompletely rescued. D-Myc is not as efficient in its rescue ability, except in the case of D-Pax2, in which nearly all flies were rescued to wild-type adults. It is concluded that developmental pathway interference through ectopic expression of transcription factors results in the inhibition of cell proliferation that is at least partially overcome by co-expression of D-Myc or D-CycE (Jiao, 2001).

Regulation of Drosophila glial cell proliferation by Merlin-Hippo signaling

Glia perform diverse and essential roles in the nervous system, but the mechanisms that regulate glial cell numbers are not well understood. This study identified and characterize a requirement for the Hippo pathway and its transcriptional co-activator Yorkie in controlling Drosophila glial proliferation. Yorkie was found to be both necessary for normal glial cell numbers and, when activated, sufficient to drive glial over-proliferation. Yorkie activity in glial cells is controlled by a Merlin-Hippo signaling pathway, whereas the upstream Hippo pathway regulators Fat, Expanded, Crumbs and Lethal giant larvae have no detectable role. Functional characterization of Merlin-Hippo signaling was extended by showing that Merlin and Hippo can be physically linked by the Salvador tumor suppressor. Yorkie promotes expression of the microRNA gene bantam in glia, and bantam promotes expression of Myc, which is required for Yorkie and bantam-induced glial proliferation. These results provide new insights into the control of glial growth, and establish glia as a model for Merlin-specific Hippo signaling. Moreover, as several of these genes have been linked to human gliomas, the results suggest that this linkage could reflect their organization into a conserved pathway for the control of glial cell proliferation (Reddy, 2011).

Merlin was first identified as the product of a human tumor suppressor gene, NF2, loss of which in peripheral glial cells results in benign tumors. Merlin has also been identified as an inhibitor of gliomas. The current observations indicate that the role of Merlin as a negative regulator of glial cell proliferation is conserved from humans to Drosophila and, thus, that Drosophila can serve as a model for understanding Merlin-dependent regulation of glial growth (Reddy, 2011).

Studies in Drosophila imaginal discs first linked Merlin to Hippo signaling, and Merlin was subsequently linked to Hippo signaling in mammalian cells, including its role in meningioma. However, the tumor suppressor activity of Merlin has also been linked to other downstream effectors in mammals, including Erb2, Src, ras, rac, TORC1 (CRTC1 -- Human Gene Nomenclature Database; see Drosophila CRTC) and CRL4 (IL17RB -- Human Gene Nomenclature Database), creating some uncertainty regarding the general importance of the linkage of Merlin to Hippo in growth control. This study found that depletion of Merlin, depletion of other tumor suppressors in the Hippo pathway, or expression of an activated form of Yki, all result in similar glial overgrowth phenotypes. Moreover, depletion of Merlin increased nuclear localization of Yki, and depletion of Yki suppressed the overgrowth phenotype of Merlin. Together, these observations clearly establish that the glial overgrowth phenotype associated with Merlin depletion in Drosophila is mediated through the Hippo signaling pathway (Reddy, 2011).

A noteworthy feature of Hippo signaling in Drosophila glial cells is that Merlin appears to be uniquely required as an upstream regulator of Hippo signaling, as the Fat-dependent, Ex-dependent and Lgl-dependent branches have no detectable role. Glia might, thus, provide an ideal model for mechanistic investigations of the Merlin branch of Hippo signaling. Fat-Hippo signaling employs Fat as a transmembrane receptor and Dachsous as its transmembrane ligand, whereas Ex-Hippo signaling appears to employ Crumbs as a transmembrane receptor and ligand. By contrast, Drosophila transmembrane proteins that mediate extracellular signaling and interact with Merlin have not yet been identified. Distinct mechanisms might also be involved in signal transduction downstream of Merlin. Although there is evidence that Ex and Merlin can both influence Hippo activity, Ex, but not Mer, can directly associate with Hpo. Conversely, Merlin, but not Ex, can interact directly with Salvador, and Merlin, Salvador and Hippo can form a trimeric complex. Moreover, the kibra loss-of-function phenotype is weaker than expanded in imaginal discs, but comparable to Merlin, and it was found that depletion of kibra also has a significant effect on glial cell proliferation. Kibra is highly expressed in mammalian brain, and alleles of KIBRA (WWC1 -- Human Gene Nomenclature Database) have been linked to human memory performance. The role of kibra in regulating glial cell numbers in Drosophila thus raise the possibility that the influence of KIBRA on human memory might reflect a role in glial cells (Reddy, 2011).

Finally, it is noted that although Hippo signaling has been investigated in several different organs in Drosophila, including imaginal discs, ovarian follicle cells, neuroepithelial cells and intestinal cells, these all involve roles in epithelial cells, in which upstream regulators of the pathway (e.g. Fat, Ex, Mer) all have a distinctive localization near adherens junctions. The identification of a requirement for Hippo signaling in glia is the first time in Drosophila that a role for the pathway has been identified in non-epithelial cells. Indeed, in previous studies it was found that Hippo signaling influences proliferation of neuroepithelial cells, but other neuronal cell types, including neuroblasts, ganglion mother cells and neurons, are insensitive to Yki (Reddy, 2011).

Considerable attention has been paid to genes for which mutation or inappropriate activation can cause over-proliferation of glial cells, resulting in glial tumors. However, less is known about the mechanisms required for normal glial growth. Through loss-of-function studies, several genes essential for normal glial cell numbers were identified, including yki, sd, ban, mad and myc. The requirement for yki, mad and sd, together with epistasis studies, identifies a requirement for active Yki in glial growth. This in turn implies that downregulation of Hippo signaling is important for normal glial growth. Understanding how this is achieved will provide further insights into the regulation of glial cell numbers (Reddy, 2011).

A requirement for Mad, together with its upstream regulator Thickveins (Tkv), in promoting retinal glial cell proliferation was has been established in previous studies. Current studies of glial cells, together with recent work in imaginal discs, emphasize that in mediating the growth-regulating activity of Hippo signaling, Yki utilizes multiple DNA-binding partners (i.e. Mad and Sd) in the same cells at the same time to regulate distinct downstream target genes required for tissue growth (Reddy, 2011).

Although Yki activity influenced glial cell numbers throughout the nervous system, direct analysis of cell proliferation by EdU labeling revealed that retinal glia were more sensitive to Yki activation at late third instar than central brain glia, and significant induction of central brain glial cell proliferation was only observed when Yki activation was combined with Myc over-expression. Further studies will be required to define the basis for this differential sensitivity, but the implication that the proliferative response to Yki is modulated by developmental stage and/or glial cell type has important implications for diseases associated with both excess and deficits of glial cells (Reddy, 2011).

These studies in Drosophila delineate functional relationships among genes involved in the control of glial cell proliferation. Mammalian homologs of Merlin, Yki and Myc have been implicated in glioma. Although a mammalian homolog of ban has not been described, other miRNAs have also been linked to glioma. These observations imply that these genes can be placed into a pathway, in which Merlin, through Hippo signaling, regulates Yki, Yki regulates ban, and ban regulates Myc. However, as expression of Myc alone did not lead to substantial overgrowth of glia, Yki and ban must also have other downstream targets important for the promotion of glial cell proliferation. Moreover, the current observations indicate that a Yki-Sd complex is also required for glial growth. In addition to the well characterized downstream target Diap1, Yki-Sd complexes in glial cells might regulate Myc directly, as suggested by studies in imaginal discs, and might regulate cell cycle genes in conjunction with E2F1 (Reddy, 2011).

The influence of activated-Yki on a ban-GFP sensor, together with the observations that yki is not required for ban-mediated overgrowth, whereas ban is required for Yki-mediated overgrowth, position ban downstream of Yki. This is consistent with studies of Hippo signaling in imaginal discs, in which ban has also been identified as a target of Yki for growth regulation. The placement of Myc downstream of Yki and ban is supported by the observation that Myc levels can be increased by expression of ban or activated-Yki, and by genetic tests that indicate that Myc is required for Yki- and ban-promoted glial overgrowth. A mechanism by which ban can regulate Myc levels, involving downregulation of a ubiquitin ligase that negatively regulates Myc, was identified recently in imaginal discs, and might also function in glial cells. Myc has been reported to downregulate Yki expression in imaginal discs and, although this study has not investigated whether a similar negative-feedback loop exists in glial cells, the synergistic enhancement of glial cell proliferation observed when Yki and Myc were co-expressed is consistent with this possibility, as the expression of both genes under heterologous promoters could bypass negative regulation of Yki by Myc (Reddy, 2011).

The Myc proto-oncogene is de-regulated or amplified in several human cancers, including gliomas. The sensitivity of Yki/ban-induced overgrowth to reduced Myc levels parallels studies of glioma models involving other signaling pathways. For example, Myc is upregulated by EGFR, and is limiting for EGFR-PI3K-induced glial cell overgrowth in a Drosophila glioma model, and p53 and Pten-driven glioma in mouse models is also Myc dependent. Considering the evidence linking Merlin and Yap to glial growth in mammals, and the identification of Myc as a downstream target of Yap in cultured cells, it is likely that Yap could also influence glial growth in mammals, in part, through regulation of Myc (Reddy, 2011).

The bantam microRNA acts through Numb to exert cell growth control and feedback regulation of Notch in tumor-forming stem cells in the Drosophila brain

Notch (N) signaling is central to the self-renewal of neural stem cells (NSCs) and other tissue stem cells. Its deregulation compromises tissue homeostasis and contributes to tumorigenesis and other diseases. How N regulates stem cell behavior in health and disease is not well understood. This study shows that Notch regulates bantam (ban) microRNA to impact cell growth, a process key to NSC maintenance and particularly relied upon by tumor-forming cancer stem cells. Notch signaling directly regulates ban expression at the transcriptional level, and ban in turn feedback regulates N activity through negative regulation of the Notch inhibitor Numb. This feedback regulatory mechanism helps maintain the robustness of N signaling activity and NSC fate. Moreover, this study shows that a Numb-Myc axis mediates the effects of ban on nucleolar and cellular growth independently or downstream of N. These results highlight intricate transcriptional as well as translational control mechanisms and feedback regulation in the N signaling network, with important implications for NSC biology and cancer biology (Wu, 2017).

By revealing the involvement of the miRNA pathway, this study highlights the complexity of the N signaling network in normal NSCs and tumor-forming cancer stem cell (CSC)-like NSCs. Previous studies implicated critical roles for both canonical and non-canonical N signaling pathways in NSCs and CSC-like NSCs, and revealed particular dependence of CSC-like NB growth on non-canonical N signaling, which involves PINK1, mTORC2, and mitochondrial quality control. The current study reveals a particular requirement for ban in CSC-like NBs induced by N hyperactivation. The CSC-like NB overproliferation induced by hyperactivation of N or N pathway component Dpn can all be assumed to be of type II NB origin, since previous studies have clearly established that Notch signaling is essential for the development and/or maintenance of type II NBs, but dispensable for type I NBs, and that hyperactivation of Notch or its downstream effector Dpn induced ectopic CSC-like NB growth by altering the lineage homeostasis of the type II but not type I NBs. It would be interesting to test whether, in addition to ban's role in canonical N signaling, there exists a link between ban and non-canonical N signaling. The data indicate that the ban-Numb signaling motif regulates NSC/CSC behavior through at least two mechanisms. On one hand, it regulates cell growth and particularly nucleolar growth, through Myc, a known regulator of cellular and nucleolar growth. Consistently, negative regulation of Myc protein level by Numb was observed through E3 ubiquitin-protein ligase, Huwe1, and the UPS. c-Myc is an essential regulator of embryonic stem cell (ESC) self-renewal and cellular reprogramming, and Myc level and stability can be controlled in stem cells through targeted degradation by the UPS, suggesting conserved mechanisms. A key function of the nucleolus is the biogenesis of ribosomes, the cellular machinery for mRNA translation, and previous studies in Drosophila have supported the critical role of nucleolar growth in NSC self-renewal and maintenance. On the other hand, the ban-Numb axis feedback regulates the activity of N by a double negative regulation, with the end result being positive feedback regulation. This feedback mechanism may help transform initial not so dramatic differences in N activity between NB and its daughter cell generated by the asymmetric segregation of Numb during NB division [33] into 'all-or-none' decision of cell fates. Feed-forward regulatory loops, both coherent and incoherent, are frequently found in gene regulatory networks, and although ban miRNA is not conserved in mammals, miRNAs have been implicated in an incoherent feed-forward loop in the Numb/Notch signaling network in colon CSCs in mammals (Wu, 2017).

Given the role of ban in a positive feedback regulation of N and the potency of N hyperactivity in inducing tumorigenesis, one may wonder why ban overexpression is not sufficient to cause tumorigenesis. As in any biological systems, feedback regulation is meant to increase the robustness and maintain homeostasis of a pathway. Feedback alone, either negative or positive, should not override the main effect of the signaling pathway. Thus, in the NB system feedback regulation by ban is built on top of the available N signaling activity in a given cell and serving to maintain N activity. Because of ban's 'fine-tuning' rather than 'on/off switching' of Numb expression, its effect on N activity during feedback regulation will also be 'fine-tuning', serving to maintain N activity in NB within a certain range. Overexpression of ban in a wild type background may not be sufficient to cause tumorigenesis because N activity is not be elevated to the level sufficient to induce brain tumor as in N-v5 overexpression condition. Consistent with this, the extent of Numb inhibition by ban is also modest, not reaching the threshold level of Numb inhibition needed to cause tumorigenesis. Consistent with the notion that feedback regulation by ban is built on top of the available N signaling activity in a given cell, and that there is dosage effect of N activity in tumorigenesis, overexpression of ban in N-v5 overexpression background further enhanced N-v5 induced tumorigenesis. It is likely that ban or other miRNAs may participate in additional regulatory mechanisms in the N signaling network in Drosophila. Of particular interest, it would be interesting to test whether miRNAs may impinge on the asymmetric cell division machinery to influence the symmetric vs. asymmetric division pattern, a key mechanism employed by NSCs and transit-amplifying IPs to balance self-renewal with differentiation (Wu, 2017).

The results emphasize the critical role of translational control mechanisms in NSCs and CSC-like NSCs. Compared to the heavily studied transcriptional control, knowledge of the translational control of NSCs and CSCs is rather limited. As fundamental regulators of mRNA translation, miRNAs can interact with both positive and negative regulators of translation to influence gene expression. Thus, miRNA activity can be regulated context-dependently at both the transcriptional and translational levels, which may account for the opposite effect of N on ban activity in the fly brain and wing disc, although the ban genomic locus is bound by Su(H) in both tissues. Whether N regulates the transcription of ban or its activity as a translational repressor in the wing disc remains to be tested. With regard to the translation of numb mRNA, the conserved RNA-binding protein (RNA-BP) Musashi has been shown to critically regulate the level of Numb protein in mammalian hematopoietic SCs and leukemia SCs. Further investigation into the potential interplay between miRNAs and RNA-BPs in the translational control of Numb in NBs and CSC-like NBs promises to reveal new mechanisms and logic in stem cell homeostasis regulation, with important implications for stem cell biology and cancer biology (Wu, 2017).

Oogenesis

dmyc is expressed in a dynamic pattern in ovaries. At early stages, high levels of DMYC transcripts accumulate in the germarium with lower levels in either stage 1 or stage 2 egg chambers. By stage 3, DMYC mRNA can be detected in all cell types of the chamber: the nurse cells, oocyte, and follicle cells. This expression pattern is maintained throughout oogenesis. dmax expression can still be detected in diminutive (see below) mutant ovaries (Gallant, 1996).

The Notch signaling pathway controls the follicle cell mitotic-to-endocycle transition in Drosophila oogenesis by stopping the mitotic cycle and promoting the endocycle. To understand how the Notch pathway coordinates this process, a functional analysis was performed of genes whose transcription is responsive to the Notch pathway at this transition. These genes include String, the G2/M regulator Cdc25 phosphatase; Hec/CdhFzr, a regulator of the APC ubiquitination complex and Dacapo, an inhibitor of the CyclinE/CDK complex. Notch activity leads to downregulation of String and Dacapo, and activation of Fzr. All three genes are independently responsive to Notch. In addition, CdhFzr, an essential gene for endocycles, is sufficient to stop mitotic cycle and promote precocious endocycles when expressed prematurely during mitotic stages. In contrast, overexpression of the growth controller Myc does not induce premature endocycles but accelerates the kinetics of normal endocycles. F-box/WD40-domain protein Ago/hCdc4 (Archipelago), a SCF-regulator is dispensable for mitosis, but crucial for endocycle progression in follicle epithelium. CycE oscillation remains critical for endocycling; continuous high level of CycE expression blocks the cell cycle in G2. The regulation of CycE levels is achieved by the function of Ago that presumably binds to auto-phosphorylated CycE and directs it to SCF-complex degradation: high levels of CycE and no endocycling is observed in ago-clones. The results support a model in which Notch activity executes the mitotic-to-endocycle switch by regulating all three major cell cycle transitions. Repression of String blocks the M-phase, activation of Fzr allows G1 progression, and repression of Dacapo assures entry into the S-phase. This study provides a comprehensive picture of the logic that external signaling pathways may use to control cell cycle transitions by the coordinated regulation of the cell cycle (Shcherbata, 2004).

The data suggest that a component regulating growth and thereby the kinetics of G1/S transition in follicle cell endocycles is the Myc oncogene instead and independent of CycD. In mammals c-Myc controls the decision to divide or not to divide and thereby functions as a crucial mediator of signals that determine organ and body size. Interestingly, overexpression of Myc in follicle cells does not affect the mitotic cycles but induces, instead, extra endocycles. Because the timing for entering and exit from the endocycles has not changed, however, increased ploidy is observed; therefore, it is suggested that the rate of endocycles is increased because of the overexpression of Myc. This finding is in accordance with recent loss-of-function analysis on myc in follicle cells, suggesting that myc mutant follicle cells can make the transition from mitosis to the endocycle, but that they can only very inefficiently support the endocycle. Therefore, both loss-of-function and overexpression experiments suggest that Myc is an essential component for the proper rate of endocycles in follicle cells (Shcherbata, 2004).

Integration of Insulin receptor/Foxo signaling and dMyc activity during muscle growth regulates body size in Drosophila

Drosophila larval skeletal muscles are single, multinucleated cells of different sizes that undergo tremendous growth within a few days. The mechanisms underlying this growth in concert with overall body growth are unknown. The size of individual muscles correlates with the number of nuclei per muscle cell and with increasing nuclear ploidy during development. Inhibition of Insulin receptor (InR; Insulin-like receptor) signaling in muscles autonomously reduces muscle size and systemically affects the size of other tissues, organs and indeed the entire body, most likely by regulating feeding behavior. In muscles, InR/Tor signaling, Foxo and dMyc (Diminutive) are key regulators of endoreplication, which is necessary but not sufficient to induce growth. Mechanistically, InR/Foxo signaling controls cell cycle progression by modulating dmyc expression and dMyc transcriptional activity. Thus, maximal dMyc transcriptional activity depends on InR to control muscle mass, which in turn induces a systemic behavioral response to allocate body size and proportions (Demontis, 2009).

Therefore, interplay between InR/Tor signaling, Foxo and dMyc activity regulates muscle growth that occurs during Drosophila larval development, in part via the induction of endoreplication. Interestingly, the extent of muscle growth is sensed systemically, regulates feeding behavior and, in turn, influences the size of other tissues and indeed the whole body. Thus, the growth of a single tissue is sensed systemically via modulating a whole-organism behavior (Demontis, 2009).

dMyc, as well as activation of InR signaling, can promote endoreplication in muscles, whereas Foxo and inhibitors of dMyc and of InR/Tor have the opposite effect. dMyc is likely to regulate the expression of genes required for multiple G-S and S-G transitions during endoreplication, similar to vertebrate Myc, which regulates key cell-cycle regulators including cyclin D2, cyclin E, and the cyclin kinase inhibitors p21 and p27 (Cdkn1a and Cdkn1b, respectively). Indeed, aberrant levels of Cyclin E block muscle growth, indicating that proper muscle growth requires tight control of the expression and activity of endoreplication genes. Further, endoreplication is also modulated by Foxo, which is activated in conditions of nutrient starvation, impaired InR/Tor signaling and by other cell stressors. Foxo presumably regulates cell cycle progression at least in part by modulating the expression of evolutionarily conserved Foxo/Myc-target genes, such as dacapo (the Drosophila p21/p27 homolog) and Cyclin E, that regulate the G1-S transition. Interestingly, Foxo and Myc might control different steps in the activation of common target genes (Demontis, 2009).

In addition, it was found that active Foxo can also inhibit dMyc protein activity and regulates dmyc gene expression. Mechanistically, Foxo could influence dMyc activity in several ways. First, it might physically interact with dMyc, although no evidence was found to support this notion. Second, Foxo could regulate the expression of genes that target dMyc for proteasomal degradation, including several ubiquitin E3 ligases that are induced by Foxo during muscle atrophy in mice and humans. However, by analyzing dMyc protein levels by western blot, no significant dMyc protein instability was found upon Foxo overexpression. Third, Foxo might promote the expression of transcriptional regulators that oppose dMyc function, including Mad/Mnt, although no substantial increase in dmnt mRNA levels was detected upon Foxo activation in muscles. Possibly, the expression of other dMyc regulators might be affected by Foxo. Future experiments will be needed to dissect the Foxo-dMyc interaction (Demontis, 2009).

Finally, by manipulating muscle growth and/or endoreplication, it was found that in muscles the ratio of cell size to nuclear size is not constant, and increased nuclear size and DNA content, indicative of ploidy, is necessary but not sufficient to drive growth. Usually, an increase in cell size is matched by an increase in nuclear size, which commonly parallels increases in nuclear ploidy. However, the current findings indicate that in muscles, dMyc-driven variation in nuclear size and ploidy is permissive but not sufficient for substantial growth, even in the presence of increased biogenesis of nucleoli and expression of genes involved in protein translation. This is different from fat body cells, in which dmyc overexpression induces endoreplication and proportional cell growth. Thus, additional instructive signals, possibly modulating protein synthesis, mitochondriogenesis, ribosome biogenesis, sarcomere assembly, and other anabolic responses must be concomitantly received to promote maximal muscle growth. Therefore, increases in cell size and nuclear ploidy are surprisingly uncoupled during muscle growth (Demontis, 2009).

Little is known about the mechanisms that control and coordinate cell, organ and body size, and in particular how muscle growth is matched with the growth of other tissues and of the entire organism. Inhibition of InR/Tor signaling and dMyc activity in muscles impairs, in addition to muscle mass, the size of the entire body and of other internal organs. Similarly, overexpression of Cyclin E in muscles also results in autonomous and systemic growth defects, indicating that, at least in some cases, modulation of muscle growth by means independent from InR signaling can be sensed systemically. In the larva, endoreplicating tissues and organs (gut, salivary glands, epidermis, fat body) are severely affected, whereas non-endoreplicating tissues (brain and imaginal discs) are less affected, indicating distinct tissue responsiveness to this regulation. Similarly, inhibition of Tor signaling in the fat body also primarily affects the size of endoreplicating tissues (Demontis, 2009).

Non-autonomous regulation of tissue size may rely on humoral factors (e.g. hormone-binding proteins, hormones, metabolites) produced by muscles in response to achieving a certain mass. However, alternative models are possible. In particular, decreased and increased larval feeding, respectively, were observed upon inhibition and activation of InR signaling in muscles. This whole-organism behavioral adaptation is possibly due to decreased and increased efficiency of smaller and bigger muscles, respectively, and to regulated expression of neuropeptides that hormonally control feeding behavior. As a consequence of the regulation of feeding behavior, nutrient uptake is decreased and larval growth is blocked in the cells of endoreplicating tissues, which are extremely sensitive to poor nutritional conditions, and to a lesser extent in non-endoreplicating tissues, which are more resistant to limited nutritional supply. In turn, increased or decreased size of non-muscle tissues arise as a consequence of abnormal feeding. Thus, muscle size coordinates with the size of other organs and of the entire body, at least in part via a systemic, behavioral response. Distinct tissues are differently sensitive to this regulation, resulting in altered body proportions (Demontis, 2009).

Understanding the mechanisms regulating muscle mass is of special interest because they underline the etiology of several human diseases. Directly relevant to these studies, both MYC and InR (INSR) signaling have been found to regulate muscle growth and maintenance in humans. Further, muscle atrophy is triggered by FOXO activation in several pathological conditions. In addition, MYC function has been implicated in heart hypertrophy, a process that is conversely regulated by FOXO (Demontis, 2009).

The findings that Foxo functionally antagonizes dMyc during the growth of Drosophila muscles suggest that these factors might also interact similarly in humans. Consistent with this hypothesis, FOXO and MYC regulate, in opposite fashions, the atrophic and hypertrophic programs in human skeletal muscles and cardiomyocytes, and display complementary gene expression and activity in these contexts (Demontis, 2009).

Finally, the finding that during larval development, inhibition of InR signaling in muscles has profound systemic effects might also reflect physiological conditions found in humans. Indeed, defective responsiveness of muscles to Insulin during type II diabetes has autonomous effects on muscle maintenance that are associated with systemic effects on the metabolism of the entire organism, contributing to the improper control of glycemia and the development of metabolic syndrome. This study has identified feeding behavior as part of the systemic response that in Drosophila senses perturbations in muscle mass. These findings might help further elucidate the signals involved in metabolic and growth homeostasis, which may be conserved across evolution (Demontis, 2009).


EVOLUTIONARY HOMOLOGS

Identification of proteins in the Myc-Max-Mad network

Accurate identification of specific groups of proteins by their amino acid sequence is an important goal in genome research. This study combines information theory with fuzzy logic search procedures to identify sequence signatures or predictive motifs for members of the Myc-Max-Mad (see Drosophila Mnt for information about Mad family members) transcription factor network. Myc is a well known oncoprotein, and this family is involved in cell proliferation, apoptosis, and differentiation. A small set of amino acid sites from the N-terminal portion of the basic helix-loop-helix (bHLH) domain is described that provide very accurate sequence signatures for the Myc-Max-Mad transcription factor network and three of its member proteins. A predictive motif involving 28 contiguous bHLH sequence elements found 337 network proteins in the GenBank NR database with no mismatches or misidentifications. This motif also identifies at least one previously unknown fungal protein with strong affinity to the Myc-Max-Mad network. Another motif found 96% of known Myc protein sequences with only a single mismatch, including sequences from genomes previously not thought to contain Myc proteins. The predictive motif for Myc is very similar to the ancestral sequence for the Myc group estimated from phylogenetic analyses. Based on available crystal structure studies, this motif is discussed in terms of its functional consequences. The results provide insight into evolutionary diversification of DNA binding and dimerization in a well characterized family of regulatory proteins and provide a method of identifying signature motifs in protein families (Atchley, 2005).

Interactions between Myc, Max and Mad

c-Myc (Myc) and Max proteins dimerize and bind DNA through basic-helix-loop-helix-leucine zipper motifs (b-HLH-LZ). Binding to Max is essential for Myc transforming activity since Myc homodimers are inactive. Mutants of Myc and Max that bind efficiently to each other but not to their wild-type partners were generated by either exchanging the HLH-leucine zipper domains or reciprocally modifying LZ dimerization specificities. Complementary mutants are sufficient to cause transformation defects on their own, but they restore Myc transforming activity when coexpressed in cells. The HLH-LZ exchange mutants also produce the effect of dominant negative activity on wild-type Myc function. In addition, wild-type max antagonizes myc function in a dose-dependent manner, presumably through competition of Max-Max and Myc-Max dimers for common target DNA sites. Therefore, Max can function as both suppressor and activator of Myc. Amati (1993) discusses a general model for the role of Myc and Max in growth control.

Given that Myc family proteins appear to function through heterodimerization with the stable, constitutively expressed bHLH-Zip protein, Max a lambda gt11 expression library was screened with radiolabeled Max proteinto determine whether Max mediates the function of regulatory proteins other than Myc. One cDNA identified encodes a new member of the bHLH-Zip protein family, Mad. Human Mad protein homodimerizes poorly but binds Max in vitro, forming a sequence-specific DNA binding complex with properties very similar to those of Myc-Max. Both Myc-Max and Mad-Max heterocomplexes are favored over Max homodimers, and, unlike Max homodimers, the DNA binding activity of the heterodimers is unaffected by CKII phosphorylation. Mad does not associate with Myc or with representative bHLH, bZip, or bHLH-Zip proteins. In vivo transactivation assays suggest that Myc-Max and Mad-Max complexes carry out opposing functions in transcription and that Max plays a central role in this network of transcription factors (Ayer, 1993a).

Mad is a basic-helix-loop-helix-zipper protein that heterodimerizes with Max in vitro. Mad-Max heterodimers recognize the same E-box-related DNA-binding sites as Myc-Max heterodimers. However, in transient transfection assays Myc and Mad influence transcription, albeit in opposite ways, through interaction with Max; Myc activates while Mad represses transcription. Mad protein is induced rapidly upon differentiation of cells of the myeloid lineage. The Mad protein is synthesized in human cells as a 35-kD nuclear phosphoprotein with an extremely short half-life (t1/2 = 15-30 min). It can be detected in vivo in a complex with Max. In an undifferentiated monocyte cell line Max is found complexed with Myc but not Mad. However, Mad-Max complexes begin to accumulate as early as 2 hr after induction of macrophage differentiation with TPA. By 48 hr following TPA treatment only Mad-Max complexes are detectable. Differentiation appears to be accompanied by a change in the composition of Max heterocomplexes. This switch in heterocomplexes results in a change in the transcriptional regulation of Myc-Max target genes required for cell proliferation (Ayer, 1993b).

Mxi1 belongs to the Mad (Mxi1) family of proteins, which function as potent antagonists of Myc oncoproteins. This antagonism relates partly to the ability of these proteins to compete with Myc for the protein Max and for consensus DNA binding sites and to recruit transcriptional co-repressors. Mad(Mxi1) proteins have been suggested to be essential in cellular growth control and/or in the induction and maintenance of the differentiated state. Consistent with these roles, mxi1 may be the tumour-suppressor gene that resides at region 24-26 of the long arm of chromosome 10. This region is a cancer hotspot, and mutations here may be involved in several cancers, including prostate adenocarcinoma. Mice lacking Mxi1 exhibit progressive, multisystem abnormalities. These mice also show increased susceptibility to tumorigenesis either following carcinogen treatment or when also deficient in Ink4a. This cancer-prone phenotype may correlate with the enhanced ability of several mxi1-deficient cell types, including prostatic epithelium, to proliferate. These results show that Mxi1 is involved in the homeostasis of differentiated organ systems; it acts as a tumour suppressor in vivo, and engages the Myc network in a functionally relevant manner (Schreiber-Agus, 1998).

Max on its own represses transcription, whereas a significant stimulation is obtained when Max is coexpressed with c-Myc. Analysis of specific mutants indicates that transcriptional activation requires both the c-Myc and the Max dimerization and DNA-binding domains, as well as the c-Myc transactivation function; transcriptional repression by Max requires both DNA binding and dimerization. Analogously, in stably transfected human B-lymphoblastoid cell lines, overexpressed c-Myc and Max synergize to cause malignant transformation, whereas overexpression of Max alone leads to growth inhibition. These results indicate that c-Myc and Max are both transcriptional regulators, but their regulation of target-gene expression and cell proliferation takes place in opposite directions, most likely as the result of the opposite effects of heterodimeric c-Myc-Max (positive) versus homodimeric Max (negative) complexes (Gu, 1993).

A method is described to design dominant-negative proteins (D-N) to the basic helix-loop-helix-leucine zipper (B-HLHZip) family of sequence-specific DNA binding transcription factors. The D-Ns specifically heterodimerize with the B-HLHZip dimerization domain of the transcription factors and abolish DNA binding in an equimolar competition. Thermal denaturation studies indicate that a heterodimer between a Myc B-HLHZip domain and a D-N consisting of a 12-amino acid sequence appended onto the Max dimerization domain (A-Max) is more stable than the Myc:Max heterodimer. One molar equivalent of A-Max can totally abolish the DNA binding activity of a Myc:Max heterodimer. This acidic extension also has been appended onto the dimerization domain of the B-HLHZip protein Mitf, a member of the transcription factor enhancer binding subfamily, to produce A-Mitf. The heterodimer between A-Mitf and the B-HLHZip domain of Mitf is more stable than the Mitf homodimer. Cell culture studies show that A-Mitf can inhibit Mitf-dependent transactivation both in acidic extension and in a dimerization-dependent manner. A-Max can inhibit Myc-dependent foci formation twice as well as the Max dimerization domain (HLHZip). This strategy of producing D-Ns may be applicable to other B-HLHZip or B-HLH proteins because it provides a method to inhibit the DNA binding of these transcription factors in a dimerization-specific manner (Krylov, 1997).

A recently identified mammalian histone deacetylase (HD1) shows homology to the yeast Rpd3 (see Drosophila Rpd3) protein, which together with Sin3 affects the transcription of several genes. Mammalian Sin3 proteins interact with the Mad components of the Myc/Max/Mad network of cell growth regulators. Mad/Max complexes may recruit mammalian Rpd3-like enzymes, thus directing histone deacetylase activity to promoters and negatively regulating cell growth. A tetrameric complex composed of Max, Mad1, Sin3B and HD1 is reported. This complex has histone deacetylase activity that can be blocked by the histone deacetylase inhibitors trichostatin A and sodium butyrate. The inhibition of cell growth by Mad1 is enhanced by Sin3B and HD1, as measured by colony formation assays. Furthermore, a Mad1-induced block of S-phase progression can be overcome by trichostatin A, as shown in microinjection experiments. It is concluded that the recruitment of a histone deacetylase by sequence-specific DNA-binding proteins provides a mechanism by which the state of acetylation of histones in nucleosomes and hence the activity of specific promoters can be influenced. The finding that Mad/Max complexes interact with Sin3 and HD1 in vivo suggests a model for the role of Mad proteins in antagonizing the function of Myc proteins (Sommer, 1997).

X-ray structures of the basic/helix-loop-helix/leucine zipper (bHLHZ) domains of Myc-Max and Mad-Max heterodimers bound to their common DNA target (Enhancer or E box hexanucleotide, 5'-CACGTG-3') have been determined at 1.9 Å and 2.0 Å resolution, respectively. E box recognition by these two structurally similar transcription factor pairs determines whether a cell will divide and proliferate (Myc-Max) or differentiate and become quiescent (Mad-Max). Deregulation of Myc has been implicated in the development of many human cancers, including Burkitt's lymphoma, neuroblastomas, and small cell lung cancers. Both quasi-symmetric heterodimers resemble the symmetric Max homodimer, albeit with marked structural differences in the coiled-coil leucine zipper regions that explain preferential homo- and hetero-meric dimerization of these three evolutionarily related DNA-binding proteins. The Myc-Max heterodimer, but not its Mad-Max counterpart, dimerizes to form a bivalent heterotetramer, which explains how Myc can upregulate expression of genes with promoters bearing widely separated E boxes (Nair, 2003).

Genetic characterization of the promoters of putative myc regulated genes has provided further evidence for a physiological role for Myc-Max heterotetramerization. Oligonucleotide microarray analysis has identified several Myc target genes that contain multiple E boxes within promoters, typically separated by at least 100 nucleotides. Given the persistence length of DNA, this separation of Myc-Max binding sites is compatible with DNA looping stabilized by bivalent Myc-Max heterotetramers bound to two cognate sequences. Moreover, in vitro binding studies of Myc-Max heterodimers recognizing the lactate dehydrogenase gene, a natural high-affinity target containing two consensus E boxes, demonstrates that Myc-Max complexes can engage multiple cognate sites. Functional studies with adjacent E boxes found within the lactate dehydrogenase promoter have shown that mutations in either of the two E boxes severely affect Myc-dependent activation of transcription. These results suggest that Myc-Max complexes can form higher order structures that are consistent with the heterotetrameric assembly observed in the cocrystal structure (Nair, 2003).

Myc and Mad family proteins play opposing roles in the control of cell growth and proliferation. The subcellular locations of complexes formed by Myc/Max/Mad family proteins were visualized using bimolecular fluorescence complementation (BiFC) analysis. Max is recruited to different subnuclear locations by interactions with Myc versus Mad family members. Complexes formed by Max with Mxi1, Mad3, or Mad4 are enriched in nuclear foci, whereas complexes formed with Myc are more uniformly distributed in the nucleoplasm. Mad4 is localized to the cytoplasm when it is expressed separately, and Mad4 is recruited to the nucleus through dimerization with Max. The cytoplasmic localization of Mad4 is determined by a CRM1-dependent nuclear export signal located near the amino terminus. The relative efficiencies of complex formation among Myc, Max, and Mad family proteins in living cells were compared using multicolor BiFC analysis. Max forms heterodimers with the basic helix-loop-helix leucine zipper (bHLHZIP) domain of Myc (bMyc) more efficiently than it forms homodimers. Replacement of two amino acid residues in the leucine zipper of Max reverses the relative efficiencies of homo- and hetero-dimerization in cells. Surprisingly, Mad3 forms complexes with Max less efficiently than bMyc, whereas Mad4 forms complexes with Max more efficiently than bMyc. The distinct subcellular locations and the differences between the efficiencies of dimerization with Max indicate that Mad3 and Mad4 are likely to modulate transcription activation by Myc at least in part through distinct mechanisms (Grinberg, 2004).

Myc, Max and cell cycle activation

Transcription of the human proto-oncogene Myc is repressed in quiescent or non-dividing cells. Upon mitogenic stimulation expression of Myc is rapidly and transiently induced, maintained throughout G1, and then declines to a basal level throughout further cell cycle transitions. Regulation of myc promoter activity critically depends on the presence of a binding site for transcription factor E2F (Drosophila homolog: E2F). Transcription from the myc P2 promoter is induced efficiently by E2F-1, but repressed by RB. Furthermore, overexpression of cyclin A strongly activates the myc promoter and this effect is further enhanced by coexpression of E2F-1 and cyclin A. Expression of G1-phase cyclin D1 leads to an E2F binding site-dependent trans-activation of the myc promoter and this activation can be abrogated by overexpression of RB. The interaction of D-type G1 cyclins with RB resembles that of the adenovirus E1A protein with RB: both can disrupt inhibitory E2F-RB complexes. These results support a model in which intervention of distinct cyclins and their respective associated kinases promote transcriptional activation of myc throughout the cell cycle either by conversion of E2F within multimeric complexes into an active transcription factor or by liberation of free functional E2F (Oswald, 1994).

Genomic sequences were isolated flanking the 5' region of the E2F2 coding sequence. Various assays demonstrate promoter activity in this sequence that reproduces the normal control of E2F2 expression during a growth stimulation. Sequence comparison reveals the presence of a variety of known transcription factor binding sites, including E-box elements (that are consensus Myc binding sites), as well as E2F binding sites. The E-box elements, which can function as Myc-responsive sites, contribute in a positive fashion to promoter function. E2F-dependent negative regulation in quiescent cells plays a significant role in the cell growth-dependent control of the promoter, similar to the regulation of the E2F1 gene promoter (Sears, 1997).

Activation of the human cyclin E-cdk2 (See Drosophila homolog Cyclin E) heterodimer in quiescent cells involves a Myc-dependent step, but involves no significant change in the amount of cyclin-cdk complex. Such activation involves the release of a 120 kDa cyclin E-cdk2 complex from a 250 kDa complex, present in serum starved cells. The 250 kDa complex involves an association of cyclin-cdk with inhibitory molecule p27. Release of p27 involves either a change of affinity for p27 or p27 degradation. An additional step in activation of cyclin-dependent kinases by c-myc is dephosphorylation of cdk2 carried out by cdc25A (Drosophila homolog: string), a transcriptional target of Myc/Max heterodimers. Induction of cdc25A by Myc is direct, as it is not inhibited by protein synthesis inhibitor cycloheximide. In the absence of adequate levels of growth factors, Cdc25A and myc share the ability to induce p53-dependent apoptosis. Myc-driven apoptosis is inhibited by cdc25A antisense oligonucleotides, suggesting that cdc25A expression might be essential for Myc-dependent apoptosis in some cells. The precise mechanism of cdc25A induction of apoptosis remains unclear (Steiner, 1995 and Galaktionov, 1996).

Ectopic expression of Mad1 inhibits the proliferative response of 3T3 cells to signaling through the colony-stimulating factor-1 (CSF-1) receptor. Mad1 also inhibits the ability of over-expressed Myc and cyclin D1 to complement the mutant CSF-1 receptor Y809F (containing a Y-to-F mutation at position 809). Cell cycle analysis of proliferating 3T3 cells transfected with Mad1 demonstrates a significant decrease in the fraction of cells in the S and G2/M phases and a concomitant increase in the fraction of G1 phase cells, indicating that Mad1 negatively influences cell cycle progression from the G1 to the S phase. Mutations in Mad1 that inhibit its activity as a transcription repressor also result in loss of Mad1 cell cycle inhibitory activity. Thus, the ability of Mad1 to inhibit cell cycle progression is tightly coupled to its function as a transcriptional repressor (Roussel, 1996).

Considerable evidence points to a role for G1 cyclin-dependent kinase (CDK) in allowing the accumulation of E2F transcription factor activity and induction of the S phase of the cell cycle. Numerous experiments have also demonstrated a critical role for both Myc and Ras activities in allowing cell-cycle progression. Inhibition of Ras activity blocks the normal growth-dependent activation of G1 CDK, prevents activation of the target genes of E2F, and results in cell-cycle arrest in G1. Ras is essential for entry into the S phase in Rb+/+ fibroblasts but not in Rb-/- fibroblasts, establishing a link between Ras and the G1 CDK/Rb/E2F pathway. However, although expression of Ras alone will not induce G1 CDK activity or S phase, coexpression of Ras with Myc allows the generation of cyclin E-dependent kinase activity and the induction of S phase, coincident with the loss of the p27 cyclin-dependent kinase inhibitor (CKI). These results suggest that Ras, along with the activation of additional pathways, is required for the generation of G1 CDK activity, and that activation of cyclin E-dependent kinase in particular depends on the cooperative action of Ras and Myc (Leone, 1997).

Retroviral expression of the cyclin-dependent kinase (CDK) inhibitor p16(INK4a) in rodent fibroblasts induces dephosphorylation of pRb, p107 and p130 and leads to G1 arrest. Prior expression of cyclin E allows S-phase entry and long-term proliferation in the presence of p16. Cyclin E prevents neither the dephosphorylation of pRb family proteins, nor their association with E2F proteins in response to p16. Thus, cyclin E can bypass the p16/pRb growth-inhibitory pathway downstream of pRb activation. Retroviruses expressing E2F-1, -2 or -3 also prevent p16-induced growth arrest but are ineffective against the cyclin E-CDK2 inhibitor p27(Kip1), suggesting that E2F cannot substitute for cyclin E activity. Thus, cyclin E possesses an E2F-independent function required to enter S-phase. However, cyclin E may not simply bypass E2F function in the presence of p16, since it restores expression of E2F-regulated genes such as cyclin A or CDC2. Finally, c-Myc bypasses the p16/pRb pathway with effects indistinguishable from those of cyclin E. It is suggested that this effect of Myc is mediated by its action upstream of cyclin E-CDK2, and occurs via the neutralization of p27(Kip1) family proteins, rather than induction of Cdc25A. These data imply that oncogenic activation of c-Myc, and possibly also of cyclin E, mimics loss of the p16/pRb pathway during oncogenesis (Alevizopoulos, 1997).

Proto-oncogenes like c-myc are thought to control exit from the cell cycle rather than progression through the cell cycle itself. A different view of Myc function is presented in the current study. Exponentially growing Rat1-MycER fibroblasts were size-fractionated by centrifugal elutriation. In these cells, activation of cyclin E- and cyclin A-dependent kinases, degradation of p27, hyperphosphorylation of retinoblastoma protein and activation of E2F occur sequentially at specific cell sizes. However, upon activation of Myc, these transitions all occur simultaneously in small cells immediately after exit from mitosis. Interestingly, Myc has no discernible effect on the cell size at which DNA replication is initiated. Even in the presence of Myc activated cell cycle proteins, cells must grow to normal size before they initiate DNA replication and replication-dependent transcription of the histone H4 gene. These data show first that Myc controls the activity of G1 cyclin-dependent kinases independent of the transition between quiescence and proliferation or from any size effect on cell growth. These data also provide evidence of at least one dominant mechanism in addition to activation of E2F and of cyclin E/cdk2 kinase, which prevents DNA replication unless a critical cell size has been reached (Pusch, 1997).

Estrogen-induced progression through G1 phase of the cell cycle is preceded by increased expression of the G1-phase regulatory proteins c-Myc and cyclin D1. To investigate the potential contribution of these proteins to estrogen action, clonal MCF-7 breast cancer cell lines were derived in which c-Myc or cyclin D1 is expressed under the control of the metal-inducible metallothionein promoter. Inducible expression of either c-Myc or cyclin D1 is sufficient for S-phase entry in cells previously arrested in G1 phase by pretreatment with ICI 182780, a potent estrogen antagonist. c-Myc expression is not accompanied by increased cyclin D1 expression or Cdk4 activation, nor is cyclin D1 induction accompanied by increases in c-Myc. Expression of c-Myc or cyclin D1 is sufficient to activate cyclin E-Cdk2 by promoting the formation of high-molecular-weight complexes lacking the cyclin-dependent kinase inhibitor p21 following estrogen treatment. Interestingly, this is accompanied by an association between active cyclin E-Cdk2 complexes and hyperphosphorylated p130 (a pRB-related pocket protein), identifying a previously undefined role for p130 in estrogen action. These data provide evidence for distinct c-Myc and cyclin D1 pathways in estrogen-induced mitogenesis, which converge on or prior to the formation of active cyclin E-Cdk2-p130 complexes and loss of inactive cyclin E-Cdk2-p21 complexes, indicating a physiologically relevant role for the cyclin E binding motifs shared by p130 and p21 (Prall, 1998).

Cyclin E-Cdk2 kinase activation is an essential step in Myc-induced proliferation. It is presumed that this requires sequestration of G1 cell cycle inhibitors p27Kip1 and p21Cip1 (Ckis) via a Myc-induced protein. Biochemical and genetic evidence is provided to show that this sequestration is mediated via induction of cyclin D1 and/or cyclin D2 protein synthesis rates. Consistent with this conclusion, primary cells from cyclin D1-/- and cyclin D2-/- mouse embryos, unlike wild-type controls, do not respond to Myc with increased proliferation, although they undergo accelerated cell death in the absence of serum. Myc sensitivity of cyclin D1-/- cells can be restored by retroviruses expressing either cyclins D1, D2 or a cyclin D1 mutant forming kinase-defective, Cki-binding cyclin-cdk complexes. At the same time Myc also induces cyclin E protein synthesis rate helping to promote a net gain of newly formed Cki-free cyclin E-Cdk2 complexes. These become active concomitantly with phosphorylation of the kinase subunit by cyclin activating kinase (CAK). The sequestration function of D cyclins thus appears essential for Myc-induced cell cycle progression but dispensable for apoptosis (Perez-Roger, 1999).

The c-Myc oncoprotein is strongly induced during the G0 to S-phase transition and is an important regulator of cell cycle entry. In contrast to c-Myc, the putative Myc antagonist Mnt is maintained at a constant level during cell cycle entry. Mnt and Myc require interaction with Max for specific DNA binding at E-box sites, but have opposing transcriptional activities. c-Myc induction during cell cycle entry leads to a transient decrease in Mnt-Max complexes and a transient switch in the ratio of Mnt-Max to c-Myc-Max on shared target genes. Mnt overexpression suppresses cell cycle entry and cell proliferation, suggesting that the ratio of Mnt-Max to c-Myc-Max is critical for cell cycle entry. Furthermore, simultaneous Cre-Lox mediated deletion of Mnt and c-Myc in mouse embryo fibroblasts rescues the cell cycle entry and proliferative block caused by c-Myc ablation alone. These results demonstrate that Mnt-Myc antagonism plays a fundamental role in regulating cell cycle entry and proliferation (Walker, 2005).

Myc interaction with Miz-1 and Myc destruction

The human proto-oncogene c-myc encodes a highly unstable transcription factor that promotes cell proliferation. Although the extreme instability of Myc plays an important role in preventing its accumulation in normal cells, little is known about how Myc is targeted for rapid destruction. An investigation has been carried out of mechanisms regulating the stability of Myc. Myc is destroyed by ubiquitin-mediated proteolysis; two elements are defined in Myc that oppositely regulate its stability: a transcriptional activation domain that promotes Myc destruction, and a region required for association with the POZ domain protein Miz-1, that stabilizes Myc. Myc is stabilized by cancer-associated and transforming mutations within its transcriptional activation domain. What is the significance of Myc stabilization by Miz-1? Miz-1 is a zinc finger/POZ domain protein that, alone, has been shown to bind to and transactivate the cyclin D1 promoter. When Miz-1 is complexed with Myc, however, a latent Miz-1 repressor function is revealed, and cyclin D1 promoter activity is attenuated. Because of this behavior, Miz-1 has been proposed as the partner protein through which Myc represses transcription of cyclin D1 and other genes in vivo. It is hypothesized that stabilization of Myc by Miz-1 relates to the function of Miz-1-Myc complexes as transcriptional repressors. Perhaps, therefore, enhanced protein stability is a common feature of transcriptional repressors, permitting the formation of recalcitrant complexes that stably attenuate gene transcription. In conclusion data reveals a complex network of interactions regulating Myc destruction, and imply that enhanced protein stability contributes to oncogenic transformation by mutant Myc proteins (Salghetti, 1999).

Deregulated expression of c-myc can induce cell proliferation in established cell lines and in primary mouse embryonic fibroblasts (MEFs), through a combination of both transcriptional activation and repression by Myc. Miz-1, a Myc-associated zinc finger transcription factor, arrests cells in G1 phase and inhibits cyclin D-associated kinase activity. Miz-1 upregulates expression of the cyclin-dependent kinases (CDK) inhibitor p15INK4b by binding to the initiator element of the p15INK4b promoter. Myc and Max form a complex with Miz-1 at the p15 initiator and inhibit transcriptional activation by Miz-1. Expression of Myc in primary cells inhibits the accumulation of p15INK4b that is associated with cellular senescence; conversely, deletion of c-myc in an established cell line activates p15INK4b expression. Alleles of c-myc that are unable to bind to Miz-1 fail to inhibit accumulation of p15INK4b messenger RNA in primary cells and are, as a consequence, deficient in immortalization (Staller, 2001).

The Myc oncoprotein represses initiator-dependent transcription through the POZ domain transcription factor Miz-1. Transactivation by Miz-1 is negatively regulated by association with topoisomerase II binding protein (TopBP1); UV irradiation downregulates expression of TopBP1 and releases Miz-1. Miz-1 binds to the p21Cip1 core promoter in vivo and is required for upregulation of p21Cip1 upon UV irradiation. Using both c-myc-/- cells and a point mutant of Myc that is deficient in Miz-1 dependent repression, it has been shown that Myc negatively regulates transcription of p21Cip1 upon UV irradiation and facilitates recovery from UV-induced cell cycle arrest through binding to Miz-1. These data implicate Miz-1 in a pathway that regulates cell proliferation in response to UV irradiation (Herold, 2002).

One conserved biological function of Myc proteins that is critical for their tumorigenic effects is their ability to upregulate transcription of E box-dependent target genes. Myc uses a conserved surface of the helix-loop-helix/leucine zipper domain for interaction with Max. A second evolutionary conserved surface on the helix-loop-helix domain of Myc proteins has now been identified that binds to Miz-1 but not to Max. Extrapolating from the crystal structure of the Max homodimer, amino acids of Myc involved in binding to Miz-1 point away from the interface with Max, consistent with data that demonstrate the presence of a trimeric Max/Myc/Miz-1 complex at the p15Ink4b initiator. It is suggested, therefore, that Myc proteins have integrated and conserved at least two distinct biochemical properties: the ability to activate through Max and the ability to repress through Miz-1. E box-dependent transactivation by Myc proteins is antagonized by Mad proteins, which also complex with Max. The residues of Myc that bind to Miz-1 are not present in Mad proteins, and Mad proteins do not signal through Miz-1. These data therefore also suggest that loss of Mad proteins is not equivalent to deregulation of Myc during tumorigenesis. The precise importance of the complex formation between Myc and Miz-1 in tumorigenesis can now be addressed with appropriate transgenic and knockin models (Herold, 2002).

In response to UV irradiation, Myc inhibits UV-induced cell cycle arrest by p53 through Miz-1. In this regard, the function of c-myc is very similar to c-jun, a well-characterized negative regulator of the mammalian UV response. Like c-jun, expression of c-myc itself can be induced by UV irradiation in fibroblasts. Furthermore, c-jun is required for the recovery from UV-induced cell cycle arrest, and c-myc-/- cells also recover very poorly from UV irradiation. Both c-jun and c-myc enhance p53-dependent apoptosis, and thus the balance between cell cycle arrest and apoptosis in response to UV irradiation is affected by the status of either gene. It is suggested therefore, that c-myc and c-jun regulate cell fate in response to UV irradiation and switch the response from cell cycle arrest to apoptosis. Recent work shows that Myc overrides G1 arrest not only in response to UV irradiation but also in response to gamma-irradiation and to DNA damage inflicted by reactive oxygen species. In both cases, the DNA damage causes a p53-dependent G1 arrest, and the damage response is overridden by Myc. It is therefore possible that inhibition of Miz-1-dependent transactivation is a general mechanism through which Myc overrides p53-dependent checkpoint functions (Herold, 2002).

Myc interaction with INI1/hSNF5

Chromatin organization plays a key role in the regulation of gene expression. The evolutionarily conserved SWI/SNF complex is one of several multiprotein complexes that activate transcription by remodeling chromatin in an ATP-dependent manner. SWI2/SNF2 is an ATPase whose homologs, BRG1 and hBRM, mediate cell-cycle arrest; the SNF5 homolog, INI1/hSNF5, appears to be a tumor suppressor. A search for INI1-interacting proteins using the two-hybrid system led to the isolation of c-MYC, a transactivator. The c-MYC-INI1 interaction has been observed both in vitro and in vivo. The c-MYC basic helix-loop-helix (bHLH) and leucine zipper (Zip) domains and the INI1 repeat 1 (Rpt1) region are required for this interaction. c-MYC-mediated transactivation is inhibited by a deletion fragment of INI1 and the ATPase mutant of BRG1/hSNF2 in a dominant-negative manner contingent upon the presence of the c-MYC bHLH-Zip domain. These results suggest that the SWI/SNF complex is necessary for c-MYC-mediated transactivation and that the c-MYC-INI1 interaction helps recruit the complex. Recruitment of the SWI/SNF complex, mediated by the interaction of INI1 with c-MYC, may facilitate the transcription of a discrete subset of c-MYC target genes, especially those involved in apoptosis, which might explain the tumor-suppressor activity of INI1 (Cheng, 1999).

The c-myc oncogene product (c-Myc) is a transcription factor that forms a complex with Max and recognizes the E-box sequence. c-Myc plays key functions in cell proliferation, differentiation and apoptosis. As for its activity towards cell proliferation, it is generally thought that c-Myc transactivates the E-box-containing genes that encode proteins essential to cell-cycle progression. Despite the characterization of candidate genes regulated by c-Myc in culture cells, these have still not been firmly recognized as real target genes for c-Myc. c-Myc has been found to directly bind to the N-terminal region of origin recognition complex-1 (ORC1), a region that is responsible for gene silencing, in a state of complex containing other ORC subunits and Max in vivo and in vitro. Furthermore, ORC1 inhibits E-box-dependent transcription activity of c-Myc by competitive binding to the C-terminal region of c-Myc with SNF5, a component of chromatin remodelling complex SNF/Swi1. These results suggest that ORC1 suppresses the transcription activity of c-Myc by its recruitment into an inactive form of chromatin during some stage of the cell cycle (Takayama, 2000).

Phosphorylation of Myc

Myc family transcription factors are destabilized by phosphorylation of a conserved amino-terminal GSK-3ß motif. In proliferating cerebellar granule neuron precursors (CGNPs), Sonic hedgehog signaling induces N-myc expression, and N-myc protein is stabilized by insulin-like growth factor-mediated suppression of GSK-3ß. N-myc phosphorylation-mediated degradation is a prerequisite for CGNP growth arrest and differentiation. This study investigated whether N-myc phosphorylation and turnover are thus linked to cell cycle exit in primary mouse CGNP cultures and the developing cerebellum. Phosphorylation-induced turnover of endogenous N-myc protein in CGNPs increases during mitosis, due to increased priming phosphorylation of N-myc for GSK-3ß. The priming phosphorylation requires the Cdk1 complex, whose cyclin subunits are indirect Sonic hedgehog targets. These findings provide a mechanism for promoting growth arrest in the final cycle of neural precursor proliferation competency, or for resetting the cell cycle in the G1 phase, by destabilizing N-myc in mitosis (Sjostrom, 2005).

Increased S54 phosphorylation is strongly associated with N-myc destabilization. Further, the mitotic kinase Cdk1, in complex with cyclins A and B1, mediates N-myc S54 phosphorylation in primary CGNPs. N-myc is thus primed for GSK-3ß-mediated phosphorylation, which promotes degradation of c-myc and other cell cycle regulatory proteins. This model is consistent with permitting primary neural precursor exit from the cell cycle before G1 is reentered. This allows differentiation to begin, in accordance with intrinsic programs (Sjostrom, 2005).

Shh directly induces N-myc expression and indirectly affects N-myc posttranslational modification, mediated by its indirect targets, cyclins A and/or B. Mitotic degradation of N-myc permits neuronal precursor cell cycle exit in the absence of Shh signaling or in the case of an intrinsic program-directed shift toward differentiation (Sjostrom, 2005).

The data indicate an increase in phosphorylation-associated N-myc degradation during mitosis, which occurs over an interval of less than 1 hr. N-myc is a short-lived protein with a half-life of approximately 40 min, while the cell cycle in mouse CGNPs at PN4 lasts 16-18 hr. How is N-myc disposed of during interphase in proliferating CGNPs? There is growing evidence that myc protein stability regulation may also involve regions outside of the amino-terminal myc box 1 domain containing T50 and S54. Interactions between the myc box 2 domain and the F box protein Skp2 promote c-myc proteolysis during the G1-to-S phase transition, indicating that myc metabolism may be regulated in a cell cycle-dependent manner. With regard to N-myc, these mechanisms have yet to be verified in primary neural precursors (Sjostrom, 2005).

GSK-3ß-mediated phosphorylation targets c-myc for degradation through a mechanism involving the F box protein Fbw7 in a variety of cell lines. A similar mechanism for N-myc degradation was suggested. Whether Fbw7, or another F box protein with an appropriate spatiotemporal expression pattern, plays a role in N-myc proteolysis in primary cells and during cerebellar development in vivo remains to be determined. To act upon N-myc at T50, GSK-3ß must first be primed by phosphorylation at S54. Basal levels of N-myc S54 phosphorylation could be mediated by nonmitotic kinases, such as Erk, outside mitosis. N-myc was also identified as a substrate for the neural-specific kinase Cdk5. However, neither Erk nor Cdk5 activity was specific for S54. It was found that the Cdk1 complex contains a potent, specific N-myc S54 kinase (Sjostrom, 2005).

Cdk1 heterodimerizes with cyclin A and cyclin B1. Cell-free in vitro assays have shown that cyclin A in complex with Cdk1 and p107 can phosphorylate GST-c-myc fusion proteins. Both cyclin A and cyclin B1 immunoprecipitate specific activity toward N-myc, and both cyclins are expressed in Shh-treated CGNPs during mitosis. Cyclin A has also been found in interphase CGNPs, consistent with its participation in Cdk2 complexes during S phase. The lack of S54 phosphorylation in G1/S-arrested cells indicates that cyclin A:Cdk2 does not phosphorylate N-myc. Although cyclin A:Cdk1 complexes may have some substrates distinct from those of cyclin B:Cdk1 complexes, it has been shown that many Cdk substrates are indifferent as to the cyclin subunit of the cyclin:Cdk1 complex. These analyses suggest that, in primary CGNPs, N-myc S54 can be targeted by either cyclin A:Cdk1 or cyclin B1:Cdk1 (Sjostrom, 2005).

Early studies reported that c-myc protein synthesis and modification is not altered during mitosis. Later studies demonstrated mitosis-specific c-myc phosphorylation. These studies were carried out in cell lines with flexible cell cycle exit and reentry capacity. This work with N-myc has been conducted in primary neuronal precursors with a defined intrinsic program for irreversible cell cycle exit and subsequent differentiation. Many previous myc turnover studies have relied on overexpression, while this study focused on regulation of endogenous N-myc stability, in primary cultures and in vivo. The finding that N-myc is largely degraded at the conclusion of the primary neural precursor cell cycle provides insight as to how enhanced stability of N-myc protein can contribute to brain tumorigenesis, by enhancing CGNPs' capacity for ongoing division. Increased activity of IGF2, which is predicted to stabilize N-myc, is associated with increased proliferation in primary neural precursors and in mouse and human medulloblastomas. Thus, future analysis of how N-myc turnover is regulated during CGNP expansion in vivo will yield greater understanding of normal brain development and brain tumor biology (Sjostrom, 2005).

Translational repression of Myc

RNA-binding proteins (RBPs) and microRNAs (miRNAs) are potent post-transcriptional regulators of gene expression. This study shows that the RBP HuR reduces c-Myc expression by associating with the c-Myc 3' untranslated region (UTR) next to a miRNA let-7-binding site. Lowering HuR or let-7 levels relieves the translational repression of c-Myc. Unexpectedly, HuR and let-7 repressed c-Myc through an interdependent mechanism; let-7 requires HuR to reduce c-Myc expression and HuR required let-7 to inhibit c-Myc expression. These findings suggest a regulatory paradigm wherein HuR inhibits c-Myc expression by recruiting let-7-loaded RISC (RNA miRNA-induced silencing complex) to the c-Myc 3'UTR (Kim, 2009).

Other Myc interactions

The Myc protein binds to and transactivates the expression of genes via E-box elements containing a central CAC(G/A)TG sequence. The transcriptional activation function of Myc is required for its ability to induce cell cycle progression, cellular transformation and apoptosis. Transactivation by Myc is under negative control by the transcription factor AP-2 (see Drosophila AP-2). AP-2 inhibits transactivation by Myc via two distinct mechanisms. First, high affinity binding sites for AP-2 overlap Myc-response elements in two bona fide target genes of Myc, prothymosin-alpha and ornithine decarboxylase. On these sites, AP-2 competes for binding of either Myc/Max heterodimers or Max/Max homodimers. The second mechanism involves a specific interaction between C-terminal domains of AP-2 and the BR/HLH/LZ domain of Myc, but not Max or Mad. Binding of AP-2 to Myc does not preclude association of Myc with Max, but impairs DNA binding of the Myc/Max complex and inhibits transactivation by Myc even in the absence of an overlapping AP-2 binding site. Taken together, these data suggest that AP-2 acts as a negative regulator of transactivation by Myc (Gaubatz, 1995).

TIP49 was originally identified as a TBP interacting protein using TBP as an in vitro affinity matrix for rat liver nuclear extracts. However, there is no observable association between TIP49 and TBP in vivo. No TBP is detected in the affinity-purified proteins that bind to the Myc N terminus. TIP49 has also been cloned in a two-hybrid screen using the replication protein 3 as bait and termed RUVBL1, due to the limited homology with RuvB. However, as with TBP, no in vivo association of TIP49 with RPA3 has been demonstrated, and there are no data implicating TIP49 for a role in DNA replication. Of relevance to the present study is the observation that a portion of the cellular TIP49 could be isolated in chromatographic fractions containing RNA polymerase II. TIP49 associates with the nuclear matrix (Wood, 2000 and references therein).

The finding that TIP49 can also bind to beta-catenin and LEF-1/TCF supports a role for TIP49 as a cofactor that is likely to function with diverse transcription factors. Despite the identification of TIP49 through binding studies with different nuclear components, no functional role for TIP49 in these systems has previously been established. The observation that the TIP49D302N allele inhibits Myc oncogenic activity demonstrates that TIP49 is an essential nuclear cofactor in at least the Myc transcription factor pathway. Since the analogous mutation is nonviable in yeast and it targets the conserved Walker B motif, the data imply that Myc-mediated oncogenesis requires the ATPase activity of TIP49. Even though the predicted ATPase-deficient TIP49 inhibits oncogenic transformation, it is not overtly toxic since there is no inhibition of drug-resistant colony formation in several cell types. It is likely that ectopic expression of the TIP49D302N protein creates only a partial loss of function within a cell and that this partial loss of function is only rate limiting when high levels of Myc activity are demanded. The inviability of the yeast strain with the analogous TIP49D311N mutation argues that the exclusive expression of the TIP49D302N protein in mammalian cells would also be incompatible with cell growth. Nevertheless, a direct role for TIP49 in the transcriptional activation of any specific Myc target gene is only inferred at this stage, but not yet readily assayed. Coexpression of TIP49wt or TIP49D302N with Myc neither augments nor inhibits the activation or repression of several reporter constructs. A similar finding has been reported for TIP49 in the beta-catenin/LEF-1 system. The activity of TIP49 may only be apparent with currently uncharacterized promoters or with chromosomal target sites that are not adequately recapitulated by DNA transfection (Wood, 2000).

The c-Myc and E2F transcription factors are among the most potent regulators of cell cycle progression in higher eukaryotes. This report describes the isolation of a novel, highly conserved 434 kDa protein, designated TRRAP, which interacts specifically with the c-Myc N terminus and has homology to the ATM/PI3-kinase family. TRRAP also interacts specifically with the E2F-1 transactivation domain. Expression of transdominant mutants of the TRRAP protein or antisense RNA blocks c-Myc- and E1A-mediated oncogenic transformation. These data suggest that TRRAP is an essential cofactor for both the c-Myc and E1A/E2F oncogenic transcription factor pathways (McMahon, 1998).

The c-Myc protein functions as a transcription factor to facilitate oncogenic transformation; however, the biochemical and genetic pathways leading to transformation remain undefined. The recently described c-Myc cofactor TRRAP recruits histone acetylase activity, which is catalyzed by the human GCN5 protein (see Drosophila Pcaf). Since c-Myc function is inhibited by recruitment of histone deacetylase activity through Mad family proteins, these opposing biochemical activities are likely to be responsible for the antagonistic biological effects of c-Myc and Mad on target genes and ultimately on cellular transformation (McMahon, 2000).

The c-Myc oncogene has been implicated in the genesis of diverse human tumors. Ectopic expression of the c-Myc gene in cultured epithelial cells causes resistance to the antiproliferative effects of TGF-ß. However, little is known about the precise mechanisms of c-Myc-mediated TGF-ß resistance. In this study, it has been revealed that c-Myc physically interacts with Smad2 and Smad3, two specific signal transducers involved in TGF-ß signaling. Through its direct interaction with Smads, c-Myc binds to the Sp1-Smad complex on the promoter of the p15Ink4B gene, thereby inhibiting the TGF-ß-induced transcriptional activity of Sp1 and Smad/Sp1-dependent transcription of the p15Ink4B gene. These results suggest that oncogenic c-Myc promotes cell growth and cancer development partly by inhibiting the growth inhibitory functions of Smads (Feng, 2002).

The cellular Bcr protein consists of an N-terminal serine/threonine kinase domain, a central guanine nucleotide exchange factor homology region and a C-terminal GTPase-activating protein domain. Bcr is a multifunctional protein that is the fusion partner for Abl (p210 Bcr-Abl) in Philadelphia chromosome positive leukemias. c-Myc has been identified as a binding partner for Bcr in both yeast and mammalian cells. Interactions between natively expressed c-Myc and Bcr have been observed in leukemic cell lines. The interaction between c-Myc and Bcr is detected in a fragment containing residues 871-910 that encompasses the small region between the PH domain and the C2 domain of Bcr. The smallest fragment of c-Myc that retains its ability to interact with the full-length Bcr clone consists of the carboxy-terminal B/HLH/Z domain. Although Bcr and Max have overlapping binding sites on c-Myc, Bcr cannot interact with Max, or with the c-Myc/Max heterodimer. Bcr expression blocks activation of c-Myc-responsive genes, as well as the transformed phenotype induced by coexpression of c-Myc and H-Ras, and this finding suggests that one function of Bcr is to limit the activity of c-Myc. However, Bcr does not block c-Myc function by preventing its nuclear localization. Interestingly, increased Bcr dosage in COS-7 and K-562 cells correlates with a reduction in c-Myc protein levels, suggesting that Bcr may in fact be limiting c-Myc activity by regulating its stability. These data indicate that Bcr is a novel regulator of c-Myc function whose disrupted expression may contribute to the high level of c-Myc protein that is observed in Bcr-Abl transformed cells (Mahon, 2003).

c-Myc promotes cellular proliferation, sensitizes cells to apoptosis and prevents differentiation. It binds cyclin T1 structurally and functionally from the positive transcription elongation factor b (P-TEFb). The cyclin-dependent kinase 9 (Cdk9) in P-TEFb then phosporylates the C-terminal domain of RNA polymerase II, which is required for the transition from initiation to elongation of eukaryotic transcription. Inhibiting P-TEFb blocks the transcription of its target genes as well as cellular proliferation and apoptosis induced by c-Myc (Kanazawa, 2003).

The c-Myc oncoprotein (Myc) controls cell fate by regulating gene transcription in association with a DNA-binding partner, Max. While Max lacks a transcription regulatory domain, the N terminus of Myc contains a transcription activation domain (TAD) that recruits cofactor complexes containing the histone acetyltransferases (HATs) GCN5 and Tip60. This study reports a novel functional interaction between Myc TAD and the p300 coactivator-acetyltransferase. p300 associates with Myc in mammalian cells and in vitro through direct interactions with Myc TAD residues 1 to 110 and acetylates Myc in a TAD-dependent manner in vivo at several lysine residues located between the TAD and DNA-binding domain. Moreover, the Myc:Max complex is differentially acetylated by p300 and GCN5 and is not acetylated by Tip60 in vitro, suggesting distinct functions for these acetyltransferases. Whereas p300 and CBP can stabilize Myc independent of acetylation, p300-mediated acetylation results in increased Myc turnover. In addition, p300 functions as a coactivator that is recruited by Myc to the promoter of the human telomerase reverse transcriptase gene; also, p300/CBP stimulates Myc TAD-dependent transcription in a HAT domain-dependent manner. These results suggest dual roles for p300/CBP in Myc regulation: as a Myc coactivator that stabilizes Myc and as an inducer of Myc instability via direct Myc acetylation (Faiola, 2005).

The c-MYC oncoprotein functions as a sequence-specific transcription factor. The ability of c-MYC to activate transcription relies in part on the recruitment of cofactor complexes containing the histone acetyltransferases mammalian GCN5 (mGCN5)/PCAF and TIP60. In addition to acetylating histones, these enzymes have been shown to acetylate other proteins involved in transcription, including sequence-specific transcription factors. This study was initiated in order to determine whether c-MYC is a direct substrate of mGCN5 and TIP60. mGCN5/PCAF and TIP60 are shown to acetylate c-MYC in vivo. By using nanoelectrospray tandem mass spectrometry to examine c-MYC purified from human cells, the major mGCN5-induced acetylation sites have been mapped. Acetylation of c-MYC by either mGCN5/PCAF or TIP60 results in a dramatic increase in protein stability. The data reported here suggest a conserved mechanism by which acetyltransferases regulate c-MYC function by altering its rate of degradation (Patel, 2005).

Mad-Sin3 complex and the modification of chromatin

Members of the Mad family of bHLHZip proteins heterodimerize with Max and function to repress the transcriptional and transforming activities of the Myc proto-oncogene. Mad:Max heterodimers repress transcription by recruiting a large multi-protein complex containing the histone deacetylases, HDAC1 and HDAC2, to DNA. The interaction between Mad proteins and HDAC1/2 is mediated by the corepressor mSin3A (see Drosophila Sin3A) and requires sequences at the amino terminus of the Mad proteins, termed the SID, for Sin3 interaction domain, and the second of four paired amphipathic alpha-helices (PAH2) in mSin3A. To better understand the requirements for the interaction between the SID and PAH2, mutagenesis and structural studies on the SID have been performed. These studies show that amino acids 8-20 of Mad1 are sufficient for SID:PAH2 interaction. Further, this minimal 13-residue SID peptide forms an amphipathic alpha-helix in solution, and residues on the hydrophobic face of the SID helix are required for interaction with PAH2. Finally, the minimal SID can function as an autonomous and portable repression domain, demonstrating that it is sufficient to target a functional mSin3A/HDAC corepressor complex (Eilers, 1999).

Gene-specific targeting of the Sin3 corepressor complex by DNA-bound repressors is an important mechanism of gene silencing in eukaryotes. The Sin3 corepressor specifically associates with a diverse group of transcriptional repressors, including members of the Mad family, that play crucial roles in development. The NMR structure of the complex formed by the PAH2 domain of mammalian Sin3A with the transrepression domain (SID) of human bHLHZip protein Mad1 reveals that both domains undergo mutual folding transitions upon complex formation generating an unusual left-handed four-helix bundle structure and an amphipathic alpha helix, respectively. The SID helix is wedged within a deep hydrophobic pocket defined by two PAH2 helices. Structure-function analyses of the Mad-Sin3 complex provide a basis for understanding the underlying mechanism(s) that lead to gene silencing (Brubaker, 2000).

Sin3 appears to function as a large protein scaffold capable of multiple protein-protein interactions. While Sin3 interacts with class I histone deacetylases (HDAC1 and HDAC2) and presumed accessory proteins such as RbAp48, SAP30, and SAP18 it also associates with a surprisingly wide range of DNA binding transcription factors, including the nuclear hormone receptors (through the N-CoR and SMRT corepressors), MeCP2, Ski, p53, Ikaros and Aiolos, REST/NRSF, MNF-beta, and the Mad family of Max binding bHLH-Zip transcriptional repressors. The activities of these proteins and their ability to interact with Sin3 are thought to be crucial for cell proliferation and differentiation (Brubaker, 2000 and references therein).

The nature and possible regulation of the specific interaction between transcription factors and Sin3 is of great interest. For nuclear hormone receptors, the interaction with N-CoR/SMRT is hormone regulated, while for 'dedicated' repressors such as the Mad protein family, the association appears to be constitutive. In the case of the Mad proteins, all four family members (Mad1, Mxi1, Mad3, and Mad4) and the related repressor, Mnt (or Rox) contain an ~30-residue, N-terminally located segment known as the Sin3 interaction domain, or SID, which is both necessary and sufficient for Sin3 association and for transcriptional repression. Deletion or specific mutation of the SID abrogates Mad repression as well as its growth inhibitory functions. Furthermore, the Mad SID is capable of conferring repression activity when fused to a heterologous DNA binding domain. Helical wheel analysis and circular dichroism (CD) studies of the Mad SID suggest that it has the potential to form an amphipathic alpha helix. Mutational analyses further demonstrate that a cluster of residues on the apolar face of the helix is essential for interaction with mammalian Sin3A (mSin3A) (Brubaker, 2000 and references therein).

Sin3 interacts with many proteins in the complex through four imperfect repeats of ~100 residues known as paired amphipathic helix (PAH) domains. The PAH domains, which were each suggested to be organized into two alpha helices separated by a flexible spacer region, are among the most evolutionarily conserved regions of the large Sin3 proteins (100-170 kDa). Indeed, these domains are important for Sin3 function as a corepressor, most likely through their independent associations with various repressors and other associated proteins. For example, PAH2 is both necessary and sufficient for interaction with the Mad proteins as well as with a newly discovered Sin3-interacting protein, Pf1. However, PAH1 associates with N-CoR and PLZF, while PAH3 binds the SAP30 protein (Brubaker, 2000 and references therein).

While previous work on repressor-Sin3 corepressor interactions has localized functionally important regions and provides hints regarding their structure, details of these important interactions have remained largely unknown. In this study, a high-resolution structure is described for the Mad1 SID bound to the PAH2 domain of mSin3A determined by NMR (nuclear magnetic resonance) methods. Mutational studies of mSin3A are presented that confirm many of the specific interactions predicted from the NMR structure. Finally, it is shown that an unrelated Sin3-interacting protein, Pf1, with an interaction domain distinct from the Mad family SID, is likely to interact with PAH2 in a manner closely resembling Mad1 SID (Brubaker, 2000).

MYC associates with TIP60 complex

The c-Myc transactivation domain was used to affinity purify tightly associated nuclear proteins. Two of these proteins were identified as TIP49 and a novel related protein called TIP48, both of which are highly conserved in evolution and contain ATPase/helicase motifs. TIP49 and TIP48 are complexed with c-Myc in vivo, and binding is dependent on a c-Myc domain essential for oncogenic activity. A missense mutation in the TIP49 ATPase motif acts as a dominant inhibitor of c-Myc oncogenic activity but does not inhibit normal cell growth, indicating that functional TIP49 protein is an essential mediator of c-Myc oncogenic transformation. The TIP49 and TIP48 ATPase/helicase proteins represent a novel class of cofactors recruited by transcriptional activation domains that function in diverse pathways (Wood, 2000).

The c-Myc oncoprotein functions as a transcription factor that can transform normal cells into tumor cells, as well as playing a direct role in normal cell proliferation. The c-Myc protein transactivates cellular promoters by recruiting nuclear cofactors to chromosomal sites through an N-terminal transactivation domain. Four different c-Myc cofactors: TRRAP, hGCN5, TIP49, and TIP48 have been identified and functionally characterized. This study presents the identification and characterization of the actin-related protein BAF53 as a c-Myc-interacting nuclear cofactor that forms distinct nuclear complexes. In addition to the human SWI/SNF-related BAF complex, BAF53 forms a complex with TIP49 and TIP48 and a separate biochemically distinct complex containing TRRAP and a histone acetyltransferase which does not contain TIP60. Using deletion mutants of BAF53, it is shown that BAF53 is critical for c-Myc oncogenic activity. These results indicate that BAF53 plays a functional role in c-Myc-interacting nuclear complexes (Park, 2002).

The transcription factor MYC binds specific DNA sites in cellular chromatin and induces the acetylation of histones H3 and H4. However, the histone acetyltransferases (HATs) that are responsible for these modifications have not yet been identified. MYC associates with TRRAP, a subunit of distinct macromolecular complexes that contain the HATs GCN5/PCAF or TIP60. Although the association of MYC with GCN5 has been shown, its interaction with TIP60 has never been analysed. This study shows that MYC associates with TIP60 and recruits it to chromatin in vivo with four other components of the TIP60 complex: TRRAP, p400, TIP48 and TIP49. Overexpression of enzymatically inactive TIP60 delays the MYC-induced acetylation of histone H4, and also reduces the level of MYC binding to chromatin. Thus, the TIP60 HAT complex is recruited to MYC-target genes and, probably with other other HATs, contributes to histone acetylation in response to mitogenic signals (Frank, 2003).

Pontin (Tip49) and Reptin (Tip48; see Drosophila Reptin) are highly conserved components of multimeric protein complexes important for chromatin remodelling and transcription. They interact with many different proteins including TATA box binding protein (TBP), beta-catenin and c-Myc and thus, potentially modulate different pathways. As antagonistic regulators of Wnt-signalling, they control wing development in Drosophila and heart growth in zebrafish. This study shows that the Xenopus xPontin and xReptin in conjunction with c-Myc regulate cell proliferation in early development. Overexpression of xPontin or xReptin results in increased mitoses and bending of embryos, which is mimicked by c-Myc overexpression. Furthermore, the knockdown of either xPontin or xReptin resulted in embryonic lethality at late gastrula stage, which is abrogated by the injection of c-Myc-RNA. The N-termini of xPontin and xReptin, which mediate the mitogenic effect were mapped to contain c-Myc interaction domains. c-Myc protein promotes cell cycle progression either by transcriptional activation through the c-Myc/Max complex or by repression of cyclin dependent kinase inhibitors (p21, p15) through c-Myc/Miz-1 interaction. Importantly, xPontin and xReptin exert their mitogenic effect through the c-Myc/Miz-1 pathway as dominant negative Miz-1 and wild-type c-Myc but not a c-Myc mutant deficient in Miz-1 binding could rescue embryonic lethality. Finally, promoter reporter studies revealed that xPontin and xReptin but not the N-terminal deletion mutants enhance p21 repression by c-Myc. It is concluded that xPontin and xReptin are essential genes regulating cell proliferation in early Xenopus embryogenesis through interaction with c-Myc. A novel function of xPontin and xReptin is proposed as co-repressors in the c-Myc/Miz-1 pathway (Etard, 2005).

Binding of Myc to DNA

The biological activity of the c-Abl protein is linked to its tyrosine kinase and DNA-binding activities. The protein, which plays a major role in the cell cycle response to DNA damage, interacts preferentially with sequences containing an AAC motif and exhibits a higher affinity for bent or bendable DNA, as is the case with high mobility group (HMG) proteins. The DNA-binding characteristics of the DNA-binding domain of human c-Abl and the HMG-D protein from Drosophila melanogaster have been compared. c-Abl binds tightly to circular DNA molecules and potentiates the interaction of DNA with HMG-D. In addition, a series of DNA molecules containing modified bases were used to determine how the exocyclic groups of DNA influence the binding of the two proteins. Interfering with the 2-amino group of purines affects the binding of the two proteins similarly. Adding a 2-amino group to adenines restricts the access of the proteins to the minor groove, whereas deleting this bulky substituent from guanines facilitates the protein-DNA interaction. In contrast, c-Abl and HMG-D respond very differently to deletion or addition of the 5-methyl group of pyrimidine bases in the major groove. Adding a methyl group to cytosines favors the binding of c-Abl to DNA but inhibits the binding of HMG-D. Conversely, deleting the methyl group from thymines promotes the interaction of the DNA with HMG-D but diminishes its interaction with c-Abl. The enhanced binding of c-Abl to DNA containing 5-methylcytosine residues may result from an increased propensity of the double helix to denature locally, coupled with a protein-induced reduction in the base stacking interaction. The results show that c-Abl has unique DNA-binding properties, quite different from those of HMG-D, and suggest an additional role for the protein kinase (David-Cordonnier, 1999).

N-Myc is a transcription factor that forms heterodimers with the protein Max and binds gene promoters by recognizing a DNA sequence, CACGTG, called E-box. The identification of N-myc target genes is an important step for understanding N-Myc biological functions in both physiological and pathological contexts. In this study, the identification of N-Myc-responsive genes through chromatin immunoprecipitation and methylation-sensitive restriction analysis is described. Results show that N-Myc is a direct regulator of several identified genes, and that methylation of the CpG dinucleotide within the E-box prevents the access of N-Myc to gene promoters in vivo. Furthermore, methylation profile of the E-box within the promoters of EGFR and CASP8, two genes directly controlled by Myc, is cell type-specific, suggesting that differential E-box methylation may contribute to generating unique patterns of Myc-dependent transcription. This study illuminates a central role of DNA methylation in controlling N-Myc occupancy at gene promoters and modulating its transcriptional activity in cancer cells (Perini, 2005).

Regulation of transcription and translation of vertebrate myc

Increasing evidence supports an important biological role for Myc in the downregulation of specific gene transcription. Recent studies suggest that c-Myc may suppress promoter activity through proteins of the basal transcription machinery. Myc protein, in combination with additional cellular factors, suppresses transcription initiation from the c-myc promoter. There is a four-fold to five-fold suppression of a c-myc P2 minimal promoter fragment upon induction of wild-type Myc protein activity, while induction of a mutant Myc protein lacking amino acids 106 to 143 required for Myc autosuppression fails to elicit this response. This assay is physiologically significant, as it reflects Myc autosuppression of the endogenous c-myc gene with regard to kinetics, dose dependency, cell type specificity, and c-Myc functional domains. Analysis of mutations within the P2 minimal promoter indicates that the cis components of Myc autosuppression could not be ascribed to any known protein-binding motifs. Myc-Max heterodimerization is obligatory for Myc autosuppression (Facchini, 1997).

Organization of DNA into chromatin has been shown to contribute to a repressed state of gene transcription. Disruption of nucleosomal structure is observed in response to gene induction, suggesting a model in which RNA polymerase II (pol II) is recruited to the promoter upon reorganization of nucleosomes. Induction of c-myc transcription correlates with the disruption of two nucleosomes in the upstream promoter region. However, this nucleosomal disruption is not necessary for the binding of pol II to the promoter. Transcriptionally engaged pol II complexes can be detected when the upstream chromatin is in a more closed configuration. Thus, upstream chromatin opening is suggested to affect activation of promoter-bound pol II rather than entry of polymerases into the promoter. Interestingly, pol II complexes are detectable in both sense and antisense transcriptional directions, but only complexes in the sense direction respond to activation signals resulting in processive transcription (Albert, 1997).

The small constitutively expressed bHLHZip protein Max is known to form sequence-specific DNA binding heterodimers with members of both the Myc and Mad families of bHLHZip proteins. Myc:Max complexes activate transcription, promote proliferation, and block terminal differentiation. In contrast, Mad:Max heterodimers act as transcriptional repressors, have an antiproliferative effect, and are induced upon differentiation in a wide variety of cell types. A novel bHLHZip Max-binding protein, Mnt, has been identified that belongs to neither the Myc nor the Mad families and that is coexpressed with Myc in a number of proliferating cell types. Mnt:Max heterodimers act as transcriptional repressors and efficiently suppress Myc-dependent activation from a promoter containing proximal CACGTG sites. Transcription repression by Mnt maps to a 13-amino-acid amino-terminal region related to the Sin3 interaction domain (SID) of Mad proteins. This region of Mnt mediates interaction with mSin3 corepressor proteins; its deletion converts Mnt from a repressor to an activator. Furthermore, wild-type Mnt suppresses Myc+Ras cotransformation of primary cells, whereas Mnt containing a SID deletion cooperates with Ras in the absence of Myc to transform cells. This suggests that Mnt and Myc regulate an overlapping set of target genes in vivo. When mnt is expressed as a transgene under control of the beta-actin promoter in mice the transgenic embryos exhibit a delay in development and die during mid-gestation, just when c- and N-Myc functions are critical. It is proposed that Mnt:Max:Sin3 complexes normally function to restrict Myc:Max activities associated with cell proliferation (Hurlin, 1997).

Interferons (IFNs) inhibit cell growth in a Stat1-dependent fashion that involves regulation of c-myc expression. IFN-gamma suppresses c-myc in wild-type mouse embryo fibroblasts, but not in Stat1-null cells, where IFNs induce c-myc mRNA rapidly and transiently, thus revealing a novel signaling pathway. Both tyrosine and serine phosphorylation of Stat1 are required for suppression. Induced expression of c-myc is likely to contribute to the proliferation of Stat1-null cells in response to IFNs. IFNs also suppress platelet-derived growth factor (PDGF)-induced c-myc expression in wild-type but not in Stat1-null cells. A gamma-activated sequence element in the promoter is necessary but not sufficient to suppress c-myc expression in wild-type cells. In PKR-null cells, the phosphorylation of Stat1 on Ser727 and transactivation are both defective, and c-myc mRNA is induced, not suppressed, in response to IFN-gamma. A role for Raf-1 in the Stat1-independent pathway is revealed by studies with geldanamycin, an HSP90-specific inhibitor, and by expression of a mutant of p50cdc37 that is unable to recruit HSP90 to the Raf-1 complex. Both agents abrogate the IFN-gamma-dependent induction of c-myc expression in Stat1-null cells (Ramana, 2000).

ROR alpha1 (Drosophila homolog Hormone receptor-like/Hr46) and RVR are orphan members of the superfamily of nuclear hormone receptors that constitutively activate and repress, respectively, gene transcription by binding to a common DNA sequence. A consensus binding site for ROR alpha1 and RVR is found in the first intron of the N-myc gene. This site is designated N-myc RORE (ROR response element). Unlike most of the intronic sequence, the region encompassing the N-myc RORE is highly conserved between human and mouse, underscoring its importance. ROR alpha1 and RVR specifically bind to the human and mouse N-myc ROREs and transactivate and transrepress, respectively, reporter constructs containing the ROREs. There is a direct modulation of an exogenously introduced N-myc gene by ROR alpha1 and RVR in COS-1 cells. This effect is mediated through the N-myc RORE, since mutation of this site abolishes the regulatory effects of both receptors. While transfection of ROR alpha1 in P19 embryonic carcinoma cells has no effect on the levels of endogenous N-myc mRNA, RVR down-regulates its expression. Mutation of the RORE increases the oncogenic potential of the N-myc gene. Concomitant expression of ROR alpha1 and wild-type N-myc results in a twofold increase in the number of transformed foci. These observations show that ablation of the RORE results in a more oncogenic form of N-myc and suggest that deregulation of the activity of the ROR alpha1 and RVR could contribute to the initiation and progression of certain neoplasias (Dussault, 1997).

The receptor-binding factor (RBF) for the avian oviduct progesterone (Pg) receptor (PR) has previously been shown to be a unique 10-kDa nuclear matrix protein that generates high affinity PR-binding sites on avian DNA. This paper describes the use of Southwestern blot and DNA gel shift analyses with RBF protein to identify a minimal 54-base pair RBF-binding element in the matrix-associated region (MAR) of the Pg-regulated c-myc gene promoter. This element contains a 5'-GC-rich domain and a 3'-AT-rich domain, the latter having a homopurine/homopyrimidine structure. The gel shift assays required the generation of an RBF-maltose fusion protein (RBF-MBP), which specifically binds this element and is supershifted when the anti-RBF polyclonal antibody is added. Computer analysis of the full-length amino acid sequence for RBF predicts a DNA-binding motif involving a beta-sheet structure at the N-terminal domain. Southern blot analyses using nuclear matrix DNA suggests that there are dual MAR sites in the c-myc promoter, which flank an intervening domain containing the RBF element. The co-transfection of this MAR sequence, containing the RBF element and cloned into a luciferase reporter vector, together with an RBF expression vector construct, into steroid treated human MCF-7 cells, results in a decrease of the c-myc promoter activity relative to control transfections containing only the parent vector of the RBF expression construct. These data suggest that a unique chromatin/nuclear matrix structure, composed of the RBF-DNA element complex, flanked by nuclear matrix attachment sites, serves to bind the PR and repress the c-myc promoter (Lauber, 1997).

The far-upstream element-binding protein (FBP) is one of several recently described factors which bind to a single strand of DNA in the 5' region of the c-myc gene. Although cotransfection of FBP increases expression from a far-upstream element-bearing c-myc promoter reporter, the mechanism of this stimulation has been heretofore unknown. Can a single-strand-binding protein function as a classical transactivator, or are these proteins restricted to stabilizing or altering the conformation of DNA in an architectural role? The carboxyl-terminal region (residues 448 to 644) of FBP is a potent transcriptional activation domain. This region contains three copies of a unique amino acid sequence motif containing tyrosine diads. Analysis of deletion mutants demonstrates that a single tyrosine motif alone (residues 609 to 644) is capable of activating transcription. The activation property of the C-terminal domain is repressed by the N-terminal 107 amino acids of FBP. These results show that FBP contains a transactivation domain that can function alone, suggesting that FBP contributes directly to c-myc transcription while bound to a single-strand site. Furthermore, activation is mediated by a new motif that can be negatively regulated by a repression domain of FBP (Duncan, 1996).

The proto-oncoprotein c-Myc and the multifunctional transcriptional regulator YY1 (Drosophila homolog: Pleiohomeotic) have been shown to interact directly in a manner that excludes Max from the complex. Since binding to Max is necessary for all known c-Myc activities, the influence of YY1 on c-Myc function has been analyzed. YY1 is shown to be a potent inhibitor of c-Myc transforming activity. The region in YY1 required for inhibition corresponds to a functional DNA-binding domain and is distinct from the domains necessary for direct binding to c-Myc. Furthermore the transactivation domain of YY1 was not necessary, suggesting that gene regulation by YY1, for example, through DNA bending or displacement of regulators from DNA, could be the cause for the negative regulation of c-Myc. This model of indirect regulation of c-Myc by YY1 is supported by the finding that although YY1 does not bind to the c-Myc transactivation domain (TAD) in vitro it is able to inhibit transactivation by Gal4-MycTAD fusion proteins in transient transfections. As for the inhibition of transformation, an intact DNA-binding domain of YY1 was necessary and sufficient for this effect. In addition, YY1 does not alter c-Myc/Max DNA binding, further supporting an indirect mode of action. These findings point to a role of YY1 as a negative regulator of cell growth with a possible involvement in tumor suppression (Austen, 1998).

Transcription activation and repression of eukaryotic genes are associated with conformational and topological changes of the DNA and chromatin, altering the spectrum of proteins associated with an active gene. Segments of the human c-myc gene possessing non-B structure in vivo were first located with probes that cleave single stranded DNA. Sites hypertensive to cleavage include the major promoters P1 and P2 of c-myc, as well as the far upstream sequence element (FUSE) and CT elements. These bind, respectively, the single-strand-specific factors FUSE-binding protein and heterogeneous nuclear ribonucleoprotein K in vitro. Active and inactive c-myc genes yield different patterns of S1 nuclease and permanganate sensitivity, indicating alternative chromatin configurations of active and silent genes. The melting of specific cis elements of active c-myc genes in vivo suggests that transcriptionally associated torsional strain might assist strand separation and facilitate factor binding. Therefore, the interaction of FUSE-binding protein and heterogeneous nuclear ribonucleoprotein K with supercoiled DNA was examined. Remarkably, both proteins recognize their respective elements torsionally strained but not as liner duplexes. Single-strand- or supercoil-dependent gene regulatory proteins may directly link alterations in DNA conformation and topology with changes in gene expression (Michelotti, 1996).

The CT element of the c-myc gene is required for promoter P1 usage and can drive expression of a heterologous promoter. Both double strand (Sp1) and single strand (hnRNP K) CT-binding proteins have been implicated as mediators of CT action. Although significant levels of CT activity persist following Sp1 immunodepletion, EGTA totally abolishes transactivation, thus implicating another metal requiring factor in CT element activity. Since hnRNP K binds to one strand of the CT element, but has no metal requirement, the opposite (purine-rich strand) was examined as a target for a metal-dependent protein. A zinc-requiring purine strand binding activity was identified as cellular nucleic acid binding protein (CNBP), a protein previously implicated in the regulation of sterol responsive genes. Two forms of CNBP differ in their relative binding to the CT- or sterol-response elements. CNBP is a bona fide regulator of the CT element by cotransfection of a CNBP expression vector that stimulates expression of a CT-driven (but not an AP1-dependent) reporter. These data suggest that hnRNP K and CNBP bind to opposite strands and co-regulate the CT element (Michelotti, 1995).

The far upstream element (FUSE) is required for proper expression of the human c-myc gene. FUSE-binding protein (FBP) binds the single-stranded far upstream element of active c-myc genes; it possesses potent transcription activation and repression domains, and is necessary for c-myc expression. The FUSE-binding protein (FBP) specifically recognizes this site and stimulates expression in a FUSE-dependent manner. The up and downregulation of c-myc and FBP are highly correlated, suggesting that FBP and c-myc transcription are linked in vivo. Moreover, interfering directly with the levels or activity of FBP extinguishes c-myc expression and arrests cell growth. FBP, first discerned from its primary structure, is a 644-amino acid protein possessing three domains. The central domain of the protein employs a set of four KH motifs to destabilize the double helix of FUSE and to bind sequence specifically with the noncoding strand. The carboxyl terminus of FBP (FBPC, amino acids 488-644) contains three copies of an unusual tyrosine-rich motif, which strongly activates transcription when fused with the GAL4 DNA-binding domain. In contrast, the amino terminus of FBP (FBPN) represses expression driven by FBPC as well as some, but not all, heterologous activators. A novel 60 kDa protein, the FBP interacting repressor (FIR), blocks activator-dependent, but not basal, transcription through TFIIH. Recruited through FBP's nucleic acid-binding domain, FIR forms a ternary complex with FBP and FUSE. FIR represses a c-myc reporter via the FUSE. The amino terminus of FIR contains an activator-selective repression domain capable of acting in cis or even in trans in vivo and in vitro. The repression domain of FIR targets only TFIIH's p89/XPB helicase, required at several stages in transcription, but not factors required for promoter selection. Thus, FIR locks TFIIH in an activation-resistant configuration that still supports basal transcription (Liu, 2000).

Can sequence specific single strand binding proteins find their cognate elements and modify transcription? Heterogeneous nuclear ribonucleoprotein K (hnRNP K) binds the single stranded sequence (CCCTCCCCA; CT-element) of the human c-myc gene in vitro. To monitor its DNA binding in vivo, the ability of hnRNP K to activate a reporter gene was amplified by fusion with the VP16 transactivation domain. This chimeric protein transactivates circular (but not linear) CT-element driven reporters, suggesting that hnRNP K recognizes a single strand region generated by negative supercoiling in circular plasmid. When CT-elements are engineered to overlap with lexA operators, addition of lexA protein, either in vivo or in vitro, abrogates hnRNP K binding most likely by preventing single strand formation. These results not only reveal hnRNP K to be a single strand DNA binding protein in vivo, but demonstrate how a segment of DNA may modify the transcriptional activity of an adjacent gene through the interconversion of duplex and single strands (Tomonaga, 1996).

beta-Catenin and gamma-catenin (plakoglobin), vertebrate homologs of Drosophila armadillo, function in cell adhesion and the Wnt signaling pathway. In colon and other cancers, mutations in the APC tumor suppressor protein or beta-catenin's amino terminus stabilize beta-catenin, enhancing its ability to activate transcription of Tcf/Lef target genes. Though beta- and gamma-catenin have analogous structures and functions and like binding to APC, evidence that gamma-catenin has an important role in cancer has been lacking. APC is shown in this study to regulate both beta- and gamma-catenin and gamma-catenin functions as an oncogene. In contrast to beta-catenin, for which only amino-terminal mutated forms transform RK3E epithelial cells, wild-type and several amino-terminal mutated forms of gamma-catenin have similar transforming activity. gamma-Catenin's transforming activity, like beta-catenin's, is dependent on Tcf/Lef function. However, in contrast to beta-catenin, gamma-catenin strongly activates c-Myc expression and c-Myc function is crucial for gamma-catenin transformation. These findings suggest APC mutations alter regulation of both beta- and gamma-catenin, perhaps explaining why the frequency of APC mutations in colon cancer far exceeds that of beta-catenin mutations. Elevated c-Myc expression in cancers with APC defects may be due to altered regulation of both beta- and gamma-catenin. Furthermore, the data imply beta- and gamma-catenin may have distinct roles in Wnt signaling and cancer via differential effects on downstream target genes (Kolligs, 2000).

The data presented here are the first to suggest that beta- and gamma-catenin may have differential effects on Tcf/Lef target genes. Specifically, it was found wild-type beta-catenin has a roughly twofold greater effect and S33Y mutated beta-catenin a roughly 15-fold greater effect than wild-type gamma-catenin in activating gene expression from a model promoter construct containing three Tcf-binding sites upstream of a minimal c-Fos promoter. In contrast, the ability of wild-type gamma-catenin to activate c-MYC reporter gene constructs is similar to that of S33Y beta-catenin. S33Y is a cancer-derived missense substitution in beta-catenin's presumptive GSK3beta phosphorylation sequences. gamma-Catenin activates endogenous c-Myc gene expression in RK3E cells more strongly than does the S33Y mutant beta-catenin protein. The underlying mechanisms for their differential effects on the reporter gene constructs and on endogenous c-Myc are not yet clear, though differences in the interactions of the distantly related amino- and carboxy-terminal domains of gamma- and beta-catenin with specific transcription factors, coactivators, and/or other chromatin-associated proteins are among the possible explanations. For instance, gamma-catenin may enhance or facilitate the binding of certain transcription factors to promoters, whereas beta-catenin may cooperate with other factors. The presence or absence of specific DNA-binding sites for certain transcription factors in regulatory elements of a particular Tcf/Lef-regulated target gene might account for its differential activation by beta- or gamma-catenin. Alternatively, beta- and gamma-catenin may differ in their ability to interact with certain chromatin remodeling proteins, some of which likely have differential effects on specific genes in vivo. Regardless of the particular mechanisms underlying their differential effects on c-Myc and potentially other target genes, the data presented here support the view that beta- and gamma-catenin are likely to have distinct but complementary roles in Wnt signaling and cancer development (Kolligs, 2000).

A prominent feature of cell differentiation is the initiation and maintenance of an irreversible cell cycle arrest with the complex involvement of the retinoblastoma (RB) family (RB, p130, p107). The HBP1 transcriptional repressor has been isolated as a potential target of the RB family in differentiated cells. By homology, HBP1 is a sequence-specific HMG transcription factor, of which LEF-1 (Drosophila homolog: Pangolin) is the best-characterized family member. Several features of HBP1 suggest an intriguing role as a transcriptional and cell cycle regulator in differentiated cells:

  1. Inspection of the HBP1 protein sequence revealed two consensus RB interaction motifs (LXCXE and IXCXE).
  2. HBP1 interaction is selective for RB and p130, but not p107. HBP1, RB, and p130 levels are all up-regulated with differentiation; in contrast, p107 levels decline.
  3. HBP1 can function as a transcriptional repressor of the promoter for N-MYC, which is a critical cell cycle and developmental gene.
  4. Because the activation of the N-MYC promoter in cycling cells required the E2F (See Drosophila E2F) transcription factor, E2F-1 and HBP1 represent opposite transcriptional signals that can be integrated within the N-MYC promoter.
  5. The expression of HBP1 leads to efficient cell cycle arrest. The arrest phenotype is manifested in the presence of optimal proliferation signals, suggesting that HBP1 exerts a dominant regulatory role.

Taken together, the results suggest that HBP1 may represent a unique transcriptional repressor with a role in initiation and establishment of cell cycle arrest during differentiation (Tevosian. 1997).

The adenomatous polyposis coli gene (APC) is a tumor suppressor gene that is inactivated in most colorectal cancers. Mutations of APC cause aberrant accumulation of beta-catenin, which then binds T cell factor-4 (Tcf-4), causing increased transcriptional activation of unknown genes. The c-MYC oncogene has been identified as a target gene in this signaling pathway. Expression of c-MYC is repressed by wild-type APC and activated by beta-catenin; these effects are mediated through Tcf-4 binding sites in the c-MYC promoter. These results provide a molecular framework for understanding the previously enigmatic overexpression of c-MYC in colorectal cancers (He, 1998).

The v-abl oncogene of Abelson murine leukemia virus encodes a deregulated form of the cellular nonreceptor tyrosine kinase. v-Abl activates c-myc transcription, and c-Myc is an essential downstream component in the v-Abl transformation program. To explore the mechanism by which v-Abl activates c-myc transcription, a cotransfection assay was developed. Transactivation of a c-myc promoter by v-Abl requires the SH1 (tyrosine kinase) and SH2 domains of v-Abl; the C-terminal domains are not required for transactivation. The assay also identifies the E2F site in the c-myc promoter as a v-Abl-responsive element. In addition, multimerized E2F sites were shown to be sufficient to confer v-Abl-dependent activation on a minimal promoter. This is the first identification of a v-Abl response element for transcriptional activation. v-Abl tyrosine kinase-dependent changes in proteins binding the c-myc E2F site have also been demonstrated, including induction of a complex containing DP1, p107, cyclin A, and cdk2. Identification of v-Abl-dependent changes in E2F-binding proteins provides an important link between v-Abl, transcription, cell cycle regulation, and control of cellular growth (Wong, 1995).

A novel human Polycomb homolog, hPc2, is more closely related to a Xenopus Pc homolog, XPc, than to a previously described human Pc homolog, CBX2 (hPc1). However, the hPc2 and CBX2/hPc1 proteins colocalize in interphase nuclei of human U-2 OS osteosarcoma cells, suggesting that the proteins are part of a common protein complex. To study the functions of the novel human Pc homolog, a mutant protein, delta hPc2, was generated, lacking an evolutionarily conserved C-terminal domain. This C-terminal domain is important for hPc2 function, since the delta hPc2 mutant protein that lacks the C-terminal domain is unable to repress gene activity. Expression of the delta hPc2 protein, but not of the wild-type hPc2 protein, results in cellular transformation of mammalian cell lines as judged by phenotypic changes, altered marker gene expression, and anchorage-independent growth. Specifically in delta hPc2-transformed cells, the expression of the c-myc proto-oncogene is strongly enhanced; serum deprivation results in apoptosis. In contrast, overexpression of the wild-type hPc2 protein results in decreased c-myc expression. These data suggest that hPc2 is a repressor of proto-oncogene activity and that interference with hPc2 function can lead to derepression of proto-oncogene transcription and subsequently to cellular transformation (Satijn, 1997).

Turnover of labile mRNAs is thought to be mediated in part by the interactions of trans-acting factors with elements withing the 3' untranslated region. Neuronal and non-neuronal cells established from neuroblastoma tumors differ in N-myc mRNA levels. There are two distinct regions within the 3'-UTR of N-myc mRNA that bind a 40kDA protein complex present in non-neuronal cells but absent from neuronal cells. The N-myc binding protein is identified as a member of the ELAV-like family of RNA-binding proteins (See Drosophila ELAV). It is likely that the ELAV-like mRNA-binding protein acts to stabilize the mRNA, and potentially regulates N-myc mRNA turnover (Chagnovich, 1996)

Mutation of the p53 tumor suppressor gene is the most common genetic alteration in human cancer, and tumors that express mutant p53 may be more aggressive and have a worse prognosis than p53-null cancers. Mutant p53 enhances tumorigenicity in the absence of a transdominant negative mechanism, and this tumor-promoting activity correlates with its ability to transactivate reporter genes in transient transfection assays. However, the mechanism by which mutant p53 functions in transactivation and its endogenous cellular targets that promote tumorigenicity are unknown. Mutant p53 is shown to be able to regulate the expression of the endogenous c-myc gene and is a potent activator of the c-myc promoter. Wild-type p53 normally represses p53. The region of mutant p53 responsiveness in the c-myc gene has been mapped to the 3' end of exon 1. The mutant p53 response region is position and orientation dependent and therefore does not function as an enhancer. Transactivation by mutant p53 requires the C terminus, which is not essential for wild-type p53 transactivation. These data suggest that it may be possible to selectively inhibit mutant p53 gain of function and consequently reduce the tumorigenic potential of cancer cells (Frazier, 1998).

Smad3 is a direct mediator of transcriptional activation by the TGFß receptor. Its target genes in epithelial cells include cyclin-dependent kinase inhibitors that generate a cytostatic reponse. This study defines how, in the same context, Smad3 can also mediate transcriptional repression of the growth-promoting gene c-myc. A complex containing Smad3, the transcription factors E2F4/5 and DP1, and the corepressor p107 preexists in the cytoplasm. In response to TGFbeta, this complex moves into the nucleus and associates with Smad4, recognizing a composite Smad-E2F site on c-myc for repression. Previously known as the ultimate recipients of cdk regulatory signals, E2F4/5 and p107 act here as transducers of TGFbeta receptor signals upstream of cdk. Smad proteins therefore mediate transcriptional activation or repression depending on their associated partners (Chen, 2002).

The liver is capable of completely regenerating itself in response to injury and after partial hepatectomy. In liver of old animals, the proliferative response is dramatically reduced, the mechanism for which is unknown. The liver specific protein, C/EBPalpha (see Drosophila Slbo), normally arrests proliferation of hepatocytes through inhibiting cyclin dependent kinases (cdks). Evidence that aging switches the liver-specific pathway of C/EBPalpha growth arrest to repression of E2F transcription. An age-specific C/EBPalpha-Rb-E2F4 complex has been identified that binds to E2F-dependent promoters and represses these genes. The C/EBPalpha-Rb-E2F4 complex occupies the c-myc promoter and blocks induction of c-myc in livers of old animals after partial hepatectomy. These results show that the age-dependent switch from cdk inhibition to repression of E2F transcription causes a loss of proliferative response in the liver because of an inability to induce E2F target genes after partial hepatectomy, providing a possible mechanism for the age-dependent loss of liver regenerative capacity (Iakova, 2003).

Rel/NF-kappaB transcription factors regulate the division and survival of B lymphocytes. B cells lacking NF-kappaB1 and c-Rel fail to increase in size upon mitogenic stimulation due to a reduction in induced c-myc expression. Mitogen-induced B cell growth, although not markedly impaired by FRAP/mTOR or MEK inhibitors, requires phosphatidylinositol 3-kinase (PI3K) activity. Inhibition of PI3K-dependent growth coincides with a block in the nuclear import of NF-kappaB1/c-Rel dimers and a failure to upregulate c-myc. In addition, PI3K has been shown to be necessary for a transcription-independent increase in c-Myc protein levels that accompanies mitogenic activation. Collectively, these findings establish a role for Rel/NF-kappaB signaling in the mitogen-induced growth of mammalian cells; such growth in B lymphocytes requires a PI3K/c-myc-dependent pathway (Grumont, 2003).

Neuronal precursor cells in the developing cerebellum require activity of the sonic hedgehog (Shh) and phosphoinositide-3-kinase (PI3K) pathways for growth and survival. Synergy between the Shh and PI3K signaling pathways are implicated in the cerebellar tumor medulloblastoma. A mechanism through which these disparate signaling pathways cooperate to promote proliferation of cerebellar granule neuron precursors is described. Shh signaling drives expression of mRNA encoding the Nmyc1 oncoprotein (previously N-myc), which is essential for expansion of cerebellar granule neuron precursors. The PI3K pathway stabilizes Nmyc1 protein via inhibition of GSK3-dependent Nmyc1 phosphorylation and degradation. The effects of PI3K activity on Nmyc1 stabilization are mimicked by insulin-like growth factor, a PI3K agonist with roles in central nervous system precursor growth and tumorigenesis. These findings indicate that Shh and PI3K signaling pathways converge on N-Myc to regulate neuronal precursor cell cycle progression. Furthermore, they provide a rationale for therapeutic targeting of PI3K signaling in medulloblastoma (Kenney, 2004).

Myc synergizes with Ras and PI3-kinase in cell transformation, yet the molecular basis for this behavior is poorly understood. Myc is shown to recruit TFIIH, P-TEFb and Mediator to the cyclin D2 and other target promoters, while the PI3-kinase pathway controls formation of the preinitiation complex and loading of RNA polymerase II. The PI3-kinase pathway involves Akt-mediated phosphorylation of FoxO transcription factors. In a nonphosphorylated state, FoxO factors inhibit induction of multiple Myc target genes, Myc-induced cell proliferation and transformation by Myc and Ras. Abrogation of FoxO function enables Myc to activate target genes in the absence of PI3-kinase activity and to induce foci formation in primary cells in the absence of oncogenic Ras. It is suggested that the cooperativity between Myc and Ras is at least in part due to the fact that Myc and FoxO proteins control distinct steps in the activation of an overlapping set of critical target genes (Bouchard, 2004).

Renal dysplasia, the major cause of childhood renal failure in humans, arises from perturbed renal morphogenesis and molecular signaling during embryogenesis. Induction of molecular crosstalk between Smad1 and ß-catenin occurs in the TgAlk3QD mouse model of renal medullary cystic dysplasia. The finding that Myc, a Smad and ß-catenin transcriptional target and effector of renal epithelial dedifferentiation, is misexpressed in dedifferentiated epithelial tubules provides a basis for investigating coordinate transcriptional control by Smad1 and ß-catenin in disease. Enhanced interactions occur between a molecular complex consisting of Smad1, ß-catenin and Tcf4 and adjacent Tcf- and Smad-binding regions located within the Myc promoter in TgAlk3QD dysplastic renal tissue, and Bmp-dependent cooperative control of Myc transcription by Smad1, ß-catenin and Tcf4. Analysis of nuclear extracts derived from TgAlk3QD and wild-type renal tissue revealed increased levels of Smad1/ß-catenin molecular complexes, and de novo formation of chromatin-associated Tcf4/Smad1 molecular complexes in TgAlk3QD tissues. Analysis of a 476 nucleotide segment of the 1490 nucleotide Myc genomic region upstream of the transcription start site demonstrated interactions between Tcf4 and the Smad consensus binding region and associations of Smad1, ß-catenin and Tcf4 with oligo-duplexes that encode the adjacent Tcf- and Smad-binding elements only in TgAlk3QD tissues. In collecting duct cells that express luciferase under the control of the 1490 nucleotide Myc genomic region, Bmp2-dependent stimulation of Myc transcription is dependent on contributions by each of Tcf4, ß-catenin and Smad1. These results provide novel insights into mechanisms by which interacting signaling pathways control transcription during the genesis of renal dysplasia (Hu, 2005)

Murine ES cells can be maintained as a pluripotent, self-renewing population by IL6 family members such as cytokine leukemia inhibitory factor (LIF) ---> STAT3-dependent signaling. The downstream effectors of this pathway have not been defined. A key target of the LIF self-renewal pathway was identified by showing that STAT3 directly regulates the expression of the Myc transcription factor. Murine ES cells express elevated levels of Myc and following LIF withdrawal, Myc mRNA levels collapse and Myc protein becomes phosphorylated on threonine 58 (T58), triggering its GSK3§ dependent degradation. Maintained expression of stable Myc (T58A) renders self-renewal and maintenance of pluripotency independent of LIF. By contrast, expression of a dominant negative form of Myc antagonizes self-renewal and promotes differentiation. Transcriptional control by STAT3 and suppression of T58 phosphorylation are crucial for regulation of Myc activity in ES cells and therefore in promoting self-renewal. Together, these results establish a mechanism for how LIF and STAT3 regulate ES cell self-renewal and pluripotency (Cartwright, 2005).

Although LIF/STAT3 signaling is crucial for murine ES cell maintenance, this pathway does not appear to have a role in human ES cell self-renewal, indicating the existence of alternate self-renewal mechanisms. A role has been defined for Wnt-dependent signaling in self-renewal of human and murine ES cells that functions independently of LIF and STAT3. Moreover, suppression of GSK3beta, an antagonist of Wnt signaling, is sufficient to maintain self-renewal and pluripotency of human and murine ES cells in the absence of LIF and Wnt. These observations signify a common mechanism of self-renewal that may be further applicable to adult stem cell populations that require Wnt-dependent signaling (Cartwright, 2005 and references therein).

Although LIF and Wnt promote self-renewal by activation of separate signaling pathways, it was reasoned that they would converge on a common target(s). It was hypothesized that Myc could be a common effector on which these signals converge because the Myc gene is a transcriptional target of STAT3 in a number of biological contexts, and signals transduced by Wnt can activate the Myc transcription through a §-catenin/TCF-dependent mechanism. Myc belongs to a family of helix-loop-helix/leucine zipper transcription factors and together with its obligatory binding partner, Max, performs roles in control of cell proliferation, transformation, growth, differentiation and apoptosis. A potential role for Myc in ES cell maintenance is suggested by two reports. (1) Expression of an RLF/L-myc minigene that frequently arises from a chromosomal translocation event in human small lung carcinomas, delays ES cell differentiation and interferes with early embryonic development. (2) Elevated Myc activity is able to block the differentiation of multiple cell lineages. These lines of evidence prompted an investigation of whether Myc plays a role in ES cell self-renewal downstream of LIF and/or Wnt. This report shows that elevated Myc activity is required for ES cell maintenance and that Myc is a key effector of the LIF/STAT3 self-renewal pathway. The data indicate that signals transduced by LIF and possibly Wnt, converge on Myc to maintain ES cell identity (Cartwright, 2005).

Shh signaling induces proliferation of many cell types during development and disease, but how Gli transcription factors regulate these mitogenic responses remains unclear. By genetically altering levels of Gli activator and repressor functions in mice, it has been demonstrated that both Gli functions are involved in the transcriptional control of N-myc and Cyclin D2 during embryonic hair follicle development. The results also indicate that additional Gli-activator-dependent functions are required for robust mitogenic responses in regions of high Shh signaling. Through posttranscriptional mechanisms, including inhibition of GSK3-β activity, Shh signaling leads to spatially restricted accumulation of N-myc and coordinated cell cycle progression. Furthermore, a temporal shift in the regulation of GSK3-β activity occurs during embryonic hair follicle development, resulting in a synergy with β-catenin signaling to promote coordinated proliferation. These findings demonstrate that Shh signaling controls the rapid and patterned expansion of epithelial progenitors through convergent Gli-mediated regulation (Mill, 2005).

The APC tumor suppressor controls the stability and nuclear export of β-catenin (β-cat), a transcriptional coactivator of LEF-1/TCF HMG proteins in the Wnt/Wg signaling pathway. β-cat and APC have opposing actions at Wnt target genes in vivo. The β-cat C-terminal activation domain associates with TRRAP/TIP60 and mixed-lineage-leukemia (MLL1/MLL2) SET1-type chromatin-modifying complexes in vitro, and β-cat promotes H3K4 trimethylation at the c-Myc gene in vivo. H3K4 trimethylation in vivo requires prior ubiquitination of H2B, and ubiquitin is found necessary for transcription initiation on chromatin but not nonchromatin templates in vitro. Chromatin immunoprecipitation experiments reveal that β-cat recruits Pygopus, Bcl-9/Legless, and MLL/SET1-type complexes to the c-Myc enhancer together with the negative Wnt regulators, APC, and βTrCP. Interestingly, APC-mediated repression of c-Myc transcription in HT29-APC colorectal cancer cells is initiated by the transient binding of APC, βTrCP, and the CtBP corepressor to the c-Myc enhancer, followed by stable binding of the TLE-1 and HDAC1 corepressors. Moreover, nuclear CtBP physically associates with full-length APC, but not with mutant SW480 or HT29 APC proteins. It is concluded that, in addition to regulating the stability of β-cat, APC facilitates CtBP-mediated repression of Wnt target genes in normal, but not in colorectal cancer cells (Sierra, 2006).

The data presented here support a model in which the APC tumor suppressor functions directly to counteract β-cat-mediated transcription at Wnt target genes in vivo. This possibility was first suggested by the finding that full-length APC cycles on and off the c-Myc enhancer in conjunction with β-cat and associated coactivators in LiCl-treated C2C12 cells. In contrast, the enhancer complex appears to be stable and does not cycle in HT29 CRC cells, which contain a Class II APC mutant protein that is unable to degrade β-cat. Most strikingly, the binding of the full-length APC protein to the c-Myc gene in HT29-APC cells correlates with the rapid disassembly of the Wnt enhancer complex in vivo and the subsequent decline in steady-state c-Myc mRNA levels, both of which significantly precede the drop in β-cat protein levels that occurs as a result of proteolytic degradation in the cytoplasm. Thus, the effect of APC on c-Myc transcription appears to be immediate and direct, and may serve to coordinate the switch between the β-cat coactivator and TLE1 corepressor complexes (Sierra, 2006).

The β-cat enhancer complex includes the Wnt coactivators Pygopus and Bcl-9/Lgs, which control the retention of β-cat in the nucleus and may also function directly in transcription. The observation that APC can also regulate nuclear transport of β-cat raises the possibility that these factors may reside within a larger regulatory complex that chaperones β-cat in and out of the nucleus and mediates its release from the DNA. Indeed, sequential ChIP (re-ChIP) data indicate that the mutant APC in HT29 colorectal cancer cells exists in a stable complex with β-cat and LEF-1 at the active c-Myc gene. This finding is unexpected because β-cat cannot bind simultaneously to APC and LEF-1, and thus, if the full-length APC is part of a larger β-cat:LEF enhancer complex, it may interact with other subunits. Alternatively, the full-length APC and β-cat may exist in different complexes that rapidly exchange at the enhancer. The current data indicate that targeting is mediated by the N-terminal half of the APC protein, and that CtBP and βTrCP appear only in conjunction with the full-length APC protein. How APC is recruited to Wnt enhancers remains an open and important question (Sierra, 2006).

The ChIP experiments also suggest that APC-mediated inhibition of c-Myc transcription in HT29 cells occurs in two steps, initiated by transient binding of APC, βTrCP, CtBP, and YY1 to the enhancer, and followed by stable binding of the TLE-1 and HDAC1 corepressors. The transient recruitment of APC and CtBP, at the time when β-cat, Bcl-9, Pygo, and other Wnt enhancer factors leave the DNA, strongly suggests a role for these factors in the exchange of Wnt coactivator and corepressor complexes. In this respect it is interesting that CtBP was shown recently to associate with APC, both in vivo and in vitro. The results confirm a high-affinity interaction between CtBP and the full-length APC protein induced in HT29-APC cells, as well as with the native (full-length) APC protein in 293 cells. Consequently, APC may function to recruit CtBP to Wnt enhancers. Although both CtBP and TLE-1 are well-established corepressors of Wnt target genes, the different functions of the two types of corepressors remain unclear, and the ChIP data suggest that they act at distinct steps. Together, these data suggest that APC counteracts β-cat function in the nucleus, as well as in the cytoplasm, and may facilitate turnover of the enhancer complex at responsive genes by recruiting βTrCP and CtBP (Sierra, 2006).

Human acute T-cell lymphoblastic leukemias and lymphomas (T-ALL) are commonly associated with gain-of-function mutations in Notch1 that contribute to T-ALL induction and maintenance. Starting from an expression-profiling screen, c-myc was identified as a direct target of Notch1 in Notch-dependent T-ALL cell lines, in which Notch accounts for the majority of c-myc expression. In functional assays, inhibitors of c-myc interfere with the progrowth effects of activated Notch1, and enforced expression of c-myc rescues multiple Notch1-dependent T-ALL cell lines from Notch withdrawal. The existence of a Notch-c-myc signaling axis was bolstered further by experiments using c-myc-dependent murine T-ALL cells, which are rescued from withdrawal of c-myc by retroviral transduction of activated Notch1. This Notch1-mediated rescue is associated with the up-regulation of endogenous murine c-myc and its downstream transcriptional targets, and the acquisition of sensitivity to Notch pathway inhibitors. Additionally, this study shows that primary murine thymocytes at the DN3 stage of development depend on ligand-induced Notch signaling to maintain c-myc expression. Together, these data implicate c-myc as a developmentally regulated direct downstream target of Notch1 that contributes to the growth of T-ALL cells (Weng, 2006; full text of article).

Notch signaling regulates myriad cellular functions by activating transcription, yet how Notch selectively activates different transcriptional targets is poorly understood. The core Notch transcriptional activation complex can bind DNA as a monomer, but it can also dimerize on DNA-binding sites that are properly oriented and spaced. However, the significance of Notch dimerization is unknown. This study shows that dimeric Notch transcriptional complexes are required for T-cell maturation and leukemic transformation but are dispensable for T-cell fate specification from a multipotential precursor. The varying requirements for Notch dimerization result from the differential sensitivity of specific Notch target genes. In particular, c-Myc and pre-T-cell antigen receptor α (Ptcra) are dimerization-dependent targets, whereas Hey1 and CD25 are not. These findings identify functionally important differences in the responsiveness among Notch target genes attributable to the formation of higher-order complexes. Consequently, it may be possible to develop a new class of Notch inhibitors that selectively block outcomes that depend on Notch dimerization (e.g., leukemogenesis) (Liu, 2010).

Replication origin associated with myc: The BAH domain facilitates the ability of human Orc1 protein to activate replication origins in vivo

Selection of initiation sites for DNA replication in eukaryotes is determined by the interaction between the origin recognition complex (ORC) and genomic DNA. In mammalian cells, this interaction appears to be regulated by Orc1, the only ORC subunit that contains a bromo-adjacent homology (BAH) domain. Since BAH domains mediate protein-protein interactions, the human Orc1 BAH domain was mutated, and the mutant proteins expressed in human cells to determine their affects on ORC function. The BAH domain was not required for nuclear localization of Orc1, association of Orc1 with other ORC subunits, or selective degradation of Orc1 during S-phase. It does, however, facilitate reassociation of Orc1 with chromosomes during the M to G1-phase transition, and it is required for binding Orc1 to the Epstein-Barr virus oriP and stimulating oriP-dependent plasmid DNA replication. Moreover, the BAH domain affects Orc1's ability to promote binding of Orc2 to chromatin as cells exit mitosis. Thus, the BAH domain in human Orc1 facilitates its ability to activate replication origins in vivo by promoting association of ORC with chromatin (Noguchi, 2006).

Eukaryotic DNA replication initiates at a large number of chromosomal origins, controlled by the ordered assembly of multiprotein replication complexes and the cell cycle-dependent activity of kinases that phosphorylate them. In cases where origins have been transposed to other chromosomal locations, they have been found to colocalize with genetically defined replicators, i.e., sequences capable of promoting DNA replication at ectopic genomic sites. In metazoan systems, replication origins or replicators are bound by homologues of proteins first characterized for the yeast Saccharomyces cerevisiae, suggesting that the basic mechanisms controlling replication initiation are conserved among eukaryotes. In S. cerevisiae, replicators typically comprise a binding site for the hexameric origin recognition complex ORC and a DNA unwinding element (DUE). ORC enables the Cdc6-, Cdt1-dependent recruitment of the MCM helicase complex to replication origins, forming a prereplication complex (pre-RC) early during the G1 phase of the cell cycle. Cyclin-dependent kinase and DDK activities promote the binding of Mcm10, Cdc45, and RPA to form preinitiation complexes and unwind the DNA template in advance of replication. The effect of kinase activity on the pre-RC is partially to disassemble ORC and release MCMs and Cdc6 from chromatin (Noguchi, 2006 and references therein).

The 2.4-kb 5' region of the human c-myc gene contains multiple transcription factor binding sites and a DUE that is unwound in vivo. The DUE is situated in a 100-bp zone containing three 10/11 matches to the S. cerevisiae ARS consensus sequence. It was initially reported that replication initiates in this region, and quantitative PCR (qPCR) has been used to define the replication initiation zone. Subsequent work has confirmed that replication initiates in the 5' flanking DNA of the c-myc gene in multiple species. The 2.4-kb c-myc core origin endows plasmids with ARS activity in vitro and shows replicator activity when moved to an ectopic chromosomal location. This region displays an ordered chromatin structure stable to chromosomal translocation, and mutational analyses have identified regions of the replicator essential for replication initiation, including the DUE (Noguchi, 2006 and references therein).

Chromatin immunoprecipitation (ChIP) was used in this work to show that the human analogs of the yeast ORC, MCM, and Cdc6 proteins bind preferentially and selectively to the c-myc replicator. The distributions of Mcm3 and Mcm7 are similar in asynchronous cells, with the greatest ChIP signal at, and upstream of, the DNA unwinding element. These distributions change in parallel in cells synchronized in G1 or M phases. By contrast, Orc1, Orc2, and Cdc6 appear to be least abundant at the DUE and each displays a different temporal pattern of replicator binding. The DNA unwinding element binding protein DUE-B, identified using the c-myc DUE as bait in a yeast one-hybrid assay, preferentially binds near the c-myc DUE in a pattern comparable to that of the MCMs in asynchronous and G1-phase cells. Furthermore, at an ectopic locus, c-myc replicator deletions that removed the DUE or altered chromatin structure suppressed DUE-B or Mcm3 binding, respectively, and eliminated origin activity. The relationship between chromatin structure, MCM binding, and origin activity is supported by the demonstration that inhibition of histone deacetylase activity by trichostatin A (TSA) causes a redistribution of Mcm3 binding similar to the broadening of the c-myc replication initiation zone. These results suggest that pre-RC proteins bind nonrandomly to the c-myc replicator and that c-myc origin activity is a function of ORC, MCM, Cdc6, and DUE-B binding to c-myc chromatin (Noguchi, 2006).

The c-Myc proto-oncogene encodes a transcription factor that is essential for cell growth and proliferation and is broadly implicated in tumorigenesis. However, the biological functions required by c-Myc to induce oncogenesis remain elusive. This study shows that c-Myc has a direct role in the control of DNA replication. c-Myc interacts with the pre-replicative complex and localizes to early sites of DNA synthesis. Depletion of c-Myc from mammalian (human and mouse) cells as well as from Xenopus cell-free extracts, which are devoid of RNA transcription, demonstrates a non-transcriptional role for c-Myc in the initiation of DNA replication. Overexpression of c-Myc causes increased replication origin activity with subsequent DNA damage and checkpoint activation. These findings identify a critical function of c-Myc in DNA replication and suggest a novel mechanism for its normal and oncogenic functions (Dominguez-Sola, 2007).

Minichromosome maintenance (MCM) proteins are part of the pre-replicative complex, a multiprotein complex essential for the assembly and activity of DNA replication origins13. Indeed, all MCM2-MCM7 subunits, ORC2, Cdc6 and Cdt1, were present in the affinity-purified Myc complex, consistent with the known interaction of Myc with MCM2 and MCM7. In contrast, proteins involved in DNA replication elongation (MCM10, RPA and PCNA) were absent. The interaction with pre-replicative complex components was also observed with N-Myc. Other proteins forming complexes with Myc, such as TRRAP18, were not found in this Myc and pre-replicative-complex-associated complex, whereas small, non-stoichiometrical amounts of Max (Myc-associated factor X) were detectable (Dominguez-Sola, 2007).

Myc and pre-replicative complex proteins co-sedimented in high molecular mass fractions (approx1.7 MDa) after glycerol density gradient sedimentation and size-exclusion chromatography of Myc-bound protein complexes. Notably, Myc is also present in a distinct set of fractions that contained the majority of Max protein that co-purified with this complex). These fractions also contained MCM5, which might be involved in other transcriptional complexes. Overall, these results identify a novel Myc-associated complex in mammalian cells that contains pre-replicative complex components and thus suggests a direct role of Myc in DNA replication (Dominguez-Sola, 2007).

It has been proposed that Myc promotes G1/S transition and DNA replication through the transcription of factors promoting S-phase entry and/or cell growth. The current results indicate that Myc control of DNA replication is not dependent on its transcriptional activity in both Xenopus extracts and mammalian cells. Nonetheless, transcriptional regulation of critical target genes may also be an important component of the overall role of Myc in regulating DNA replication initiation. Notably, the transactivation domain of Myc is required to control both DNA replication initiation and transcriptional activity, suggesting that Myc may use a common molecular mechanism to facilitate both DNA transactions. This mechanism might involve Myc-dependent chromatin modifications such as histone acetylation, which might also be implicated in the selection of replication origins (Dominguez-Sola, 2007).

The results indicate that Myc deregulation generates DNA damage and may promote genomic instability by inducing DNA replication stress, strengthening previous observations. This notion is also supported by the dependence on Werner RecQ helicase for Myc-driven proliferation (Grandori, 2003), and by the requirement for RecQ helicases during replication stress. These observations can explain the occurrence of genomic alterations, such as gene amplification and illegitimate replication of some loci, that are consistently associated with Myc deregulation during tumorigenesis. However, in contrast with other oncogenes that may cause DNA re-replication when deregulated, overexpression of Myc increases the number of active replication origins in the absence of detectable re-replication (Dominguez-Sola, 2007).

The results also suggest that the p53-dependent G2/M checkpoint and subsequent apoptosis observed in mammalian cells carrying deregulated Myc alleles may be due to DNA damage generated predominantly during S phase. Frequent p53 inactivation in tumours carrying deregulated Myc genes may then reflect selection for tumoural cells with disabled checkpoint responses. Thus, these results suggest that Myc may exert its oncogenic function, at least in part, by promoting origin activity, thereby inducing replication stress and genomic instability (Dominguez-Sola, 2007).

Myc degradation

Myc is an oncoprotein transcription factor that plays a prominent role in cancer. Like many transcription factors, Myc is an unstable protein that is destroyed by ubiquitin (Ub)-mediated proteolysis. The oncoprotein and Ub ligase Skp2 regulate Myc ubiquitylation and stability. Because of the growing number of Ub ligases that function as transcriptional coactivators, it has been speculated that Skp2 might also regulate Myc's transcriptional activity. Consistent with this model, Skp2 has been shown to be a transcriptional coactivator for Myc, recognizing an essential element within the Myc activation domain and activating Myc target genes. These data suggest that Skp2 functions to connect Myc activity and destruction, and reveal an unexpected oncoprotein connection that may play an important role in controlling cell growth in normal and cancer cells (Kim, 2003).

The ability of Skp2 to stimulate the transcriptional activity of Myc reveals a previously unanticipated function for Skp2 -- regulation of gene expression. This activity places Skp2 in an emerging group of Ub ligases that are transcriptional coactivators and suggests that one way in which Skp2 regulates cell growth is via transcriptional control of Myc and perhaps other transcription factors. In this regard, it is interesting that Skp2 also targets the transcription factor E2F-1 for destruction. It has been proposed that Skp2-mediated destruction of E2F-1 allows cells to exit from S phase, but, on the basis of these findings, it is tempting to speculate that Skp2 may also stimulate E2F's transcriptional activity. Interestingly, the Skp2-E2F-1 interaction may also be phosphorylation independent, although there is no obvious homology between the Skp2-interacting sites on E2F-1 and Myc (Kim, 2003 and references therein).

It has been argued that Skp2 controls cell proliferation through its ability to target destruction of the CDK inhibitor p27. This, however, cannot be the only essential function of Skp2 in mammalian cell growth control, because although Skp2 collaborates with Ras to induce cellular transformation, Ras alone will not transform p27-deficient fibroblasts. The observation that Skp2 stimulates Myc transcriptional activity reveals a second pathway through which Skp2 controls cell growth. It is suggested that, as cells approach S phase, increasing levels of Skp2 not only target destruction of p27 but also serve to transiently activate the Myc protein. The transient activation of Myc synergizes with p27 downregulation to enforce the commitment of cells to enter S phase. As the cell cycle proceeds and Skp2 levels drop, the self-limiting action of activator licensing results in Myc destruction and attenuation of the signal to proliferate (Kim, 2003).

It is suggested that the ability of Skp2 to both activate Myc and destroy p27 is likely to have a significant role in the development of cancer. It is intriguing to note that loss of p27 has been shown to cooperate with Myc overexpression to drive lymphomagenesis in mice, suggesting that p27 can antagonize Myc function. Perhaps, therefore, one reason why Skp2 is overexpressed in cancer is that it confers a unique growth advantage, not only stimulating Myc's transcriptional activity but at the same time destroying a potential inhibitor of Myc function (Kim, 2003).

The transcription regulatory oncoprotein c-Myc controls genes involved in cell growth, apoptosis, and oncogenesis. c-Myc is turned over very quickly through the ubiquitin/proteasome pathway. Skp2 is shown to interact with c-Myc and participates in c-Myc ubiquitylation and degradation. The interaction between Skp2 and c-Myc occurs during the G1 to S phase transition of the cell cycle in normal lymphocytes. Surprisingly, Skp2 enhances c-Myc-induced S phase transition and activates c-Myc target genes in a Myc-dependent manner. Further, Myc-induced transcription is Skp2 dependent, suggesting interdependence between c-Myc and Skp2 in activation of transcription. Moreover, Myc-dependent association of Skp2, ubiquitylated proteins, and subunits of the proteasome to a c-Myc target promoter has been demonstrated in vivo. The results suggest that Skp2 is a transcriptional cofactor for c-Myc and indicate a close relationship between transcription activation and transcription factor ubiquitination (von der Lehr, 2003).

In what way could E3 ligase activity stimulate c-Myc-induced transcription? One possibility is that SCFSkp2 ubiquitylates and degrades negative regulators of transcription at the promoter. This could also be part of an autoregulatory loop, where the Myc activator protein needs to be eliminated at some step in order to complete the transcription cycle. Another possibility is that ubiquitin modifications of c-Myc or other substrates at the promoter play a nonproteolytic function in, for instance, protein-protein interactions of importance for transcription. It remains to be determined whether degradation of c-Myc is a necessary step for activation of transcription. This general model has been proposed in the 'licensing' hypothesis, linking transcription factor activity to their destruction in order to maintain stringent control of transcription activation in cells (von der Lehr, 2003).

Cerebellar granule cells are the most abundant neurons in the brain, and granule cell precursors (GCPs) are a common target of transformation in the pediatric brain tumor medulloblastoma. Proliferation of GCPs is regulated by the secreted signaling molecule Sonic hedgehog (Shh), but the mechanisms by which Shh controls proliferation of GCPs remain inadequately understood. DNA microarrays have been used to identify targets of Shh in these cells; Shh was found to activate a program of transcription that promotes cell cycle entry and DNA replication. Among the genes most robustly induced by Shh are cyclin D1 and N-myc. N-myc transcription is induced in the presence of the protein synthesis inhibitor cycloheximide, so it appears to be a direct target of Shh. Retroviral transduction of N-myc into GCPs induces expression of cyclin D1, E2F1, and E2F2, and promotes proliferation. Moreover, dominant-negative N-myc substantially reduces Shh-induced proliferation, indicating that N-myc is required for the Shh response. Finally, cyclin D1 and N-myc are overexpressed in murine medulloblastoma. These findings suggest that cyclin D1 and N-myc are important mediators of Shh-induced proliferation and tumorigenesis (Oliver, 2003).

Rapid Myc protein turnover is critical for maintaining basal levels of Myc activity in normal cells and a prompt response to changing growth signals. A new Myc-interacting factor, TRPC4AP (transient receptor potential cation channel, subfamily C, member 4-associated protein)/TRUSS (tumor necrosis factor receptor-associated ubiquitous scaffolding and signaling protein) has been characterized, that is the receptor for a DDB1 (damage-specific DNA-binding protein 1)-CUL4 (Cullin 4) E3 ligase complex for selective Myc degradation through the proteasome. TRPC4AP/TRUSS binds specifically to the Myc C terminus and promotes its ubiquitination and destruction through the recognition of evolutionarily conserved domains in the Myc N terminus. TRPC4AP/TRUSS suppresses Myc-mediated transactivation and transformation in a dose-dependent manner. TRPC4AP/TRUSS expression was found to be strongly down-regulated in most cancer cell lines, leading to Myc protein stabilization. These studies identify a novel pathway targeting Myc degradation that is suppressed in cancer cells (Choi, 2010).

Ubiquitylation of the amino terminus of Myc by SCFβ-TrCP antagonizes SCFFbw7-mediated turnover

The SCFFbw7 ubiquitin ligase mediates growth-factor-regulated turnover of the Myc oncoprotein. This study shows that SCFβ-TrCP binds to Myc by means of a characteristic phosphodegron and ubiquitylates Myc; this results in enhanced Myc stability. SCFFbw7 and SCFβ-TrCP can exert these differential effects through polyubiquitylation of the amino terminus of Myc. Whereas SCFFbw7 with the Cdc34 ubiquitin-conjugating enzyme specifically requires lysine 48 (K48) of ubiquitin, SCFβ-TrCP uses the UbcH5 ubiquitin-conjugating enzyme to form heterotypic polyubiquitin chains on Myc. Ubiquitylation of Myc by SCFβ-TrCP is required for Myc-dependent acceleration of cell cycle progression after release from an arrest in S phase. Therefore, alternative ubiquitylation events at the N terminus can lead to the ubiquitylation-dependent stabilization of Myc (Popov, 2010).

Deregulated expression of the c-myc proto-oncogene occurs in multiple human tumours, and many experiments with transgenic animals document the oncogenic potential of enhanced c-myc expression. c-myc encodes a nuclear transcription factor, Myc, which can both activate and repress transcription. One of the central functions of Myc is to enhance expression of a broad spectrum of genes involved in nucleotide biogenesis, ribosomal biogenesis and translation. In addition, Myc induces the expression of several cyclins and suppresses the transcription of cyclin-dependent kinase inhibitors. By means of both mechanisms, Myc promotes exit from quiescence and stimulates progression through G1 phase. Elevated expression of Myc also accelerates progression through S phase of the cell cycle. Conversely, depletion or loss of Myc delays cell cycle progression through S and G2 phases (Popov, 2010).

Myc protein is unstable and is subject to continuous ubiquitylation and degradation in the proteasome. At least four ubiquitin ligases have been identified that ubiquitylate Myc and regulate its turnover. The binding motif is known for one of them, SCFFbw7 (SCF stands for Skp1/Cul1/F-box protein). The SCFFbw7 complex recognizes Myc that is phosphorylated at threonine 58 (T58) by glycogen synthase kinase 3 (Gsk3). Because Gsk3 is inactivated by Akt-dependent phosphorylation, degradation by SCFFbw7 links Myc turnover to growth-factor-dependent signalling. Several mechanisms disrupt the Fbw7-dependent degradation of Myc in human tumours; for example, point mutations of T58 occur in plasmacytoma, and mutations in FBW7 are found in multiple human tumours. Less is known about the recognition of Myc by the other ubiquitin ligases: the F-box ubiquitin ligase SCFSkp2 and the Truss/Ddb1/Cul4 complex bind to the carboxy terminus of Myc; SCFSkp2 also binds to MycBoxII, a short sequence that is essential for all biological functions of Myc. The Hect-domain ubiquitin ligase HectH9 (ARF-BP1, Huwe1) binds to both N-Myc and c-Myc and mediates the turnover of N-Myc. Skp2 and HectH9 also positively affect Myc function, because they are required for the activation and repression of a subset of Myc target genes (Popov, 2010).

Degradation of Myc by SCFFbw7 has been implicated in controlling Myc stability in G1 phase. In contrast, little is known about the regulation of Myc stability in S and G2 phases. Because mammalian cells are less dependent on external growth factors after passage through the restriction point in late G1, it is possible that Myc is protected from SCFFbw7-mediated degradation during later phases of the cell cycle. For example, interaction of Aurora A with the N-Myc protein in neuroblastoma cells antagonizes its degradation by SCFFbw7 in G2 phase. This study shows that the SCFFbw7 and SCFβ-TrCP ubiquitin ligases assemble functionally distinct polyubiquitin chains on the N terminus of Myc and that ubiquitylation by SCFβ-TrCP thereby attenuates the degradation of Myc (Popov, 2010).

The TRIM-NHL protein TRIM32 activates microRNAs and prevents self-renewal in mouse neural progenitors

In the mouse neocortex, neural progenitor cells generate both differentiating neurons and daughter cells that maintain progenitor fate. This study shows that the TRIM-NHL protein TRIM32 regulates protein degradation and microRNA activity to control the balance between those two daughter cell types. In both horizontally and vertically dividing progenitors, TRIM32 becomes polarized in mitosis and is concentrated in one of the two daughter cells. TRIM32 overexpression induces neuronal differentiation while inhibition of TRIM32 causes both daughter cells to retain progenitor cell fate. TRIM32 ubiquitinates and degrades the transcription factor c-Myc but also binds Argonaute-1 and thereby increases the activity of specific microRNAs. Let-7 is one of the TRIM32 targets and is required and sufficient for neuronal differentiation. TRIM32 is the mouse ortholog of Drosophila Brat and Mei-P26 and might be part of a protein family that regulates the balance between differentiation and proliferation in stem cell lineages (Schwamborn, 2009).

The data suggest that the increased levels of TRIM32 in one of the two daughter cells contribute to the decision of this cell to undergo neuronal differentiation. Like Brat, TRIM32 localizes asymmetrically in mitosis. Brat is localized by binding to Miranda, which, in turn, is recruited to the basal side by the protein Lgl and excluded from the apical side by aPKC (Knoblich, 2008). In fly neuroblasts, aPKC promotes self-renewal whereas Lgl inhibits proliferation. Although Miranda is not conserved, mouse Lgl and aPKC have similar effects on neural progenitor proliferation. In Lgl knockout mice, neural precursors overproliferate and eventually die by apoptosis. Removing one of the two aPKC mouse homologs does not affect the rate of neurogenesis, but depletion of its binding partner Par-3 results in premature cell-cycle exit of cortical progenitors. Despite these similarities, the precise mechanism by which TRIM32 localizes may be quite distinct. In Drosophila, the apical Par-3/6/aPKC complex directs the basal localization of Brat and Miranda but also orients the mitotic spindle along the apical-basal axis. In mice, however, the vast majority of progenitor divisions do not occur along the apical-basal axis. TRIM32 is asymmetric even in those planar divisions and provides a suitable explanation for how unequal fates can be generated independently of cleavage plane orientation. Therefore, the relevance of TRIM32 segregation is independent of the somewhat conflicting results that have been reported for the fraction of horizontal versus vertical divisions. Since TRIM32 asymmetry does not follow the polarity set up by Par-3/6/aPKC, however, it is likely that it is established by mechanisms distinct from Drosophila (Schwamborn, 2009).

What could those mechanisms be? TRIM32 often concentrates in the retracting basal fiber, a structure that is not present in Drosophila neuroblasts. TRIM32 might be present in the cytoplasm of the fiber and could be retained in the basal part of the cell during mitosis, when the fiber becomes extremely thin and its cytoplasm flows into the dividing progenitor. This would explain why TRIM32 is asymmetric even when the spindle is not oriented along the apical-basal axis. Since TRIM32-GFP expression prevents mitosis even at low levels, this observation cannot be verified by live imaging. The model would predict that the cell inheriting the basal fiber preferentially undergoes neuronal differentiation. This is in good agreement with some previous live-imaging studies, but other studies have actually proposed that the fiber is maintained in mitosis and serves as a guide for migration of the newly formed neuron. At the moment, it cannot be excluded that other mechanisms contribute to the asymmetric localization of TRIM32 (Schwamborn, 2009).

How does TRIM32 affect proliferation and differentiation? The data suggest that TRIM32 acts through two distinct pathways. Through its N-terminal RING finger, TRIM32 ubiquitinylates c-Myc and targets it for proteasome-mediated degradation. High levels of c-Myc are important for the ability of NSCs to self-renew and make NSCs relatively easy targets for reprogramming into ES cells. Furthermore, the bFGF–SHP2–ERK–c-Myc–Bmi-1 pathway is critical for the self-renewal capacity of neural progenitor cells, and Myc overexpression is known to promote neural progenitor proliferation in the mouse CNS. Therefore, a TRIM32-mediated reduction in the levels of c-Myc may well serve as a first step to induce neuronal differentiation. In agreement with this, overexpression of c-Myc in GFAP-positive astrocytes promotes formation of less differentiated Nestin-positive progenitor-like cells while a conditional ablation of the c-Myc ortholog N-Myc in mouse neuronal progenitor cells dramatically increases neuronal differentiation (Schwamborn, 2009).

Through its C-terminal NHL domain, TRIM32 acts as a potent activator of certain microRNAs. Although Drosophila Mei-P26 also binds Ago1, it inhibits rather than enhances microRNAs, and the mechanisms by which TRIM32 and its invertebrate homologs regulate microRNAs may actually be quite distinct. This is consistent with the observation that microRNAs support self-renewal in Drosophila stem cells while they potentiate differentiation in mammalian stem cells. In particular, Let-7a has an antiproliferative effect, and its expression reduces tumor growth and can prevent self-renewal in breast cancer cells. In NSCs, Let-7a is expressed and upregulated during differentiation. It is interesting to note that one of the targets for Let-7a is Myc. Protein degradation and concomitant translational inhibition through microRNAs might be the key strategy through which TRIM32 induces differentiation in NSCs (Schwamborn, 2009).

Although brat and mei-P26 mutant flies develop tumors, TRIM32 has not been described as a tumor suppressor. In fact, several reports have even suggested that TRIM32 might induce rather than prevent tumor formation. TRIM32 is mutated in patients carrying limb girdle muscular dystrophy type 2H. Since TRIM32 expression is upregulated during myogenic differentiation, the muscular dystrophy in these patients could be explained by a differentiation defect in the satellite cell lineage analogous to the one found in NSC lineages. TRIM32 has also been described as a gene potentially responsible for Bardet-Biedl syndrome and therefore has also been named BBS11. Distinct TRIM32 mutations are responsible for the two diseases, but none of them seems to cause cancer since an increase in tumor formation is not described for any of the two diseases. Since TRIM32 is a bifunctional molecule, mutating only the RING or the NHL domain might not be sufficient to prevent the antiproliferative function of TRIM32. In Drosophila, tumors only form in a small subset of brat mutant neuroblasts (Bowman, 2008). In other neuroblasts, redundancy with other tumor suppressors prevents overproliferation. Should a similar degree of redundancy exist in vertebrates, this might explain why TRIM32 is not a common target for oncogenic mutations. A similar lack of a human tumor phenotype has been shown for the Drosophila tumor suppressor Lgl. In Drosophila, lgl mutant neuroblasts overproliferate and form brain tumors. In mice, however, lgl mutant neural progenitors overproliferate initially but then die by apoptosis. A vertebrate-specific mechanism that prevents tumorigenesis in response to stem cell overproliferation could provide an alternative explanation for the lack of tumor formation when TRIM32 function is compromised. Although such a mechanism has been suggested previously the underlying mechanism remains unclear (Schwamborn, 2009).

These data establish TRIM-NHL proteins as a family of conserved stem cell regulators. The fact that Mei-P26 regulates stem cell proliferation in Drosophila ovaries (Neumuller, 2008) suggests that the function of this protein family might extend way beyond the brain. If this is the case, the presence of a catalytically active RING finger domain that could be inhibited by pharmaceutical compounds might make these proteins attractive targets for the manipulation of stem cell proliferation and the stimulation of regeneration in vivo (Schwamborn, 2009).

Transcriptional targets of MYC

The c-Myc protein is involved in cell proliferation, differentiation and apoptosis though heterodimerization with Max to form a transcriptionally active sequence-specific DNA binding complex. By means of sequential immunoprecipitation of chromatin using anti-Max and anti-Myc antibodies, a Myc-regulated gene and genomic sites occupied by Myc-Max in vivo have been identified. Four of 27 sites recovered by this procedure correspond to the highest affinity 'canonical' CACGTG sequence. However, the most common in vivo binding sites belong to the group of 'non-canonical' E box-related binding sites previously identified by in vitro selection. Several of the genomic fragments isolated contain transcribed sequences, including one, MrDb, encoding an evolutionarily conserved RNA helicase of the DEAD box family. The corresponding mRNA is induced following activation of a Myc-estrogen receptor fusion protein (Myc-ER) in the presence of a protein synthesis inhibitor, consistent with this helicase gene being a direct target of Myc-Max. As for c-Myc, the expression of MrDb is induced upon proliferative stimulation of primary human fibroblasts as well as B cells and down-regulated during terminal differentiation of HL60 leukemia cells. These results indicate that (1) Myc-Max heterodimers interact in vivo with a specific set of E box-related DNA sequences, and (2) that Myc is likely to activate multiple target genes, including a highly conserved DEAD box protein. Therefore, Myc may exert its effects on cell behavior through proteins that affect RNA structure and metabolism (Grandori, 1996). MrDb is a homolog of the Drosophila gene pitchoune.

Cad is the trifunctional enzyme carbamoyl-phosphate synthase/aspartate transcarbamoylase/dihydroorotase, which is required for the first three rate-limiting steps of pyrimidine biosynthesis. The expression of virtually all proposed c-Myc target genes is unchanged in cells containing a homozygous null deletion of c-myc. Two noteworthy exceptions are the gene cad, which has reduced log phase expression and serum induction in c-myc null cells, and the growth arrest gene gadd45, which is derepressed by c-myc knockout. Thus, cad and gadd45 are the only proposed targets of c-Myc that may contribute to the dramatic slow growth phenotype of c-myc null cells. These results demonstrate that a loss-of-function approach is critical for the evaluation of potential c-Myc target genes (Bush, 1998).

The c-Myc protein is a site-specific DNA-binding transcription factor that is up-regulated in a number of different cancers. Binding of Myc correlates with increased transcription of the cad promoter. The mechanism by which Myc mediates transcriptional activation of the cad gene has been investigated. Using a chromatin immunoprecipitation assay, high levels of RNA polymerase II were found bound to the cad promoter in quiescent NIH 3T3 cells and in differentiated U937 cells, even though the promoter is inactive. However, chromatin immunoprecipitation with an antibody that recognizes the hyperphosphorylated form of the RNA polymerase II carboxyl-terminal domain (CTD) reveals that phosphorylation of the CTD does correlate with c-Myc binding and cad transcription. The c-Myc transactivation domain interacts with cdk9 and cyclin T1, components of the CTD kinase P-TEFb. Furthermore, activator bypass experiments have shown that direct recruitment of cyclin T1 to the cad promoter can substitute for c-Myc to activate the promoter. In summary, these results suggest that c-Myc activates transcription of cad by stimulating promoter clearance and elongation, perhaps via recruitment of P-TEFb (Eberhardy, 2001).

Telomere maintenance has been proposed as an essential prerequisite to human tumor development. The telomerase enzyme is itself a marker for tumor cells, but the genetic alterations that activate the enzyme during neoplastic transformation have remained a mystery. Myc induces telomerase in both normal human mammary epithelial cells (HMECs) and normal human diploid fibroblasts. Myc increases expression of hEST2 (hTRT/TP2), the limiting subunit of telomerase, and both Myc and hEST2 can extend the life span of HMECs. The ability of Myc to activate telomerase may contribute to its ability to promote tumor formation (Wang, 1998).

E-cadherin plays a pivotal role in the biogenesis of the first epithelium during development, and its down-regulation is associated with metastasis of carcinomas. Inactivation of RB family proteins by simian virus 40 large T antigen (LT) in MDCK epithelial cells results in a mesenchymal conversion associated with invasiveness and a down-regulation of c-Myc. Reexpression of RB or c-Myc in such cells allows the reexpression of epithelial markers, including E-cadherin. Both RB and c-Myc specifically activate transcription of the E-cadherin promoter in epithelial cells but not in NIH 3T3 mesenchymal cells. This transcriptional activity is mediated in both cases by the transcription factor AP-2. In vitro AP-2 and RB interaction involves the N-terminal domain of AP-2 and the oncoprotein binding domain and C-terminal domain of RB. In vivo physical interaction between RB and AP-2 has been demonstrated in MDCK and HaCat cells. In LT-transformed MDCK cells, LT, RB, and AP-2 were all coimmunoprecipitated by each of the corresponding antibodies, and a mutation of the RB binding domain of the oncoprotein inhibits its binding to both RB and AP-2. Taken together, these results suggest that there is a tripartite complex between LT, RB, and AP-2 and that the physical and functional interactions between LT and AP-2 are mediated by RB. Moreover, they define RB and c-Myc as coactivators of AP-2 in epithelial cells and shed new light on the significance of the LT-RB complex, linking it to the dedifferentiation processes occurring during tumor progression. These data confirm the important role for RB and c-Myc in the maintenance of the epithelial phenotype and reveal a novel mechanism of gene activation by c-Myc (Batsche, 1998).

A set of c-Myc-responsive genes has been identified in the Rat1a fibroblast through the application of cDNA representational difference analysis (RDA) to cDNAs isolated from nonadherent Rat1a and Rat1a-myc cells. In this system, c-Myc overexpression is sufficient to induce the transformed phenotype of anchorage-independent growth. Twenty differentially expressed cDNAs have been identified, several of which represent novel cDNA sequences. One of the novel cDNAs identified in this screen, termed rcl, is (1) directly stimulated by c-Myc; (2) stimulated in the in vivo growth system of regenerating rat liver, as is c-myc, and (3) elevated in human lymphoid cells that overexpress c-myc. The Rcl protein was found to be a 23-kDa nuclear protein. Ectopic expression of the protein encoded by the rcl cDNA induces anchorage-independent growth in Rat1a fibroblasts, albeit to a diminished extent compared to ectopic c-Myc expression. These data suggest a role for rcl during cellular proliferation and c-Myc-mediated transformation (Lewis, 1997).

The c-Myc protein activates transcription as part of a heteromeric complex with Max. However, Myc-transformed cells are characterized by loss of expression of several genes, suggesting that Myc may also repress gene expression. Two-hybrid cloning identifies a novel POZ domain Zn finger protein (Miz-1; Myc-interacting Zn finger protein-1) that specifically interacts with Myc, but not with Max or USF. Miz-1 binds to start sites of both the adenovirus major late promoter and the cyclin D1 promoter; it activates transcription from both promoters. Miz-1 has a potent growth arrest function. Binding of Myc to Miz-1 requires the helix-loop-helix domain of Myc and a short amphipathic helix located in the carboxy-terminus of Miz-1. Expression of Myc inhibits transactivation, overcomes Miz-1-induced growth arrest and renders Miz-1 insoluble in vivo. These processes depend on the association of Myc and Miz-1, and on the integrity of the POZ domain of Miz-1, suggesting that Myc binding activates a latent inhibitory function for this domain. Fusion of a nuclear localization signal induces efficient nuclear transport of Miz-1 and impairs the ability of Myc to overcome transcriptional activation and growth arrest by Miz-1. These data suggest a model for how gene repression by Myc may occur in vivo (Peukert, 1997).

Cell proliferation is regulated by the induction of growth promoting genes and the suppression of growth inhibitory genes. Malignant growth can result from the altered balance of expression of these genes in favor of cell proliferation. Induction of the transcription factor, c-Myc, promotes cell proliferation and transformation by activating growth promoting genes, including the ODC and cdc25A genes. c-Myc transcriptionally represses the expression of a growth arrest gene, gas1. A conserved Myc structure, Myc box 2, is required for repression of gas1, and for Myc induction of proliferation and transformation, but not for activation of ODC. Activation of a Myc-estrogen receptor fusion protein by 4-hydroxytamoxifen is sufficient to repress gas1 gene transcription. These findings suggest that transcriptional repression of growth arrest genes, including gas1, is one step in the promotion of cell growth by Myc (Lee, 1997).

A problem common to many investigators is uncertainty as to which member of a family of DNA-binding transcription factors regulates a specific promoter in intact cells. Determining target gene specificity requires both an analysis of protein binding to the endogenous promoter as well as a characterization of the functional consequences of transcription factor binding. By using a formaldehyde crosslinking procedure and Gal4 fusion proteins, the timing and functional consequences of the binding of Myc and upstream stimulatory factor (USF)1 to endogenous cellular genes has been analyzed. The endogenous cad promoter (cad is carbamoyl-phosphate synthase/aspartate carbamoyltransferase/dihydroorotase, a growth responsive gene) can be immunoprecipitated with antibodies against Myc and USF1. Although both Myc and USF1 can bind to cad, the cad promoter can respond only to the Myc transactivation domain. The amount of Myc bound to the cad promoter fluctuates in a growth-dependent manner. Thus, these data on both DNA binding and promoter activity in intact cells suggest that cad is a Myc target gene. In addition, Myc binding can occur at many sites in vivo but the position of the binding site determines the functional consequences of this binding. These data indicate that a post-DNA-binding mechanism determines Myc target gene specificity. Importantly, the feasibility of analyzing the binding of site-specific transcription factors in vivo to single copy mammalian genes has been demonstrated (Boyd, 1998).

The lactate dehydrogenase A (LDH-A) gene, whose product participates in normal anerobic glycolysis and is frequently increased in human cancers, has been identified as a c-Myc-responsive gene. It was of interest, therefore, to compare the effect of glucose deprivation in c-Myc-transformed and nontransformed cells. Glucose deprivation or treatment with the glucose antimetabolite 2-deoxyglucose causes nontransformed cells to arrest in the G0/G1 phase of the cell cycle. In contrast, c-Myc-transformed fibroblasts, lymphoblastoid, or lung carcinoma cells undergo extensive apoptosis. Ectopic expression of LDH-A alone in Rat1a fibroblasts is sufficient to induce apoptosis with glucose deprivation but not with serum withdrawal, suggesting that LDH-A mediates the unique apoptotic effect of c-Myc when glycolysis is blocked. The apoptosis caused by glucose deprivation is blocked by Bcl-2 expression but appears to be independent of wild-type p53 activity. These studies provide insights on the coupling of glucose metabolism and the cell cycle in c-Myc-transformed cells and may in the future be exploited for cancer therapeutics (Shim, 1998).

To identify genes regulated by N-myc, subtraction of whole embryo cDNA was carried out between wild type and N-myc-deficient mutant mice. Six cDNA clones were isolated as representing genes expressed at higher levels in the mutant embryos and two as those expressed at lower levels. One of them, Ndr1, coding for 43 kDa cytoplasmic protein, was studied in detail. The Ndr1 gene is augmented 20-fold in the mutant embryos at 10.5 days of development, which is indicative of repression by N-myc. An inverse relationship exists between the expression of N-myc and Ndr1 in various developing tissues of the wild type embryos. In the early stage of differentiation of these tissues, when N-myc expression is high, Ndr1 expression is low or undetectable, and later when N-myc activity diminishes, Ndr1 expression is augmented concomitantly with the occurrence of terminal differentiation. To establish the direct link between N-myc activity and the Ndr1 regulation, the Ndr1 gene was cloned and analyzed. The Ndr1 promoter activity is down-regulated by N-myc, and more strongly by the combination of N-myc and Max in the cotransfection assay. This repressive effect is mediated by the promoter region within 52 base pairs from the transcription start site but direct binding of N-myc:Max to the promoter sequence has not been demonstrated. This failure is analogous to other cases reported for transcriptional repression by c-myc. c-myc also represses Ndr1 promoter activity similar to N-myc. The effect of N-myc:Max is sensitive to Trichostatin A, indicating involvement of histone deacetylase activity in repression of the Ndr1 promoter. The strategy adopted in identifying target genes should prove widely applicable when animals mutant for given transcription factors are available (Shimono, 1999).

Cell number is regulated by maintaining a balance between cell proliferation and cell death through apoptosis. Key regulators of this balance include the oncogene product c-Myc, which promotes either entry into the cell cycle or apoptosis. Although the mechanism of c-Myc-induced apoptosis remains unclear, it is susceptible to regulation by survival factors and can proceed through the interaction of Fas ligand (FasL) with its receptor, Fas. Activated T lymphocytes are eliminated by an apoptotic process known as activation-induced cell death (AICD), which requires the transcriptional induction of FasL expression and sustained levels of c-Myc. The FasL promoter can be driven by c-Myc overexpression, and functional inhibitors of Myc and its binding partner, Max, inhibit the transcriptional activity of the FasL promoter. A non-canonical binding site (ATTCTCT) was identified for c-Myc-Max heterodimers in the FasL promoter, which, when mutated, abolishes activity in response to c-Myc. Exchange of the canonical c-Myc responsive elements (CACGTG) in the ornithine decarboxylase (ODC) promoter with the non-canonical sequence in the FasL promoter generates an ODC-FasL promoter that is significantly more responsive to c-Myc than the wild-type ODC promoter. These findings identify a precise physiological role for c-Myc in the induction of apoptosis as a transcriptional regulator of the FasL gene (Kasibhatla, 2000).

Why should FasL expression in lymphocytes be linked to the expression and function of c-Myc? Only activated, proliferating cells, and not resting lymphocytes, express c-Myc. Since clonal expansion forms the basis of immune responses, it is only the proliferating cells that may represent a threat to the body should they happen to be auto-reactive (or even hyper-reactive, since these will also cause extensive bystander damage). Re-stimulation of activated cells therefore induces FasL, which in turn serves to check cellular expansion by induction of apoptosis. Alternatively (and non-exclusively), activated, proliferating lymphocytes that take on effector functions do so, in part, through the expression of FasL (except in this case they are resistant to Fas-mediated death), which functions to kill Fas-expressing cells with which they come in contact. By limiting this expression to proliferating cells, it serves as a fail-safe mechanism to ensure that once the cells cease to express c-Myc (for example, at the cessation of the response) expression of this lethal molecule will also cease (Kasibhatla, 2000).

c-Myc plays a key role in the cell cycle dependent control of the PDGF ß-receptor mRNA. The mouse platelet-derived growth factor (PDGF) ß-receptor promoter contains a CCAAT motif, and NF-Y plays an essential role in its transcription. NF-Y is a trimeric CCAAT-binding factor with histone fold subunits (NF-YB/NF-YC) and bipartite activation domains located on NF-YA and NF-YC. Coexpression of c-Myc represses PDGF ß-receptor luciferase reporter activity, and the CCAAT motif in the promoter is indispensable for this repression. c-Myc binds NF-Y subunits, YB and YC. The in vitro-translated c-Myc also binds the glutathione S-transferase (GST)-NF-YB fusion protein and GST-NF-YC, but not GST-NF-YA. The most C-terminal region of HAP domains of NF-YB and NF-YC, and the Myc homology boxes, but not the C-terminal bHLHZip domain, are indispensable for the coimmunoprecipitation, and also for the repression of PDGF ß-receptor. c-Myc binds NF-Y complex without affecting the efficiency of NF-Y binding to DNA. However, the expression of Myc represses the transcriptional activation of NF-YC when fused to the GAL4 DNA binding domain. Furthermore, this repression is seen only when Myc homology boxes are present, and NF-YC contains the c-Myc binding region (Izumi, 2001).

Myc transcription factor induces transcription of the E2F1, E2F2, and E2F3 genes. Using primary mouse embryo fibroblasts deleted for individual E2F genes, it has now been shown that Myc-induced S phase and apoptosis requires distinct E2F activities. The ability of Myc to induce S phase is impaired in the absence of either E2F2 or E2F3 but not E2F1 or E2F4. In contrast, the ability of Myc to induce apoptosis is markedly reduced in cells deleted for E2F1 but not E2F2 or E2F3. From this data, it is proposed that the induction of specific E2F activities is an essential component in the Myc pathways that control cell proliferation and cell fate decisions (Leone, 2001).

Deregulated Myc expression results in both the induction of S phase and the induction of apoptosis if survival factors are limiting. How these processes are linked is not well understood. A variety of possible Myc target genes have been identified, including the recent description of Id2. Like E2F2 and E2F3, Id2 appears to be essential for Myc-induced cell proliferation but not for Myc-induced apoptosis. In addition, these studies have also identified a role for Id2 in the control of Rb function, since the loss of Id2 function can partially suppress the phenotype resulting from loss of Rb. As such, it has been proposed that one role for Myc in the stimulation of cell growth is the induction of Id2, leading to inactivation of Rb. Nevertheless, since the loss of Id2 does not fully suppress an Rb null phenotype (mice die at birth), and previous work has shown a suppression of Rb phenotype as a result of loss of either E2F1, E2F2, or E2F3 function, it seems likely that the control of E2F proteins and interactions with Id2 are both important for Rb function. Moreover, given the ability of Myc to induce both Id2 and E2Fs, it is concluded that the induction of both groups of activities is likely to be an important function of Myc (Leone, 2001).

In addition to results showing the requirement for distinct E2Fs to mediate Myc-induced S phase versus apoptosis, other work has also suggested that distinct downstream events mediate these two functions of Myc. In particular, cdk activation has been shown to be necessary for the Myc-mediated induction of proliferation but not apoptosis. Although the lack of a cdk requirement for Myc-induced apoptosis might suggest an E2F-independent event, since E2F accumulation is normally regulated by Rb through cdk-mediated phosphorylation of Rb, previous work has demonstrated an ability of Myc to induce E2F1 accumulation in the absence of cdk activity, presumably by bypassing the normal Rb control. Thus, Myc-induced apoptosis could bypass the need for the cell cycle machinery by directly activating E2F1 (Leone, 2001).

Myc oncoproteins promote cell cycle progression in part through the transcriptional up-regulation of the cyclin D2 gene. Myc is bound to the cyclin D2 promoter in vivo. Binding of Myc induces cyclin D2 expression and histone acetylation at a single nucleosome in a MycBoxII/TRRAP-dependent manner. TRRAP is a component of TIP60 and PCAF/GCN5 histone acetyl transferase (HAT) complexes (see Drosophila Pcaf). Down-regulation of cyclin D2 mRNA expression in differentiating HL60 cells is preceded by a switch of promoter occupancy from Myc/Max to Mad/Max complexes, loss of TRRAP binding, increased HDAC1 binding, and histone deacetylation. Thus, recruitment of TRRAP and regulation of histone acetylation are critical for transcriptional activation by Myc (Bouchard, 2001).

The aim of this study was to resolve the role of MBII (an effector domain of Myc that binds TRRAP) and TRRAP in gene activation by Myc, using an endogenous target gene of Myc, cyclin D2, as model. Upon binding to the cyclin D2 promoter, Myc recruits TRRAP and induces the preferential acetylation of histone H4 at a single nucleosome. Conversely, loss of endogenous Myc binding correlates with histone deacetylation and loss of TRRAP binding during the TPA-induced differentiation of a human promyelocytic cell line, HL60. The integrity of MBII is required for TRRAP recruitment, histone acetylation, and transcriptional activation at the cyclin D2 locus. Therefore, previous suggestions that MBII has no role in transcriptional activation based on transient reporter assays need to be reevaluated. Deletion of the entire N terminus of Myc up to MBII (s-Myc) renders Myc unable to induce cell cycle progression and expression of either cyclin A or cyclin D2 in 3T3 fibroblasts, consistent with recent results that the N terminus of Myc is required for regulation of proliferation and induction of gene expression in a cell-type-dependent manner. Most likely, this is because stable association with TRRAP requires sequences in the N terminus of Myc in addition to MBII (Bouchard, 2001 and references therein).

Mad proteins are thought to antagonize the function of Myc by recruiting a repressor complex that contains histone deacetylase activity. Observations suggest that this model applies to the cyclin D2 promoter: (1) repression of the cyclin D2 promoter by Mad1 requires the integrity of an N-terminal domain, which mediates recruitment of histone deacetylase complexes through interaction with Sin3A (see Drosophila Sin3A); (2) during HL60 differentiation, Mad1 and HDAC1 are corecruited to the cyclin D2 promoter, correlating with histone deacetylation of both histones H3 and H4 at the cyclin D2 promoter. Taken together, these data strongly support a model in which endogenous Myc/Max and Mad/Max complexes contribute to the regulation of transcription of the cyclin D2 gene through their antagonistic effects on histone acetylation. In addition, these findings show the functional relevance of the switch between Myc/Max and Mad/Max complexes during differentiation of hematopoietic cells. Recent work on the gene encoding the catalytic subunit of telomerase, htert, suggests that this model also may apply to this promoter (Bouchard, 2001 and references therein).

Up-regulation of the CAD (carbamoyl phosphate synthase, aspartate transcarbamylase, dihydroorotase) gene by Myc does not involve changes in histone acetylation. Instead, high levels of histone acetylation at the promoter were found in both quiescent and proliferating cells, showing that Myc can control at least one step in addition to histone acetylation to promote active transcription. Additional proteins have been identified that bind to different domains of Myc and that are candidates for such an activity: for example, the C terminus of Myc binds to Ini1, a component of the Swi/Snf family of chromatin-remodeling complexes. Clearly, a detailed analysis of the role of Myc in activation of individual promoters will be required before the role of each interaction in Myc biology can be resolved fully (Bouchard, 2001 and references therein).

The Period2 gene plays a key role in controlling circadian rhythm in mice. Mice deficient in the mPer2 gene are cancer prone. After gamma radiation, these mice show a marked increase in tumor development and reduced apoptosis in thymocytes. The core circadian genes are induced by gamma radiation in wild-type mice but not in mPer2 mutant mice. Temporal expression of genes involved in cell cycle regulation and tumor suppression, such as Cyclin D1, Cyclin A, Mdm-2, and Gadd45alpha, is deregulated in mPer2 mutant mice. In particular, the transcription of c-myc is controlled directly by circadian regulators and is deregulated in the mPer2 mutant. BMAL1/NPAS2 or BMAL1/CLOCK heterodimers likely repress transcription of c-myc through E box-mediated reactions in the P1 promoter, and mPer2 can suppress c-myc expression indirectly through stimulating Bmal1 transcription. Deregulation of Bmal1 in mPer2m/m cells, therefore, results in c-myc overexpression. These studies suggest that the mPer2 gene functions in tumor suppression by regulating DNA damage-responsive pathways (Fu, 2002).

Based on recent discoveries from c-myc studies and the results of this study, a model is proposed for the role of mPer2 in tumor suppression. In this model, the loss of mPer2 function results in dampened Bmal1 expression and decreased intracellular levels of BMAL1/NPAS2 or BMAL1/CLOCK heterodimers, leading to the derepression of c-myc throughout 24 hr L/D cycles. Overexpression of c-myc causes genomic DNA damage and eventually leads to hyperplasia and tumor development. Following gamma radiation, the loss of mPer2 function partially impairs p53-mediated apoptosis, leading to accumulation of damaged cells. However, the mPer2m/m cells, expressing c-myc at elevated levels, can still progress through cell cycle in the presence of genomic DNA damage, resulting in the high incidence of tumor development after gamma radiation (Fu, 2002).

Activation of the tumor suppressor p53 by DNA damage induces either cell cycle arrest or apoptotic cell death. The cytostatic effect of p53 is mediated by transcriptional activation of the cyclin-dependent kinase (CDK) inhibitor p21Cip1, whereas the apoptotic effect is mediated by transcriptional activation of mediators including PUMA and PIG3. What determines the choice between cytostasis and apoptosis is not clear. The transcription factor Myc is shown to be a principal determinant of this choice. Myc is directly recruited to the p21Cip1 promoter by the DNA-binding protein Miz-1. This interaction blocks p21Cip1 induction by p53 and other activators. As a result Myc switches, from cytostatic to apoptotic, the p53-dependent response of colon cancer cells to DNA damage. Myc does not modify the ability of p53 to bind to the p21Cip1 or PUMA promoters, but selectively inhibits bound p53 from activating p21Cip1 transcription. By inhibiting p21Cip1 expression Myc favors the initiation of apoptosis, thereby influencing the outcome of a p53 response in favor of cell death (Seoane, 2002).

Several conclusions can be drawn from these results. Myc selectively targets p21Cip1 in the p53 transcriptional program, sparing the ability of p53 to induce the expression of PUMA or PIG3. Myc does not alter the ability of p53 to bind to the p21Cip1 promoter but inhibits p21Cip1 transcriptional activation by promoter-bound p53. In the presence of p21, p53 can still bind to the PUMA promoter and induce the accumulation of its product, but apoptosis is not achieved. Thus, the p21-dependent block in apoptosis maps to a step downstream of the DNA damage-p53-PUMA pathway. The mechanism for this provocative observation is not obvious. These results suggest a model in which Myc selectively prevents p53-dependent transcriptional activation of p21Cip1, enabling pro-apoptotic factors such as PUMA to execute a cell death program. Thus, these results define, in mechanistic terms, how one element of the cellular context, that is, the level of Myc activity, can determine the outcome of the p53 response. Although it remains to be seen whether repression of p21Cip1 would be beneficial in cancer treatment, the mechanism proposed here suggests ways to influence the cell's response to stresses that result in activation of p53 (Seoane, 2002).

The c-myc proto-oncogene encodes a ubiquitous transcription factor involved in the control of cell growth and implicated in inducing tumorigenesis. Understanding the function of c-Myc and its role in cancer depends upon the identification of c-Myc target genes. Nijmegen breakage syndrome (NBS) is a chromosomal-instability syndrome associated with cancer predisposition, radiosensitivity, and chromosomal instability. The NBS gene product, NBS1 (p95 or nibrin: Drosophila homolog Nbs), is a part of the hMre11 complex, a central player associated with double-strand break (DSB) repair. NBS1 contains domains characteristic for proteins involved in DNA repair, recombination, and replication. This study shows that c-Myc directly activates NBS1. c-Myc-mediated induction of NBS1 gene transcription occurs in different tissues, is independent of cell proliferation, and is mediated by a c-Myc binding site in the intron 1 region of NBS1 gene. Overexpression of NBS1 in Rat1a cells increased cell proliferation. These results indicate that NBS1 is a direct transcriptional target of c-Myc and links the function of c-Myc to the regulation of DNA DSB repair pathway operating during DNA replication (Chiang, 2003).

In hypoxic cells, HIF-1alpha escapes from oxygen-dependent proteolysis and binds to the hypoxia-responsive element (HRE) for transcriptional activation of target genes involved in angiogenesis and glycolysis. The G1 checkpoint gene p21(cip1)is activated by HIF-1alpha with a novel mechanism that involves the HIF-1alpha PAS domains to displace Myc binding from p21(cip1) promoter. This HIF-1alpha-Myc pathway may account for up- and down-regulation of other hypoxia-responsive genes that lack the HRE. Moreover, the role of HIF-1alpha in cell cycle control indicates a dual, yet seemingly conflicting, nature of HIF-1alpha: promoting cell growth and arrest in concomitance. It is speculated that a dynamic balance between the two processes is achieved by a 'stop-and-go' strategy to maintain cell growth and survival. Tumor cells may adopt such a scheme to evade the killing by chemotherapeutic agents (Koshiji, 2004).

The glutamate transporter gene, EAAT2/GLT-1, is induced by epidermal growth factor (EGF) and downregulated by tumor necrosis factor alpha (TNFalpha). While TNFalpha is generally recognized as a positive regulator of NF-kappaB-dependent gene expression, its ability to control transcriptional repression is not well characterized. Additionally, the regulation of NF-kappaB by EGF is poorly understood. Both TNFalpha-mediated repression and EGF-mediated activation of EAAT2 expression require NF-kappaB. EGF activates NF-kappaB independently of signaling to IkappaB. Furthermore, TNFalpha can abrogate IKKbeta- and p65-mediated activation of EAAT2. These results suggest that NF-kappaB can intrinsically activate EAAT2 and that TNFalpha mediates repression through a distinct pathway also requiring NF-kappaB. Consistently, it was found that N-myc is recruited to the EAAT2 promoter with TNFalpha and that N-myc-binding sites are required for TNFalpha-mediated repression. Moreover, N-myc overexpression inhibits both basal and p65-induced activation of EAAT2. These data highlight the remarkable specificity of NF-kappaB activity to regulate gene expression in response to diverse cellular signals and have implications for glutamate homeostasis and neurodegenerative disease (Sitcheran, 2005).

The ability of NF-kappaB to regulate EAAT2 expression has important implications for the regulation of glutamate homeostasis in the CNS. To prevent the overstimulation of neuronal glutamate receptors that can trigger excitotoxic mechanisms and cell death, extracellular concentrations of excitatory amino acids are tightly controlled by transport systems on both neurons and glial cells. EAAT2 is critical for rapid clearance of synaptically released glutamate for proper neurotransmission. Accumulation of excessive glutamate levels in neuronal synapses can lead to excitotoxic neuronal death, which has been implicated in the pathogenesis of numerous neurodegenerative diseases, as well as CNS injury resulting from stroke and ischemia. Notably, these conditions have been associated with increased NF-kappaB activity, and reduced EAAT2 expression is observed after brain injury and in patients with Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis and multiple sclerosis. Interestingly, EAAT2 may also have a role in development, as work in Drosophila demonstrates that this transporter is involved in terminal glial cell differentiation. Moreover, reduced glial cell populations are observed in mice lacking the EGF receptor suggesting that EGF signaling is also important for glial cell differentiation. Based on the work in this study, it is proposed that positive regulation of EAAT2 by NF-kappaB in response to EGF may promote glial cell differentiation and uptake of synaptic glutamate by glial cells, whereas TNF-mediated inhibition of EAAT2 by NF-kappaB may contribute to glutamate toxicity and cell death in neuroinflammation and disease (Sitcheran, 2005).

Myc, transcriptional activation and repression and chromatin

The protein encoded by the c-MYC proto-oncogene is a transcription factor that can both activate and repress the expression of target genes, but few of its transcriptional targets have been identified. c-MYC is shown to repress the expression of the heavy subunit of the protein ferritin (H-ferritin), which sequesters intracellular iron, and to stimulate the expression of the iron regulatory protein-2 (IRP2), which increases the intracellular iron pool. Down-regulation of the expression of the H-ferritin gene is required for cell transformation by c-MYC. These results indicate that c-MYC coordinately regulates genes controlling intracellular iron concentrations and that this function is essential for the control of cell proliferation and transformation by c-MYC. The finding that c-MYC controls iron metabolism is consistent with observations that iron chelation leads to growth arrest and decreased synthesis of the cell cycle regulators cdc2 and cyclin A, whereas increased iron availability up-regulates the activity of ribonucleotide reductase (Wu, 1999a).

The MYC proto-oncogene encodes a ubiquitous transcription factor (c-MYC) involved in the control of cell proliferation and differentiation. Deregulated expression of c-MYC caused by gene amplification, retroviral insertion, or chromosomal translocation is associated with tumorigenesis. The function of c-MYC and its role in tumorigenesis are poorly understood because few c-MYC targets have been identified. c-MYC has a direct role in induction of the activity of telomerase, the ribonucleoprotein complex expressed in proliferating and transformed cells, in which it preserves chromosome integrity by maintaining telomere length. c-MYC activates telomerase by inducing expression of its catalytic subunit, telomerase reverse transcriptase (TERT). Telomerase complex activity is dependent on TERT, a specialized type of reverse transcriptase. TERT and c-MYC are expressed in normal and transformed proliferating cells, downregulated in quiescent and terminally differentiated cells, and both can induce immortalization when constitutively expressed in transfected cells. Consistent with the recently reported association between MYC overexpression and induction of telomerase activity, the TERT promoter contains numerous c-MYC-binding sites that mediate TERT transcriptional activation. c-MYC-induced TERT expression is rapid and independent of cell proliferation and additional protein synthesis, consistent with direct transcriptional activation of TERT. These results indicate that TERT is a target of c-MYC activity and identify a pathway linking cell proliferation and chromosome integrity in normal and neoplastic cells (Wu, 1999b).

Transcription repression by the basic region-helix-loop-helix-zipper (bHLHZip) protein Mad1 requires DNA binding as a ternary (three protein) complex with Max and mSin3A or mSin3B, the mammalian orthologs of the S. cerevisiae transcriptional corepressor SIN3. The interaction between Mad1 and mSin3 is mediated by three potential amphipathic alpha-helices: one in the N terminus of Mad (mSin interaction domain, or SID) and two within the second paired amphipathic helix domain (PAH2) of mSin3A. Mutations that alter the structure of the SID inhibit in vitro interaction between Mad and mSin3 and inactivate Mad's transcriptional repression activity. A 35-residue region containing the SID represents a dominant repression domain whose activity can be transferred to a heterologous DNA binding region. A fusion protein comprising the Mad1 SID linked to a Ga14 DNA binding domain mediates repression of minimal as well as complex promoters dependent on Ga14 DNA binding sites. In addition, the SID represses the transcriptional activity of linked VP16 and c-Myc transactivation domains. When fused to a full-length c-Myc protein, the Mad1 SID specifically represses both c-Myc's transcriptional and transforming activities. Fusions between the GAL DNA binding domain and full-length mSin3 are also capable of repression. The association between Mad1 and mSin3 is not only dependent on the helical SID but is also dependent on both putative helices of the mSin3 PAH2 region, suggesting that stable interaction requires all three helices. These data indicate that the SID is necessary and sufficient for transcriptional repression mediated by the Mad protein family and that SID repression is dominant over several distinct transcriptional activators (Ayer, 1996).

Documented interactions among members of the Myc superfamily support a yin-yang model for the regulation of Myc-responsive genes in which transactivation-competent Myc-Max heterodimers are opposed by repressive Mxi1-Max or Mad-Max complexes. Analysis of mouse mxi1 has led to the identification of two mxi1 transcript forms possessing open reading frames that differ in their capacity to encode a short amino-terminal alpha-helical domain. The presence of this segment dramatically augments the suppressive potential of Mxi1 and allows for association with a mammalian protein that is structurally homologous to the yeast transcriptional repressor SIN3. These findings provide a mechanistic basis for the antagonistic actions of Mxi1 on Myc activity that appears to be mediated in part through the recruitment of a putative transcriptional repressor (Schreiber-Agus, 1995).

The bHLH-ZIP protein Mad heterodimerizes with Max as a sequence-specific transcriptional repressor. Mad is rapidly induced upon differentiation, and the associated switch from Myc-Max to Mad-Max heterocomplexes seem to repress genes normally activated by Myc-Max. Two related mammalian cDNAs have been identified that encode Mad-binding proteins. Both possess sequence homology with the yeast transcription repressor Sin3, including four conserved paired amphipathic helix (PAH) domains. mSin3A and mSin3B bind specifically to Mad and the related protein Mxi1. Mad-Max and mSin3 form ternary complexes in solution that specifically recognize the Mad-Max E box-binding site. Mad-mSin3 association requires PAH2 of mSin3A/mSin3B and the first 25 residues of Mad, which contains a putative amphipathic alpha-helical region. Point mutations in this region eliminate interaction with mSin3 proteins and block Mad transcriptional repression. It is suggested that Mad-Max represses transcription by tethering mSin3 to DNA as corepressors and that a transcriptional repression mechanism is conserved from yeast to mammals (Ayer, 1995).

mSin3A, a corepressor that binds to the transcription factor Mad and appears to tether MAD to histone deacetylase, in contrast to other repressors, is not detected in the complex between Retinoblastoma protein and histone deacetylase. Interaction between domain A and B in the Rb pocket forms a site for association with histone deacetylase. Recruitment of histone deacetylase by either Rb or Mad results in a decrease in acetylated histone H3 associated with the promoter in vivo, consistent with the idea that this recruitment indeed results in deacetylation of histones bound to the promoter. This Rb-mediated recruitment of histone deacetylase can only repress a subset of promoters and transcription factors. Repression of the adenovirus major late promoter by Rb and Mad is dependent on histone deacetylase activity, while repression of the tyrosine kinase promoter and the SV40 enhancer by Rb is independent of histone deacetylase activity (Luo, 1998).

Normal mammalian growth and development are highly dependent on the regulation of the expression and activity of the Myc family of transcription factors. Mxi1-mediated inhibition of Myc activity requires interaction with mammalian Sin3A or Sin3B proteins, which are purported to act as scaffolds for additional co-repressor factors. The identification of two such Sin3-associated factors, the nuclear receptor co-repressor (N-CoR) and histone deacetylase (HD1), provides a basis for Mxi1/Sin3-induced transcriptional repression and tumour suppression. The involvement of histone deacetylase suggests that the silencing function of Mxi1 involves a modification of chromatin involving deacetylation, converting chromatin into a form that impedes the interaction of the transcriptional apparatus with promoter regions (Alland, 1997).

Members of the Mad family of bHLH-Zip proteins heterodimerize with Max to repress transcription in a sequence-specific manner. Transcriptional repression by Mad:Max heterodimers is mediated by ternary complex formation with either of the corepressors mSin3A or mSin3B. mSin3A is an in vivo component of large, heterogeneous multiprotein complexes and is tightly and specifically associated with at least seven polypeptides. Two of the mSin3A-associated proteins, p50 and p55, are highly related to the histone deacetylase HDAC1. The mSin3A immunocomplexes possess histone deacetylase activity that is sensitive to the specific deacetylase inhibitor trapoxin. mSin3A-targeted repression is reduced by trapoxin treatment, suggesting that histone deacetylation mediates transcriptional repression through Mad-Max-mSin3A multimeric complexes (Hassig, 1997).

Transcriptional repression by mammalian nuclear receptors has been correlated with the binding of the putative co-repressor, N-CoR to nuclear receptors. A complex has been identified that contains N-CoR, the Mad presumptive co-repressor mSin3, and the histone deacetylase mRPD3, and which is required for both nuclear receptor- and Mad-dependent repression, but not for repression by transcription factors of the ets-domain family. mSin3 and mRPD3 are required to mediate thyroid-hormone receptor mediated repression. These data predict that the ligand-induced switch of heterodimeric nuclear receptors from repressor to activator functions involves the exchange of complexes containing histone deacetylases with those that have histone acetylase activity. This work provides a molecular mechanism for integrating chromatin remodelling and interactions with the core transcriptional machinery. A model is presented for the reversal of repression. Upon binding of activating ligands, the co-repressor complex dissociates from nuclear receptors and is replaced by a co-activator complex containing Creb binding protein (CBP, a factor with intrinsic histone acetylation activity), the histone chaperone P/CAF, NCoA-1/SRC-1 (a steroid receptor coactivator), and a factor termed p/CIP (Heinzel, 1997).

Transcriptional repression by Mad-Max heterodimers requires interaction of Mad with the corepressors mSin3A/B. Sin3p, the S. cerevisiae homolog of mSin3, functions in the same pathway as Rpd3p, a protein related to two recently identified mammalian histone deacetylases, HDAC1 and HDAC2. mSin3A and HDAC1/2 are associated in vivo. HDAC2 binding requires a conserved region of mSin3A capable of mediating transcriptional repression. Mad1 forms a complex with mSin3 and HDAC2 that contains histone deacetylase activity. Trichostatin A, an inhibitor of histone deacetylases, abolishes Mad repression. It is proposed that Mad-Max functions by recruiting the mSin3-HDAC corepressor complex that deacetylates nucleosomal histones, producing alterations in chromatin structure that block transcription (Laherty, 1997).

The protooncogene MYC plays an important role in the regulation of cellular proliferation, differentiation, and apoptosis and has been implicated in a variety of human tumors. MYC and the closely related MYCN encode highly conserved nuclear phosphoproteins (Myc and NMyc) that apparently function as transcription factors in the cell. A large and highly conserved nuclear protein has been identified that interacts directly with the transcriptional activating domain of Myc (designated "protein associated with Myc" or Pam). Pam contains an extended amino acid sequence with similarities to a protein known as regulator of chromosome condensation (RCC1), which may play a role in the function of chromatin. RCC1 contains a motif of 50-60 aa that is repeated seven times in tandem. These repeats form a seven-bladed propeller structure as determined recently by X-ray crystallography. A similar 7-fold repeat is present in Pam, but is divided into two elements (RHD-1 and RHD-2) by an insertion of 134 aa after the fourth repeat. The insertion between RHD-1 and RHD-2 contains a C-terminal region of 55 aa that is rich in basic amino acids. Such a short basic region (40-50 aa) is also present in RCC1 proteins at their N termini. These N termini are important for chromatin binding. Although most RCC1 proteins end with a repeated element, the Drosophila RCC1 protein BJ1 has a substantial C-terminal extension, which has limited homology to chromatin proteins such as Xenopus histone-binding protein N1/N2. Pam contains a similar region, situated in the midst of a serine-rich domain and in the vicinity of the Myc-binding domain, but relatively distant from RHD-1/2. The gene encoding Pam (PAM) is expressed in all of the human tissue examined, but expression is exceptionally abundant in brain and thymus. Pam binds specifically to Myc, but not NMyc. The region in Myc required for binding to Pam includes a domain that is essential for the function of Myc and that is frequently mutated in Burkitt's lymphomas. PAM is located within a 300-kb region on chromosome 13q22 (Guo, 1998).

The Myc protein binds DNA and activates transcription by mechanisms that are still unclear. Chromatin immunoprecipitation (ChIP) was used to evaluate Myc-dependent changes in histone acetylation at seven target loci. Upon serum stimulation of Rat1 fibroblasts, Myc associates with chromatin, histone H4 becomes locally hyperacetylated, and gene expression is induced. These responses are lost or severely impaired in Myc-deficient cells, but are restored by adenoviral delivery of Myc simultaneous with mitogenic stimulation. When targeted to chromatin in the absence of mitogens, Myc directly induces H4 acetylation. In addition, Myc recruits TRRAP to chromatin, consistent with a role for this cofactor in histone acetylation. Finally, unlike serum, Myc alone is very inefficient in inducing expression of most target genes. Myc therefore governs a step, most likely H4 acetylation, that is required but not sufficient for transcriptional activation. It is proposed that Myc acts as a permissive factor, allowing additional signals to activate target promoters (Frank, 2001).

A new quantitative proteomics technology has been applied to the analysis of the function of the Myc oncoprotein in mammalian cells. Employing isotope-coded affinity tag (ICATTM) reagent labeling and tandem mass spectrometry, the global pattern of protein expression in rat myc-null cells has been compared with that of myc-plus cells (myc-null cells in which myc has been introduced) to generate a differential protein expression catalog. Expression differences among many functionally related proteins were identified, including reduction of proteases, induction of protein synthesis pathways and upregulation of anabolic enzymes in myc-plus cells, which are predicted to lead to increased cell mass (cell growth). In addition, reduction in the levels of adhesion molecules, actin network proteins and Rho pathway proteins were observed in myc-plus cells, leading to reduced focal adhesions and actin stress fibers as well as altered morphology. These effects are dependent on the highly conserved Myc Box II region. These results reveal a novel cytoskeletal function for Myc and indicate the feasibility of quantitative whole-proteome analysis in mammalian cells (Shiio, 2002).

Accumulating evidence suggests that Myc influences cell growth (defined as an increase in cell mass). Myc overexpression was shown to increase cell size both in Drosophila and mammalian cells. Using global protein expression analysis, a more comprehensive view of the mode of action of Myc on cell growth has been obtained. In Myc(+) cells there is an increase in many proteins implicated in protein biosynthesis, a decrease in different proteases and an increase in several anabolic enzymes. Consistent with the observed augmentation in the levels of many ribosomal protein subunits, proteins implicated in rRNA processing and assembly (fibrillarin, Nop56, Nop58, Bop1, DDX5, DDX17 and DDX21), and a translation initiation factor (eIF2B), the rate of protein synthesis was increased by nearly 3-fold in Myc(+) cells when compared with Myc(-) cells. The increased levels of anabolic enzymes (such as fatty acid synthase, adenylate kinase and cad) should result in increased synthesis of fatty acids, nucleotides, amino acids and ATP. Collectively, the increase in these biomolecules may account for the growth stimulatory effects of Myc. The Rho pathway has also been shown to negatively regulate cell and organism size, raising the possibility that downregulation of the Rho pathway by Myc plays some role in cell growth in addition to the well established role in cytoskeletal organization (Shiio, 2002).

The Myc transcription factor is an essential mediator of cell growth and proliferation through its ability to both positively and negatively regulate transcription. The mechanisms by which Myc silences gene expression are not well understood. The current model is that Myc represses transcription through functional interference with transcriptional activators. Myc is shown to bind the corepressor DNA CpG methyltransferase Dnmt3a and associate with DNA methyltransferase activity in vivo. In cells with reduced Dnmt3a levels, specific reactivation of the Myc-repressed p21Cip1 gene is seen, whereas the expression of Myc-activated E-boxes genes is unchanged. In addition, it was found that Myc can target Dnmt3a selectively to the promoter of p21Cip1. Myc is known to be recruited to the p21Cip1 promoter by the DNA-binding factor Miz-1. Consistent with this, Myc and Dnmt3a form a ternary complex with Miz-1 and this complex can corepress the p21Cip1 promoter. Finally, it is shown that DNA methylation is required for Myc-mediated repression of p21Cip1. These data identify a new mechanism by which Myc can silence gene expression not only by passive functional interference but also by active recruitment of corepressor proteins. Furthermore, these findings suggest that targeting of DNA methyltransferases by transcription factors is a wide and general mechanism for the generation of specific DNA methylation patterns within a cell (Brenner, 2005).

Myc and growth

The c-Myc oncoprotein plays an important role in the growth and proliferation of normal and neoplastic cells. To execute these actions, c-Myc is thought to regulate functionally diverse sets of genes that directly govern cellular mass and progression through critical cell cycle transitions. Several lines of evidence are provided that c-Myc promotes ubiquitin-dependent proteolysis by directly activating expression of the Cul1 gene, encoding a critical component of the ubiquitin ligase SCFSKP2. The cell cycle inhibitor p27kip1 is a known target of the SCFSKP2 complex, and Myc-induced Cul1 expression matches well with the kinetics of declining p27kip1 protein. Enforced Cul1 expression or antisense neutralization of p27kip1 is capable of overcoming the slow-growth phenotype of c-Myc null primary mouse embryonic fibroblasts (MEFs). In reconstitution assays, the addition of in vitro translated Cul1 protein alone is able to restore p27kip1 ubiquitination and degradation in lysates derived from c-myc/MEFs or density-arrested human fibroblasts. These functional and biochemical data provide a direct link between c-Myc transcriptional regulation and ubiquitin-mediated proteolysis and together support the view that c-Myc promotes G1 exit in part via Cul1-dependent ubiquitination and degradation of the CDK inhibitor, p27kip1 (O'Hagan, 2000).

Activated lymphocytes must increase in size and duplicate their contents (cell growth) before they can divide. The molecular events that control cell growth in proliferating lymphocytes and other metazoan cells are still unclear. Transgenesis has been utilized to provide evidence suggesting that the basic helix-loop-helix-zipper (bHLHZ) transcriptional repressor Mad1, considered to be an antagonist of Myc function, inhibits lymphocyte expansion, maturation and growth following pre-T-cell receptor (pre-TCR) and TCR stimulation. Furthermore, cDNA microarray technology was used to determine that of the genes repressed by Mad1, the majority (77%) are involved in cell growth, which correlates with a decrease in size of Mad1 transgenic thymocytes. Over 80% of the genes repressed by Mad1 have previously been found to be induced by Myc. These results suggest that a balance between Myc and Mad levels may normally modulate lymphocyte proliferation and development in part by controlling expression of growth-regulating genes (Iritani, 2002).

c-Myc promotes cell growth and transformation by ill-defined mechanisms. cmyc-/- mice die by embryonic day 10.5 (E10.5) with defects in growth and in cardiac and neural development. The lethality of cmyc-/- embryos is also associated with profound defects in vasculogenesis and primitive erythropoiesis. Furthermore, cmyc-/- embryonic stem (ES) and yolk sac cells are compromised in their differentiative and growth potential. These defects are intrinsic to c-Myc, and are in part associated with a requirement for c-Myc for the expression of vascular endothelial growth factor (VEGF), since VEGF can partially rescue these defects. However, c-Myc is also required for the proper expression of other angiogenic factors in ES and yolk sac cells, including angiopoietin-2, and the angiogenic inhibitors thrombospondin-1 and angiopoietin-1. Finally, cmyc-/- ES cells are dramatically impaired in their ability to form tumors in immune-compromised mice, and the small tumors that sometimes develop are poorly vascularized. Therefore, c-Myc function is also necessary for the angiogenic switch that is indispensable for the progression and metastasis of tumors. These findings support the model wherein c-Myc promotes cell growth and transformation, as well as vascular and hematopoietic development, by functioning as a master regulator of angiogenic factors (Baudino, 2002).

Hedgehog pathway activation is required for expansion of specific neuronal precursor populations during development and is etiologic in the human cerebellar tumor, medulloblastoma. Sonic hedgehog (Shh) signaling upregulates expression of the proto-oncogene Nmyc in cultured cerebellar granule neuron precursors (CGNPs) in the absence of new protein synthesis. The temporal-spatial expression pattern of Nmyc, but not other Myc family members, precisely coincides with regions of hedgehog proliferative activity in the developing cerebellum and is observed in medulloblastomas of Patched (Ptch) heterozygous mice. Overexpression of Nmyc promotes cell-autonomous G1 cyclin upregulation and CGNP proliferation independent of Shh signaling. Furthermore, Myc antagonism in vitro significantly decreases proliferative effects of Shh in cultured CGNPs. Together, these findings identify Nmyc as a direct target of the Shh pathway that functions to regulate cell cycle progression in cerebellar granule neuron precursors (Kenney, 2003).

The activity of adult stem cells is essential to replenish mature cells constantly lost due to normal tissue turnover. By a poorly understood mechanism, stem cells are maintained through self-renewal while concomitantly producing differentiated progeny. Genetic evidence is provided for an unexpected function of the c-Myc protein in the homeostasis of hematopoietic stem cells (HSCs). Conditional elimination of c-Myc activity in the bone marrow (BM) results in severe cytopenia and accumulation of HSCs in situ. Mutant HSCs self-renew and accumulate due to their failure to initiate normal stem cell differentiation. Impaired differentiation of c-Myc-deficient HSCs is linked to their localization in the differentiation preventative BM niche environment, and correlates with up-regulation of N-cadherin and a number of adhesion receptors, suggesting that release of HSCs from the stem cell niche requires c-Myc activity. Accordingly, enforced c-Myc expression in HSCs represses N-cadherin and integrins leading to loss of self-renewal activity at the expense of differentiation. Endogenous c-Myc is differentially expressed and induced upon differentiation of long-term HSCs. Collectively, these data indicate that c-Myc controls the balance between stem cell self-renewal and differentiation, presumably by regulating the interaction between HSCs and their niche (Wilson, 2004).

The stem cell niche is defined as a subset of tissue cells and extracellular substrates that can harbor one or more stem cells controlling their self-renewal and progeny production in vivo. Retention of stem cells in the niche is thought to be accomplished by stem cell niche and stem-cell extracellular matrix (ECM)-ligand interactions. It has been shown in the Drosophila ovary that DE-cadherin-mediated anchoring of germ line and somatic stem cells to the niche is essential for their maintenance. Putative niches have also been identified in vertebrates, including the bulge region in the skin epidermis and the stem cell-bearing base of intestinal crypts. In the BM, HSCs are located at the endosteal lining of the BM cavities, and recent studies show that specialized spindle-shaped N-cadherin+ osteoblasts (SNO) are a key component of the BM stem cell niche. HSCs are thought to be anchored to SNO cells via a homotypic N-cadherin interaction (Wilson, 2004 and references therein).

Although c-myc is the first proto-oncogene described to control stem cell homeostasis, some of its target genes and proteins that collaborate with Myc during tumorigenesis have recently been implicated in stem cell function. For example, the polycomb protein Bmi-1 collaborates with c-Myc during lymphomagenesis, and has been shown to be essential for maintenance of adult HSCs. The CDK inhibitor p21CIP, which is repressed by c-Myc, controls HSC proliferation, and is furthermore required for maintenance of long-term self-renewal. This suggests that part of c-Myc`s effects in HSCs could be mediated by p21 repression. Because c-Myc has been postulated to be an effector of canonical Wnt signaling and also appears to be connected to the Ang-1/Tie2-signaling pathway (referring to angiopoietin and its receptor), this protein is now evolving as a ringmaster in regulating adult stem cell function in vivo. It is thus crucial to elucidate which signaling pathways are responsible for the tight control of c-Myc expression in stem/progenitor cells. Irrespective of what niche signals fine tune c-Myc expression during the constantly changing conditions of BM homeostasis in vivo, it appears that this oncoprotein is a key element that fulfils the function of a homeostat, determining the balance between stem cell self-renewal and differentiation (Wilson, 2004 and references therein).

Myc, development and differentiation

To directly assess c-myc function in processes of cellular proliferation, differentiation, and embryogenesis, both heterozygous and homozygous c-myc mutant ES cell lines were generated using homologous recombination in embryonic stem cells. The mutation is a null allele at the protein level. Mouse chimeras from seven heterozygous cell lines transmitted the mutant allele to their offspring. The analysis of embryos from two clones has shown that the mutation is lethal in homozygotes between 9.5 and 10.5 days of gestation. The embryos are generally smaller and retarded in development compared with their littermates. Pathologic abnormalities include the heart, pericardium, neural tube, and delay or failure in the turning of the embryo. Heterozygous females have reduced fertility owing to embryonic resorption before 9.5 days of gestation in 14% of implanted embryos. c-Myc protein appears to be necessary for embryonic survival beyond 10.5 days of gestation; however, it appears to be dispensable for cell division both in ES cell lines and in the embryo before that time (Davis, 1993).

A leaky mutation has been generated in N-myc (the neural myc isoform) by gene targeting in embryonic stem cells. In this allele, the neo(r) gene was inserted into the first intron of N-myc, in such a way that alternative splicing around this insertion could result in the generation of a normal N-myc transcript in addition to a mutant transcript. Mice homozygous for this mutation die immediately after birth owing to their inability to oxygenate blood. Histological examination reveals a marked underdevelopment in the lung airway epithelium, resulting in a decreased respiratory surface area. Analysis of N-myc expression in wild-type and homozygous mutant embryonic lungs suggests that N-myc is required for the proliferation of the lung epithelium in response to local inductive signals emanating from the lung mesenchyme. Homozygous mutant embryos are slightly smaller than normal and also had a marked reduction in spleen size, whereas other tissues that normally express N-myc appeared to be unaffected by the mutation. Molecular analysis reveals that normal N-myc transcripts are found in tissues from homozygous mutant embryos. Different tissues expressed the normal N-myc transcript at different levels, relative to those observed in wild-type embryos, with the lowest levels being observed in the lungs (Moens, 1992).

To investigate liver development in mice, an N-myc mutant mouse line with abnormal liver development was used. N-myc mutant embryos die between 11.5 and 12.5 days postcoitum, most probably from heart failure. At 11.5 days of gestation, extensive apoptosis restricted to the hepatocytes occurs in N-myc mutant liver, when compared to wild-type samples. The number of hematopoietic cells is reduced in the mutant liver. During early liver organogenesis, the N-myc gene is expressed in tissues involved in the induction and the differentiation of the hepatocytes. At 11.5 days of development, both c-myc and N-myc genes are expressed in the liver. While c-myc is expressed at a high level in the organ per se, N-myc expression is mostly confined to the peripheral layer of the liver that will generate the Glisson's capsule. Taken together, the expression pattern of N-myc in the liver and the specific apoptosis of hepatocytes observed in N-myc mutants indicate that N-myc is required for hepatocyte survival and suggest that it is involved in the genetic cascade leading to normal liver development (Giroux, 1998).

The highest expression of the N-myc gene occurs during embryonic organogenesis in the mouse ontogeny, with the peak of expression around embryonic day 9.5. Homozygous N-myc-deficient mice, produced by germline transmission of a disrupted allele in ES cells, develop normally to day 10.5, indicating dispensability of N-myc expression in the earlier period, but later lin their development, they accumulate organogenic abnormalities and die around day 11.5. The most notable abnormalities are found in the limb bud, visceral organs (lung, stomach, liver and heart) and the central/peripheral nervous systems; all abnormalities are highly correlated with the site of N-myc expression. The limb buds and the lungs excised from N-myc-deficient mutant embryos were placed in culture to allow their continued development to stages beyond the point of death of the embryos. Analyses indicates that the mutant limbs fail to develop distal structures and the development of bronchi from the trachea is defective in the lungs. The latter defect is largely corrected by addition of fetal calf serum to the culture medium, suggesting that an activity missing in the mutant lung is replenished by a component of the serum. The phenotype of N-myc-deficient mutant embryos indicates requirement of the N-myc function in many instances of tissue interactions in organogenesis and also in cell-autonomous regulation of tissue maturation (Sawai, 1993).

Expression of the N-myc gene has also been examined in embryos during postimplantation development using RNA in situ hybridization. Tissue- and cell-specific patterns of expression unique to N-myc were observed, as compared with the related c-myc gene. N-myc transcripts become progressively restricted to specific cell types, primarily to epithelial tissues including those of the developing nervous system and those in developing organs characterized by epithelio-mesenchymal interaction. In contrast, c-myc transcripts are confined to the mesenchymal compartments. These data suggest that c-myc and N-myc proteins may interact with different substrates in performing their function during embryogenesis and suggest further that there are linked regulatory mechanisms for normal expression in the embryo. The N-myc locus was mutated via homologous recombination in embryonic stem (ES) cells and the mutated allele was then introduced into the mouse germ line. Live-born heterozygotes are under-represented but appear normal. Homozygous mutant embryos die prenatally at approximately 11.5 days of gestation. Histologic examination of homozygous mutant embryos indicates that several developing organs are affected. These include the central and peripheral nervous systems, mesonephros, lung, and gut. Thus, N-myc function is required during embryogenesis, and the pathology observed is consistent with the normal pattern of N-myc expression. Examination of c-myc expression in mutant embryos indicates the existence of coordinate regulation of myc genes during mouse embryogenesis (Stanton, 1992).

During neural crest development in avian embryos, transcription factor N-myc is initially expressed in the entire cell population. The expression is then turned off in the period following colonization in ganglion and nerve cord areas except for the cells undergoing neuronal differentiation. This is also recapitulated in the culture of Japanese quail neural crest; the cells expressing N-myc eventually coincided with those expressing neurofilaments. These findings suggested that N-myc is involved in regulation of neuronal differentiation in the neural crest cell population. In fact, transient overexpression of N-myc in the neural crest culture by transfection results in a remarkable promotion of neuronal differentiation. An experimental procedure was developed to examine the effect of exogenous N-myc expression in the neural crest cells in embryos. Neural crest cell clusters still attached to the neural tube were excised from Japanese quail embryos, transfected and grafted into chicken host embryos. Using this chimera technique, the consequence of transient high N-myc could be analyzed during the early phase of neural crest migration. Two effects were demonstrated in the embryos:

  1. High N-myc expression provokes massive ventral migration of the neural crest population.
  2. Those cells that migrated to the ganglion-forming areas undergo neuronal differentiation with the cell type determined by the nature of the ganglion.
Thus, N-myc is involved in regulation of the neural crest fate in two different aspects: ventral migration and neuronal differentiation (Wakamatsu, 1997).

The epidermis contains two types of proliferative keratinocyte: stem cells, with unlimited self-renewal capacity, and transit amplifying cells, those daughters of stem cells destined to withdraw from the cell cycle and terminally differentiate after a few rounds of division. In a search for factors that regulate exit from the stem cell compartment, c-Myc was constitutively expressed in primary human keratinocytes by use of wild-type and steroid-activatable constructs. In contrast to its role in other cell types, activation of c-Myc in keratinocytes causes a progressive reduction in growth rate, without inducing apoptosis, and a marked stimulation of terminal differentiation. Keratinocytes can be enriched for stem or transit amplifying cells on the basis of beta1 integrin expression. By the use of this method to fractionate cells prior to c-Myc activation, it is found that c-Myc acts selectively on stem cells, driving them into the transit amplifying compartment. As a result, activation of c-Myc in epidermis reconstituted on a dermal equivalent leads to premature execution of the differentiation program. The transcriptional regulatory domain of c-Myc is required for these effects because a deletion within that domain acts as a dominant-negative mutation. These results reveal a novel biological role for c-Myc and provide new insights into the mechanism regulating epidermal stem cell fate (Gandarillas, 1997).

Mad-Max heterodimers have been shown to antagonize Myc transforming activity by a mechanism requiring multiple protein-protein and protein-DNA interactions. However, the mechanism by which Mad functions in differentiation is unknown. Mad functions by an active repression mechanism to antagonize the growth-promoting function(s) of Myc and bring about a transition from cellular proliferation to differentiation. Exogenously expressed c-Myc blocks inducer-mediated differentiation of murine erythroleukemia cells without disrupting the induction of endogenous Mad; rather, high levels of c-Myc prevent a heterocomplex switch from growth-promoting Myc-Max to growth-inhibitory Mad-Max. Cotransfection of a constitutive c-myc with a zinc-inducible mad1 results in clones expressing both genes, whereby a switch from proliferation to differentiation can be modulated. Whereas cells grown in N'N'-hexamethylene bisacetamide in the absence of zinc fail to differentiate, addition of zinc up-regulates Mad expression by severalfold and differentiation proceeds normally. Coimmunoprecipitation analysis reveals that Mad-Max complexes are in excess of Myc-Max in these cotransfectants. The Sin-binding, basic region, and leucine zipper motifs are each required for Mad to function during a molecular switch from proliferation to differentiation (Cultraro, 1997).

Two novel Mad1- and Mxi1-related proteins, Mad3 and Mad4, interact with both Max and mSin3 and repress transcription from a promoter containing CACGTG binding sites. Both Mad3 and Mad4 inhibit c-Myc dependent cell transformation. An examination of the expression patterns of all mad genes during murine embryogenesis reveals that mad1, mad3 and mad4 are expressed primarily in growth-arrested differentiating cells. mxi1 is also expressed in differentiating cells, but is co-expressed with either c-myc, N-myc, or both in proliferating cells of the developing central nervous system and the epidermis. In the developing central nervous system and epidermis, downregulation of myc genes occurs concomitant with upregulation of mad family genes. These expression patterns, together with the demonstrated ability of Mad family proteins to interfere with the proliferation promoting activities of Myc, suggest that the regulated expression of Myc and Mad family proteins function in a concerted fashion to regulate cell growth in differentiating tissues (Hurlin, 1995).

The switch from transcriptionally activating MYC-MAX to transcriptionally repressing MAD1-MAX protein heterodimers has been correlated with the initiation of terminal differentiation in many cell types. To investigate the function of MAD1-MAX dimers during differentiation, the Mad1 gene was disrupted by homologous recombination in mice. Analysis of hematopoietic differentiation in homozygous mutant animals reveals that cell cycle exit of granulocytic precursors is inhibited following the colony-forming cell stage, resulting in increased proliferation and delayed terminal differentiation of low proliferative potential cluster-forming cells. Surprisingly, the numbers of terminally differentiated bone marrow and peripheral blood granulocytes are essentially unchanged in Mad1 null mice. This imbalance between the frequencies of precursor and mature granulocytes is correlated with a compensatory decrease in granulocytic cluster-forming cell survival under apoptosis-inducing conditions. Recovery of the peripheral granulocyte compartment following bone marrow ablation is significantly enhanced in Mad1 knockout mice. Two Mad1-related genes, Mxi1 and Mad3, are expressed ectopically in adult spleen, indicating that functional redundancy and cross-regulation between MAD family members may allow for apparently normal differentiation in the absence of MAD1. These findings demonstrate that MAD1 regulates cell cycle withdrawal during a late stage of granulocyte differentiation, and suggest that the relative levels of MYC versus MAD1 mediate a balance between cell proliferation and terminal differentiation (Foley, 1998).

Members of the myc family of cellular oncogenes have been implicated as transcriptional regulators in pathways that govern cellular proliferation and death. In addition, N-myc and c-myc are essential for completion of murine embryonic development. However, the basis for the evolutionary conservation of the myc gene family has remained unclear. To elucidate this issue, mice in which the endogenous c-myc coding sequences have been replaced with N-myc coding sequences were generated. Strikingly, mice homozygous for this replacement mutation can survive into adulthood and reproduce. Moreover, when expressed from the c-myc locus, N-myc is similarly regulated and functionally complementary to c-myc in the context of various cellular growth and differentiation processes. Therefore, the myc gene family must have evolved, to a large extent, to facilitate differential patterns of expression (Malynn, 2000).

The neural crest, a population of multipotent progenitor cells, is a defining feature of vertebrate embryos. Neural crest precursor cells arise at the neural plate border in response to inductive signals, but much remains to be learned about the molecular mechanisms underlying their induction. The protooncogene c-Myc is an essential early regulator of neural crest cell formation in Xenopus. c-myc is localized at the neural plate border prior to the expression of early neural crest markers, such as the bHLH protein slug. A morpholino-mediated knockdown of c-Myc protein results in the absence of neural crest precursor cells and a resultant loss of neural crest derivatives. These effects are not dependent upon changes in cell proliferation or cell death. Instead, these findings reveal an important and unexpected role for c-Myc in the specification of cell fates in the early ectoderm (Bellmeyer, 2003).

To assess the critical role of Wnt signals in intestinal crypts, transgenic mice were generated ectopically expressing Dickkopf1 (Dkk1), a secreted Wnt inhibitor. Epithelial proliferation is greatly reduced coincidentally with the loss of crypts. Although enterocyte differentiation appears unaffected, secretory cell lineages are largely absent. Disrupted intestinal homeostasis is reflected by an absence of nuclear ß-catenin, inhibition of c-myc expression, and subsequent up-regulation of p21CIP1/WAF1. Thus, these data are the first to establish a direct requirement for Wnt ligands in driving proliferation in the intestinal epithelium, and also define an unexpected role for Wnts in controlling secretory cell differentiation (Pinto, 2003).

The growth inhibitory cytokine TGF-beta enforces homeostasis of epithelia by activating processes such as cell cycle arrest and apoptosis. Id2 expression is often highest in proliferating epithelial cells and declines during differentiation. Recently, Id2 expression has been found to depend on Myc-Max transcriptional complexes. TGF-beta signaling inhibits Id2 expression in human and mouse epithelial cell lines from different tissue origins. Furthermore, the observed Id2 down-regulation by TGF-beta in mouse mammary epithelial cells occurs without a concurrent drop in c-Myc levels. However, sustained Id2 repression in these cells and in human keratinocytes coincides with induction of the Myc antagonistic repressors Mad2 and Mad4, decreased formation of Myc-Max heterodimers and the replacement of Myc-Max complexes with Mad-Max complexes on the Id2 promoter. These results argue that induction of Mad expression and Id2 down-regulation are important events during the TGF-beta cytostatic program in epithelial cells (Siegel, 2003).

The Mnt gene (see Drosophila Mnt) encodes a Mad-family bHLH transcription factor located on human 17p13.3. Mnt is one of 20 genes deleted in a heterozygous fashion in Miller-Dieker syndrome (MDS), a contiguous gene syndrome that consists of severe neuronal migration defects and craniofacial dysmorphic features. Mnt can inhibit Myc-dependent cell transformation and is hypothesized to counterbalance the effects of c-Myc on growth and proliferation in vivo by competing with Myc for binding to Max and by repressing target genes activated by Myc/Max heterodimers. Unlike the related Mad family members, Mnt is expressed ubiquitously and Mnt/Max heterodimers are found in proliferating cells that contain Myc/Max heterodimers, suggesting a unique role for Mnt during proliferation. To examine the role of Mnt in vivo, mice with null (MntKO) and loxP-flanked conditional knock-out (MntCKO) alleles of Mnt were produced. Virtually all MntKO/KO mutants in a mixed (129S6 x NIH Black Swiss) or inbred (129S6) genetic background died perinatally. Mnt-deficient embryos exhibit small size throughout development and show reduced levels of c-Myc and N-Myc. In addition, 37% of the mixed background mutants displayed cleft palate as well as retardation of skull development, a phenotype not observed in the inbred mutants. These results demonstrate an important role for Mnt in embryonic development and survival, and suggest that Mnt may play a role in the craniofacial defects displayed by MDS patients (Toyo-oka, 2004).

Neural crest cells, a population of proliferative, migratory, tissue-invasive stem cells, are a defining feature of vertebrate embryos. These cells arise at the neural plate border during a time in development when precursors of the central nervous system and the epidermis are responding to the extracellular signals that will ultimately dictate their fates. Neural crest progenitors, by contrast, must be maintained in a multipotent state until after neural tube closure. Although the molecular mechanisms governing this process have yet to be fully elucidated, recent work has suggested that Myc functions to prevent premature cell fate decisions in neural crest forming regions of the early ectoderm. The small HLH protein Id3 is a Myc target that plays an essential role in the formation and maintenance of neural crest stem cells. A morpholino-mediated 'knockdown' of Id3 protein results in embryos that lack neural crest. Moreover, forced expression of Id3 maintains the expression of markers of the neural crest progenitor state beyond the time when they would normally be downregulated and blocks the differentiation of neural crest derivatives. These results shed new light on the mechanisms governing the formation and maintenance of a developmentally and clinically important cell population (Light, 2005).

Following their emigration from the neural tube, early migratory neural crest cells initially retain their stem cell-like characteristics, including the potential to contribute to the sensory neuronal, autonomic neuronal, glial, smooth muscle and ectomesenchymal lineages. However, these cells soon begin responding to signals that direct their development into specific neural crest derivatives, as evidenced by the downregulation of pan-neural crest markers expressed by the early progenitor population, and the expression of markers characteristic of specific differentiating lineages. Enforced misexpression of Id3 in the migratory neural crest population maintains the expression of markers characteristic of the progenitor state, and delays or prevents the differentiation of neural crest derivatives. For example, Id3-expressing cells sustain robust expression of Sox10, a factor that has itself been implicated in the maintenance of stem cell identity, and Slug, an important regulatory protein expressed by all neural crest precursor cells, long beyond the time that most neural crest cells on the control side of the embryo have downregulated these factors. Importantly, Id3 expression does not appear to irreversibly alter the potential of these cells. Once their pool of Id3 has turned over, neural crest cells are capable of responding to signals that direct the formation of specific derivatives such as melanocytes. These findings suggest a model in which Id family proteins expressed in the neural crest progenitor pool help control the timing with which these cells respond to differentiative signals during normal development. An alternative explanation of these findings, however, in which Id3 dictates the outcome of neural crest cell fate determination in a dose-dependant fashion, cannot br formally rule out. Future studies might profitably explore whether controlling the timing of release from Id3 activity can lead to excess recruitment of neural crest progenitors to fates other than melanocytes (Light, 2005).

Understanding how lung progenitor cells balance proliferation against differentiation is relevant to clinical disorders such as bronchopulmonary dysplasia of premature babies and lung cancer. Previous studies have established that lung development is severely disrupted in mouse mutants with reduced levels of the proto-oncogene Nmyc, but the precise mechanisms involved have not been explored. Nmyc expression in the embryonic lung is normally restricted to a distal population of undifferentiated epithelial cells, a high proportion of which are in the S phase of the cell cycle. Overexpression of NmycEGFP in the epithelium under the control of surfactant protein C (Sftpc) regulatory elements expands the domain of S phase cells and upregulates numerous genes associated with growth and metabolism, as shown by transcriptional microarray. In addition, there is marked inhibition of differentiation, coupled with an expanded domain of expression of Sox9 protein, which is also normally restricted to the distal epithelial compartment. By contrast, conditional deletion of Nmyc leads to reduced proliferation, epithelial differentiation and high levels of apoptosis in both epithelium and mesenchyme. Unexpectedly, about 50% of embryos in which only one copy of Nmyc is deleted die perinatally, with similarly abnormal lungs. A model is proposed in which Nmyc is essential in the developing lung for maintaining a distal population of undifferentiated, proliferating progenitor cells (Okubo, 2005).

The developing limb serves as a paradigm for studying pattern formation and morphogenetic cell death. Conditional deletion of N-Myc (Mycn) in the developing mouse limb leads to uniformly small skeletal elements and profound soft-tissue syndactyly. The small skeletal elements are associated with decreased proliferation of limb bud mesenchyme and small cartilaginous condensations, and syndactyly is associated with a complete absence of interdigital cell death. Although Myc family proteins have pro-apoptotic activity, N-Myc is not expressed in interdigital cells undergoing programmed cell death. Evidence is provided indicating that the lack of interdigital cell death and associated syndactyly is related to an absence of interdigital cells marked by expression of Fgfr2 and Msx2. Thus, instead of directly regulating interdigital cell death, it is proposed that N-Myc is required for the proper generation of undifferentiated mesenchymal cells that become localized to interdigital regions and trigger digit separation when eliminated by programmed cell death. These results provide new insight into mechanisms that control limb development and suggest that defects in the formation of N-Myc-dependent interdigital tissue may be a root cause of common syndromic forms of syndactyly (Ota, 2007).

Myc family members play crucial roles in regulating cell proliferation, size, differentiation, and survival during development. N-myc is expressed in retinal progenitor cells, where it regulates proliferation in a cell-autonomous manner. In addition, N-myc coordinates the growth of the retina and eye. Specifically, the retinas of N-myc-deficient mice are hypocellular but are precisely proportioned to the size of the eye. N-myc represses the expression of the cyclin-dependent kinase inhibitor p27Kip1 but acts independently of cyclin D1, the major D-type cyclin in the developing mouse retina. Acute inactivation of N-myc leads to increased expression of p27Kip1, and simultaneous inactivation of p27Kip1 and N-myc rescues the hypocellular phenotype in N-myc-deficient retinas. N-myc is not required for retinal cell fate specification, differentiation, or survival. These data represent the first example of a role for a Myc family member in retinal development and the first characterization of a mouse model in which the hypocellular retina is properly proportioned to the other ocular structures. It is proposed that N-myc lies upstream of the cell cycle machinery in the developing mouse retina and thus coordinates the growth of both the retina and eye through extrinsic cues (Martins, 2008).

Self-renewal and proliferation of neural stem cells and the decision to initiate neurogenesis are crucial events directing brain development. The ubiquitin ligase Huwe1 operates upstream of the N-Myc-DLL3-Notch pathway to control neural stem cell activity and promote neurogenesis. Conditional inactivation of the Huwe1 gene in the mouse brain caused neonatal lethality associated with disorganization of the laminar patterning of the cortex. These defects stemmed from severe impairment of neurogenesis associated with uncontrolled expansion of the neural stem cell compartment. Loss- and gain-of-function experiments in the mouse cortex demonstrated that Huwe1 restrains proliferation and enables neuronal differentiation by suppressing the N-Myc-DLL3 cascade. Notably, human high-grade gliomas carry focal hemizygous deletions of the X-linked Huwe1 gene (Drosophila homolog: CG8184) in association with amplification of the N-myc locus. These results indicate that Huwe1 balances proliferation and neurogenesis in the developing brain and that this pathway is subverted in malignant brain tumors (Zhao, 2009).

The expansion of the neural stem cell compartment elicited by loss of Huwe1 becomes progressively more evident as neural development proceeds and Huwe1−/− neural stem/progenitor cells fail to exit cell cycle and commence neuronal differentiation. The deregulated proliferative activity conferred by loss of Huwe1 together with abnormal cell morphology and loss of 'crowd control' ultimately lead to severe perturbation of neuronal differentiation and disorganization of brain architecture. During relatively early stages of neurogenesis (before E14.5), cell cycle timing is not affected by mutation of Huwe1. However, loss of Huwe1 severely impairs the lengthening of the cell cycle that accompanies the progressive shift from proliferation to differentiation during late neurogenesis (Zhao, 2009).

The phenotype of the Huwe1 mutant brain in the mouse is complementary to that caused by inactivation of transcription factors that expand the neural stem cell compartment and inhibit neurogenesis (N-Myc and Notch). The N-Myc protein markedly accumulated in the Huwe1 null brain and this effect preceded the phenotypic defects. To unravel the identity of the downstream signaling events triggered by aberrant N-Myc in Huwe1 null brain, a computational approach was designed to dissect and interrogate the activity of transcription factors following modulation of candidate regulators in a specific cellular context. From this approach, the Notch ligand DLL3 emerged as one of the strongest inferred N-Myc targets in the brain, and it was confirmed that, during neural development, Huwe1 negatively regulates expression of DLL3 in an N-Myc-dependent fashion. Based on this information, DLL3 was experimentally validated as a direct transcriptional target of N-Myc and, most importantly, it was discovered that the hyperproliferation and neuronal differentiation defects resulting from knocking out Huwe1 in the cortex are fully reversed by silencing the expression of DLL3 in vivo. Thus, the N-Myc-DLL3 cascade is restrained by Huwe1 to set the timing of cell cycle withdrawal and neuronal differentiation in the developing brain. Although these results are consistent with DLL3 activating Notch1 in the neural stem cell compartment at midgestation, in other systems DLL3 might also behave as inhibitor of Notch1, possibly through competition with other Notch ligands (Zhao, 2009).

The mTORC1/S6K1 pathway regulates glutamine metabolism through the eIF4B-dependent control of c-Myc translation

Growth-promoting signaling molecules, including the mammalian target of rapamycin complex 1 (mTORC1; see Drosophila Tor), drive the metabolic reprogramming of cancer cells required to support their biosynthetic needs for rapid growth and proliferation. Glutamine is catabolyzed to alpha-ketoglutarate (alphaKG), a tricarboxylic acid (TCA) cycle intermediate, through two deamination reactions, the first requiring glutaminase (GLS) to generate glutamate and the second occurring via glutamate dehydrogenase (GDH) or transaminases. Activation of the mTORC1 pathway has been shown previously to promote the anaplerotic entry of glutamine to the TCA cycle via GDH. Moreover, mTORC1 activation also stimulates the uptake of glutamine, but the mechanism is unknown. It is generally thought that rates of glutamine utilization are limited by mitochondrial uptake via GLS, suggesting that, in addition to GDH, mTORC1 could regulate GLS. This study demonstrates that mTORC1 positively regulates GLS and glutamine flux through this enzyme. mTORC1 controls GLS levels through the S6K1-dependent (see Drosophila S6K) regulation of c-Myc. Molecularly, S6K1 enhances Myc translation efficiency by modulating the phosphorylation of eukaryotic initiation factor eIF4B (see Drosophila eIF4B), which is critical to unwind its structured 5' untranslated region (5'UTR). Finally, these data show that the pharmacological inhibition of GLS is a promising target in pancreatic cancers expressing low levels of PTEN (Csibi, 2014).

FGF-dependent metabolic control of vascular development

Blood and lymphatic vasculatures are intimately involved in tissue oxygenation and fluid homeostasis maintenance. Assembly of these vascular networks involves sprouting, migration and proliferation of endothelial cells. Recent studies have suggested that changes in cellular metabolism are important to these processes. Although much is known about vascular endothelial growth factor (VEGF)-dependent regulation of vascular development and metabolism, little is understood about the role of fibroblast growth factors (FGFs) in this context. This study identified FGF receptor (FGFR; see Drosophila Breathless) signalling as a critical regulator of vascular development. This is achieved by FGF-dependent control of c-MYC (MYC; see Drosophila Myc) expression that, in turn, regulates expression of the glycolytic enzyme hexokinase 2 (HK2; see Drosophila Hexokinase A). A decrease in HK2 levels in the absence of FGF signalling inputs results in decreased glycolysis, leading to impaired endothelial cell proliferation and migration. Pan-endothelial- and lymphatic-specific Hk2 knockouts phenocopy blood and/or lymphatic vascular defects seen in Fgfr1/Fgfr3 double mutant mice, while HK2 overexpression partly rescues the defects caused by suppression of FGF signalling. Thus, FGF-dependent regulation of endothelial glycolysis is a pivotal process in developmental and adult vascular growth and development (Yu, 2017).

Myc and neural development

Wnt signaling and its downstream target N-myc regulate basal progenitors in the developing neocortex

Basal progenitors (also called non-surface dividing or intermediate progenitors) have been proposed to regulate the number of neurons during neocortical development through expanding cells committed to a neuronal fate, although the signals that govern this population have remained largely unknown. This study shows that N-myc mediates the functions of Wnt signaling in promoting neuronal fate commitment and proliferation of neural precursor cells in vitro. Wnt signaling and N-myc also contribute to the production of basal progenitors in vivo. Expression of a stabilized form of beta-catenin, a component of the Wnt signaling pathway, or of N-myc increased the numbers of neocortical basal progenitors, whereas conditional deletion of the N-myc gene reduced these and, as a likely consequence, the number of neocortical neurons. These results reveal that Wnt signaling via N-myc is crucial for the control of neuron number in the developing neocortex (Kuwahara, 2010).

Wnt signaling and its downstream target N-Myc play a key role in the production of basal progenitors. Expression of N-myc or stabilized β-catenin increases, while conditional gene deletion of N-myc decreases the numbers of basal progenitors found in the developing neocortex, as determined by the numbers of Tbr2-positive cells and non-surface dividing cells. The increase in basal progenitors by the Wnt-N-myc axis can be ascribed to either: (1) differentiation of apical progenitors into basal progenitors; or (2) proliferation (and survival) of basal progenitors, or both. The observation that retroviral expression of stabilized β-catenin or N-myc in the neocortex reduced the number of apical progenitors while increasing that of the basal progenitors supports a role for the former mechanism (Kuwahara, 2010).

Members of the Myc family have been reported to be involved in differentiation processes in other cell types, including epithelial, neural crest and hematopoietic stem cells, although previous reports have not directly demonstrated that Myc is involved in fate commitment by a lineage-tracing analysis. In this study, the clonal analysis suggests that N-myc instructs commitment of NPC fate into the neuronal lineage at the expense of the glial lineage and reduces multipotent neurosphere-forming NPCs. This function of N-myc is similar to the reported function of Wnt signaling (Kuwahara, 2010).

It is not known what transcriptional targets of N-myc are involved in instructing neurogenesis. Possible candidates include the proneural gene Ngn1, as deletion of N-myc was observed to cause a decrease in the level of Ngn1 mRNA in the developing neocortex. As Ngn1 is also a direct target of the β-catenin/Tcf transcription complex, it would be interesting to examine the interaction between N-myc and these transcription factors on the Ngn1 promoter. The Myc family has also been shown to function in the regulation of the global chromatin state, in addition to its function as a classical transcription factor; thus it is possible that mechanisms other than direct target gene activation are also involved in N-myc regulation of neurogenesis and proliferation of NPCs (Kuwahara, 2010).

This study also provides evidence that N-myc is directly regulated by the β-catenin/Tcf transcription complex and mediates the functions of Wnt signaling to stimulate neocortical NPC proliferation and differentiation: (1) Wnt3a treatment and stabilized β-catenin expression induced N-myc expression, whereas expression of a dominant-negative form of Tcf3 reduced N-myc expression in NPC cultures; (2) misexpression of stabilized β-catenin in the ventral telencephalon induced ectopic N-myc expression in vivo; (3) N-myc is expressed in the developing neocortex in a pattern similar to that of a Tcf reporter transgene; (4) Tcf3 directly binds to a Tcf-consensus site 1.6 kb upstream of the N-myc gene; (5) Wnt stimulation of proliferation and differentiation in NPC cultures was abrogated by deletion of the N-myc gene. These results provide evidence that N-myc is a key downstream mediator of Wnt-β-catenin signaling in the developing neocortex. It is of note that N-myc is not the only downstream target responsible for the functions of Wnt signaling in the neocortex (Kuwahara, 2010).

The Wnt-β-catenin pathway exerts multiple functions in a context-dependent manner. For instance, persistent expression of stabilized β-catenin in NPCs results in overproliferation of apical progenitors and horizontal/tangential expansion of the cortex in addition to the reduction of Tbr2-positive basal progenitors. However, when the same stabilized β-catenin was expressed by retroviral infection in a small proportion of NPCs located at the VZ, it had the opposite effect: increasing the numbers of basal progenitors and decreasing the number of apical progenitors. This difference does not appear to be due to the differential requirement of N-myc, as N-myc gene deletion rescued both proliferative and differentiating effects of activation of β-catenin. This difference might be rather due to the aberrant brain architecture generated in the β-catenin-δEx3 mice (mutant for β-catenin), to other non-cell autonomous effects of β-catenin or to differences in the levels or timing of active β-catenin expression. Indeed, different levels of active β-catenin expression result in different outcomes in hair follicle stem cells (Kuwahara, 2010).

Although it has previously been postulated that β-catenin exerts its different functions via distinct targets, this study observed that both the proliferating and neurogenic functions of Wnt-β-catenin signaling in the developing neocortex are mediated in common by N-myc. It is noteworthy that c-Myc can also exert distinct functions depending on its expression levels, such as in epithelial stem cells, raising the possibility that the levels of N-myc might determine the cellular output. Importantly, heterozygous mutation of N-MYC (MYCN) in humans causes Feingold syndrome, comprising several defects including microcephaly, supporting the notion that the levels of N-myc in the nervous system are crucial for determining the neuronal number and brain size. It is also possible that N-myc alters its function in a developmental-stage-dependent manner. This possibility is consistent with a previous finding that canonical Wnt signaling promotes proliferation of neocortical neural precursor cells at a relatively early stage (E10.5) but promotes their differentiation at a relatively late stage (E13.5) (Kuwahara, 2010).

Which Wnt ligands are responsible for the activation of N-myc and consequent regulation of basal progenitors in the developing brain? Wnt7a is expressed in NPCs at the VZ and might be important for increase in cells localized in the SVZ. Wnt7b, which is expressed in the deep-layer neurons (neurons at the layer VI), might elicit a feed-forward signal to increase the number of basal progenitors that in turn contribute to the generation of the upper-layer neurons. It is plausible that extracellular signals other than Wnt ligands are also involved in the activation of N-myc and regulation of basal progenitors. N-myc is induced by Shh signaling in cerebellar granule cells, and a recent report shows that Shh protein is localized in the IMZ of the neocortex and contributes to the production of basal progenitors. Growth factors expressed in NPCs such as Fgf2 and epidermal growth factor (Egf) might also participate in the activation of N-myc. Growth factor receptors activate the PI3K (Pik3r1 - Mouse Genome Informatics) pathway, which induces phosphorylation and stabilization of N-myc protein. In addition, Egfr as well as Frs2, an adaptor of Fgfr/Egfr, have been shown to regulate the production of basal progenitors. The RNA-binding protein HuC/D is another candidate that could regulate N-myc function in basal progenitors, as it binds to and stabilizes N-myc mRNA and is localized in the SVZ (Kuwahara, 2010).

As a mechanism of neocortical expansion during animal evolution, the increase of basal progenitors is considered to be a key event, given that basal progenitors increase the number of neurons from a given number of apical progenitors through extra cell division and that the number of basal progenitors dramatically increases during animal evolution. The observation in this study that N-myc deletion decreases Tbr2-positive cells and non-surface dividing cells without marked reduction of Pax6-positive cells supports the notion that Wnt signaling, via N-myc, promotes differentiation from apical progenitors to basal progenitors and promotes indirect neurogenesis. It would be interesting to investigate possible roles of this signaling pathway in the neocortical expansion during animal evolution in future studies (Kuwahara, 2010).

Otx2 is a target of N-myc and acts as a suppressor of sensory development in the mammalian cochlea

Transcriptional regulatory networks are essential during the formation and differentiation of organs. The transcription factor N-myc is required for proper morphogenesis of the cochlea and to control correct patterning of the organ of Corti. The Otx2 gene, a mammalian orthologue of the Drosophila orthodenticle homeobox gene, is a crucial target of N-myc during inner ear development. Otx2 expression is lost in N-myc mouse mutants, and N-myc misexpression in the chick inner ear leads to ectopic expression of Otx2. Furthermore, Otx2 enhancer activity is increased by N-myc misexpression, indicating that N-myc may directly regulate Otx2. Inactivation of Otx2 in the mouse inner ear leads to ectopic expression of prosensory markers in non-sensory regions of the cochlear duct. Upon further differentiation, these domains give rise to an ectopic organ of Corti, together with the re-specification of non-sensory areas into sensory epithelia, and the loss of Reissner's membrane. Therefore the Otx2-positive domain of the cochlear duct shows a striking competence to develop into a mirror-image copy of the organ of Corti. Taken together, the work shows that Otx2 acts downstream N-myc and is essential for patterning and the spatial restriction of the sensory domain of the mammalian cochlea (Vendrell, 2015).

Myc, proliferation, apoptosis and tumorigenesis

The transactivation of TCF target genes induced by Wnt pathway mutations constitutes the primary transforming event in colorectal cancer (CRC). Disruption of ß-catenin/TCF-4 activity in CRC cells induces a rapid G1 arrest and blocks a genetic program that is physiologically active in the proliferative compartment of colon crypts. Coincidently, an intestinal differentiation program is induced. The TCF-4 target gene c-MYC plays a central role in this switch by direct repression of the p21CIP1/WAF1 promoter. Following disruption of ß-catenin/TCF-4 activity, the decreased expression of c-MYC releases p21CIP1/WAF1 transcription, which in turn mediates G1 arrest and differentiation. Thus, the ß-catenin/TCF-4 complex constitutes the master switch that controls proliferation versus differentiation in healthy and malignant intestinal epithelial cells (van de Wetering, 2002).

c-MYC plays a central role in the proliferative capacity of many cancers, including CRC. tHE data imply that c-MYC blocks the expression of the cell cycle inhibitor p21CIP1/WAF1. The region responsible for p21CIP1/WAF1 regulation has been mapped to a 200 bp fragment of the proximal promoter. The presence of MIZ-1 and c-MYC on this promoter suggests that c-MYC-mediated repression of p21CIP1/WAF1 occurs by a mechanism resembling c-MYC control of p15INK4b, i.e., through preventing promoter activation by the transcription factor MIZ-1. Decreased expression of c-MYC would allow MIZ-1 to activate p21CIP1/WAF1 transcription. The complementarity in the expression of c-MYC and p21CIP1/WAF1 in the intestine supports this mechanism (van de Wetering, 2002).

The carboxyl terminus of c-Myc, containing the basic region (B) and helix-loop-helix/leucine zipper (HLH/LZ) domain, is necessary and sufficient for sequence-specific DNA binding and heterodimerization with Max. The amino terminus, containing two highly conserved regions termed Myc box (Mb) I and II, is necessary for transcriptional activation and repression. Both the transactivation domain (TAD) and the BHLH/LZ domain are necessary for biological activity. The c-MycS proteins arise from a leaking scanning mechanism and initiate at two closely spaced downstream AUG codons, yielding c-Myc proteins lacking ~100 amino-terminal amino acids, including the highly conserved MbI region. Synthesis of c-MycS increases to levels comparable to c-Myc2 during rapid cell growth, and constitutively high levels of c-MycS synthesis are found in some tumor cell lines. Transcriptional activation by c-Myc through specific E box elements is thought to be essential for its biological role. However, c-MycS is unable to activate transcription through these elements and yet retains the ability to stimulate proliferation, induce anchorage-independent growth, and induce apoptosis. In addition, c-MycS retains the ability to repress transcription of several specific promoters. Furthermore, c-MycS can rescue the c-myc null phenotype in fibroblasts with homozygous deletion of c-myc. Taken together, these data argue against the paradigm that all of the biological functions of c-Myc are mediated by transcriptional activation of specific target genes through E box elements (Xiao, 1998).

Cyclin E-Cdk2 kinase activation is an essential step in Myc-induced proliferation. It is presumed that this requires sequestration of G1 cell cycle inhibitors p27Kip1 and p21Cip1 (Ckis) via a Myc-induced protein. This sequestration is shown to be mediated by the protein synthesis rate of induction of cyclin D1 and/or cyclin D2. Consistent with this, primary cells from cyclin D1-/- and cyclin D2-/- mouse embryos, unlike wild-type controls, do not respond to Myc with increased proliferation, although they undergo accelerated cell death in the absence of serum. Myc sensitivity of cyclin D1-/- cells can be restored by retroviruses expressing either cyclins D1, D2 or a cyclin D1 mutant that forms kinase-defective, Cki-binding cyclin-cdk complexes. Thus, the sequestration function of D cyclins appears essential for Myc-induced cell cycle progression but dispensable for apoptosis (Perez-Roger, 1999).

The rate of the induction of cyclin D1 and/or cyclin D2 protein synthesis leads to the preferential association of p27Kip1 and p21Cip1 with cyclin D-Cdk complexes. At the same time Myc also induces cyclin E protein synthesis; the rates of induction help to promote a net gain of newly formed Cki-free cyclin E-Cdk2 complexes. These complexes become active concomitant with phosphorylation of the kinase subunit by CAK. Consistent with this model of dynamic equilibrium, cyclin E-Cdk2 kinase activity can be controlled by changes in the rates of cyclin D synthesis. Moreover, as shown with a cyclin D mutant that forms kinase-defective Cki-binding cyclin D-Cdk complexes, this link between cyclins D-Cdk and cyclin E-Cdk2 is independent of cyclin D-Cdk activity, but correlates with the ability of cyclin D-Cdk complexes to bind or sequester Ckis. This is strongly supported by the fact that the deficiency of cyclin D1-/- mouse embryo cells to respond to Myc with increased proliferation is restored by expression of the same cyclin D mutant. Consistent with these findings, transient over-expression of either catalytically inactive cyclin D-Cdk, or cyclin E-Cdk2 complexes can rescue the cell cycle inhibitory effect of a dominant-negative Mad-Myc chimera. It is concluded that due to the nature of physical interactions between cyclin D-Cdks and the cell cycle inhibitors p27Kip1 and p21Cip1, cyclin D-Cdk complexes can fulfil a dual function as cell cycle kinases and as buffers for sequestration or release of cell cycle inhibitors (Perez-Roger, 1999 and references therein).

The ability of c-MycS to repress transcription suggests that repression of growth inhibitory genes, such as gadd45 and gas1, remains viable as an alternative model for c-Myc molecular function. Although the transactivation-defective c-MycS protein can function in several biological assays and can substitute for the full-length c-Myc2 in myc null cells, c-MycS may not be able to function as full-length c-Myc2 in all assays. For example, c-MycS does not appear to cooperate with Ras in the transformation of rat embryo fibroblasts (REFs). One explanation for these results is that the Myc/Ras cotransformation of REF cells requires transactivation of specific myc target genes through EMS sites that are not required for stimulation of proliferation, apoptosis, or anchorage-independent growth. Perhaps the ability of c-Myc2 to immortalize, which may be distinct from its ability to stimulate proliferation or induce apoptosis, is required to render REF cells susceptible to transformation by Ras, as Ras has been shown to induce senescence. However, one caveat in the interpretation of these negative results is that in transient transfection assays c-MycS is expressed severalfold less in REF and other cells compared to c-Myc2. The finding of new c-Myc target genes and perhaps new DNA-binding sites will also determine whether c-MycS has any transactivation capabilities. Comparison of c-Myc2 and c-MycS allows the separation of the transcriptional activation and repression abilities of c-Myc and will allow further insight into the molecular basis for the complex and diverse biological functions of c-Myc (Xiao, 1998).

Activation of the Ras/Raf/ERK pathway extends the half-life of the Myc protein and thus enhances the accumulation of Myc activity. Investigated were two N-terminal phosphorylation sites in Myc, Thr 58 and Ser 62, known to be regulated by mitogen stimulation. Phosphorylation of these two residues is critical for determining the stability of Myc. Phosphorylation of Ser 62 is required for Ras-induced stabilization of Myc, likely mediated through the action of ERK. Conversely, phosphorylation of Thr 58, likely mediated by GSK-3 but dependent on the prior phosphorylation of Ser 62, is associated with degradation of Myc. Further analysis demonstrates that the Ras-dependent PI-3K pathway is also critical for controlling Myc protein accumulation, likely through the control of GSK-3 activity. These observations thus define a synergistic role for multiple Ras-mediated phosphorylation pathways in the control of Myc protein accumulation during the initial stage of cell proliferation (Sears, 2000).

The amino acid sequence surrounding Ser 62 represents a consensus ERK recognition sequence, and evidence has been presented that ERK can mediate the phosphorylation of Myc at Ser 62. Mutation of Ser 62 prevents mitogen- and Ras-induced stabilization of Myc. Moreover, phosphorylation at Ser 62 is enhanced under conditions where Myc is stabilized. The importance of Ser 62 in the control of Myc stability is seen in the strict requirement for the stabilization of Myc by Ras, but seen from work that has demonstrated an impaired transforming function when Ser 62 is altered. In contrast, phosphorylation at Thr 58 coincides with a decreased stability of Myc and mutations that prevent Thr 58 phosphorylation lead to stable Myc protein. Once again, this coincides with work that has shown that alteration of Thr 58 enhances the transforming activity of Myc and that mutations at this site are common in Myc proteins derived from tumors. Various lines of work suggest that the GSK-3 protein kinase is most likely responsible for the phosphorylation of Myc at Thr 58. Thr 58 lies within an established consensus, and GSK-3 has been shown to phosphorylate Thr 58 in Myc in vitro. However, unlike ERK, which is tightly regulated by cell growth, the level of GSK-3 protein is constant and does not fluctuate with cell growth. Nevertheless, despite the continual presence of GSK-3 protein, the activity of the kinase is regulated during the initial phase of cell proliferation. In particular, GSK-3 activity is inhibited through the action of PI-3K/AKT. Thus, as Ras initiates the PI-3K/AKT pathway, GSK-3 activity is held in check, preventing the phosphorylation of Thr 58. Only when AKT activity declines would GSK-3 then have the capacity to phosphorylate Thr 58 to induce the degradation of Myc. Thus, Ras activation elicits two responses within the cell that can cooperate to enhance Myc stability: a direct effect of ERK and an indirect effect of AKT (Sears, 2000 and references therein).

Ski is a component of the histone deacetylase complex required for transcriptional repression by Mad and thyroid hormone receptor. The basic helix-loop-helix (bHLH) proteins of the Mad family act as transcriptional repressors after heterodimerization with Max. N-CoR is required for Mad-induced transcriptional repression. The same target sequence of Mad/Max, the so-called E-box, is also recognized by a heterodimer of Myc/Max that activates transcription. It is believed that transcriptional activation of a group of target genes by Myc/Max enhances cellular proliferation or transformation, whereas transcriptional repression of the same target genes by Mad/Max leads to suppression of proliferation or induction of terminal differentiation in a wide range of cell types. The N-CoR/SMRT complex containing mSin3 and histone deacetylase (HDAC) mediates transcriptional repression by nuclear hormone receptors and Mad. The oncogene v-ski was originally identified in avian Sloan-Kettering viruses, and found to transform chicken embryo fibroblasts. Overexpression of either c-ski or v-ski induces either transformation or muscle differentiation of quail embryo fibroblasts, depending on the growth conditions. Furthermore, v-ski transgenic mice have increased muscle mass caused by hypertrophy of type II fast muscle fibers. The capacity of ski to induce both transformation (growth) and differentiation, which is usually associated with the cessation of growth, is an intriguing paradox. The human c-ski proto-oncogene product (c-Ski) is a 728-amino-acid nuclear protein. Recombinant c-Ski protein purified from Escherichia coli cannot directly bind to DNA, but c-Ski in nuclear extracts from mammalian cell cultures binds to DNA, suggesting that c-Ski binds only to DNA when associated with other proteins. The amino- and carboxy-terminal regions of c-Ski possess a cysteine-rich and a coiled-coil region, respectively, and both regions contribute additionally to indirect DNA binding by c-Ski. The v-Ski protein lacks 292 amino acids from the carboxyl terminus of c-Ski, but still contains the amino-terminal cysteine-rich region. The amino-terminal region is responsible for both the cellular transformation and myogenesis capacity of ski. The ski gene family comprises two members, ski and sno (ski-related novel gene) and both have been shown to share clear homology in their amino- and carboxy-terminal regions. Although it was speculated that Ski/Sno proteins are involved in transcriptional repression of specific target genes, their function remains unknown (Nomura, 1999 and references).

The proteins encoded by the ski proto-oncogene family directly bind to N-CoR/SMRT and mSin3A, and form a complex with HDAC. c-Ski and its related gene product Sno are required for transcriptional repression by Mad and thyroid hormone receptor (TRbeta). The oncogenic form, v-Ski, which lacks the mSin3A-binding domain, acts in a dominant-negative fashion, and abrogates transcriptional repression by Mad and TRbeta. In ski-deficient mouse embryos, the ornithine decarboxylase gene, whose expression is normally repressed by Mad-Max, is expressed ectopically. These results show that Ski is a component of the HDAC complex and that Ski is required for the transcriptional repression mediated by this complex. The involvement of c-Ski in the HDAC complex indicates that the function of the HDAC complex is important for oncogenesis (Nomura, 1999).

Study of the transformation capacity of various forms of c-Ski indicate that the amino-terminal cysteine-rich region is responsible for cellular transformation, however, the mechanism of transformation has remained obscure. The results presented here indicate that the amino-terminal region, which is needed for cellular transformation, is responsible for interaction with N-CoR/SMRT. Furthermore, v-Ski and the carboxy-truncated form of c-Ski lack the carboxy-terminal mSin3A-binding domain; they abrogate transcriptional repression by Mad by functioning in a dominant-negative fashion. Transcriptional activation by Myc causes cell proliferation, whereas transcriptional repression by Mad inhibits cell proliferation. Therefore, Mad is thought to act as a tumor suppressor, and in fact, one of the mad-related genes, mxi1, acts as a tumor suppressor using mutant mice. Therefore, abrogation of Mad-induced transcriptional repression by v-Ski may lead to induction of Myc target genes and cellular transformation (Nomura, 1999 and references).

To address the role of N-myc in neurogenesis and in nervous system tumors, N-myc expression was conditionally disrupted in neuronal progenitor cells (NPCs) with a nestin-Cre transgene. Null mice display ataxia, behavioral abnormalities, and tremors that correlate with a twofold decrease in brain mass that disproportionately affects the cerebellum (sixfold reduced in mass) and the cerebral cortex, both of which show signs of disorganization. In control mice at E12.5, a domain of high N-Myc protein expression is detected in the rapidly proliferating cerebellar primordium. Targeted deletion of N-myc results in severely compromised proliferation as shown by a striking decrease in S phase and mitotic cells as well as in cells expressing the Myc target gene cyclin D2, whereas apoptosis is unaffected. Null progenitor cells also have comparatively high levels of the cdk inhibitors p27Kip1 and p18Ink4c, whereas p15Ink4b, p21Cip1, and p19Ink4d levels are unaffected. Many null progenitors also exhibit altered nuclear morphology and size. In addition, loss of N-myc disrupts neuronal differentiation as evidenced by ectopic staining of the neuron specific marker ßTUBIII in the cerebrum. Furthermore, in progenitor cell cultures derived from null embryonic brain, a dramatic increase is observed in neuronal differentiation compared with controls. Thus, N-myc is essential for normal neurogenesis, regulating NPC proliferation, differentiation, and nuclear size. Its effects on proliferation and differentiation appear due, at least in part, to down-regulation of a specific subset of cyclin-dependent kinase inhibitors (Knoepfler, 2002).

Upon activation, cell surface death receptors, Fas/APO-1/CD95 and tumor necrosis factor receptor-1 (TNFR-1), are attached to cytosolic adaptor proteins, which in turn recruit caspase-8 (MACH/FLICE/Mch5) to activate the interleukin-1 beta-converting enzyme (ICE)/CED-3 family protease (caspase) cascade (see Drosophila Caspase 1). However, it remains unknown whether these apoptotic proteases are generally involved in apoptosis triggered by other stimuli, such as Myc and p53. This study suggests that a death protease cascade consisting of caspases and serine proteases plays an essential role in Myc-mediated apoptosis. When Rat-1 fibroblasts stably expressing either s-Myc or c-Myc are induced to undergo apoptosis by serum deprivation, a caspase-3 (CPP32)-like protease activity that cleaves a specific peptide substrate (Ac-DEVD-MCA) appears in the cell lysates. Induction of s-Myc- and c-Myc-mediated apoptotic cell death is effectively prevented by caspase inhibitors such as Z-Asp-CH2-DCB and Ac-DEVD-CHO. Exposing the cells to a serine protease inhibitor also significantly inhibits s-Myc- and c-Myc-mediated apoptosis and the appearance of the caspase-3-like protease activity in vivo. However, the inhibitor does not directly inhibit caspase-3-like protease activity in the apoptotic cell lysates in vitro. Together, these results indicate that caspase-3-like proteases play a critical role in both s-Myc- and c-Myc-mediated apoptosis and that caspase-3-like proteases function downstream of the protease-sensitive step in the signaling pathway of Myc-mediated apoptosis (Kagaya, 1997).

Induction of apoptosis by oncogenes like c-myc may be important in restraining the emergence of neoplasia. However, the mechanism by which c-myc induces apoptosis is unknown. CD95 (also termed Fas or APO-1) is a cell surface transmembrane receptor of the tumor necrosis factor receptor family that activates an intrinsic apoptotic suicide program in cells upon binding either its ligand CD95L or antibody. c-myc-induced apoptosis is shown to require interaction on the cell surface between CD95 and its ligand. c-Myc acts downstream of the CD95 receptor by sensitizing cells to the CD95 death signal. IGF-I signaling and Bcl-2 suppress c-myc-induced apoptosis by also acting downstream of CD95. These findings link two apoptotic pathways previously thought to be independent and establish the dependency of Myc on CD95 signaling for its killing activity (Hueber, 1997).

Pim-1 oncoprotein is a serine/threonine kinase that can closely cooperate with c-Myc in lymphomagenesis, as does Bcl-2. Although the molecular mechanism of this cooperative transformation remains unknown, it is speculated that (similar to Bcl-2) Pim-1 contributes to transformation by inhibiting apoptosis. In this study, therefore, the effect of Pim-1 expression was examined on c-Myc-mediated apoptosis of Rat-1 fibroblasts triggered by serum deprivation. Rather than inhibiting apoptosis, Pim-1 expression stimulates c-Myc-mediated apoptosis in Rat-1 fibroblasts. Pim-1 stimulates c-Myc-mediated apoptosis through an enhancement of the c-Myc-mediated activation of caspase-3 (CPP32)-like proteases, since the suppression of this activity by a specific caspase inhibitor abolishes the apoptosis stimulation by Pim-1. A kinase-defective Pim-1 mutant fails to stimulate c-Myc-mediated apoptosis; Pim-1 expression alone, in the absence of c-Myc overexpression, does not induce apoptosis of serum-deprived Rat-1 cells, indicating that the kinase activity of Pim-1 and the activated c-Myc signaling pathway are required for apoptosis stimulation by Pim-1. Together, these results suggest that Pim-1 oncoprotein stimulates as a serine/threonine kinase the death signaling elicited by c-Myc at a step upstream of caspase-3-like protease activation in Rat-1 fibroblasts. These results also suggest that Pim-1 kinase might function cooperatively with c-Myc through the phosphorylation of a factor(s) that regulates the common signaling pathway involved in c-Myc-mediated apoptosis and transformation (Mochizuki, 1997).

Nuclear factor kappaB (NF-kappaB) appears to participate in the excitotoxin-induced apoptosis of striatal medium spiny neurons. To elucidate molecular mechanisms by which this transcription factor contributes to NMDA receptor-triggered apoptotic cascades in vivo, rats were given the NMDA receptor agonist quinolinic acid (QA) by intrastriatal infusion, and the role of NF-kappaB in the induction of apoptosis-related genes and gene products was evaluated. QA administration induces time-dependent NF-kappaB nuclear translocation. The nuclear NF-kappaB protein after QA treatment is comprised mainly of p65 and c-Rel subunits as detected by gel supershift assay. Levels of c-Myc and p53 mRNA and protein are markedly increased at the time of QA-induced NF-kappaB nuclear translocation. Immunohistochemical analysis shows that c-Myc and p53 induction occurs in the excitotoxin-sensitive medium-sized striatal neurons. NF-kappaB nuclear translocation is blocked in a dose-dependent manner by the cell-permeable recombinant peptide NF-kappaB SN50, but not by the NF-kappaB SN50 control peptide. NF-kappaB SN50 significantly inhibits the QA-induced elevation in levels of c-Myc and p53 mRNA and protein. Pretreatment or posttreatment with NF-kappaB SN50, but not the control peptide, also substantially reduces the intensity of QA-induced internucleosomal DNA fragmentation. The results suggest that NF-kappaB may promote an apoptotic response in striatal medium-sized neurons to excitotoxic insult through upregulation of c-Myc and p53. This study also provides evidence indicating a unique signaling pathway from the cytoplasm to the nucleus, which regulates p53 and c-Myc levels in these neurons during apoptosis (Qin, 1999).

Human monocytic leukemia U937 cells readily undergo apoptosis when they are treated with TNF-alpha, anti-Fas antibody and anticancer drugs, such as etoposide and Ara-C. To study the mechanism of apoptosis, a novel apoptosis-resistant variant, UC, was developed from U937 cells. The UC cells show resistance to apoptosis induced by TNF-alpha, anti-Fas antibody, etoposide and Ara-C. Somatic cell hybridization between U937 and UC shows that apoptosis-resistance to TNF-alpha in UC is genetically recessive, while resistance to etoposide is dominant, suggesting that UC has at least two different mutations functionally involved in apoptosis. Mechanistic analysis reveals that UC cells express reduced amounts of c-Myc. Transfection of the c-myc gene into UC cells restores the sensitivity of the cells to undergo apoptosis induced by TNF-alpha and anti-Fas, which attributes apoptosis-resistance in this circumstance to the reduced expression of c-Myc. In contrast, c-myc transfection into UC cells can not restore their sensitivity to etoposide- and Ara-C-induced apoptosis, arguing against the role of c-myc in chemotherapy-induced apoptosis. However, treating the parental U937 cells with antisense oligonucleotides designed to reduce c-Myc expression renders the cells resistant to etoposide-induced apoptosis as well as to TNF-alpha-induced apoptosis. These results indicate that the reduced expression of c-Myc in UC is strongly associated with the resistance to etoposide-induced apoptosis. The finding that c-myc transfection into UC cannot restore the sensitivity to etoposide-induced apoptosis, suggests UC could have a second mutation that confers resistance to etoposide-induced apoptosis in a genetically dominant manner. Taken together, these present results indicate that c-Myc plays a role in cellular susceptibility to death receptor-mediated and chemotherapy-induced apoptosis (Dong, 1997).

The INK4a-ARF locus is a common target of deletion and mutation in human cancers, possibly second in frequency only to p53. The INK4a tumor suppressor locus encodes p16INK4a, an inhibitor of cyclin D-dependent kinases, and p19ARF, an alternative reading frame protein that also blocks cell proliferation. Establishment of primary mouse embryo fibroblasts (MEFs) as continuously growing cell lines is normally accompanied by loss of the p53 or p19(ARF) tumor suppressors, which act in a common biochemical pathway. Given their apparent immortalizing functions, it seems paradoxical that myc and E1A are also potent inducers of apoptosis. The sensitivity of rodent fibroblasts to myc- or E1A-induced apoptosis correlates directly with the levels of oncoprotein expression and is greatly potentiated by depriving cells of extracellular survival factors. Both Myc and E1A can induce p53 stabilization and trigger p53-dependent transcription. Several lines of evidence indicate that p53 mediates apoptosis by myc and E1A in primary fibroblasts, with p53 loss rendering cells highly resistant to their deleterious effects. For cells overexpressing myc to grow, programmed cell death must be actively suppressed. Therefore, myc overexpression should provide a strong selective pressure for events that dismantle apoptotic signaling pathways. myc rapidly activates ARF and p53 gene expression in primary MEFs and triggers replicative crisis by inducing apoptosis. MEFs that survive myc overexpression sustain p53 mutation or ARF loss during the process of establishment and become immortal. MEFs lacking ARF or p53 exhibit an attenuated apoptotic response to myc and rapidly give rise to cell lines that proliferate in chemically defined medium lacking serum. Therefore, ARF regulates a p53-dependent checkpoint that safeguards cells against hyperproliferative, oncogenic signals (Zindy, 1998).

Overexpression of the MYC protooncogene has been implicated in the genesis of diverse human tumors. Tumorigenesis induced by MYC has been attributed to sustained effects on proliferation and differentiation. MYC may also contribute to tumorigenesis by destabilizing the cellular genome. A transient excess of MYC activity increases tumorigenicity of Rat1A cells by at least 50-fold. The increase in tumorigenicity persists for >30 days after the return of MYC activity to normal levels. The brief surfeit of MYC activity is accompanied by evidence of genomic instability, including karyotypic abnormalities, gene amplification, and hypersensitivity to DNA-damaging agents. MYC also induced genomic destabilization in normal human fibroblasts, although these cells do not become tumorigenic. Stimulation of Rat1A cells with MYC accelerates their passage through G1/S. Moreover, MYC can force normal human fibroblasts to transit G1 and S after treatment with N-(phosphonoacetyl)-L-aspartate (PALA) at concentrations that normally lead to arrest in S phase by checkpoint mechanisms. Instead, the cells subsequently appear to arrest in G2. It is suggest that the accelerated passage through G1 is mutagenic but that the effect of MYC permits a checkpoint response only after G2 has been reached. Thus, MYC may contribute to tumorigenesis through a dominant mutator effect (Felsher, 1999a).

The targeted repair of mutant protooncogenes or the inactivation of their gene products may be a specific and effective therapy for human neoplasia. To examine this possibility, the tetracycline regulatory system has been used to generate transgenic mice that conditionally express the MYC protooncogene in hematopoietic cells. Sustained expression of the MYC transgene culminates in the formation of malignant T cell lymphomas and acute myleoid leukemias. The subsequent inactivation of the transgene causes regression of established tumors. Tumor regression is associated with rapid proliferative arrest, differentiation and apoptosis of tumor cells, and resumption of normal host hematopoiesis. It is concluded that even though tumorigenesis is a multistep process, remediation of a single genetic lesion may be sufficient to reverse malignancy (Felsher, 1999b).

The protooncogene c-myc regulates cell growth, differentiation, and apoptosis, and its aberrant expression is frequently observed in human cancer. However, the consequences of activating c-Myc in an adult tissue, in which these cellular processes are part of normal homeostasis, remain unknown. In order to activate the protein in adult tissue, expression of a switchable form of the c-Myc protein was targeted to the skin epidermis, a well characterized homeostatic tissue. Activation of c-MycER in adult suprabasal epidermis rapidly triggers proliferation and disrupts differentiation of postmitotic keratinocytes. Sustained activation of c-Myc is sufficient to induce papillomatosis together with angiogenesis -- changes that resemble hyperplastic actinic keratosis, a commonly observed human precancerous epithelial lesion. All these premalignant changes spontaneously regress upon deactivation of c-MycER (Pelengaris, 1999).

The bmi-1 and myc oncogenes collaborate strongly in murine lymphomagenesis, but previously, the basis for this collaboration has not been understood. The ink4a-ARF tumor suppressor locus is a critical downstream target of the Polycomb-group transcriptional repressor Bmi-1. Part of Myc's ability to induce apoptosis depends on induction of p19arf. Down-regulation of ink4a-ARF by Bmi-1 underlies its ability to cooperate with Myc in tumorigenesis. Heterozygosity for bmi-1 inhibits lymphomagenesis in Eµ-myc mice by enhancing c-Myc-induced apoptosis. Increased apoptosis is observed in bmi-1 -/- lymphoid organs. This apoptosis can be rescued by deletion of ink4a-ARF or overexpression of bcl2. Furthermore, Bmi-1 collaborates with Myc in enhancing proliferation and transformation of primary embryo fibroblasts (MEFs) in an ink4a-ARF dependent manner, by prohibiting Myc-mediated induction of p19arf and apoptosis. Strong collaboration is observed between the Eµ-myc transgene and heterozygosity for ink4a-ARF. This heterozygosity is accompanied by loss of the wild-type ink4a-ARF allele and formation of highly aggressive B-cell lymphomas. Together, these results reinforce the critical role of Bmi-1 as a dose-dependent regulator of ink4a-ARF, which in its turn acts to prevent tumorigenesis upon activation of oncogenes such as c-myc (Jacobs, 1999).

N-myc is a transcription factor expressed in the developing metanephric kidney and other organs. In mice, complete disruption of the N-myc gene results in fetal death on the first day of renal organogenesis. In addition to the null N-myc allele, others have generated a hypomorphic N-myc allele. In this study, combinations of these mutant genes were used to demonstrate that reduction in N-myc protein levels correlate with fewer developing glomeruli and collecting ducts in embryonic kidney explants. Histological sections reveal that the mutant kidneys are hypoplastic with normal developing structures. The data indicate that the hypoplasia is due to a reduction in proliferation rather than an increase in apoptosis. Thus, N-myc loss causes a decrease in numbers of ureteric bud tips and developing glomeruli in explants and hypoplastic kidneys in vivo, in a dose-dependent manner (Bates, 2000).

Ids regulate differentiation1 through sequestration of basic helix-loop-helix (bHLH) transcription factors, and the consequent inhibition of their ability to bind DNA. Although all Id proteins are viewed as positive regulators of cell-cycle progression, this role has been firmly established only for one member of the Id family, Id2. Only Id2, and not the other members of the family, Id1 and Id3, is able to disrupt the antiproliferative effects of tumor suppressor proteins of the Rb family (the 'pocket' proteins: Rb, p107 and p130), thus allowing cell-cycle progression. This function correlates with the ability of Id2, but not Id1 and Id3, to associate physically with active, hypophosphorylated forms of the pocket proteins in vitro and in vivo. By inactivating Rb, Id2 is also able to abolish the function of another growth-inhibitory protein, p16, that operates upstream of Rb (Lasorella, 2000).

The Rb-null phenotype is lethal by embryonic day 14.5 because of widespread proliferation, defective differentiation and apoptosis in the nervous system and haematopoietic precursors. Since Id2 is expressed in these cell types at the time that Rb-null embryos die1, it is hypothesized that, if Id2 is a natural target of Rb, manifestation of the Rb-mutant phenotype might require intact Id2 (Lasorella, 2000).

Disruption of the Rb pathway (which also includes cyclin D, cdk4/6 and p16) is a hallmark of cancer and it is widely accepted that normal Rb function must be removed, one way or another, in all human tumors. Therefore, it was of interest to determine whether tumor cells deregulate Id2 to bypass the Rb pathway. Correct expression of Id2 is essential to regulate proliferation and differentiation of the neural crest, thus neural crest precursor cells might be sensitive to inappropriate expression of Id2. In humans, neoplastic transformation of neural crest precursors during embryogenesis causes neuroblastoma. Interestingly, genetic alterations of Rb, cyclin D, cdk4/6 or p16 are absent in neuroblastoma. The genetic hallmark of neuroblastoma is amplification of the gene for a member of the Myc family of proto-oncogenes, N-myc. Resembling enforced expression of Id2, Myc overexpression is sufficient to bypass the Rb-p16 growth-inhibitory pathway, in spite of persistent hypophosphorylated Rb. Consequently, Myc activation may release the pressure to mutate components of the Rb-p16 pathway during tumorigenesis (Lasorella, 2000).

Id2-Rb double knockout embryos survive to term with minimal or no defects in neurogenesis and hematopoiesis, but they die at birth from severe reduction of muscle tissue. In neuroblastoma Id2 is overexpressed in cells carrying extra copies of the N-myc gene. In these cells, Id2 is in molar excess of the active form of Rb. The overexpression of Id2 results from transcriptional activation by oncoproteins of the Myc family. Cell-cycle progression induced by Myc oncoproteins requires inactivation of Rb by Id2. Thus, a dual connection links Id2 and Rb: during normal cell-cycle, Rb prohibits the action of Id2 on its natural targets, but oncogenic activation of the Myc-Id2 transcriptional pathway overrides the tumor-suppressor function of Rb (Lasorella, 2000).

Overexpression of the proto-oncogene c-myc has been implicated in the genesis of diverse human tumors. c-Myc seems to regulate diverse biological processes, but its role in tumorigenesis and normal physiology remains enigmatic. An allelic series of mice has been generated in which c-myc expression is incrementally reduced to zero. Fibroblasts from these mice show reduced proliferation and after complete loss of c-Myc function they exit the cell cycle. Myc activity is not needed for cellular growth but does determine the percentage of activated T cells that re-enter the cell cycle. In vivo, reduction of c-Myc levels results in reduced body mass owing to multiorgan hypoplasia, in contrast to Drosophila c-myc mutants, which are smaller as a result of hypotrophy. Drosophila myc substitutes for c-myc in fibroblasts, indicating they have similar biological activities. This suggests there may be fundamental differences in the mechanisms by which mammals and insects control body size. It is proposed that in mammals c-Myc controls the decision to divide or not to divide and thereby functions as a crucial mediator of signals that determine organ and body size (Trumpp, 2001).

Although the data thus appear to suggest that Myc has different functions in Drosophila and mice, this is contradicted by evidence that despite their relatively weak sequence homology, both c-Myc and dMyc proteins have similar biological activities. The results showing that dmyc expression can at least partially rescue the proliferation defect in c-myc-deficient mouse fibroblasts support this view and further suggest that in mouse cells dMyc can control target genes normally regulated by c-Myc. The difference between flies and mice may therefore lie in the identity of the target genes that are controlled by Myc activity in each organism, or the way in which those target genes are integrated in the genetic circuitry of that organism. The opposite phenotypes, organ hypotrophy in Drosophila versus hypoplasia in mice, may therefore result from differences in the way invertebrates and mammals regulate tissue and body size. Among insect species, organ and body size differences appear to be a function of cell number and cell size, whereas among mammalian species they are almost exclusively due to variations in cell number. This may be due to a tighter coupling of cell growth and cell division in mammals than in insects. Such a link would maintain average cell size in an expanding population and would thus make tissue size determination in mammals a function of the number of cell divisions and hence a function of Myc activity (Trumpp, 2001).

Myc overexpression is a hallmark of human cancer and promotes transformation by facilitating immortalization. This function has been linked to the ability of c-Myc to induce the expression of the catalytic subunit of telomerase, telomerase reverse transcriptase (TERT), since ectopic expression of TERT immortalizes some primary human cell types. c-Myc up-regulates telomerase activity in primary mouse embryonic fibroblasts (MEFs) and myeloid cells. Paradoxically, Myc overexpression also triggers the ARF-p53 apoptotic program, which is activated when MEFs undergo replicative crises following culture ex vivo. The rare immortal variants that arise from these cultures generally suffer mutations in p53 or delete Ink4a/ARF, and Myc greatly increases the frequency of these events. Alternative reading frame (ARF)- and p53-null MEFs have increased telomerase activity, as do variant immortal clones that bypass replicative crisis. Similarly, immortal murine NIH-3T3 fibroblasts and myeloid 32D.3 and FDC-P1.2 cells do not express ARF and have robust telomerase activity. However, Myc overexpression in these immortal cells results in remarkably discordant regulation of TERT and telomerase activity. Furthermore, in MEFs and 32D.3 cells, TERT expression and telomerase activity are regulated independently of endogenous c-Myc. Thus, the regulation of TERT and telomerase activity is complex and is also regulated by factors other than Myc, ARF, or p53 (Drissi, 2001).

To explore the role of c-Myc in carcinogenesis, a reversible transgenic model of pancreatic ß cell oncogenesis has been developed using a switchable form of the c-Myc protein. Activation of c-Myc in adult, mature ß cells induces uniform ß cell proliferation but is accompanied by overwhelming apoptosis that rapidly erodes ß cell mass. Thus, the oncogenic potential of c-Myc in ß cells is masked by apoptosis. Upon suppression of c-Myc-induced ß cell apoptosis by coexpression of Bcl-xL, c-Myc triggers rapid and uniform progression into angiogenic, invasive tumors. Subsequent c-Myc deactivation induces rapid regression associated with vascular degeneration and ß cell apoptosis. The data indicate that highly complex neoplastic lesions can be both induced and maintained in vivo by a simple combination of two interlocking molecular lesions. Recent studies with other switchable oncogene transgenic models reinforce the notion that incapacitating the driving oncogenic lesion can lead to expeditious regression of tumors induced in many different tissues by c-Myc, or even T antigens. At least in principle, therefore, the complexity of the tumor phenotype need not be instructed by an equivalent complexity of genetic or epigenetic alteration. Rather, cancers may be underpinned by only a modest number of interdependent, pleiotropic lesions that present themselves as mission-critical targets for effective cancer therapies (Pelengaris, 2002).

In most postmitotic neurons, expression or activation of proteins that stimulate cell cycle progression or DNA replication results in apoptosis. One potential exception to this generalization is neuroblastoma (NB), a tumor derived from the sympathoadrenal lineage. NBs often express high levels of N-myc, a proto-oncogene that can potently activate key components of the cell cycle machinery. In postmitotic sympathetic neurons, N-myc can induce S-phase entry while protecting neurons from death caused by aberrant cell cycle reentry. Specifically, these experiments demonstrate that expression of N-myc at levels similar to those in NBs causes sympathetic neurons to reenter S-phase, as monitored by 5-bromo-2-deoxyuridine incorporation and expression of cell cycle regulatory proteins, and rescues them from apoptosis induced by withdrawal of their obligate survival factor, nerve growth factor. The N-myc-induced cell cycle entry, but not enhanced survival, is inhibited by coexpression of a constitutively hypophosphorylated form of the retinoblastoma tumor suppressor protein, suggesting that these two effects of N-myc are mediated by separate pathways. In contrast, N-myc does not cause S-phase entry in postmitotic cortical neurons. Thus, N-myc both selectively causes sympathetic neurons to reenter the cell cycle and protects them from apoptosis, potentially contributing to their transformation to NBs (Wartiovaara, 2002).

How does N-myc mediate this S-phase entry? The results demonstrating that coexpression of hypophosphorylated pRb rescues the BrdU incorporation argues that N-myc mediates this effect via pRb. Such an effect could be mediated by direct interactions between N-myc and pRb, and it could also be indirectly mediated via an N-myc-induced increase in levels of the inhibitory basic helix-loop-helix protein, Id2, which binds to hypophosphorylated pRb and inhibits its ability to lock cells out of S-phase. An additional, potentially related mechanism involves N-myc-mediated downregulation of the cyclin-dependent kinase inhibitor p27, which in fibroblasts is essential for induction of cyclin E-cdk2 kinase activity, but not for S-phase entry. Although the data presented here do not distinguish between these alternative explanations, it has been observed that p27 levels are decreased and Id2 levels increased in sympathetic neurons overexpressing N-myc, suggesting that decreased p27 may collaborate with increased Id2 to trigger S-phase entry (Wartiovaara, 2002).

Results reported here also indicate that N-myc overexpression does not induce S-phase entry in cortical neurons, suggesting that sympathetic and cortical neurons are locked out of the cell cycle via distinct mechanisms. Such a difference could be predicted by considering the development of these two populations of neurons. Cortical neurons, like most CNS neurons, induce neuronal gene expression and undergo terminal mitosis at the same time. Perturbation of this progenitor-to-postmitotic neuron transition, for example, via functional inhibition of the pRb family or via overexpression of Id2, leads to cellular apoptosis; in no conditions yet reported do cortical cells divide while expressing a neuronal phenotype. In contrast, sympathetic neuroblasts transition through a stage in which they express a neuronal phenotype while still dividing, suggesting that the nature of terminal mitosis differs in sympathetic versus CNS neurons. In that regard, findings may indicate that the mechanisms locking most CNS neurons out of the cell cycle are much more stringent than for sympathetic neurons (Wartiovaara, 2002).

A somewhat surprising finding reported here is that, coincident with S-phase entry, N-myc promotes enhanced survival of sympathetic neurons in the absence of NGF. This is particularly surprising in light of findings indicating that aberrant cell cycle entry is one of the major mechanisms whereby NGF withdrawal causes sympathetic neuron apoptosis. In particular, NGF withdrawal causes increased cyclin D1 expression, and inhibition of cdk4 and -6, both of which phosphorylate and activate pRb, is sufficient to delay NGF withdrawal-induced apoptosis. However, in this regard, NGF withdrawal does not cause enhanced BrdU incorporation and hypophosphorylated pRb is not, by itself, sufficient to rescue sympathetic neurons from apoptosis. Of themselves, these findings do not necessarily argue against a role for cell cycle dysregulation in NGF withdrawal-induced apoptosis, although they do demonstrate that this dysregulation does not actually lead to S-phase reentry. Instead, the data suggest that N-myc-induced survival mechanisms may be 'dominant' to any apoptotic signals deriving from the coincident aberrant reentry into S-phase. Interestingly, data presented here suggest (but do not definitively establish) that one such N-myc-mediated mechanism may involve downregulation of p75NTR (Wartiovaara, 2002).

N-myc is a true oncogene with overexpression in the sympathetic chain and adrenal medulla of transgenic mice that results, via unknown mechanisms, in malignant neuroblastoma. The experimental and clinical data showing a strong correlation between N-myc gene amplification and poor outcome in neuroblastoma suggest that N-myc is involved in the malignant transformation of developing sympathetic precursors or neurons, or both. On the basis of data showing that N-myc can promote S-phase entry and survival of 'postmitotic' sympathetic neurons, a model is suggested in which N-myc contributes to malignant neuroblastoma by either stopping sympathetic neuroblasts from exiting the cell cycle or by collaborating with other risk factors to actually transform postmitotic neurons and cause them to reenter the cell cycle (Wartiovaara, 2002).

Overexpression of c-Myc or E2F1 sensitizes host cells to various types of apoptosis. Overexpressed c-Myc or E2F1 induces accumulation of reactive oxygen species (ROS) and thereby enhances serum-deprived apoptosis in NIH3T3 and Saos-2. During serum deprivation, MnSOD mRNA is induced by NF-kappaB in mock-transfected NIH3T3, while this induction was inhibited in NIH3T3 overexpressing c-Myc or E2F1. In these clones, E2F1 inhibits NF-kappaB activity by binding to its subunit p65 in competition with a heterodimeric partner p50. In addition to overexpressed E2F1, endogenous E2F1 released from Rb is also found to inhibit NF-kappaB activity in a cell cycle-dependent manner by using E2F1+/+ and E2F1-/- murine embryonic fibroblasts. These results indicate that E2F1 promotes apoptosis by inhibiting NF-kappaB activity (Tanaka, 2002).

Oncogene overexpression activates p53 by a mechanism posited to involve uncharacterized hyperproliferative signals. This study was carried out to determine whether such signals produce metabolic perturbations that generate DNA damage, a known p53 inducer. Biochemical, cytological, cell cycle, and global gene expression analyses revealed that brief c-Myc activation can induce DNA damage prior to S phase in normal human fibroblasts. Damage correlates with induction of reactive oxygen species (ROS) without induction of apoptosis. Deregulated c-Myc partially disables the p53-mediated DNA damage response, enabling cells with damaged genomes to enter the cycle, resulting in poor clonogenic survival. An antioxidant reduces ROS, decreases DNA damage and p53 activation, and improves survival. It is proposed that oncogene activation can induce DNA damage and override damage controls, thereby accelerating tumor progression via genetic instability (Vafa, 2002).

The cyclin-dependent kinase (CDK) inhibitors p21Cip1 and p27Kip1 are induced in response to anti-proliferative stimuli and block G1/S-phase progression through the inhibition of CDK2. Although the cyclin E-CDK2 pathway is often deregulated in tumors, the relative contribution of p21Cip1 and p27Kip1 to tumorigenesis is still unclear. The MYC transcription factor is an important regulator of the G1/S transition and its expression is frequently altered in tumors. It has been suggested that p27Kip1 is a crucial G1 target of MYC. In mice, deficiency for p27Kip1 but not p21Cip1 results in decreased survival to retrovirally-induced lymphomagenesis. Importantly, in such p27Kip1 deficient lymphomas an increased frequency of Myc activation is observed. p27Kip1 deficiency also collaborates with MYC overexpression in transgenic lymphoma models. Thus, in vivo, the capacity of MYC to promote tumor growth is fully retained and even enhanced upon p27Kip1 loss. In lymphocytes, MYC overexpression and p27Kip1 deficiency independently stimulate CDK2 activity and augment the fraction of cells in S phase, in support of their distinct roles in tumorigenesis (Martins, 2002).

Myc and E2f1 promote cell cycle progression, but overexpression of either can trigger p53-dependent apoptosis. Mice expressing an Eμ-Myc transgene in B lymphocytes develop lymphomas, the majority of which sustain mutations of either Arf (a tumor suppressor whose product inhibits Mdm2, thereby stabilizing p53) or p53. Eμ-Myc transgenic mice lacking one or both E2f1 alleles exhibit a slower onset of lymphoma development associated with increased expression of the cyclin-dependent kinase inhibitor p27Kip1 and a reduced S phase fraction in precancerous B cells. In contrast, Myc-induced apoptosis and the frequency of Arf and p53 mutations in lymphomas were unaffected by E2f1 loss. Therefore, Myc does not require E2f1 to induce Arf, p53, or apoptosis in B cells, but depends upon E2f1 to accelerate cell cycle progression and downregulate p27Kip1 (Baudino, 2003).

The MYC oncoprotein is a transcription factor that coordinates cell growth and division. MYC overexpression exacerbates genomic instability and sensitizes cells to apoptotic stimuli. MYC directly stimulates transcription of the human Werner syndrome gene, WRN, which encodes a conserved RecQ helicase. Loss-of-function mutations in WRN lead to genomic instability, an elevated cancer risk, and premature cellular senescence. The overexpression of MYC in WRN syndrome fibroblasts or after WRN depletion from control fibroblasts leads to rapid cellular senescence that can not be suppressed by hTERT expression. It is proposed that WRN up-regulation by MYC may promote MYC-driven tumorigenesis by preventing cellular senescence (Grandori, 2003).

Alterations in c-myc oncogene expression have been implicated in the pathogenesis of several human cancers, including Burkitt and diffuse large B-cell lymphomas, breast and prostate cancer, colon cancer, melanoma, and multiple myeloma. The proteins encoded by MYC transcriptional target genes appear to regulate cell-cycle progression and cell growth while sensitizing cells to apoptotic stimuli. MYC may also be able to promote tumorigenesis by up-regulating the expression of genes such as hTERT that play a role in cellular immortalization or the escape from senescence. It was reasoned that MYC might modulate the expression of other genes that control cellular senescence, and thus determined whether the gene encoding the Werner syndrome RecQ helicase protein is a MYC transcriptional target (Grandori, 2003).

Werner syndrome (WRN) is an uncommon, autosomal recessive genetic instability syndrome that results from loss-of-function mutations in the chromosome 8p12-p11.2 WRN gene. The WRN phenotype resembles premature aging, and includes genomic instability, an elevated risk of malignancy, and accelerated cellular senescence. Genetic instability following loss of the 162-kD WRN RecQ helicase protein reflects the physiologic role of WRN in mitotic recombination and repair. Conversely, the elevated levels of WRN observed in immortalized and human tumor cell lines may help insure continuous cell proliferation. In order to delineate potential interactions between MYC and WRN in tumorigenesis, whether WRN expression is modulated by MYC was determined, and cellular responses to MYC overexpression in the absence of WRN were monitored. The results indicate that WRN expression appears to be required to avoid cellular senescence upon MYC up-regulation in hTERT-immortalized fibroblasts (Grandori, 2003).

Mnt is a Max-interacting transcriptional repressor that has been hypothesized to function as a Myc antagonist. To investigate Mnt function the Mnt gene was deleted in mice. Since mice lacking Mnt are born severely runted and typically die within several days of birth, mouse embryo fibroblasts (MEFs) derived from these mice and conditional Mnt knockout mice were used in this study. In the absence of Mnt, MEFs prematurely enter the S phase of the cell cycle and proliferated more rapidly than Mnt+/+ MEFs. Defective cell cycle control in the absence of Mnt is linked to upregulation of Cdk4 and cyclin E and the Cdk4 gene appears to be a direct target of Mnt-Myc antagonism. Like MEFs that overexpress Myc, Mnt-/- MEFs are prone to apoptosis, efficiently escape senescence and can be transformed with oncogenic Ras alone. Consistent with Mnt functioning as a tumor suppressor, conditional inactivation of Mnt in breast epithelium leads to adenocarinomas. These results demonstrate a unique negative regulatory role for Mnt in governing key Myc functions associated with cell proliferation and tumorigenesis (Hurlin, 2003).

Epidemiological findings suggest that the consequences of a given oncogenic stimulus vary depending upon the developmental state of the target tissue at the time of exposure. This is particularly evident in the mammary gland, where both age at exposure to a carcinogenic stimulus and the timing of a first full-term pregnancy can markedly alter the risk of developing breast cancer. Analogous to this, the biological consequences of activating oncogenes, such as MYC, can be influenced by cellular context both in terms of cell lineage and cellular environment. In light of this, it was hypothesized that the consequences of aberrant MYC activation in the mammary gland might be determined by the developmental state of the gland at the time of MYC exposure. To test this hypothesis directly, a doxycycline-inducible transgenic mouse model was used to overexpress MYC during different stages of mammary gland development. Using this model, it was found that the ability of MYC to inhibit postpartum lactation is due entirely to its activation within a specific 72-hour window during mid-pregnancy; by contrast, MYC activation either prior to or following this 72-hour window has little or no effect on postpartum lactation. Surprisingly, it was found that MYC does not block postpartum lactation by inhibiting mammary epithelial differentiation, but rather by promoting differentiation and precocious lactation during pregnancy, which in turn leads to premature involution of the gland. This developmental stage-specific ability of MYC to promote mammary epithelial differentiation is tightly linked to its ability to downregulate caveolin 1 and activate Stat5 in a developmental stage-specific manner. These findings provide unique in vivo molecular evidence for developmental stage-specific effects of oncogene activation, as well as the first evidence linking MYC with activation of the Jak2-Stat5 signaling pathway (Blakely, 2005).

ß-catenin signaling is heavily involved in organogenesis. This study investigated how pancreas differentiation, growth and homeostasis are affected following inactivation of an endogenous inhibitor of ß-catenin, adenomatous polyposis coli (Apc). In adult mice, Apc-deficient pancreata are enlarged, solely as a result of hyperplasia of acinar cells, which accumulate ß-catenin, with the sparing of islets. Expression of a target of ß-catenin, the proto-oncogene c-myc (Myc), is increased in acinar cells lacking Apc, suggesting that c-myc expression is essential for hyperplasia. In support of this hypothesis, it was found that conditional inactivation of c-myc in pancreata lacking Apc completely reverse the acinar hyperplasia. Apc loss in organs such as the liver, colon and kidney, as well as experimental misexpression of c-myc in pancreatic acinar cells, lead to tumor formation with high penetrance. Surprisingly, pancreas tumors failed to develop following conditional pancreas Apc inactivation. In Apc-deficient acini of aged mice, these studies revealed a cessation of their exaggerated proliferation and a reduced expression of c-myc, in spite of the persistent accumulation of ß-catenin. In conclusion, this work shows that ß-catenin modulation of c-myc is an essential regulator of acinar growth control, and unveils an unprecedented example of Apc requirement in the pancreas that is both temporally restricted and cell-specific. This provides new insights into the mechanisms of tumor pathogenesis and tumor suppression in the pancreas (Strom, 2007).

Inhibition of protein phosphatase 2A (PP2A) activity has been identified as a prerequisite for the transformation of human cells. However, the molecular mechanisms by which PP2A activity is inhibited in human cancers are currently unclear. In this study, a cellular inhibitor of PP2A with oncogenic activity is described. The protein, designated Cancerous Inhibitor of PP2A (CIP2A), interacts directly with the oncogenic transcription factor c-Myc, inhibits PP2A activity toward c-Myc serine 62 (S62), and thereby prevents c-Myc proteolytic degradation. In addition to its function in c-Myc stabilization, CIP2A promotes anchorage-independent cell growth and in vivo tumor formation. The oncogenic activity of CIP2A is demonstrated by transformation of human cells by overexpression of CIP2A. Importantly, CIP2A is overexpressed in two common human malignancies, head and neck squamous cell carcinoma (HNSCC) and colon cancer. Thus, these data show that CIP2A is a human oncoprotein that inhibits PP2A and stabilizes c-Myc in human malignancies (Junttila, 2007).

FoxO transcription factors play critical roles in cell cycle control and cellular stress responses, and abrogation of FoxO function promotes focus formation by Myc in vitro. Stable introduction of a dominant-negative FoxO moiety (dnFoxO) into Eµ-myc transgenic hematopoietic stem cells accelerates lymphoma development in recipient mice by attenuating Myc-induced apoptosis. When expressed in Eµ-myc; p53+/- progenitor cells, dnFoxO alleviates the pressure to inactivate the remaining p53 allele in upcoming lymphomas. Expression of the p53 upstream regulator p19Arf (alternative reading frame of p16INK, also called p14arf in humans and p19arf in mice) is virtually undetectable in most dnFoxO-positive Myc-driven lymphomas. It was found that FoxO proteins bind to a distinct site within the Ink4a/Arf locus and activate Arf expression. Moreover, constitutive Myc signaling induces a marked increase in nuclear FoxO levels and stimulates binding of FoxO proteins to the Arf locus. These data demonstrate that FoxO factors mediate Myc-induced Arf expression and provide direct genetic evidence for their tumor-suppressive capacity (Bouchard, 2007).

The FoxO subclass of forkhead-box transcription factors (consisting of FoxO1 (FKHR), FoxO3a (FKHRL1), FoxO4 (AFX), and FoxO6) regulates numerous cellular functions including proliferation, stress sensitivity, and survival; it has also been implicated in the regulation of organism life span. The members of this family activate gene expression via interaction with a specific DNA sequence, and known targets include the cell cycle regulating Kip1, the proapoptotic Bim, the DNA damage-responsive Gadd45a, and the oxidative stress-protective manganese superoxide dismutase genes. In addition, FoxO proteins can repress several cell cycle promoting genes (e.g., cyclin D1 and cyclin D2) in a manner that might be independent of direct DNA binding (Bouchard, 2007 and references therein).

In response to growth factor signaling and to oxidative stress, FoxO proteins are post-translationally modified by phosphorylation, acetylation, and ubiquitination; collectively, these modifications regulate FoxOs’ subcellular localization, transcriptional activity, and stability. Notably, all FoxO proteins are inhibited by protein kinase B/Akt-mediated phosphorylation that promotes their nuclear export and subsequent proteolytic degradation via ubiquitination by the SCFSkp2 complex. As a consequence, FoxO proteins mediate the induction of p27Kip1 and Bim expression in response to inhibition of the phosphatidylinositol-3-OH (PI3)-kinase/Akt pathway (Bouchard, 2007 and references therein).

Conditional codeletion of the FoxO1, FoxO3, and FoxO4 alleles uncovers a context-dependent cancer-prone phenotype characterized by thymic lymphomas forming in some and hemangiomas developing in most animals after a long latency, suggesting that FoxO proteins exert their tumor-suppressive capability in the presence of additional oncogenic mutations. In support of this view, Akt-mediated phosphorylation of FoxO proteins has been identified as the critical PI3-kinase signaling component that substitutes for oncogenic Ras in Myc-induced proliferation and focus formation in vitro. Furthermore, constitutive Akt signaling cooperates with Myc to accelerate B-cell lymphomagenesis; however, it remains unclear whether Akt-mediated phosphorylation of FoxO proteins contributes to Eµ-myc transgenic lymphoma formation in this setting (Bouchard, 2007).

Proapoptotic Arf/p53 signaling is known as the pivotal Myc-induced tumor-suppressive barrier. Eµ-myc transgenic mice lacking one p53 allele develop lymphomas that inactivate the remaining wild-type allele. Likewise, Eµ-myc; Arf+/- or Eµ-myc; Ink4a/Arf+/- mice produce tumors that lack expression of p19Arf. Primary Arf deletions protect cells from acquiring p53 mutations during lymphoma development. Similarly, introduction of strictly anti-apoptotic genes such as bcl2 or a dominant-negative form of caspase 9 into Eµ-myc; p53+/- hematopoietic stem cells alleviates the pressure to inactivate p53, thereby underscoring apoptosis as the critical p53-governed tumor suppressor function in Myc-driven lymphomagenesis (Bouchard, 2007).

Previous work has shown that p53 and FoxO3a share target genes and that FoxO3a can activate transcription via p53 sites, suggesting a potential collaboration of FoxO3a and p53 in tumor suppression. Although a direct interaction between FoxO3a and p53 proteins has been demonstrated under conditions of overexpression, the observed collaboration would be consistent with an as-yet-unidentified FoxO target acting upstream of p53. This study reports that FoxO factors elicit their tumor-suppressive potential as critical inducers of Arf during Myc-driven lymphomagenesis, providing further evidence for a close link between the FoxO and p53 tumor suppressor pathways (Bouchard, 2007).

Studying the early stages of cancer can provide important insight into the molecular basis of the disease. A preneoplastic stage was identified in the patched (ptc) mutant mouse, a model for the brain tumor medulloblastoma. Preneoplastic cells (PNCs) are found in most ptc mutants during early adulthood, but only 15% of these animals develop tumors. Although PNCs are found in mice that develop tumors, the ability of PNCs to give rise to tumors has never been demonstrated directly, and the fate of cells that do not form tumors remains unknown. Using genetic fate mapping and orthotopic transplantation, definitive evidence was provide that PNCs give rise to tumors, and the predominant fate of PNCs that do not form tumors is differentiation. Moreover, N-myc, a gene commonly amplified in medulloblastoma, can dramatically alter the fate of PNCs, preventing differentiation and driving progression to tumors. Importantly, N-myc allows PNCs to grow independently of hedgehog signaling, making the resulting tumors resistant to hedgehog antagonists. These studies provide the first direct evidence that PNCs can give rise to tumors, and demonstrate that identification of genetic changes that promote tumor progression is critical for designing effective therapies for cancer (Kessler, 2009).

The Myc protein suppresses the transcription of several cyclin-dependent kinase inhibitors (CKIs) via binding to POZ domain/zinc finger transciption factor Miz1. Whether this interaction is important for Myc's ability to induce or maintain tumorigenesis is not known. This study shows that the oncogenic potential of a point mutant of Myc (MycV394D) that is selectively deficient in binding to Miz1 is greatly attenuated. Binding of Myc to Miz1 is continuously required to repress CKI expression and inhibit accumulation of trimethylated histone H3 at Lys 9 (H3K9triMe), a hallmark of cellular senescence, in T-cell lymphomas. Lymphomas that arise express high amounts of transforming growth factor beta-2 (TGFbeta-2) and TGFbeta-3. Upon Myc suppression, TGFbeta signaling is required to induce CKI expression and cellular senescence and suppress tumor recurrence. Binding of Myc to Miz1 is required to antagonize growth suppression and induction of senescence by TGFbeta. Since lymphomas express high levels of TGFbeta, they are poised to elicit an autocrine program of senescence upon Myc inactivation, demonstrating that TGFbeta is a key factor that establishes oncogene addiction of T-cell lymphomas (van Riggelen, 2010).

Myc is required for induction of pluripotent stem cells from mouse fibroblasts

Differentiated cells can be reprogrammed to an embryonic-like state by transfer of nuclear contents into oocytes or by fusion with embryonic stem (ES) cells. Little is known about factors that induce this reprogramming. This study demonstrates induction of pluripotent stem cells from mouse embryonic or adult tail tip fibroblasts (TTFs) by introducing four factors, Oct3/4, Sox2, c-Myc, and Klf4, under ES cell culture conditions. Unexpectedly, Nanog was dispensable. These cells, which have been designated iPS (induced pluripotent stem) cells, exhibit the morphology and growth properties of ES cells and express ES cell marker genes. Subcutaneous transplantation of iPS cells into nude mice resulted in tumors containing a variety of tissues from all three germ layers. Following injection into blastocysts, iPS cells contributed to mouse embryonic development. These data demonstrate that pluripotent stem cells can be directly generated from fibroblast cultures by the addition of only a few defined factors (Takahashi, 2007).

Oct3/4, Sox2, and Nanog have been shown to function as core transcription factors in maintaining pluripotency. Among the three, it was found that Oct3/4 and Sox2 are essential for the generation of iPS cells. c-Myc and Klf4 were also identified as essential factors. These two tumor-related factors could not be replaced by other oncogenes including E-Ras, Tcl1, β-catenin, and Stat3 (Takahashi, 2007).

The c-Myc protein has many downstream targets that enhance proliferation and transformation, many of which may have roles in the generation of iPS cells. Of note, c-Myc associates with histone acetyltransferase (HAT) complexes, including TRRAP, which is a core subunit of the TIP60 (see Drosophila Tip60) and GCN5 HAT complexes, CREB binding protein (CBP), and p300. Within the mammalian genome, there may be up to 25,000 c-Myc binding sites, many more than the predicted number of Oct3/4 and Sox2 binding sites. c-Myc protein may induce global histone acetylation, thus allowing Oct3/4 and Sox2 to bind to their specific target loci. Klf4 has been shown to repress p53 directly, and p53 protein has been shown to suppress Nanog during ES cell differentiation. iPS cells showed levels of p53 protein lower than those in MEFs. Thus, Klf4 might contribute to activation of Nanog and other ES cell-specific genes through p53 repression. Alternatively, Klf4 might function as an inhibitor of Myc-induced apoptosis through the repression of p53 in this system. In contrast, Klf4 activates p21CIP1, thereby suppressing cell proliferation. This antiproliferation function of Klf4 might be inhibited by c-Myc, which suppresses the expression of p21CIP1. The balance between c-Myc and Klf4 may be important for the generation of iPS cells (Takahashi, 2007).

One question that remains concerns the origin of the iPS cells. With the retroviral expression system, it is estimated that only a small portion of cells expressing the four factors become iPS cells. The low frequency suggests that rare tissue stem/progenitor cells that coexisted in the fibroblast cultures might have given rise to the iPS cells. Indeed, multipotent stem cells have been isolated from skin. These studies showed that ~0.067% of mouse skin cells are stem cells. One explanation for the low frequency of iPS cell derivation is that the four factors transform tissue stem cells. However, it was found that the four factors induced iPS cells with comparably low efficiency even from bone marrow stroma, which should be more enriched in mesenchymal stem cells and other multipotent cells. Furthermore, cells induced by the three factors were nullipotent. DNA microarray analyses suggested that iPS-MEF4 cells and iPS-MEF3 cells have the same origin. These results do not favor multipotent tissue stem cells as the origin of iPS cells (Takahashi, 2007).

There are several other possibilities for the low frequency of iPS cell derivation. First, the levels of the four factors required for generation of pluripotent cells may have narrow ranges, and only a small portion of cells expressing all four of the factors at the right levels can acquire ES cell-like properties. Consistent with this idea, a mere 50% increase or decrease in Oct3/4 proteins induces differentiation of ES cells. iPS clones overexpressed the four factors when RNA levels were analyzed, but their protein levels were comparable to those in ES cells, suggesting that the iPS clones possess a mechanism (or mechanisms) that tightly regulates the protein levels of the four factors. It is speculated that high amounts of the four factors are required in the initial stage of iPS cell generation, but, once they acquire ES cell-like status, too much of the factors are detrimental for self-renewal. Only a small portion of transduced cells show such appropriate transgene expression. Second, generation of pluripotent cells may require additional chromosomal alterations, which take place spontaneously during culture or are induced by some of the four factors. Although the iPS-TTFgfp4 clones had largely normal karyotypes, the existence of minor chromosomal alterations cannot be ruled out. Site-specific retroviral insertion may also play a role. Southern blot analyses showed that each iPS clone has ~20 retroviral integrations. Some of these may have caused silencing or fusion with endogenous genes. Further studies will be required to determine the origin of iPS cells (Takahashi, 2007).

Another unsolved question is whether the four factors identified play roles in reprogramming induced by fusion with ES cells or nuclear transfer into oocytes. Since the four factors are expressed in ES cells at high levels, it is reasonable to speculate that they are involved in the reprogramming machinery that exists in ES cells. These result is also consistent with the finding that the reprogramming activity resides in the nucleus, but not in the cytoplasm, of ES cells. However, iPS cells were not identical to ES cells, as shown by the global gene-expression patterns and DNA methylation status. It is possible that additional important factors have been missed. One such candidate is ECAT1, although its forced expression in iPS cells did not consistently upregulate ES cell marker genes (Takahashi, 2007).

More obscure are the roles of the four factors, especially Klf4 and c-Myc, in the reprogramming observed in oocytes. Both Klf4 and c-Myc are dispensable for preimplantation mouse development. Furthermore, c-myc is not detected in oocytes. In contrast, L-myc is expressed maternally in oocytes. Klf17 and Klf7, but not Klf4, are found in expressed sequence-tag libraries derived from unfertilized mouse eggs. Klf4 and c-Myc might be compensated by these related proteins. It is highly likely that other factors are also required to induce complete reprogramming and totipotency in oocytes (Takahashi, 2007).

Reprogramming of human somatic cells to pluripotency with defined factors

Pluripotency pertains to the cells of early embryos that can generate all of the tissues in the organism. Embryonic stem cells are embryo-derived cell lines that retain pluripotency and represent invaluable tools for research into the mechanisms of tissue formation. Recently, murine fibroblasts have been reprogrammed directly to pluripotency by ectopic expression of four transcription factors (Oct4, Sox2, Klf4 and Myc) to yield induced pluripotent stem (iPS) cells. Using these same factors, iPS cells were derived from fetal, neonatal and adult human primary cells, including dermal fibroblasts isolated from a skin biopsy of a healthy research subject. Human iPS cells resemble embryonic stem cells in morphology and gene expression and in the capacity to form teratomas in immune-deficient mice. These data demonstrate that defined factors can reprogramme human cells to pluripotency, and establish a method whereby patient-specific cells might be established in culture (Park, 2008).

Myc function in plants

Jasmonates (JA) are important regulators of plant defense responses that activate expression of many wound-induced genes including the tomato proteinase inhibitor II (pin2) and leucine aminopeptidase (LAP) genes. Elements required for JA induction of the LAP gene are all present in the -317 to -78 proximal promoter region. Using yeast one-hybrid screening, the bHLH-leu zipper JAMYC2 and JAMYC10 proteins were identified, specifically recognizing a T/G-box AACGTG motif in this promoter fragment. Mutation of the G-box element decreases JA-responsive LAP promoter expression. Expression of JAMYC2 and JAMYC10 is induced by JA, with a kinetics that precedes that of the LAP or pin2 transcripts. JAMYC overexpression enhanced JA-induced expression of these defense genes in potato, but did not result in constitutive transcript accumulation. Using footprinting assays, an additional protected element was identified, located directly adjacent to the T/G-box motif. Mutation of this element abolishes JA response, showing that recognition of this duplicated element is also required for gene expression. Knockout mutants in the AtMYC2 homolog gene of Arabidopsis are insensitive to JA and exhibit a decreased activation of the JA-responsive genes AtVSP and JR1. Activation of the PDF1.2 and b-CHI, ethylene/JA-responsive genes, is, however, increased in these mutants. These results show that the JAMYC/AtMYC2 transcription factors function as members of a MYC-based regulatory system conserved in dicotyledonous plants with a key role in JA-induced defense gene activation (Boter, 2004).

Myc, proliferation, apoptosis and tumorigenesis

The transactivation of TCF target genes induced by Wnt pathway mutations constitutes the primary transforming event in colorectal cancer (CRC). Disruption of ß-catenin/TCF-4 activity in CRC cells induces a rapid G1 arrest and blocks a genetic program that is physiologically active in the proliferative compartment of colon crypts. Coincidently, an intestinal differentiation program is induced. The TCF-4 target gene c-MYC plays a central role in this switch by direct repression of the p21CIP1/WAF1 promoter. Following disruption of ß-catenin/TCF-4 activity, the decreased expression of c-MYC releases p21CIP1/WAF1 transcription, which in turn mediates G1 arrest and differentiation. Thus, the ß-catenin/TCF-4 complex constitutes the master switch that controls proliferation versus differentiation in healthy and malignant intestinal epithelial cells (van de Wetering, 2002).

c-MYC plays a central role in the proliferative capacity of many cancers, including CRC. tHE data imply that c-MYC blocks the expression of the cell cycle inhibitor p21CIP1/WAF1. The region responsible for p21CIP1/WAF1 regulation has been mapped to a 200 bp fragment of the proximal promoter. The presence of MIZ-1 and c-MYC on this promoter suggests that c-MYC-mediated repression of p21CIP1/WAF1 occurs by a mechanism resembling c-MYC control of p15INK4b, i.e., through preventing promoter activation by the transcription factor MIZ-1. Decreased expression of c-MYC would allow MIZ-1 to activate p21CIP1/WAF1 transcription. The complementarity in the expression of c-MYC and p21CIP1/WAF1 in the intestine supports this mechanism (van de Wetering, 2002).

The carboxyl terminus of c-Myc, containing the basic region (B) and helix-loop-helix/leucine zipper (HLH/LZ) domain, is necessary and sufficient for sequence-specific DNA binding and heterodimerization with Max. The amino terminus, containing two highly conserved regions termed Myc box (Mb) I and II, is necessary for transcriptional activation and repression. Both the transactivation domain (TAD) and the BHLH/LZ domain are necessary for biological activity. The c-MycS proteins arise from a leaking scanning mechanism and initiate at two closely spaced downstream AUG codons, yielding c-Myc proteins lacking ~100 amino-terminal amino acids, including the highly conserved MbI region. Synthesis of c-MycS increases to levels comparable to c-Myc2 during rapid cell growth, and constitutively high levels of c-MycS synthesis are found in some tumor cell lines. Transcriptional activation by c-Myc through specific E box elements is thought to be essential for its biological role. However, c-MycS is unable to activate transcription through these elements and yet retains the ability to stimulate proliferation, induce anchorage-independent growth, and induce apoptosis. In addition, c-MycS retains the ability to repress transcription of several specific promoters. Furthermore, c-MycS can rescue the c-myc null phenotype in fibroblasts with homozygous deletion of c-myc. Taken together, these data argue against the paradigm that all of the biological functions of c-Myc are mediated by transcriptional activation of specific target genes through E box elements (Xiao, 1998).

Cyclin E-Cdk2 kinase activation is an essential step in Myc-induced proliferation. It is presumed that this requires sequestration of G1 cell cycle inhibitors p27Kip1 and p21Cip1 (Ckis) via a Myc-induced protein. This sequestration is shown to be mediated by the protein synthesis rate of induction of cyclin D1 and/or cyclin D2. Consistent with this, primary cells from cyclin D1-/- and cyclin D2-/- mouse embryos, unlike wild-type controls, do not respond to Myc with increased proliferation, although they undergo accelerated cell death in the absence of serum. Myc sensitivity of cyclin D1-/- cells can be restored by retroviruses expressing either cyclins D1, D2 or a cyclin D1 mutant that forms kinase-defective, Cki-binding cyclin-cdk complexes. Thus, the sequestration function of D cyclins appears essential for Myc-induced cell cycle progression but dispensable for apoptosis (Perez-Roger, 1999).

The rate of the induction of cyclin D1 and/or cyclin D2 protein synthesis leads to the preferential association of p27Kip1 and p21Cip1 with cyclin D-Cdk complexes. At the same time Myc also induces cyclin E protein synthesis; the rates of induction help to promote a net gain of newly formed Cki-free cyclin E-Cdk2 complexes. These complexes become active concomitant with phosphorylation of the kinase subunit by CAK. Consistent with this model of dynamic equilibrium, cyclin E-Cdk2 kinase activity can be controlled by changes in the rates of cyclin D synthesis. Moreover, as shown with a cyclin D mutant that forms kinase-defective Cki-binding cyclin D-Cdk complexes, this link between cyclins D-Cdk and cyclin E-Cdk2 is independent of cyclin D-Cdk activity, but correlates with the ability of cyclin D-Cdk complexes to bind or sequester Ckis. This is strongly supported by the fact that the deficiency of cyclin D1-/- mouse embryo cells to respond to Myc with increased proliferation is restored by expression of the same cyclin D mutant. Consistent with these findings, transient over-expression of either catalytically inactive cyclin D-Cdk, or cyclin E-Cdk2 complexes can rescue the cell cycle inhibitory effect of a dominant-negative Mad-Myc chimera. It is concluded that due to the nature of physical interactions between cyclin D-Cdks and the cell cycle inhibitors p27Kip1 and p21Cip1, cyclin D-Cdk complexes can fulfil a dual function as cell cycle kinases and as buffers for sequestration or release of cell cycle inhibitors (Perez-Roger, 1999 and references therein).

The ability of c-MycS to repress transcription suggests that repression of growth inhibitory genes, such as gadd45 and gas1, remains viable as an alternative model for c-Myc molecular function. Although the transactivation-defective c-MycS protein can function in several biological assays and can substitute for the full-length c-Myc2 in myc null cells, c-MycS may not be able to function as full-length c-Myc2 in all assays. For example, c-MycS does not appear to cooperate with Ras in the transformation of rat embryo fibroblasts (REFs). One explanation for these results is that the Myc/Ras cotransformation of REF cells requires transactivation of specific myc target genes through EMS sites that are not required for stimulation of proliferation, apoptosis, or anchorage-independent growth. Perhaps the ability of c-Myc2 to immortalize, which may be distinct from its ability to stimulate proliferation or induce apoptosis, is required to render REF cells susceptible to transformation by Ras, as Ras has been shown to induce senescence. However, one caveat in the interpretation of these negative results is that in transient transfection assays c-MycS is expressed severalfold less in REF and other cells compared to c-Myc2. The finding of new c-Myc target genes and perhaps new DNA-binding sites will also determine whether c-MycS has any transactivation capabilities. Comparison of c-Myc2 and c-MycS allows the separation of the transcriptional activation and repression abilities of c-Myc and will allow further insight into the molecular basis for the complex and diverse biological functions of c-Myc (Xiao, 1998).

Activation of the Ras/Raf/ERK pathway extends the half-life of the Myc protein and thus enhances the accumulation of Myc activity. Investigated were two N-terminal phosphorylation sites in Myc, Thr 58 and Ser 62, known to be regulated by mitogen stimulation. Phosphorylation of these two residues is critical for determining the stability of Myc. Phosphorylation of Ser 62 is required for Ras-induced stabilization of Myc, likely mediated through the action of ERK. Conversely, phosphorylation of Thr 58, likely mediated by GSK-3 but dependent on the prior phosphorylation of Ser 62, is associated with degradation of Myc. Further analysis demonstrates that the Ras-dependent PI-3K pathway is also critical for controlling Myc protein accumulation, likely through the control of GSK-3 activity. These observations thus define a synergistic role for multiple Ras-mediated phosphorylation pathways in the control of Myc protein accumulation during the initial stage of cell proliferation (Sears, 2000).

The amino acid sequence surrounding Ser 62 represents a consensus ERK recognition sequence, and evidence has been presented that ERK can mediate the phosphorylation of Myc at Ser 62. Mutation of Ser 62 prevents mitogen- and Ras-induced stabilization of Myc. Moreover, phosphorylation at Ser 62 is enhanced under conditions where Myc is stabilized. The importance of Ser 62 in the control of Myc stability is seen in the strict requirement for the stabilization of Myc by Ras, but seen from work that has demonstrated an impaired transforming function when Ser 62 is altered. In contrast, phosphorylation at Thr 58 coincides with a decreased stability of Myc and mutations that prevent Thr 58 phosphorylation lead to stable Myc protein. Once again, this coincides with work that has shown that alteration of Thr 58 enhances the transforming activity of Myc and that mutations at this site are common in Myc proteins derived from tumors. Various lines of work suggest that the GSK-3 protein kinase is most likely responsible for the phosphorylation of Myc at Thr 58. Thr 58 lies within an established consensus, and GSK-3 has been shown to phosphorylate Thr 58 in Myc in vitro. However, unlike ERK, which is tightly regulated by cell growth, the level of GSK-3 protein is constant and does not fluctuate with cell growth. Nevertheless, despite the continual presence of GSK-3 protein, the activity of the kinase is regulated during the initial phase of cell proliferation. In particular, GSK-3 activity is inhibited through the action of PI-3K/AKT. Thus, as Ras initiates the PI-3K/AKT pathway, GSK-3 activity is held in check, preventing the phosphorylation of Thr 58. Only when AKT activity declines would GSK-3 then have the capacity to phosphorylate Thr 58 to induce the degradation of Myc. Thus, Ras activation elicits two responses within the cell that can cooperate to enhance Myc stability: a direct effect of ERK and an indirect effect of AKT (Sears, 2000 and references therein).

Ski is a component of the histone deacetylase complex required for transcriptional repression by Mad and thyroid hormone receptor. The basic helix-loop-helix (bHLH) proteins of the Mad family act as transcriptional repressors after heterodimerization with Max. N-CoR is required for Mad-induced transcriptional repression. The same target sequence of Mad/Max, the so-called E-box, is also recognized by a heterodimer of Myc/Max that activates transcription. It is believed that transcriptional activation of a group of target genes by Myc/Max enhances cellular proliferation or transformation, whereas transcriptional repression of the same target genes by Mad/Max leads to suppression of proliferation or induction of terminal differentiation in a wide range of cell types. The N-CoR/SMRT complex containing mSin3 and histone deacetylase (HDAC) mediates transcriptional repression by nuclear hormone receptors and Mad. The oncogene v-ski was originally identified in avian Sloan-Kettering viruses, and found to transform chicken embryo fibroblasts. Overexpression of either c-ski or v-ski induces either transformation or muscle differentiation of quail embryo fibroblasts, depending on the growth conditions. Furthermore, v-ski transgenic mice have increased muscle mass caused by hypertrophy of type II fast muscle fibers. The capacity of ski to induce both transformation (growth) and differentiation, which is usually associated with the cessation of growth, is an intriguing paradox. The human c-ski proto-oncogene product (c-Ski) is a 728-amino-acid nuclear protein. Recombinant c-Ski protein purified from Escherichia coli cannot directly bind to DNA, but c-Ski in nuclear extracts from mammalian cell cultures binds to DNA, suggesting that c-Ski binds only to DNA when associated with other proteins. The amino- and carboxy-terminal regions of c-Ski possess a cysteine-rich and a coiled-coil region, respectively, and both regions contribute additionally to indirect DNA binding by c-Ski. The v-Ski protein lacks 292 amino acids from the carboxyl terminus of c-Ski, but still contains the amino-terminal cysteine-rich region. The amino-terminal region is responsible for both the cellular transformation and myogenesis capacity of ski. The ski gene family comprises two members, ski and sno (ski-related novel gene) and both have been shown to share clear homology in their amino- and carboxy-terminal regions. Although it was speculated that Ski/Sno proteins are involved in transcriptional repression of specific target genes, their function remains unknown (Nomura, 1999 and references).

The proteins encoded by the ski proto-oncogene family directly bind to N-CoR/SMRT and mSin3A, and form a complex with HDAC. c-Ski and its related gene product Sno are required for transcriptional repression by Mad and thyroid hormone receptor (TRbeta). The oncogenic form, v-Ski, which lacks the mSin3A-binding domain, acts in a dominant-negative fashion, and abrogates transcriptional repression by Mad and TRbeta. In ski-deficient mouse embryos, the ornithine decarboxylase gene, whose expression is normally repressed by Mad-Max, is expressed ectopically. These results show that Ski is a component of the HDAC complex and that Ski is required for the transcriptional repression mediated by this complex. The involvement of c-Ski in the HDAC complex indicates that the function of the HDAC complex is important for oncogenesis (Nomura, 1999).

Study of the transformation capacity of various forms of c-Ski indicate that the amino-terminal cysteine-rich region is responsible for cellular transformation, however, the mechanism of transformation has remained obscure. The results presented here indicate that the amino-terminal region, which is needed for cellular transformation, is responsible for interaction with N-CoR/SMRT. Furthermore, v-Ski and the carboxy-truncated form of c-Ski lack the carboxy-terminal mSin3A-binding domain; they abrogate transcriptional repression by Mad by functioning in a dominant-negative fashion. Transcriptional activation by Myc causes cell proliferation, whereas transcriptional repression by Mad inhibits cell proliferation. Therefore, Mad is thought to act as a tumor suppressor, and in fact, one of the mad-related genes, mxi1, acts as a tumor suppressor using mutant mice. Therefore, abrogation of Mad-induced transcriptional repression by v-Ski may lead to induction of Myc target genes and cellular transformation (Nomura, 1999 and references).

To address the role of N-myc in neurogenesis and in nervous system tumors, N-myc expression was conditionally disrupted in neuronal progenitor cells (NPCs) with a nestin-Cre transgene. Null mice display ataxia, behavioral abnormalities, and tremors that correlate with a twofold decrease in brain mass that disproportionately affects the cerebellum (sixfold reduced in mass) and the cerebral cortex, both of which show signs of disorganization. In control mice at E12.5, a domain of high N-Myc protein expression is detected in the rapidly proliferating cerebellar primordium. Targeted deletion of N-myc results in severely compromised proliferation as shown by a striking decrease in S phase and mitotic cells as well as in cells expressing the Myc target gene cyclin D2, whereas apoptosis is unaffected. Null progenitor cells also have comparatively high levels of the cdk inhibitors p27Kip1 and p18Ink4c, whereas p15Ink4b, p21Cip1, and p19Ink4d levels are unaffected. Many null progenitors also exhibit altered nuclear morphology and size. In addition, loss of N-myc disrupts neuronal differentiation as evidenced by ectopic staining of the neuron specific marker ßTUBIII in the cerebrum. Furthermore, in progenitor cell cultures derived from null embryonic brain, a dramatic increase is observed in neuronal differentiation compared with controls. Thus, N-myc is essential for normal neurogenesis, regulating NPC proliferation, differentiation, and nuclear size. Its effects on proliferation and differentiation appear due, at least in part, to down-regulation of a specific subset of cyclin-dependent kinase inhibitors (Knoepfler, 2002).

Upon activation, cell surface death receptors, Fas/APO-1/CD95 and tumor necrosis factor receptor-1 (TNFR-1), are attached to cytosolic adaptor proteins, which in turn recruit caspase-8 (MACH/FLICE/Mch5) to activate the interleukin-1 beta-converting enzyme (ICE)/CED-3 family protease (caspase) cascade (see Drosophila Caspase 1). However, it remains unknown whether these apoptotic proteases are generally involved in apoptosis triggered by other stimuli, such as Myc and p53. This study suggests that a death protease cascade consisting of caspases and serine proteases plays an essential role in Myc-mediated apoptosis. When Rat-1 fibroblasts stably expressing either s-Myc or c-Myc are induced to undergo apoptosis by serum deprivation, a caspase-3 (CPP32)-like protease activity that cleaves a specific peptide substrate (Ac-DEVD-MCA) appears in the cell lysates. Induction of s-Myc- and c-Myc-mediated apoptotic cell death is effectively prevented by caspase inhibitors such as Z-Asp-CH2-DCB and Ac-DEVD-CHO. Exposing the cells to a serine protease inhibitor also significantly inhibits s-Myc- and c-Myc-mediated apoptosis and the appearance of the caspase-3-like protease activity in vivo. However, the inhibitor does not directly inhibit caspase-3-like protease activity in the apoptotic cell lysates in vitro. Together, these results indicate that caspase-3-like proteases play a critical role in both s-Myc- and c-Myc-mediated apoptosis and that caspase-3-like proteases function downstream of the protease-sensitive step in the signaling pathway of Myc-mediated apoptosis (Kagaya, 1997).

Induction of apoptosis by oncogenes like c-myc may be important in restraining the emergence of neoplasia. However, the mechanism by which c-myc induces apoptosis is unknown. CD95 (also termed Fas or APO-1) is a cell surface transmembrane receptor of the tumor necrosis factor receptor family that activates an intrinsic apoptotic suicide program in cells upon binding either its ligand CD95L or antibody. c-myc-induced apoptosis is shown to require interaction on the cell surface between CD95 and its ligand. c-Myc acts downstream of the CD95 receptor by sensitizing cells to the CD95 death signal. IGF-I signaling and Bcl-2 suppress c-myc-induced apoptosis by also acting downstream of CD95. These findings link two apoptotic pathways previously thought to be independent and establish the dependency of Myc on CD95 signaling for its killing activity (Hueber, 1997).

Pim-1 oncoprotein is a serine/threonine kinase that can closely cooperate with c-Myc in lymphomagenesis, as does Bcl-2. Although the molecular mechanism of this cooperative transformation remains unknown, it is speculated that (similar to Bcl-2) Pim-1 contributes to transformation by inhibiting apoptosis. In this study, therefore, the effect of Pim-1 expression was examined on c-Myc-mediated apoptosis of Rat-1 fibroblasts triggered by serum deprivation. Rather than inhibiting apoptosis, Pim-1 expression stimulates c-Myc-mediated apoptosis in Rat-1 fibroblasts. Pim-1 stimulates c-Myc-mediated apoptosis through an enhancement of the c-Myc-mediated activation of caspase-3 (CPP32)-like proteases, since the suppression of this activity by a specific caspase inhibitor abolishes the apoptosis stimulation by Pim-1. A kinase-defective Pim-1 mutant fails to stimulate c-Myc-mediated apoptosis; Pim-1 expression alone, in the absence of c-Myc overexpression, does not induce apoptosis of serum-deprived Rat-1 cells, indicating that the kinase activity of Pim-1 and the activated c-Myc signaling pathway are required for apoptosis stimulation by Pim-1. Together, these results suggest that Pim-1 oncoprotein stimulates as a serine/threonine kinase the death signaling elicited by c-Myc at a step upstream of caspase-3-like protease activation in Rat-1 fibroblasts. These results also suggest that Pim-1 kinase might function cooperatively with c-Myc through the phosphorylation of a factor(s) that regulates the common signaling pathway involved in c-Myc-mediated apoptosis and transformation (Mochizuki, 1997).

Nuclear factor kappaB (NF-kappaB) appears to participate in the excitotoxin-induced apoptosis of striatal medium spiny neurons. To elucidate molecular mechanisms by which this transcription factor contributes to NMDA receptor-triggered apoptotic cascades in vivo, rats were given the NMDA receptor agonist quinolinic acid (QA) by intrastriatal infusion, and the role of NF-kappaB in the induction of apoptosis-related genes and gene products was evaluated. QA administration induces time-dependent NF-kappaB nuclear translocation. The nuclear NF-kappaB protein after QA treatment is comprised mainly of p65 and c-Rel subunits as detected by gel supershift assay. Levels of c-Myc and p53 mRNA and protein are markedly increased at the time of QA-induced NF-kappaB nuclear translocation. Immunohistochemical analysis shows that c-Myc and p53 induction occurs in the excitotoxin-sensitive medium-sized striatal neurons. NF-kappaB nuclear translocation is blocked in a dose-dependent manner by the cell-permeable recombinant peptide NF-kappaB SN50, but not by the NF-kappaB SN50 control peptide. NF-kappaB SN50 significantly inhibits the QA-induced elevation in levels of c-Myc and p53 mRNA and protein. Pretreatment or posttreatment with NF-kappaB SN50, but not the control peptide, also substantially reduces the intensity of QA-induced internucleosomal DNA fragmentation. The results suggest that NF-kappaB may promote an apoptotic response in striatal medium-sized neurons to excitotoxic insult through upregulation of c-Myc and p53. This study also provides evidence indicating a unique signaling pathway from the cytoplasm to the nucleus, which regulates p53 and c-Myc levels in these neurons during apoptosis (Qin, 1999).

Human monocytic leukemia U937 cells readily undergo apoptosis when they are treated with TNF-alpha, anti-Fas antibody and anticancer drugs, such as etoposide and Ara-C. To study the mechanism of apoptosis, a novel apoptosis-resistant variant, UC, was developed from U937 cells. The UC cells show resistance to apoptosis induced by TNF-alpha, anti-Fas antibody, etoposide and Ara-C. Somatic cell hybridization between U937 and UC shows that apoptosis-resistance to TNF-alpha in UC is genetically recessive, while resistance to etoposide is dominant, suggesting that UC has at least two different mutations functionally involved in apoptosis. Mechanistic analysis reveals that UC cells express reduced amounts of c-Myc. Transfection of the c-myc gene into UC cells restores the sensitivity of the cells to undergo apoptosis induced by TNF-alpha and anti-Fas, which attributes apoptosis-resistance in this circumstance to the reduced expression of c-Myc. In contrast, c-myc transfection into UC cells can not restore their sensitivity to etoposide- and Ara-C-induced apoptosis, arguing against the role of c-myc in chemotherapy-induced apoptosis. However, treating the parental U937 cells with antisense oligonucleotides designed to reduce c-Myc expression renders the cells resistant to etoposide-induced apoptosis as well as to TNF-alpha-induced apoptosis. These results indicate that the reduced expression of c-Myc in UC is strongly associated with the resistance to etoposide-induced apoptosis. The finding that c-myc transfection into UC cannot restore the sensitivity to etoposide-induced apoptosis, suggests UC could have a second mutation that confers resistance to etoposide-induced apoptosis in a genetically dominant manner. Taken together, these present results indicate that c-Myc plays a role in cellular susceptibility to death receptor-mediated and chemotherapy-induced apoptosis (Dong, 1997).

The INK4a-ARF locus is a common target of deletion and mutation in human cancers, possibly second in frequency only to p53. The INK4a tumor suppressor locus encodes p16INK4a, an inhibitor of cyclin D-dependent kinases, and p19ARF, an alternative reading frame protein that also blocks cell proliferation. Establishment of primary mouse embryo fibroblasts (MEFs) as continuously growing cell lines is normally accompanied by loss of the p53 or p19(ARF) tumor suppressors, which act in a common biochemical pathway. Given their apparent immortalizing functions, it seems paradoxical that myc and E1A are also potent inducers of apoptosis. The sensitivity of rodent fibroblasts to myc- or E1A-induced apoptosis correlates directly with the levels of oncoprotein expression and is greatly potentiated by depriving cells of extracellular survival factors. Both Myc and E1A can induce p53 stabilization and trigger p53-dependent transcription. Several lines of evidence indicate that p53 mediates apoptosis by myc and E1A in primary fibroblasts, with p53 loss rendering cells highly resistant to their deleterious effects. For cells overexpressing myc to grow, programmed cell death must be actively suppressed. Therefore, myc overexpression should provide a strong selective pressure for events that dismantle apoptotic signaling pathways. myc rapidly activates ARF and p53 gene expression in primary MEFs and triggers replicative crisis by inducing apoptosis. MEFs that survive myc overexpression sustain p53 mutation or ARF loss during the process of establishment and become immortal. MEFs lacking ARF or p53 exhibit an attenuated apoptotic response to myc and rapidly give rise to cell lines that proliferate in chemically defined medium lacking serum. Therefore, ARF regulates a p53-dependent checkpoint that safeguards cells against hyperproliferative, oncogenic signals (Zindy, 1998).

Overexpression of the MYC protooncogene has been implicated in the genesis of diverse human tumors. Tumorigenesis induced by MYC has been attributed to sustained effects on proliferation and differentiation. MYC may also contribute to tumorigenesis by destabilizing the cellular genome. A transient excess of MYC activity increases tumorigenicity of Rat1A cells by at least 50-fold. The increase in tumorigenicity persists for >30 days after the return of MYC activity to normal levels. The brief surfeit of MYC activity is accompanied by evidence of genomic instability, including karyotypic abnormalities, gene amplification, and hypersensitivity to DNA-damaging agents. MYC also induced genomic destabilization in normal human fibroblasts, although these cells do not become tumorigenic. Stimulation of Rat1A cells with MYC accelerates their passage through G1/S. Moreover, MYC can force normal human fibroblasts to transit G1 and S after treatment with N-(phosphonoacetyl)-L-aspartate (PALA) at concentrations that normally lead to arrest in S phase by checkpoint mechanisms. Instead, the cells subsequently appear to arrest in G2. It is suggest that the accelerated passage through G1 is mutagenic but that the effect of MYC permits a checkpoint response only after G2 has been reached. Thus, MYC may contribute to tumorigenesis through a dominant mutator effect (Felsher, 1999a).

The targeted repair of mutant protooncogenes or the inactivation of their gene products may be a specific and effective therapy for human neoplasia. To examine this possibility, the tetracycline regulatory system has been used to generate transgenic mice that conditionally express the MYC protooncogene in hematopoietic cells. Sustained expression of the MYC transgene culminates in the formation of malignant T cell lymphomas and acute myleoid leukemias. The subsequent inactivation of the transgene causes regression of established tumors. Tumor regression is associated with rapid proliferative arrest, differentiation and apoptosis of tumor cells, and resumption of normal host hematopoiesis. It is concluded that even though tumorigenesis is a multistep process, remediation of a single genetic lesion may be sufficient to reverse malignancy (Felsher, 1999b).

The protooncogene c-myc regulates cell growth, differentiation, and apoptosis, and its aberrant expression is frequently observed in human cancer. However, the consequences of activating c-Myc in an adult tissue, in which these cellular processes are part of normal homeostasis, remain unknown. In order to activate the protein in adult tissue, expression of a switchable form of the c-Myc protein was targeted to the skin epidermis, a well characterized homeostatic tissue. Activation of c-MycER in adult suprabasal epidermis rapidly triggers proliferation and disrupts differentiation of postmitotic keratinocytes. Sustained activation of c-Myc is sufficient to induce papillomatosis together with angiogenesis -- changes that resemble hyperplastic actinic keratosis, a commonly observed human precancerous epithelial lesion. All these premalignant changes spontaneously regress upon deactivation of c-MycER (Pelengaris, 1999).

The bmi-1 and myc oncogenes collaborate strongly in murine lymphomagenesis, but previously, the basis for this collaboration has not been understood. The ink4a-ARF tumor suppressor locus is a critical downstream target of the Polycomb-group transcriptional repressor Bmi-1. Part of Myc's ability to induce apoptosis depends on induction of p19arf. Down-regulation of ink4a-ARF by Bmi-1 underlies its ability to cooperate with Myc in tumorigenesis. Heterozygosity for bmi-1 inhibits lymphomagenesis in Eµ-myc mice by enhancing c-Myc-induced apoptosis. Increased apoptosis is observed in bmi-1 -/- lymphoid organs. This apoptosis can be rescued by deletion of ink4a-ARF or overexpression of bcl2. Furthermore, Bmi-1 collaborates with Myc in enhancing proliferation and transformation of primary embryo fibroblasts (MEFs) in an ink4a-ARF dependent manner, by prohibiting Myc-mediated induction of p19arf and apoptosis. Strong collaboration is observed between the Eµ-myc transgene and heterozygosity for ink4a-ARF. This heterozygosity is accompanied by loss of the wild-type ink4a-ARF allele and formation of highly aggressive B-cell lymphomas. Together, these results reinforce the critical role of Bmi-1 as a dose-dependent regulator of ink4a-ARF, which in its turn acts to prevent tumorigenesis upon activation of oncogenes such as c-myc (Jacobs, 1999).

N-myc is a transcription factor expressed in the developing metanephric kidney and other organs. In mice, complete disruption of the N-myc gene results in fetal death on the first day of renal organogenesis. In addition to the null N-myc allele, others have generated a hypomorphic N-myc allele. In this study, combinations of these mutant genes were used to demonstrate that reduction in N-myc protein levels correlate with fewer developing glomeruli and collecting ducts in embryonic kidney explants. Histological sections reveal that the mutant kidneys are hypoplastic with normal developing structures. The data indicate that the hypoplasia is due to a reduction in proliferation rather than an increase in apoptosis. Thus, N-myc loss causes a decrease in numbers of ureteric bud tips and developing glomeruli in explants and hypoplastic kidneys in vivo, in a dose-dependent manner (Bates, 2000).

Ids regulate differentiation1 through sequestration of basic helix-loop-helix (bHLH) transcription factors, and the consequent inhibition of their ability to bind DNA. Although all Id proteins are viewed as positive regulators of cell-cycle progression, this role has been firmly established only for one member of the Id family, Id2. Only Id2, and not the other members of the family, Id1 and Id3, is able to disrupt the antiproliferative effects of tumor suppressor proteins of the Rb family (the 'pocket' proteins: Rb, p107 and p130), thus allowing cell-cycle progression. This function correlates with the ability of Id2, but not Id1 and Id3, to associate physically with active, hypophosphorylated forms of the pocket proteins in vitro and in vivo. By inactivating Rb, Id2 is also able to abolish the function of another growth-inhibitory protein, p16, that operates upstream of Rb (Lasorella, 2000).

The Rb-null phenotype is lethal by embryonic day 14.5 because of widespread proliferation, defective differentiation and apoptosis in the nervous system and haematopoietic precursors. Since Id2 is expressed in these cell types at the time that Rb-null embryos die1, it is hypothesized that, if Id2 is a natural target of Rb, manifestation of the Rb-mutant phenotype might require intact Id2 (Lasorella, 2000).

Disruption of the Rb pathway (which also includes cyclin D, cdk4/6 and p16) is a hallmark of cancer and it is widely accepted that normal Rb function must be removed, one way or another, in all human tumors. Therefore, it was of interest to determine whether tumor cells deregulate Id2 to bypass the Rb pathway. Correct expression of Id2 is essential to regulate proliferation and differentiation of the neural crest, thus neural crest precursor cells might be sensitive to inappropriate expression of Id2. In humans, neoplastic transformation of neural crest precursors during embryogenesis causes neuroblastoma. Interestingly, genetic alterations of Rb, cyclin D, cdk4/6 or p16 are absent in neuroblastoma. The genetic hallmark of neuroblastoma is amplification of the gene for a member of the Myc family of proto-oncogenes, N-myc. Resembling enforced expression of Id2, Myc overexpression is sufficient to bypass the Rb-p16 growth-inhibitory pathway, in spite of persistent hypophosphorylated Rb. Consequently, Myc activation may release the pressure to mutate components of the Rb-p16 pathway during tumorigenesis (Lasorella, 2000).

Id2-Rb double knockout embryos survive to term with minimal or no defects in neurogenesis and hematopoiesis, but they die at birth from severe reduction of muscle tissue. In neuroblastoma Id2 is overexpressed in cells carrying extra copies of the N-myc gene. In these cells, Id2 is in molar excess of the active form of Rb. The overexpression of Id2 results from transcriptional activation by oncoproteins of the Myc family. Cell-cycle progression induced by Myc oncoproteins requires inactivation of Rb by Id2. Thus, a dual connection links Id2 and Rb: during normal cell-cycle, Rb prohibits the action of Id2 on its natural targets, but oncogenic activation of the Myc-Id2 transcriptional pathway overrides the tumor-suppressor function of Rb (Lasorella, 2000).

Overexpression of the proto-oncogene c-myc has been implicated in the genesis of diverse human tumors. c-Myc seems to regulate diverse biological processes, but its role in tumorigenesis and normal physiology remains enigmatic. An allelic series of mice has been generated in which c-myc expression is incrementally reduced to zero. Fibroblasts from these mice show reduced proliferation and after complete loss of c-Myc function they exit the cell cycle. Myc activity is not needed for cellular growth but does determine the percentage of activated T cells that re-enter the cell cycle. In vivo, reduction of c-Myc levels results in reduced body mass owing to multiorgan hypoplasia, in contrast to Drosophila c-myc mutants, which are smaller as a result of hypotrophy. Drosophila myc substitutes for c-myc in fibroblasts, indicating they have similar biological activities. This suggests there may be fundamental differences in the mechanisms by which mammals and insects control body size. It is proposed that in mammals c-Myc controls the decision to divide or not to divide and thereby functions as a crucial mediator of signals that determine organ and body size (Trumpp, 2001).

Although the data thus appear to suggest that Myc has different functions in Drosophila and mice, this is contradicted by evidence that despite their relatively weak sequence homology, both c-Myc and dMyc proteins have similar biological activities. The results showing that dmyc expression can at least partially rescue the proliferation defect in c-myc-deficient mouse fibroblasts support this view and further suggest that in mouse cells dMyc can control target genes normally regulated by c-Myc. The difference between flies and mice may therefore lie in the identity of the target genes that are controlled by Myc activity in each organism, or the way in which those target genes are integrated in the genetic circuitry of that organism. The opposite phenotypes, organ hypotrophy in Drosophila versus hypoplasia in mice, may therefore result from differences in the way invertebrates and mammals regulate tissue and body size. Among insect species, organ and body size differences appear to be a function of cell number and cell size, whereas among mammalian species they are almost exclusively due to variations in cell number. This may be due to a tighter coupling of cell growth and cell division in mammals than in insects. Such a link would maintain average cell size in an expanding population and would thus make tissue size determination in mammals a function of the number of cell divisions and hence a function of Myc activity (Trumpp, 2001).

Myc overexpression is a hallmark of human cancer and promotes transformation by facilitating immortalization. This function has been linked to the ability of c-Myc to induce the expression of the catalytic subunit of telomerase, telomerase reverse transcriptase (TERT), since ectopic expression of TERT immortalizes some primary human cell types. c-Myc up-regulates telomerase activity in primary mouse embryonic fibroblasts (MEFs) and myeloid cells. Paradoxically, Myc overexpression also triggers the ARF-p53 apoptotic program, which is activated when MEFs undergo replicative crises following culture ex vivo. The rare immortal variants that arise from these cultures generally suffer mutations in p53 or delete Ink4a/ARF, and Myc greatly increases the frequency of these events. Alternative reading frame (ARF)- and p53-null MEFs have increased telomerase activity, as do variant immortal clones that bypass replicative crisis. Similarly, immortal murine NIH-3T3 fibroblasts and myeloid 32D.3 and FDC-P1.2 cells do not express ARF and have robust telomerase activity. However, Myc overexpression in these immortal cells results in remarkably discordant regulation of TERT and telomerase activity. Furthermore, in MEFs and 32D.3 cells, TERT expression and telomerase activity are regulated independently of endogenous c-Myc. Thus, the regulation of TERT and telomerase activity is complex and is also regulated by factors other than Myc, ARF, or p53 (Drissi, 2001).

To explore the role of c-Myc in carcinogenesis, a reversible transgenic model of pancreatic ß cell oncogenesis has been developed using a switchable form of the c-Myc protein. Activation of c-Myc in adult, mature ß cells induces uniform ß cell proliferation but is accompanied by overwhelming apoptosis that rapidly erodes ß cell mass. Thus, the oncogenic potential of c-Myc in ß cells is masked by apoptosis. Upon suppression of c-Myc-induced ß cell apoptosis by coexpression of Bcl-xL, c-Myc triggers rapid and uniform progression into angiogenic, invasive tumors. Subsequent c-Myc deactivation induces rapid regression associated with vascular degeneration and ß cell apoptosis. The data indicate that highly complex neoplastic lesions can be both induced and maintained in vivo by a simple combination of two interlocking molecular lesions. Recent studies with other switchable oncogene transgenic models reinforce the notion that incapacitating the driving oncogenic lesion can lead to expeditious regression of tumors induced in many different tissues by c-Myc, or even T antigens. At least in principle, therefore, the complexity of the tumor phenotype need not be instructed by an equivalent complexity of genetic or epigenetic alteration. Rather, cancers may be underpinned by only a modest number of interdependent, pleiotropic lesions that present themselves as mission-critical targets for effective cancer therapies (Pelengaris, 2002).

In most postmitotic neurons, expression or activation of proteins that stimulate cell cycle progression or DNA replication results in apoptosis. One potential exception to this generalization is neuroblastoma (NB), a tumor derived from the sympathoadrenal lineage. NBs often express high levels of N-myc, a proto-oncogene that can potently activate key components of the cell cycle machinery. In postmitotic sympathetic neurons, N-myc can induce S-phase entry while protecting neurons from death caused by aberrant cell cycle reentry. Specifically, these experiments demonstrate that expression of N-myc at levels similar to those in NBs causes sympathetic neurons to reenter S-phase, as monitored by 5-bromo-2-deoxyuridine incorporation and expression of cell cycle regulatory proteins, and rescues them from apoptosis induced by withdrawal of their obligate survival factor, nerve growth factor. The N-myc-induced cell cycle entry, but not enhanced survival, is inhibited by coexpression of a constitutively hypophosphorylated form of the retinoblastoma tumor suppressor protein, suggesting that these two effects of N-myc are mediated by separate pathways. In contrast, N-myc does not cause S-phase entry in postmitotic cortical neurons. Thus, N-myc both selectively causes sympathetic neurons to reenter the cell cycle and protects them from apoptosis, potentially contributing to their transformation to NBs (Wartiovaara, 2002).

How does N-myc mediate this S-phase entry? The results demonstrating that coexpression of hypophosphorylated pRb rescues the BrdU incorporation argues that N-myc mediates this effect via pRb. Such an effect could be mediated by direct interactions between N-myc and pRb, and it could also be indirectly mediated via an N-myc-induced increase in levels of the inhibitory basic helix-loop-helix protein, Id2, which binds to hypophosphorylated pRb and inhibits its ability to lock cells out of S-phase. An additional, potentially related mechanism involves N-myc-mediated downregulation of the cyclin-dependent kinase inhibitor p27, which in fibroblasts is essential for induction of cyclin E-cdk2 kinase activity, but not for S-phase entry. Although the data presented here do not distinguish between these alternative explanations, it has been observed that p27 levels are decreased and Id2 levels increased in sympathetic neurons overexpressing N-myc, suggesting that decreased p27 may collaborate with increased Id2 to trigger S-phase entry (Wartiovaara, 2002).

Results reported here also indicate that N-myc overexpression does not induce S-phase entry in cortical neurons, suggesting that sympathetic and cortical neurons are locked out of the cell cycle via distinct mechanisms. Such a difference could be predicted by considering the development of these two populations of neurons. Cortical neurons, like most CNS neurons, induce neuronal gene expression and undergo terminal mitosis at the same time. Perturbation of this progenitor-to-postmitotic neuron transition, for example, via functional inhibition of the pRb family or via overexpression of Id2, leads to cellular apoptosis; in no conditions yet reported do cortical cells divide while expressing a neuronal phenotype. In contrast, sympathetic neuroblasts transition through a stage in which they express a neuronal phenotype while still dividing, suggesting that the nature of terminal mitosis differs in sympathetic versus CNS neurons. In that regard, findings may indicate that the mechanisms locking most CNS neurons out of the cell cycle are much more stringent than for sympathetic neurons (Wartiovaara, 2002).

A somewhat surprising finding reported here is that, coincident with S-phase entry, N-myc promotes enhanced survival of sympathetic neurons in the absence of NGF. This is particularly surprising in light of findings indicating that aberrant cell cycle entry is one of the major mechanisms whereby NGF withdrawal causes sympathetic neuron apoptosis. In particular, NGF withdrawal causes increased cyclin D1 expression, and inhibition of cdk4 and -6, both of which phosphorylate and activate pRb, is sufficient to delay NGF withdrawal-induced apoptosis. However, in this regard, NGF withdrawal does not cause enhanced BrdU incorporation and hypophosphorylated pRb is not, by itself, sufficient to rescue sympathetic neurons from apoptosis. Of themselves, these findings do not necessarily argue against a role for cell cycle dysregulation in NGF withdrawal-induced apoptosis, although they do demonstrate that this dysregulation does not actually lead to S-phase reentry. Instead, the data suggest that N-myc-induced survival mechanisms may be 'dominant' to any apoptotic signals deriving from the coincident aberrant reentry into S-phase. Interestingly, data presented here suggest (but do not definitively establish) that one such N-myc-mediated mechanism may involve downregulation of p75NTR (Wartiovaara, 2002).

N-myc is a true oncogene with overexpression in the sympathetic chain and adrenal medulla of transgenic mice that results, via unknown mechanisms, in malignant neuroblastoma. The experimental and clinical data showing a strong correlation between N-myc gene amplification and poor outcome in neuroblastoma suggest that N-myc is involved in the malignant transformation of developing sympathetic precursors or neurons, or both. On the basis of data showing that N-myc can promote S-phase entry and survival of 'postmitotic' sympathetic neurons, a model is suggested in which N-myc contributes to malignant neuroblastoma by either stopping sympathetic neuroblasts from exiting the cell cycle or by collaborating with other risk factors to actually transform postmitotic neurons and cause them to reenter the cell cycle (Wartiovaara, 2002).

Overexpression of c-Myc or E2F1 sensitizes host cells to various types of apoptosis. Overexpressed c-Myc or E2F1 induces accumulation of reactive oxygen species (ROS) and thereby enhances serum-deprived apoptosis in NIH3T3 and Saos-2. During serum deprivation, MnSOD mRNA is induced by NF-kappaB in mock-transfected NIH3T3, while this induction was inhibited in NIH3T3 overexpressing c-Myc or E2F1. In these clones, E2F1 inhibits NF-kappaB activity by binding to its subunit p65 in competition with a heterodimeric partner p50. In addition to overexpressed E2F1, endogenous E2F1 released from Rb is also found to inhibit NF-kappaB activity in a cell cycle-dependent manner by using E2F1+/+ and E2F1-/- murine embryonic fibroblasts. These results indicate that E2F1 promotes apoptosis by inhibiting NF-kappaB activity (Tanaka, 2002).

Oncogene overexpression activates p53 by a mechanism posited to involve uncharacterized hyperproliferative signals. This study was carried out to determine whether such signals produce metabolic perturbations that generate DNA damage, a known p53 inducer. Biochemical, cytological, cell cycle, and global gene expression analyses revealed that brief c-Myc activation can induce DNA damage prior to S phase in normal human fibroblasts. Damage correlates with induction of reactive oxygen species (ROS) without induction of apoptosis. Deregulated c-Myc partially disables the p53-mediated DNA damage response, enabling cells with damaged genomes to enter the cycle, resulting in poor clonogenic survival. An antioxidant reduces ROS, decreases DNA damage and p53 activation, and improves survival. It is proposed that oncogene activation can induce DNA damage and override damage controls, thereby accelerating tumor progression via genetic instability (Vafa, 2002).

The cyclin-dependent kinase (CDK) inhibitors p21Cip1 and p27Kip1 are induced in response to anti-proliferative stimuli and block G1/S-phase progression through the inhibition of CDK2. Although the cyclin E-CDK2 pathway is often deregulated in tumors, the relative contribution of p21Cip1 and p27Kip1 to tumorigenesis is still unclear. The MYC transcription factor is an important regulator of the G1/S transition and its expression is frequently altered in tumors. It has been suggested that p27Kip1 is a crucial G1 target of MYC. In mice, deficiency for p27Kip1 but not p21Cip1 results in decreased survival to retrovirally-induced lymphomagenesis. Importantly, in such p27Kip1 deficient lymphomas an increased frequency of Myc activation is observed. p27Kip1 deficiency also collaborates with MYC overexpression in transgenic lymphoma models. Thus, in vivo, the capacity of MYC to promote tumor growth is fully retained and even enhanced upon p27Kip1 loss. In lymphocytes, MYC overexpression and p27Kip1 deficiency independently stimulate CDK2 activity and augment the fraction of cells in S phase, in support of their distinct roles in tumorigenesis (Martins, 2002).

Myc and E2f1 promote cell cycle progression, but overexpression of either can trigger p53-dependent apoptosis. Mice expressing an Eμ-Myc transgene in B lymphocytes develop lymphomas, the majority of which sustain mutations of either Arf (a tumor suppressor whose product inhibits Mdm2, thereby stabilizing p53) or p53. Eμ-Myc transgenic mice lacking one or both E2f1 alleles exhibit a slower onset of lymphoma development associated with increased expression of the cyclin-dependent kinase inhibitor p27Kip1 and a reduced S phase fraction in precancerous B cells. In contrast, Myc-induced apoptosis and the frequency of Arf and p53 mutations in lymphomas were unaffected by E2f1 loss. Therefore, Myc does not require E2f1 to induce Arf, p53, or apoptosis in B cells, but depends upon E2f1 to accelerate cell cycle progression and downregulate p27Kip1 (Baudino, 2003).

The MYC oncoprotein is a transcription factor that coordinates cell growth and division. MYC overexpression exacerbates genomic instability and sensitizes cells to apoptotic stimuli. MYC directly stimulates transcription of the human Werner syndrome gene, WRN, which encodes a conserved RecQ helicase. Loss-of-function mutations in WRN lead to genomic instability, an elevated cancer risk, and premature cellular senescence. The overexpression of MYC in WRN syndrome fibroblasts or after WRN depletion from control fibroblasts leads to rapid cellular senescence that can not be suppressed by hTERT expression. It is proposed that WRN up-regulation by MYC may promote MYC-driven tumorigenesis by preventing cellular senescence (Grandori, 2003).

Alterations in c-myc oncogene expression have been implicated in the pathogenesis of several human cancers, including Burkitt and diffuse large B-cell lymphomas, breast and prostate cancer, colon cancer, melanoma, and multiple myeloma. The proteins encoded by MYC transcriptional target genes appear to regulate cell-cycle progression and cell growth while sensitizing cells to apoptotic stimuli. MYC may also be able to promote tumorigenesis by up-regulating the expression of genes such as hTERT that play a role in cellular immortalization or the escape from senescence. It was reasoned that MYC might modulate the expression of other genes that control cellular senescence, and thus determined whether the gene encoding the Werner syndrome RecQ helicase protein is a MYC transcriptional target (Grandori, 2003).

Werner syndrome (WRN) is an uncommon, autosomal recessive genetic instability syndrome that results from loss-of-function mutations in the chromosome 8p12-p11.2 WRN gene. The WRN phenotype resembles premature aging, and includes genomic instability, an elevated risk of malignancy, and accelerated cellular senescence. Genetic instability following loss of the 162-kD WRN RecQ helicase protein reflects the physiologic role of WRN in mitotic recombination and repair. Conversely, the elevated levels of WRN observed in immortalized and human tumor cell lines may help insure continuous cell proliferation. In order to delineate potential interactions between MYC and WRN in tumorigenesis, whether WRN expression is modulated by MYC was determined, and cellular responses to MYC overexpression in the absence of WRN were monitored. The results indicate that WRN expression appears to be required to avoid cellular senescence upon MYC up-regulation in hTERT-immortalized fibroblasts (Grandori, 2003).

Mnt is a Max-interacting transcriptional repressor that has been hypothesized to function as a Myc antagonist. To investigate Mnt function the Mnt gene was deleted in mice. Since mice lacking Mnt are born severely runted and typically die within several days of birth, mouse embryo fibroblasts (MEFs) derived from these mice and conditional Mnt knockout mice were used in this study. In the absence of Mnt, MEFs prematurely enter the S phase of the cell cycle and proliferated more rapidly than Mnt+/+ MEFs. Defective cell cycle control in the absence of Mnt is linked to upregulation of Cdk4 and cyclin E and the Cdk4 gene appears to be a direct target of Mnt-Myc antagonism. Like MEFs that overexpress Myc, Mnt-/- MEFs are prone to apoptosis, efficiently escape senescence and can be transformed with oncogenic Ras alone. Consistent with Mnt functioning as a tumor suppressor, conditional inactivation of Mnt in breast epithelium leads to adenocarinomas. These results demonstrate a unique negative regulatory role for Mnt in governing key Myc functions associated with cell proliferation and tumorigenesis (Hurlin, 2003).

Epidemiological findings suggest that the consequences of a given oncogenic stimulus vary depending upon the developmental state of the target tissue at the time of exposure. This is particularly evident in the mammary gland, where both age at exposure to a carcinogenic stimulus and the timing of a first full-term pregnancy can markedly alter the risk of developing breast cancer. Analogous to this, the biological consequences of activating oncogenes, such as MYC, can be influenced by cellular context both in terms of cell lineage and cellular environment. In light of this, it was hypothesized that the consequences of aberrant MYC activation in the mammary gland might be determined by the developmental state of the gland at the time of MYC exposure. To test this hypothesis directly, a doxycycline-inducible transgenic mouse model was used to overexpress MYC during different stages of mammary gland development. Using this model, it was found that the ability of MYC to inhibit postpartum lactation is due entirely to its activation within a specific 72-hour window during mid-pregnancy; by contrast, MYC activation either prior to or following this 72-hour window has little or no effect on postpartum lactation. Surprisingly, it was found that MYC does not block postpartum lactation by inhibiting mammary epithelial differentiation, but rather by promoting differentiation and precocious lactation during pregnancy, which in turn leads to premature involution of the gland. This developmental stage-specific ability of MYC to promote mammary epithelial differentiation is tightly linked to its ability to downregulate caveolin 1 and activate Stat5 in a developmental stage-specific manner. These findings provide unique in vivo molecular evidence for developmental stage-specific effects of oncogene activation, as well as the first evidence linking MYC with activation of the Jak2-Stat5 signaling pathway (Blakely, 2005).

ß-catenin signaling is heavily involved in organogenesis. This study investigated how pancreas differentiation, growth and homeostasis are affected following inactivation of an endogenous inhibitor of ß-catenin, adenomatous polyposis coli (Apc). In adult mice, Apc-deficient pancreata are enlarged, solely as a result of hyperplasia of acinar cells, which accumulate ß-catenin, with the sparing of islets. Expression of a target of ß-catenin, the proto-oncogene c-myc (Myc), is increased in acinar cells lacking Apc, suggesting that c-myc expression is essential for hyperplasia. In support of this hypothesis, it was found that conditional inactivation of c-myc in pancreata lacking Apc completely reverse the acinar hyperplasia. Apc loss in organs such as the liver, colon and kidney, as well as experimental misexpression of c-myc in pancreatic acinar cells, lead to tumor formation with high penetrance. Surprisingly, pancreas tumors failed to develop following conditional pancreas Apc inactivation. In Apc-deficient acini of aged mice, these studies revealed a cessation of their exaggerated proliferation and a reduced expression of c-myc, in spite of the persistent accumulation of ß-catenin. In conclusion, this work shows that ß-catenin modulation of c-myc is an essential regulator of acinar growth control, and unveils an unprecedented example of Apc requirement in the pancreas that is both temporally restricted and cell-specific. This provides new insights into the mechanisms of tumor pathogenesis and tumor suppression in the pancreas (Strom, 2007).

Inhibition of protein phosphatase 2A (PP2A) activity has been identified as a prerequisite for the transformation of human cells. However, the molecular mechanisms by which PP2A activity is inhibited in human cancers are currently unclear. In this study, a cellular inhibitor of PP2A with oncogenic activity is described. The protein, designated Cancerous Inhibitor of PP2A (CIP2A), interacts directly with the oncogenic transcription factor c-Myc, inhibits PP2A activity toward c-Myc serine 62 (S62), and thereby prevents c-Myc proteolytic degradation. In addition to its function in c-Myc stabilization, CIP2A promotes anchorage-independent cell growth and in vivo tumor formation. The oncogenic activity of CIP2A is demonstrated by transformation of human cells by overexpression of CIP2A. Importantly, CIP2A is overexpressed in two common human malignancies, head and neck squamous cell carcinoma (HNSCC) and colon cancer. Thus, these data show that CIP2A is a human oncoprotein that inhibits PP2A and stabilizes c-Myc in human malignancies (Junttila, 2007).

FoxO transcription factors play critical roles in cell cycle control and cellular stress responses, and abrogation of FoxO function promotes focus formation by Myc in vitro. Stable introduction of a dominant-negative FoxO moiety (dnFoxO) into Eµ-myc transgenic hematopoietic stem cells accelerates lymphoma development in recipient mice by attenuating Myc-induced apoptosis. When expressed in Eµ-myc; p53+/- progenitor cells, dnFoxO alleviates the pressure to inactivate the remaining p53 allele in upcoming lymphomas. Expression of the p53 upstream regulator p19Arf (alternative reading frame of p16INK, also called p14arf in humans and p19arf in mice) is virtually undetectable in most dnFoxO-positive Myc-driven lymphomas. It was found that FoxO proteins bind to a distinct site within the Ink4a/Arf locus and activate Arf expression. Moreover, constitutive Myc signaling induces a marked increase in nuclear FoxO levels and stimulates binding of FoxO proteins to the Arf locus. These data demonstrate that FoxO factors mediate Myc-induced Arf expression and provide direct genetic evidence for their tumor-suppressive capacity (Bouchard, 2007).

The FoxO subclass of forkhead-box transcription factors (consisting of FoxO1 (FKHR), FoxO3a (FKHRL1), FoxO4 (AFX), and FoxO6) regulates numerous cellular functions including proliferation, stress sensitivity, and survival; it has also been implicated in the regulation of organism life span. The members of this family activate gene expression via interaction with a specific DNA sequence, and known targets include the cell cycle regulating Kip1, the proapoptotic Bim, the DNA damage-responsive Gadd45a, and the oxidative stress-protective manganese superoxide dismutase genes. In addition, FoxO proteins can repress several cell cycle promoting genes (e.g., cyclin D1 and cyclin D2) in a manner that might be independent of direct DNA binding (Bouchard, 2007 and references therein).

In response to growth factor signaling and to oxidative stress, FoxO proteins are post-translationally modified by phosphorylation, acetylation, and ubiquitination; collectively, these modifications regulate FoxOs’ subcellular localization, transcriptional activity, and stability. Notably, all FoxO proteins are inhibited by protein kinase B/Akt-mediated phosphorylation that promotes their nuclear export and subsequent proteolytic degradation via ubiquitination by the SCFSkp2 complex. As a consequence, FoxO proteins mediate the induction of p27Kip1 and Bim expression in response to inhibition of the phosphatidylinositol-3-OH (PI3)-kinase/Akt pathway (Bouchard, 2007 and references therein).

Conditional codeletion of the FoxO1, FoxO3, and FoxO4 alleles uncovers a context-dependent cancer-prone phenotype characterized by thymic lymphomas forming in some and hemangiomas developing in most animals after a long latency, suggesting that FoxO proteins exert their tumor-suppressive capability in the presence of additional oncogenic mutations. In support of this view, Akt-mediated phosphorylation of FoxO proteins has been identified as the critical PI3-kinase signaling component that substitutes for oncogenic Ras in Myc-induced proliferation and focus formation in vitro. Furthermore, constitutive Akt signaling cooperates with Myc to accelerate B-cell lymphomagenesis; however, it remains unclear whether Akt-mediated phosphorylation of FoxO proteins contributes to Eµ-myc transgenic lymphoma formation in this setting (Bouchard, 2007).

Proapoptotic Arf/p53 signaling is known as the pivotal Myc-induced tumor-suppressive barrier. Eµ-myc transgenic mice lacking one p53 allele develop lymphomas that inactivate the remaining wild-type allele. Likewise, Eµ-myc; Arf+/- or Eµ-myc; Ink4a/Arf+/- mice produce tumors that lack expression of p19Arf. Primary Arf deletions protect cells from acquiring p53 mutations during lymphoma development. Similarly, introduction of strictly anti-apoptotic genes such as bcl2 or a dominant-negative form of caspase 9 into Eµ-myc; p53+/- hematopoietic stem cells alleviates the pressure to inactivate p53, thereby underscoring apoptosis as the critical p53-governed tumor suppressor function in Myc-driven lymphomagenesis (Bouchard, 2007).

Previous work has shown that p53 and FoxO3a share target genes and that FoxO3a can activate transcription via p53 sites, suggesting a potential collaboration of FoxO3a and p53 in tumor suppression. Although a direct interaction between FoxO3a and p53 proteins has been demonstrated under conditions of overexpression, the observed collaboration would be consistent with an as-yet-unidentified FoxO target acting upstream of p53. This study reports that FoxO factors elicit their tumor-suppressive potential as critical inducers of Arf during Myc-driven lymphomagenesis, providing further evidence for a close link between the FoxO and p53 tumor suppressor pathways (Bouchard, 2007).

Studying the early stages of cancer can provide important insight into the molecular basis of the disease. A preneoplastic stage was identified in the patched (ptc) mutant mouse, a model for the brain tumor medulloblastoma. Preneoplastic cells (PNCs) are found in most ptc mutants during early adulthood, but only 15% of these animals develop tumors. Although PNCs are found in mice that develop tumors, the ability of PNCs to give rise to tumors has never been demonstrated directly, and the fate of cells that do not form tumors remains unknown. Using genetic fate mapping and orthotopic transplantation, definitive evidence was provide that PNCs give rise to tumors, and the predominant fate of PNCs that do not form tumors is differentiation. Moreover, N-myc, a gene commonly amplified in medulloblastoma, can dramatically alter the fate of PNCs, preventing differentiation and driving progression to tumors. Importantly, N-myc allows PNCs to grow independently of hedgehog signaling, making the resulting tumors resistant to hedgehog antagonists. These studies provide the first direct evidence that PNCs can give rise to tumors, and demonstrate that identification of genetic changes that promote tumor progression is critical for designing effective therapies for cancer (Kessler, 2009).

The Myc protein suppresses the transcription of several cyclin-dependent kinase inhibitors (CKIs) via binding to POZ domain/zinc finger transciption factor Miz1. Whether this interaction is important for Myc's ability to induce or maintain tumorigenesis is not known. This study shows that the oncogenic potential of a point mutant of Myc (MycV394D) that is selectively deficient in binding to Miz1 is greatly attenuated. Binding of Myc to Miz1 is continuously required to repress CKI expression and inhibit accumulation of trimethylated histone H3 at Lys 9 (H3K9triMe), a hallmark of cellular senescence, in T-cell lymphomas. Lymphomas that arise express high amounts of transforming growth factor beta-2 (TGFbeta-2) and TGFbeta-3. Upon Myc suppression, TGFbeta signaling is required to induce CKI expression and cellular senescence and suppress tumor recurrence. Binding of Myc to Miz1 is required to antagonize growth suppression and induction of senescence by TGFbeta. Since lymphomas express high levels of TGFbeta, they are poised to elicit an autocrine program of senescence upon Myc inactivation, demonstrating that TGFbeta is a key factor that establishes oncogene addiction of T-cell lymphomas (van Riggelen, 2010).

The ubiquitous deregulation of Myc in human cancers makes it an intriguing therapeutic target, a notion supported by recent studies in Ras-driven lung tumors showing that inhibiting endogenous Myc triggers ubiquitous tumor regression. However, neither the therapeutic mechanism nor the applicability of Myc inhibition to other tumor types driven by other oncogenic mechanisms is established. This study shows that inhibition of endogenous Myc also triggers ubiquitous regression of tumors in a simian virus 40 (SV40)-driven pancreatic islet tumor model. Such regression is presaged by collapse of the tumor microenvironment and involution of tumor vasculature. Hence, in addition to its diverse intracellular roles, endogenous Myc serves an essential and nonredundant role in coupling diverse intracellular oncogenic pathways to the tumor microenvironment, further bolstering its credentials as a pharmacological target (Sodir, 2011).

Cancer cells frequently depend on chromatin regulatory activities to maintain a malignant phenotype. This study shows that leukemia cells require the mammalian SWI/SNF chromatin remodeling complex for their survival and aberrant self-renewal potential. While Brg1, an ATPase subunit of SWI/SNF, is known to suppress tumor formation in several cell types, this study found that leukemia cells instead rely on Brg1 to support their oncogenic transcriptional program, which includes Myc as one of its key targets. To account for this context-specific function, a cluster of lineage-specific enhancers located 1.7 Mb downstream from Myc was identified that are occupied by SWI/SNF as well as the BET protein Brd4. Brg1 is required at these distal elements to maintain transcription factor occupancy and for long-range chromatin looping interactions with the Myc promoter. Notably, these distal Myc enhancers coincide with a region that is focally amplified in approximately 3% of acute myeloid leukemias. Together, these findings define a leukemia maintenance function for SWI/SNF that is linked to enhancer-mediated gene regulation, providing general insights into how cancer cells exploit transcriptional coactivators to maintain oncogenic gene expression programs (Shi, 2013).

CDK9-mediated transcription elongation is required for MYC addiction in hepatocellular carcinoma

One-year survival rates for newly diagnosed hepatocellular carcinoma (HCC) are <50%, and unresectable HCC carries a dismal prognosis owing to its aggressiveness and the undruggable nature of its main genetic drivers. By screening a custom library of shRNAs directed toward known drug targets in a genetically defined Myc-driven HCC model, cyclin-dependent kinase 9 (Cdk9) was identified as required for disease maintenance. Pharmacological or shRNA-mediated CDK9 inhibition led to robust anti-tumor effects that correlated with MYC expression levels and depended on the role that both CDK9 and MYC exert in transcription elongation. These results establish CDK9 inhibition as a therapeutic strategy for MYC-overexpressing liver tumors and highlight the relevance of transcription elongation in the addiction of cancer cells to MYC (Huang, 2014).

MYC activation and BCL2L11 silencing by a tumour virus through the large-scale reconfiguration of enhancer-promoter hubs

Lymphomagenesis in the presence of deregulated MYC requires suppression of MYC-driven apoptosis, often through downregulation of the pro-apoptotic BCL2L11 gene (Bim; see Drosophila Death executioner Bcl-2). Transcription factors (EBNAs) encoded by the lymphoma-associated Epstein-Barr virus (EBV) activate MYC and silence BCL2L11. This study shows that the EBNA2 transactivator activates multiple MYC enhancers and reconfigures the MYC locus to increase upstream and decrease downstream enhancer-promoter interactions. EBNA2 recruits the BRG1 ATPase of the SWI/SNF remodeller (see Drosophila Brahma) to MYC enhancers, and BRG1 is required for enhancer-promoter interactions in EBV-infected cells. At BCL2L11, a haematopoietic enhancer hub was identified that is inactivated by the EBV repressors EBNA3A and EBNA3C through recruitment of the H3K27 methyltransferase EZH2 (see Drosophila Enhancer of zeste). Reversal of enhancer inactivation using an EZH2 inhibitor upregulates BCL2L11 and induces apoptosis. EBV therefore drives lymphomagenesis by hijacking long-range enhancer hubs and specific cellular co-factors. EBV-driven MYC enhancer activation may contribute to the genesis and localisation of MYC-Immunoglobulin translocation breakpoints in Burkitt's lymphoma (Wood, 2016).

Transactivation domain of human c-Myc Is essential to alleviate poly(Q)-mediated neurotoxicity in Drosophila disease models

Polyglutamine (poly(Q)) disorders, such as Huntington's disease (HD) and spinocerebellar ataxias, represent a group of neurological disorders which arise due to an atypically expanded poly(Q) tract in the coding region of the affected gene. Pathogenesis of these disorders inside the cells begins with the assembly of these mutant proteins in the form of insoluble inclusion bodies (IBs), which progressively sequester several vital cellular transcription factors and other essential proteins, and finally leads to neuronal dysfunction and apoptosis. Earlier studies have shown that targeted upregulation of Drosophila myc (dmyc) dominantly suppresses the poly(Q) toxicity in Drosophila. The present study examines the ability of the human c-myc proto-oncogene and also identifies the specific c-Myc isoform which drives the mitigation of poly(Q)-mediated neurotoxicity, so that it could be further substantiated as a potential drug target. This study reports that similar to dmyc, tissue-specific induced expression of human c-myc also suppresses poly(Q)-mediated neurotoxicity by an analogous mechanism. Among the three isoforms of c-Myc, the rescue potential was maximally manifested by the full-length c-Myc2 protein, followed by c-Myc1, but not by c-MycS which lacks the transactivation domain. This study suggests that strategies focussing on the transactivation domain of c-Myc could be a very useful approach to design novel drug molecules against poly(Q) disorders (Raj, 2017).


REFERENCES

Search PubMed for articles about Drosophila Myc

Albert, T., et al. (1997). Nucleosomal structures of c-myc promoters with transcriptionally engaged RNA polymerase II. Mol. Cell. Biol. 17: 4363-4371. PubMed Citation: 9234694

Alevizopoulos, K, et al. (1997). Cyclin E and c-Myc promote cell proliferation in the presence of p16(INK4a) and of hypophosphorylated retinoblastoma family proteins. EMBO J. 16(17): 5322-5333. 9311992

Alland, L., et al. (1997). Role for N-CoR and histone deacetylase in Sin3-mediated transcriptional repression. Nature 387 (6628): 49-55. PubMed Citation: 9139821

Amati, B., et al. (1993). Oncogenic activity of the c-Myc protein requires dimerization with Max. Cell 72: 233-245. PubMed Citation: 8425220

Amati, B. and Land, H. (1994). Myc-Max-Mad: a transcription factor network controlling cell cycle progression, differentiation and death. Curr. Opin. Genet. Dev. 4: 102-108. PubMed Citation: 8193530

Amcheslavsky, A., Ito, N., Jiang, J. and Ip, Y. T. (2011). Tuberous sclerosis complex and Myc coordinate the growth and division of Drosophila intestinal stem cells. J. Cell Biol. 193(4): 695-710. PubMed Citation: 21555458

Antonucci, L., D'Amico, D., Di Magno, L., Coni, S., Di Marcotullio, L., Cardinali, B., Gulino, A., Ciapponi, L. and Canettieri, G. (2014). CNBP regulates wing development in Drosophila melanogaster by promoting IRES-dependent translation of dMyc. Cell Cycle 13: 434-439. PubMed ID: 24275942

Atchley, W. R. and Fernandes, A. D. (2005). Sequence signatures and the probabilistic identification of proteins in the Myc-Max-Mad network. Proc. Natl. Acad. Sci. 102(18): 6401-6. 15851686

Ayer, D. E., Kretzner, L. and Eisenman, R. N. (1993a). Mad: a heterodimeric partner for Max that antagonizes Myc transcriptional activity. Cell 72: 211-222. PubMed Citation: 8425218

Ayer, D. E., and Eisenman, R. N. (1993b). A switch from Myc:Max to Mad:Max heterocomplexes accompanies monocyte/macrophage differentiation. Genes Dev. 7: 2110-19. PubMed Citation: 16408280

Ayer, D. E., Lawrence, Q. A., and Eisenman, R. N. (1995). Mad-Max transcriptional repression is mediated by ternary complex formation with mammalian homologs of yeast repressor Sin3. Cell 80 (5): 767-776. PubMed Citation: 7889570

Ayer, D. E., et al. (1996). Mad proteins contain a dominant transcription repression domain. Mol. Cell. Biol. 16 (10): 5772-5781. PubMed Citation: 8816491

Bates, C. M., et al. (2000). Role of N-myc in the developing mouse kidney. Dev. Biol. 222: 317-325. 10837121

Batsche E., et al. (1998). RB and c-Myc activate expression of the E-cadherin gene in epithelial cells through interaction with transcription factor AP-2. Mol. Cell. Biol. 18(7): 3647-3658. PubMed Citation: 9632747

Baudino, T. A., et al (2002). c-Myc is essential for vasculogenesis and angiogenesis during development and tumor progression. Genes Dev. 16: 2530-2543. 12368264

Baudino, T. A., et al. (2003). Myc-mediated proliferation and lymphomagenesis, but not apoptosis, are compromised by E2f1 loss. Molec. Cell 11: 905-914. 12718877

Bauer, A., et al. (2000). Pontin52 and reptin52 function as antagonistic regulators of beta-catenin signalling activity. EMBO J. 19(22): 6121-30. 11080158

Bellmeyer, A., Krase, J., Lindgren, J. and LaBonne, C. (2003). The protooncogene c-Myc is an essential regulator of neural crest formation in Xenopus. Dev. Cell 4: 827-839. 12791268

Bellosta, P., et al. (2005). Myc interacts genetically with Tip48/Reptin and Tip49/Pontin to control growth and proliferation during Drosophila development. Proc. Natl. Acad. Sci. 102(33): 11799-804. 16087886

Benassayag, C., et al. (2005). Human c-Myc isoforms differentially regulate cell growth and apoptosis in Drosophila melanogaster. Molec. Cell. Biol. 25: 9897-9909. 16260605

Benevolenskaya, E. V., Murray, H. L., Branton, P., Young, R. A., and Kaelin, W. G. (2005). Binding of pRB to the PHD protein RBP2 promotes cellular differentiation. Mol. Cell 18: 623-635. Medline abstract: 15949438

Blakely, C. M., Sintasath, L., D'Cruz, C. M., Hahn, K. T., Dugan, K. D., Belka, G. K. and Chodosh, L. A. (2005). Developmental stage determines the effects of MYC in the mammary epithelium. Development 132(5): 1147-60. 15689376

Boter, M., et al. (2004). Conserved MYC transcription factors play a key role in jasmonate signaling both in tomato and Arabidopsis. Genes Dev. 18: 1577-1591. 15231736

Bouchard, C., et al, (2001). Regulation of cyclin D2 gene expression by the Myc/Max/Mad network: Myc-dependent TRRAP recruitment and histone acetylation at the cyclin D2 promoter. Genes Dev. 15: 2042-2047. 11511535

Bouchard, C., et al. (2007). FoxO transcription factors suppress Myc-driven lymphomagenesis via direct activation of Arf. Genes Dev. 21(21): 2775-87. PubMed citation: 17974917

Bouchard, C., Marquardt, J., Bras, A., Medema, R. H. and Eilers, M. (2004). Myc-induced proliferation and transformation require Akt-mediated phosphorylation of FoxO proteins. EMBO J. 23(14):2830-40. 15241468

Bowman, S. K., et al. (2008). The tumor suppressors Brat and Numb regulate transit-amplifying neuroblast lineages in Drosophila. Dev. Cell 14: 535-546. PubMed Citation: 18342578

Boyd, K. E., et al. (1998). c-Myc target gene specificity is determined by a post-DNA binding mechanism. Proc. Natl. Acad. Sci. 95(23): 13887-92. PubMed Citation: 9811896

Brenner, C., et al. (2005). Myc represses transcription through recruitment of DNA methyltransferase corepressor. EMBO J. 24: 336-346. 15616584

Brubaker, K., et al. (2000). Solution structure of the interacting domains of the Mad-Sin3 complex: Implications for recruitment of a chromatin-modifying complex. Cell 103: 655-665. PubMed Citation: 11106735

Bush, A., et al. (1998). c-myc null cells misregulate cad and gadd45 but not other proposed c-Myc targets. Genes Dev. 12(24): 3797-802. PubMed Citation: 9869632

Cartwright, P., McLean, C., Sheppard, A., Rivett, D., Jones, K. and Dalton, S. (2005). LIF/STAT3 controls ES cell self-renewal and pluripotency by a Myc-dependent mechanism. Development 132: 885-896. 15673569

Cerosaletti, K., Wright, J. and Concannon, P. (2006). Active role for nibrin in the kinetics of atm activation. Mol. Cell. Biol. 26: 1691-1699. 16478990

Chagnovich, D. and Cohn, S. L. (1996). Binding of a 40-kAa protein to the N-myc 3'-untranslated region correlates with enhanced N-myc expression in human neuroblastoma. J. Biol. Chem. 271: 22580-86. PubMed Citation: 8969225

Chan, S. W. and Hong, W. J. (2001). Retinoblastoma-binding protein 2 (Rbp2) potentiates nuclear hormone receptor-mediated transcription. J. Biol. Chem. 276: 28402-28412. Medline abstract: 11358960

Chanu, S. I. and Sarkar, S. (2016). Targeted Downregulation of dMyc Suppresses Pathogenesis of Human Neuronal Tauopathies in Drosophila by Limiting Heterochromatin Relaxation and Tau Hyperphosphorylation. Mol Neurobiol. PubMed ID: 27000837

Chen, C.-R., et al. (2002). E2F4/5 and p107 as smad cofactors linking the TGFß receptor to c-myc repression. Cell 110: 19-32. 12150994

Cheng, S. W., et al. (1999). c-MYC interacts with INI1/hSNF5 and requires the SWI/SNF complex for transactivation function. Nat. Genet. 22(1): 102-5. PubMed Citation: 10319872

Chiang, Y. C., et al. (2003). c-Myc directly regulates the transcription of the NBS1 gene involved in DNA double-strand break repair. J. Biol. Chem. 278(21): 19286-91. 12637527

Choi, S. H., et al. (2010). Myc protein is stabilized by suppression of a novel E3 ligase complex in cancer cells. Genes Dev. 24(12): 1236-41. PubMed Citation: 20551172

Cordero, J. B., Stefanatos, R. K., Scopelliti, A., Vidal, M. and Sansom, O. J. (2012a). Inducible progenitor-derived Wingless regulates adult midgut regeneration in Drosophila. EMBO J 31: 3901-3917. PubMed ID: 22948071

Cordero, J. B., Stefanatos, R. K., Myant, K., Vidal, M. and Sansom, O. J. (2012b). Non-autonomous crosstalk between the Jak/Stat and Egfr pathways mediates Apc1-driven intestinal stem cell hyperplasia in the Drosophila adult midgut. Development 139: 4524-4535. PubMed ID: 23172913

Csibi, A., Lee, G., Yoon, S. O., Tong, H., Ilter, D., Elia, I., Fendt, S. M., Roberts, T. M. and Blenis, J. (2014). The mTORC1/S6K1 pathway regulates glutamine metabolism through the eIF4B-cependent control of c-Myc translation. Curr Biol 24: 2274-2280. PubMed ID: 25220053

Cultraro, C. M., Bino, T. and Segal. S. (1997). Function of the c-Myc antagonist Mad1 during a molecular switch from proliferation to differentiation. Mol. Cell. Biol. 17: 2353-59. PubMed Citation: 9111304

David-Cordonnier, M. H., et al. (1999). The DNA-binding domain of human c-Abl tyrosine kinase promotes the interaction of a HMG chromosomal protein with DNA. Nucleic Acids Res. 27(11): 2265-70. PubMed Citation: 10325413

Davis, A. C., et al. (1993). A null c-myc mutation causes lethality before 10.5 days of gestation in homozygotes and reduced fertility in heterozygous female mice. Genes Dev. 7: 671-682. PubMed Citation: 8458579

de la Cova, C., et al. (2004). Drosophila Myc regulates organ size by inducing cell competition. Cell 117: 107-116. 15066286

Delanoue, R., Slaidina, M. and Léopold, P. (2010). The steroid hormone ecdysone controls systemic growth by repressing dMyc function in Drosophila fat cells. Dev. Cell 18(6): 1012-21. PubMed Citation: 20627082

Demontis, F. and Perrimon, N. (2009). Integration of Insulin receptor/Foxo signaling and dMyc activity during muscle growth regulates body size in Drosophila. Development 136(6): 983-93. PubMed Citation: 19211682

Dong, J., Naito, M. and Tsuruo, T. (1997). c-Myc plays a role in cellular susceptibility to death receptor-mediated and chemotherapy-induced apoptosis in human monocytic leukemia U937 cells. Oncogene 15(6): 639-647. PubMed Citation: 9264404

Dominguez-Sola, D., et al. (2007). Non-transcriptional control of DNA replication by c-Myc. Nature 448: 445-451. PubMed Citation: 17597761

Downs, K. M., Martin, G. R., and Bishop, J. M. (1989). Contrasting patterns of myc and N-myc expression during gastrulation of the mouse embryo. Genes Dev. 3: 860-869. PubMed Citation: 2663644

Drissi, R., Zindy, F., Roussel, M. F. and Cleveland, J. L. (2001). c-Myc-mediated regulation of telomerase activity is disabled in immortalized cells. J. Biol. Chem. 276(32): 29994-30001. 11402027

Dugan, K. A., Wood, M. A. and Cole, M. D. (2002). TIP49, but not TRRAP, modulates c-Myc and E2F1 dependent apoptosis. Oncogene 21(38): 5835-43. 12185582

Duman-Scheel, M., Johnston, L. A. and Du, W. (2004). Repression of dMyc expression by Wingless promotes Rbf-induced G1 arrest in the presumptive Drosophila wing margin. Proc. Natl. Acad. Sci. 101(11): 3857-62. 15001704

Duncan, R., et al. (1996). A unique transactivation sequence motif is found in the carboxyl-terminal domain of the single-strand-binding protein FBP. Mol. Cell. Biol. 16: 2274-2282. PubMed Citation: 8628294

Dupre, A., Boyer-Chatenet, L. and Gautier J. (2006). Two-step activation of ATM by DNA and the Mre11-Rad50-Nbs1 complex. Nat. Struct. Mol. Biol. 13(5): 451-7. PubMed Citation: 16622404

Dussault, I. and Giguere, V. (1997). Differential regulation of the N-myc proto-oncogene by ROR alpha and RVR, two orphan members of the superfamily of nuclear hormone receptors. Mol Cell Biol 17 (4): 1860-1867. PubMed Citation: 9121434

Eberhardy, S. R. and Farnham, P. J. (2001). c-Myc mediates activation of the cad promoter via a post-RNA polymerase II recruitment mechanism. J. Biol. Chem. 276: 48562-48571. 11673469

Eilers, A. L., et al. (1999). A 13-amino acid amphipathic alpha-helix is required for the functional interaction between the transcriptional repressor Mad1 and mSin3A. J. Biol. Chem. 274(46): 32750-6. PubMed Citation: 10551834

Etard, C., et al. (2005). Pontin and Reptin regulate cell proliferation in early Xenopus embryos in collaboration with c-Myc and Miz-1. Mech. Dev. 122(4): 545-56. Medline abstract: 15804567

Facchini, L. M., et al. (1997). The Myc Negative Autoregulation Mechanism Requires Myc-Max Association and Involves the c-myc P2 Minimal Promoter. Mol. Cell. Biol. 17: 100-114. PubMed Citation: 8972190

Faiola, F., et al. (2005). Dual regulation of c-Myc by p300 via acetylation-dependent control of Myc protein turnover and coactivation of Myc-induced transcription. Mol. Cell. Biol. 25(23): 10220-34. 16287840

Felsher, D. W. and Bishop, J. M. (1999a). Transient excess of MYC activity can elicit genomic instability and tumorigenesis. Proc. Natl. Acad. Sci. 96(7): 3940-4. PubMed Citation: 10097142

Felsher, D. W. and Bishop, J. M. (1999b). Reversible tumorigenesis by MYC in hematopoietic lineages. Molecular Cell 4: 199-207. PubMed Citation: 10488335

Feng, X. H., et al. (2002). Direct interaction of c-Myc with Smad2 and Smad3 to inhibit TGF-ß-mediated induction of the CDK inhibitor p15Ink4B. Mol. Cell 9: 133-143. 11804592

Fero, M. L., et al. (1996). A syndrome of multiorgan hyperplasia with features of gigantism, tumorigenesis, and female sterility in p27Kip1-deficient mice. Cell 85: 733-744. PubMed Citation: 8646781

Foley, K. P., et al. (1998). Targeted disruption of the MYC antagonist MAD1 inhibits cell cycle exit during granulocyte differentiation. EMBO J. 17: 774-785. PubMed Citation: 9451002

Frank, S. R., et al. (2001). Binding of c-Myc to chromatin mediates mitogen-induced acetylation of histone H4 and gene activation. Genes Dev. 15: 2069-2082. 11511539

Frank, S. R., et al. (2003). MYC recruits the TIP60 histone acetyltransferase complex to chromatin. EMBO Rep. 4(6): 575-80. Medline abstract: 12776177

Frazier, M. W., et al. (1998). Activation of c-myc gene expression by tumor-derived p53 mutants requires a discrete C-terminal domain. Mol. Cell. Biol. 18(7): 3735-3743. PubMed Citation: 9632756

Fu, L., et al. (2002). The circadian gene Period2 plays an important role in tumor suppression and DNA damage response in vivo. Cell 111: 41-50. 12507431

Galaktionov, K., Chen, X. and Beach, D. (1996). Cdc25 cell-cycle phosphatase as a target of c-myc. Nature 382: 511-517. PubMed Citation: 8700224

Gallant, P., et al. (1996). Myc and Max homologs in Drosophila. Science 274: 1523-1527. PubMed Citation: 8929412

Galletti, M., S. et al. (2009). Identification of domains responsible for ubiquitin-dependent degradation of dMyc by glycogen synthase kinase 3beta and casein kinase 1 kinases. Mol. Cell. Biol. 29: 3424-3434. PubMed Citation: 19364825

Gandarillas, A. and Watt, F. M. (1997). c-Myc promotes differentiation of human epidermal stem cells. Genes Dev. 11(21): 2869-2882. PubMed Citation: 9353256

Garoia, F., Grifoni, D., Trotta, V., Guerra, D., Pezzoli, M. C. and Cavicchi, S. (2005). The tumor suppressor gene fat modulates the EGFR-mediated proliferation control in the imaginal tissues of Drosophila melanogaster. Mech. Dev. 122(2): 175-87. 15652705

Gaubatz, S., et al. (1995). Transcriptional activation by Myc is under negative control by the transcription factor AP-2. EMBO J. 14(7): 1508-19. 7729426

Géminard, C., Rulifson, E. J. and Léopold, P. (2009). Remote control of insulin secretion by fat cells in Drosophila. Cell Metab. 10: 199-207. PubMed Citation: 19723496

Gerlach, J. M., Furrer, M., Gallant, M., Birkel, D., Baluapuri, A., Wolf, E. and Gallant, P. (2017). PAF1 complex component Leo1 helps recruit Drosophila Myc to promoters. Proc Natl Acad Sci U S A 114(44): E9224-e9232. PubMed ID: 29078288

Gildea, J. J., Lopez, R. and Shearn, A. (2000). A screen for new trithorax group genes identified little imaginal discs, the Drosophila melanogaster homologue of human retinoblastoma binding protein 2. Genetics 156(2): 645-63. Medline abstract: 11014813

Giroux, S. and Charron, J. (1998). Defective development of the embryonic liver in N-myc-deficient mice. Dev. Biol. 195(1): 16-28. PubMed ID: 9520320

Gomez-Roman, N., Grandori, C., Eisenman, R. N. and White, R. J. (2003). Direct activation of RNA polymerase III transcription by c-Myc. Nature 421(6920): 290-4. PubMed Citation: 12529648

Goodliffe, J. M., Wieschaus, E. and Cole, M. D. (2005). Polycomb mediates Myc autorepression and its transcriptional control of many loci in Drosophila. Genes Dev. 19(24): 2941-6. 16357214

Grandori, C., et al. (1996). Myc-Max heterodimers activate a DEAD box gene and interact with multiple E box-related sites in vivo. EMBO J. 15(16): 4344-57

Grandori, C., Cowley, S. M., James, L. P. and Eisenman, R. N. (2000). The Myc/Max/Mad network and the transcriptional control of cell behavior. Annu. Rev. Cell Dev. Biol. 16: 653-699. 11031250

Grandori, C., et al. (2003). Werner syndrome protein limits MYC-induced cellular senescence. Genes Dev. 17: 1569-1574. 12842909

Gregory, M. A. and Hann, S. R. (2000). c-Myc proteolysis by the ubiquitin-proteasome pathway: stabilization of c-Myc in Burkitt's lymphoma cells. Mol. Cell. Biol. 20: 2423-2435. 10713166

Gregory, M. A., Qi, Y. and Hann, S. R. (2003). Phosphorylation by glycogen synthase kinase-3 controls c-myc proteolysis and subnuclear localization. J. Biol. Chem. 278: 51606-51612. 14563837

Grewal, S. S., Evans, J. R. and Edgar, B. A (2007). Drosophila TIF-IA is required for ribosome synthesis and cell growth and is regulated by the TOR pathway. J. Cell Biol. 179: 1105-1113. PubMed Citation: 18086911

Grewal, S. S. (2009). Insulin/TOR signaling in growth and homeostasis: a view from the fly world. Int J Biochem Cell Biol 41: 1006-1010. PubMed Citation: 18992839

Grinberg, A. V., Hu, C. D. and Kerppola, T. K. (2004). Visualization of Myc/Max/Mad family dimers and the competition for dimerization in living cells. Mol. Cell. Biol. 24(10): 4294-308. 15121849

Grumont, R. J., Strasser, A. and Gerondakis, S. (2003). B cell growth is controlled by Phosphatidylinosotol 3-kinase-dependent induction of Rel/NF-kappaB regulated c-myc transcription. Mol. Cell 10: 1283-1294. 12504005

Gu, W., et al. (1993). Opposite regulation of gene transcription and cell proliferation by c-Myc and Max. Proc. Natl. Acad. Sci. 90: 2935-39

Guo, Q., et al. (1998). Identification of a large myc-binding protein that contains RCC1-like repeats. Proc. Natl. Acad. Sci. 95(16): 9172-9177

Hassig, C. A., et al. (1997). Histone deacetylase activity is required for full transcriptional repression by mSin3A. Cell 89 (3): 341-347

He T.-C., et al. (1998). Identification of c-MYC as a target of the APC pathway. Science 281(5382): 1509-1512

Heinzel, T., et al. (1997). A complex containing N-CoR, mSin3 and histone deacetylase mediates transcriptional repression. Nature 387: 43-48

Herold, S., et al. (2002). Negative regulation of the mammalian UV response by Myc through association with Miz-1. Molec. Cell 10: 509-521. 12408820

Herter, E. K., Stauch, M., Gallant, M., Wolf, E., Raabe, T. and Gallant, P. (2015). snoRNAs are a novel class of biologically relevant Myc targets. BMC Biol 13: 25. PubMed ID: 25888729

Hirning, U., et al. (1991). A comparative analysis of N-myc and c-myc expression and cellular proliferation in mouse organogenesis. Mech. Dev. 33, 119-25

Hovhanyan, A., Herter, E. K., Pfannstiel, J., Gallant, P. and Raabe, T. (2014). Drosophila Mbm is a nucleolar Myc and CK2 target required for ribosome biogenesis and cell growth of central brain neuroblasts. Mol Cell Biol 34(10):1878-91. PubMed ID: 24615015

Hu, M. C. and Rosenblum, N. D. (2005). Smad1, ß-catenin and Tcf4 associate in a molecular complex with the Myc promoter in dysplastic renal tissue and cooperate to control Myc transcription. Development 132: 215-225. 15576399

Huang, C. H., Lujambio, A., Zuber, J., Tschaharganeh, D. F., Doran, M. G., Evans, M. J., Kitzing, T., Zhu, N., de Stanchina, E., Sawyers, C. L., Armstrong, S. A., Lewis, J. S., Sherr, C. J. and Lowe, S. W. (2014). CDK9-mediated transcription elongation is required for MYC addiction in hepatocellular carcinoma. Genes Dev 28: 1800-1814. PubMed ID: 25128497

Hueber, A. O. L., et al. (1997). Requirement for the CD95 receptor-ligand pathway in c-myc-induced apoptosis. Science 278(5341): 1305-1309

Hurlin, P. J., et al. (1995). Mad3 and Mad4: novel Max-interacting transcriptional repressors that suppress c-myc dependent transformation and are expressed during neural and epidermal differentiation. EMBO J. 14: 5646-5659. 8521822

Hurlin, P. J., QuÈva, C. and Eisenman, R. N. (1997). Mnt, a novel Max-interacting protein is coexpressed with Myc in proliferating cells and mediates repression at Myc binding sites. Genes Dev. 11(1): 44-58. 9000049

Hurlin, P. J., et al. (2003). Deletion of Mnt leads to disrupted cell cycle control and tumorigenesis. EMBO J. 22: 4584-4596. 12970171

Iakova, P., Awad, S. S., and Timchenko, N. A. (2003). Aging reduces proliferative capacities of liver by switching pathways of C/EBPalpha growth arrest. Cell 113: 495-506. 12757710

Iritani, B. M., et al. (2002). Modulation of T-lymphocyte development, growth and cell size by the Myc antagonist and transcriptional repressor Mad1. EMBO J. 21: 4820-4830. 12234922

Izumi, H., et al. (2001). Mechanism for the transcriptional repression by c-Myc on PDGF ß-receptor J. Cell Sci. 114: 1533-1544. 11282029

Jacobs, J. J. L., et al. (1999). Bmi-1 collaborates with c-Myc in tumorigenesis by inhibiting c-Myc-induced apoptosis via INK4a/ARF. Genes Dev. 13: 2678-2690.

Jiao, R., et al. (2001). Headless flies generated by developmental pathway interference. Development 128: 3307-3319. 11546747

Jin, Z., Kirilly, D., Weng, C., Kawase, E., Song, X., Smith, S., Schwartz, J. and Xie, T. (2008). Differentiation-defective stem cells outcompete normal stem cells for niche occupancy in the Drosophila ovary. Cell Stem Cell 2: 39-49. PubMed Citation: 18371420

Junttila, M. R., et al. (2007). CIP2A inhibits PP2A in human malignancies. Cell 130(1): 51-62. PubMed citation; Online text

Kagaya, S., et al. (1997). A functional role for death proteases in s-Myc- and c-Myc-mediated apoptosis. Mol. Cell. Biol. 17(11): 6736-6745. PubMed Citation: 9343438

Kamoshida, Y., et al. (2012). Ecdysone receptor (EcR) suppresses lipid accumulation in the Drosophila fat body via transcription control. Biochem. Biophys. Res. Commun. 421(2): 203-7. PubMed Citation: 22503687

Kanazawa, S., Soucek, L., Evan, G., Okamoto, T., and Peterlin, B. M. (2003). c-Myc recruits P-TEFb for transcription, cellular proliferation and apoptosis. Oncogene 22: 5707-5711. 12944920

Kasibhatla, S., et al. (2000). A 'non-canonical' DNA-binding element mediates the response of the Fas-ligand promoter to c-Myc. Curr. Biol. 10: 1205-1208. 11050389

Kenney, A. M., Cole, M. D. and Rowitch, D. H. (2003). Nmyc upregulation by sonic hedgehog signaling promotes proliferation in developing cerebellar granule neuron precursors. Development 130: 15-28. 12441288

Kenney, A. M., Widlund, H. R. and Rowitch, D. H. (2004). Hedgehog and PI-3 kinase signaling converge on Nmyc1 to promote cell cycle progression in cerebellar neuronal precursors. Development 131: 217-228 . 14660435

Kessler, J. D., et al. (2009). N-myc alters the fate of preneoplastic cells in a mouse model of medulloblastoma. Genes Dev. 23(2): 157-70. PubMed Citation: 19171780

Kharazmi, J., Moshfegh, C. and Brody, T. (2012). Identification of cis-regulatory elements in the dmyc gene of Drosophila melanogaster. Gene Regul. Syst. Bio. 6: 15-42. PubMed Citation: 22267917

Kim, H. H., et al. (2009). HuR recruits let-7/RISC to repress c-Myc expression. Genes Dev. 23(15): 1743-8. PubMed Citation: 19574298

Kim, S. Y., et al. (2003). Skp2 regulates Myc protein stability and activity. Molec. Cell 11: 1177-1188. 12769843

Klose, R. J., Yamane, K., Bae, Y. J., Zhang, D. Z., Erdjument-Bromage, H., Tempst, P., Wong, J. M., and Zhang, Y. (2006). The transcriptional repressor JHDM3A demethylates trimethyl histone H3 lysine 9 and lysine 36. Nature 442: 312-316. Medline abstract: 16732292

Knoblich, J. A. (2008). Mechanisms of asymmetric stem cell division. Cell 132: 583-597. PubMed Citation: 18295577

Kolligs, F. T., et al. (2000). gamma-Catenin is regulated by the APC tumor suppressor and its oncogenic activity is distinct from that of beta-catenin. Genes Dev. 14: 1319-1331. PubMed Citation: 10837025

Koshiji, M. and Huang, L. E. (2004). Dynamic balancing of the dual nature of HIF-1alpha for cell survival. Cell Cycle 3(7): 853-4. 15190211

Knoepfler, P. S., Cheng, P. F. and Eisenman, R. N. (2002). N-myc is essential during neurogenesis for the rapid expansion of progenitor cell populations and the inhibition of neuronal differentiation. Genes Dev. 16: 2699-2712. 12381668

Krylov, D., et al. (1997). A general method to design dominant negatives to B-HLHZip proteins that abolish DNA binding. Proc. Natl. Acad. Sci. 94(23): 12274-12279. PubMed Citation: 9356439

Kuo, Y., Huang, H., Cai, T. and Wang, T. (2015). Target of Rapamycin Complex 2 regulates cell growth via Myc in Drosophila. Sci Rep 5: 10339. PubMed ID: 25999153

Kuwahara, A., et al. (2010). Wnt signaling and its downstream target N-myc regulate basal progenitors in the developing neocortex. Development. 2010 Apr;137(7):1035-44. PubMed Citation: 20215343

Laherty, C. D., et al. (1997). Histone deacetylases associated with the mSin3 corepressor mediate Mad transcriptional repression. Cell 89, 349-356. PubMed Citation: 9150134

Lasorella, A., et al. (2000). Id2 is a retinoblastoma protein target and mediates signaling by Myc oncoproteins. Nature 407: 592-598. PubMed Citation: 11034201

Lauber, A. H., et al. (1997). A DNA-binding element for a steroid receptor-binding factor is flanked by dual nuclear matrix DNA attachment sites in the c-myc gene promoter. J. Biol. Chem. 272(39): 24657-24665. PubMed Citation: 9305935

Lee, J. E., et al (2015). Defective Hfp-dependent transcriptional repression of dMYC is fundamental to tissue overgrowth in Drosophila XPB models. Nat Commun 6: 7404. PubMed ID: 26074141

Lee, T. C., et al. (1997). Myc represses transcription of the growth arrest gene gas1. Proc. Natl. Acad. Sci. 94(24): 12886-12891. PubMed Citation: 9371770

Leone, G., et al. (1997). Myc and Ras collaborate in inducing accumulation of active cyclin E/Cdk2 and E2F. Nature 387 (6631): 422-426. PubMed Citation: 9163430

Leone, G., et al. (2001). Myc requires distinct E2F activities to induce S phase and apoptosis. Mol. Cell 8: 105-113. 11511364

Levayer, R., Hauert, B. and Moreno, E. (2015). Cell mixing induced by myc is required for competitive tissue invasion and destruction. Nature 524: 476-480. PubMed ID: 26287461

Lewis, B. C., et al. (1997). Identification of putative c-Myc-responsive genes: characterization of rcl, a novel growth-related gene. Mol. Cell. Biol. 17(9): 4967-4978

Li, L., Anderson, S., Secombe, J. and Eisenman, R. N. (2013). The Drosophila ubiquitin-specific protease Puffyeye regulates dMyc-mediated growth. Development 140: 4776-4787. PubMed ID: 24173801

Li, S., Jiang, C., Pan, J., Wang, X., Jin, J., Zhao, L., Pan, W., Liao, G., Cai, X., Li, X., Xiao, J., Jiang, J. and Wang, P. (2014). Regulation of c-Myc protein stability by proteasome activator REGγ. Cell Death Differ 24(6):1741-54. PubMed ID: 25412630

Light, W., Vernon, A. E., Lasorella, A., Iavarone, A. and Labonne, C. (2005). Xenopus Id3 is required downstream of Myc for the formation of multipotent neural crest progenitor cells. Development 132(8): 1831-1841. 15772131

Liu, H., et al. (2010). Notch dimerization is required for leukemogenesis and T-cell development. Genes Dev. 24(21): 2395-407. PubMed Citation: 20935071

Liu, J., et al. (2000). The FBP interacting repressor targets TFIIH to inhibit activated transcription. Molec. Cell 5: 331-341. PubMed Citation: 10882074

Liu J., et al. (2006). The FUSE/FBP/FIR/TFIIH system is a molecular machine programming a pulse of c-myc expression. EMBO J. 25: 2119-2130. PubMed Citation: 16628215

Lu, P. J., Sundquist, K., Baeckstrom, D., Poulsom, R., Hanby, A., Meier-Ewert, S., Jones, T., Mitchell, M., Pitha-Rowe, P., Freemont, P., et al. (1999). A novel gene (PLU-1) containing highly conserved putative DNA chromatin binding motifs is specifically up-regulated in breast cancer. J. Biol. Chem. 274: 15633-15645. Medline abstract: 10336460

Luo, R. X., Postigo, A. A. and Dean, D. C. (1998). Rb interacts with histone deacetylase to repress transcription. Cell 92:463-473

Mahon, G. M., et al. (2003). The c-Myc oncoprotein interacts with Bcr. Curr. Biol. 13: 437-441. 12620195

Maines, J. Z., Stevens, L. M., Tong, X. and Stein, D. (2004). Drosophila dMyc is required for ovary cell growth and endoreplication. Development 131: 775-786. 14724122

Malynn, B. A., et al. (2000). N-myc can functionally replace c-myc in murine development, cellular growth, and differentiation. Genes Dev. 14: 1390-1399

Mao, S. F., Neale, G .A. M. and Goorha, R. M. (1997). T-cell oncogene rhombotin-2 interacts with retinoblastoma-binding protein 2. Oncogene 14: 1531-1539. Medline abstract: 9129143

Marinho, J., Casares, F. and Pereira, P. S. (2011). The Drosophila Nol12 homologue viriato is a dMyc target that regulates nucleolar architecture and is required for dMyc-stimulated cell growth. Development 138: 349-357. PubMed ID: 21177347

Marshall L. (2008). Elevated RNA polymerase III transcription drives proliferation and oncogenic transformation. Cell Cycle 7(21): 3327-9. PubMed Citation: 18971635

Marshall, L., Rideout, E. J. and Grewal, S. S. (2012). Nutrient/TOR-dependent regulation of RNA polymerase III controls tissue and organismal growth in Drosophila. EMBO J. 31(8):1916-30. PubMed Citation: 22367393

Martins, C. P. and Berns. A. (2002). Loss of p27Kip1 but not p21Cip1 decreases survival and synergizes with MYC in murine lymphomagenesis. EMBO J. 21: 3739-3748. 12110586

Martins, R. A., et al. (2008). N-myc coordinates retinal growth with eye size during mouse development. Genes Dev. 22(2): 179-93. PubMed citation: 18198336

Matsushita, K., et al. (2006). An essential role of alternative splicing of c-myc suppressor FUSE-binding protein-interacting repressor in carcinogenesis. Cancer Res. 66: 1409-1417. PubMed Citation: 16452196

McMahon, S. B., Van, B. H., Dugan, K. A., Copeland, T. D. and Cole, M. D. (1998). The novel ATM-related protein TRRAP is an essential cofactor for the c-Myc and E2F oncoproteins. Cell 94: 363-374

McMahon, S. B., Wood, M. A. and Cole, M. D. (2000). The essential cofactor TRRAP recruits the histone acetyltransferase hGCN5 to c-Myc. Mol. Cell. Biol. 20(2): 556-62.

Michelotti, E. F., et al. (1995). Cellular nucleic acid binding protein regulates the CT element of the human c-myc protooncogene. J. Biol. Chem. 270: 9494-9499

Michelotti, G. A. (1996). Multiple single-stranded cis elements are associated with activated chromatin of the human c-myc gene in vivo. Mol. Cell. Biol. 16: 2656-2669

Mitchell, N. C., et al. (2010). Hfp inhibits Drosophila myc transcription and cell growth in a TFIIH/Hay-dependent manner. Development 137(17): 2875-84. PubMed Citation: 20667914

Mill, P., et al. (2005). Shh controls epithelial proliferation via independent pathways that converge on N-Myc. Dev. Cell 9(2): 293-303. 16054035

Moberg, K. H., Bell, D. W., Wahrer, D. C., Haber, D. A. and Hariharan, I. K. (2001). Archipelago regulates Cyclin E levels in Drosophila and is mutated in human cancer cell lines. Nature 413(6853): 311-6. 11565033

Moberg, K. H., Mukherjee, A., Veraksa, A., Artavanis-Tsakonas, S. and Hariharan, I. K. (2004). The Drosophila F box protein archipelago regulates dMyc protein levels in vivo. Curr. Biol. 14(11): 965-74. 15182669

Mochizuki, T., et al. (1997). Pim-1 kinase stimulates c-Myc-mediated death signaling upstream of caspase-3 (CPP32)-like protease activation. Oncogene 15(12): 1471-1480

Moens, C. B., et al. (1992). A targeted mutation reveals a role for N-myc in branching morphogenesis in the embryonic mouse lung. Genes Dev 6: 691-704

Mukherjee, T., Hombria, J. C. and Zeidler, M. P. (2005). Opposing roles for Drosophila JAK/STAT signalling during cellular proliferation. Oncogene 24: 2503-2511. PubMed ID: 15735706

Nagy, P., Varga, A., Pircs, K., Hegedűs, K. and Juhász, G. (2013). Myc-driven overgrowth requires unfolded protein response-mediated induction of autophagy and antioxidant responses in Drosophila melanogaster. PLoS Genet 9: e1003664. PubMed ID: 23950728

Nair, S. K. and Burley, S. K. (2003). X-Ray structures of Myc-Max and Mad-Max recognizing DNA: Molecular bases of regulation by proto-oncogenic transcription factors. Cell 112: 193-205. 12553908

Neufeld, T. P., Tang, A. H., Rubin, G. M. (1998). A genetic screen to identify components of the sina signaling pathway in Drosophila eye development. Genetics 148(1): 277-286

Neumuller, R. A., et al. (2009). Mei-P26 regulates microRNAs and cell growth in the Drosophila ovarian stem cell lineage. Nature 454: 241-245. PubMed Citation: 18528333

Noguchi, K., Vassilev, A., Ghosh, S., Yates, J. L. and Depamphilis, M. L. (2006). The BAH domain facilitates the ability of human Orc1 protein to activate replication origins in vivo. EMBO J. 25(22): 5372-82. 17066079

Nomura, T., et al. (1999). Ski is a component of the histone deacetylase complex required for transcriptional repression by mad and thyroid hormone receptor. Genes Dev. 13(4): 412-23

O'Hagan, R. C., et al. (2000). Myc-enhanced expression of Cul1 promotes ubiquitin-dependent proteolysis and cell cycle progression. Genes Dev. 14: 2185-2191

Okubo, T., Knoepfler, P. S., Eisenman, R. N. and Hogan, B. L. (2005). Nmyc plays an essential role during lung development as a dosage-sensitive regulator of progenitor cell proliferation and differentiation. Development 132(6): 1363-74. 15716345

Oliver, T. G., et al. (2003). Transcriptional profiling of the Sonic hedgehog response: a critical role for N-myc in proliferation of neuronal precursors. Proc. Natl. Acad. Sci. 100(12): 7331-6. 12777630

Orian, A., et al. (2003). Genomic binding by the Drosophila Myc, Max, Mad/Mnt transcription factor network. Genes Dev. 17: 1101-1114. 12695332

Orian, A., et al. (2007). A Myc-Groucho complex integrates EGF and Notch signaling to regulate neural development. Proc. Natl. Acad. Sci. 104(40): 15771-6. PubMed Citation: 17898168

Oswald, F., et al. (1994). E2F-dependent regulation of human MYC: trans-activation by cyclins D1 and A overrides tumour suppressor protein functions. Oncogene 9: 2029-36

Ota, S., Zhou, Z. Q., Keene, D. R., Knoepfler, P. and Hurlin. P. J. (2007). Activities of N-Myc in the developing limb link control of skeletal size with digit separation. Development 134(8): 1583-92. Medline abstract: 17360777

Parisi, F., et al. (2011). Drosophila insulin and target of rapamycin (TOR) pathways regulate GSK3 beta activity to control Myc stability and determine Myc expression in vivo. BMC Biol. 9: 65. PubMed Citation: 21951762

Park, I. H., et al. (2008). Reprogramming of human somatic cells to pluripotency with defined factors. Nature 451(7175): 141-6. PubMed citation: 18157115

Park, J., et al. (2002). BAF53 forms distinct nuclear complexes and functions as a critical c-Myc-interacting nuclear cofactor for oncogenic transformation. Mol. Cell Biol. 22: 1307-1316. Medline abstract: 11839798

Peukert, K., et al. (1997). An alternative pathway for gene regulation by Myc. EMBO J. 16(18): 5672-5686

Pelengaris, S., et al. (1999). Reversible activation of c-Myc in skin: induction of a complex neoplastic phenotype by a single oncogenic lesion. Mol. Cell 3(5): 565-77

Pelengaris, S., Khan, M. and Evan, G. I. (2002). Suppression of Myc-induced apoptosis in ß cells exposes multiple oncogenic properties of Myc and triggers carcinogenic progression. Cell 109: 321-334. 12015982

Pennetta, G. and Pauli, D. (1998). The Drosophila Sin3 gene encodes a widely distributed transcription factor essential for embryonic viability. Dev. Genes Evol. 208(9): 531-6

Perez-Roger, I., et al. (1999). Cyclins D1 and D2 mediate Myc-induced proliferation via sequestration of p27Kip1 and p21Cip1. EMBO J. 18: 5310-5320

Perini, G., et al. (2005). In vivo transcriptional regulation of N-Myc target genes is controlled by E-box methylation. Proc. Natl. Acad. Sci. 102(34): 12117-22. 16093321

Perrin, L., et al.(2003). Modulo is a target of Myc selectively required for growth of proliferative cells in Drosophila. Mech. Dev. 120: 645-655. 12834864

Peyrefitte, S., Kahn, D. and Haenlin, M. (2001). New members of the Drosophila Myc transcription factor subfamily revealed by a genome-wide examination for basic helix-loop-helix genes. Mech. Dev. 104: 99-104. 11404084

Pierce, S. B., et al. (2004). dMyc is required for larval growth and endoreplication in Drosophila. Development 131: 2317-2327. 15128666

Pierce, S. B., Yost, C., Anderson, S. A., Flynn, E. M., Delrow, J. and Eisenman, R. N. (2008). Drosophila growth and development in the absence of dMyc and dMnt. Dev. Biol. 315(2): 303-16. PubMed Citation: 18241851

Pinto, D., et al. (2003). Canonical Wnt signals are essential for homeostasis of the intestinal epithelium. Genes Dev. 17: 1709-1713. 12865297

Popov, N., et al. (2010). Ubiquitylation of the amino terminus of Myc by SCFβ-TrCP antagonizes SCFFbw7-mediated turnover. Nat. Cell Biol. 12(10): 973-81. PubMed Citation: 20852628

Prall, O. W. J., et al. (1998). c-Myc or cyclin D1 mimics estrogen effects on cyclin E-cdk2 activation and cell cycle reentry. Mol. Cell. Biol. 18(8): 4499-4508

Prendergast, G. C. and Ziff, E. B. (1992). A new bind for Myc. Trends Genet. 8: 91-96

Prober, D. A. and Edgar, B. A. (2000). Ras1 promotes cellular growth in the Drosophila wing. Cell 100: 435-446

Prober, D. A. and Edgar, B. A. (2002). Interactions between Ras1, dMyc, and dPI3K signaling in the developing Drosophila wing. Genes Dev. 16: 2286-2299. 12208851

Pusch, O., et al. (1997). Activation of c-Myc uncouples DNA replication from activation of G1-cyclin-dependent kinases. Oncogene 15(6): 649-656

Qian, J., Zhang, Z., Liang, J., Ge, Q., Duan, X., Ma, F. and Li, F. (2011). The full-length transcripts and promoter analysis of intergenic microRNAs in Drosophila melanogaster. Genomics 97: 294-303. Pubmed: 21333734

Qin, Z. H., et al. (1999). Nuclear factor kappaB nuclear translocation upregulates c-Myc and p53 expression during NMDA receptor-mediated apoptosis in rat striatum. J. Neurosci. 19(10): 4023-33

Quinn, L. M., Dickins, R. A., Coombe, M., Hime, G. R., Bowtell, D. D. and Richardson, H. (2004). Drosophila Hfp negatively regulates dmyc and stg to inhibit cell proliferation. Development 131(6): 1411-23. 14993190

Raj, K. and Sarkar, S. (2017). Transactivation domain of human c-Myc Is essential to alleviate poly(Q)-mediated neurotoxicity in Drosophila disease models. J Mol Neurosci 62(1):55-66. PubMed ID: 28316031

Ramana, C. V., et al. (2000). Regulation of c-myc expression by IFN- through Stat1-dependent and -independent pathways. EMBO J. 19: 263-272. PubMed ID: 10637230

Reddy, B. V. and Irvine, K. D. (2011). Regulation of Drosophila glial cell proliferation by Merlin-Hippo signaling. Development 138(23): 5201-12. PubMed Citation: 22069188

Rhiner, C., et al. (2009). Persistent competition among stem cells and their daughters in the Drosophila ovary germline niche. Development 136(6): 995-1006. PubMed Citation: 19211674

Rodrigues, A. B., Zoranovic, T., Ayala-Camargo, A., Grewal, S., Reyes-Robles, T., Krasny, M., Wu, D. C., Johnston, L. A. and Bach, E. A. (2012). Activated STAT regulates growth and induces competitive interactions independently of Myc, Yorkie, Wingless and ribosome biogenesis. Development 139: 4051-4061. PubMed ID: 22992954

Rottbauer, W., et al. (2002). Reptin and pontin antagonistically regulate heart growth in zebrafish embryos. Cell 111(5): 661-72. 12464178

Roussel, M. F., et al. (1996). Inhibition of cell proliferation by the Mad1 transcriptional repressor. Mol. Cell. Biol. 16: 2796-2801

Salghetti, S. E., Kim, S. Y. and Tansey, W. P. (1999). Destruction of Myc by ubiquitin-mediated proteolysis: cancer-associated and transforming mutations stabilize Myc. EMBO J. 18: 717-726. 9927431

San Juan, B. P., Andrade-Zapata, I. and Baonza, A. (2012). The bHLH factors Dpn and members of the E(spl) complex mediate the function of Notch signalling regulating cell proliferation during wing disc development. Biol Open 1: 667-676. PubMed ID: 23213460

Satijn, D. P., et al. (1997). Interference with the expression of a novel human polycomb protein, hPc2, results in cellular transformation and apoptosis. Mol. Cell. Biol. 17(10): 6076-6086

Sawai, S., et al. (1993). Defects of embryonic organogenesis resulting from targeted disruption of the N-myc gene in the mouse. Development 117: 1445-55

Schreiber-Agus, N., et al. (1995). An amino-terminal domain of Mxi1 mediates anti-Myc oncogenic activity and interacts with a homolog of the yeast transcriptional repressor SIN3. Cell 80 (5): 777-786

Schreiber-Agus, N., et al. (1998). Role of Mxi1 in ageing organ systems and the regulation of normal and neoplastic growth. Nature 393: 483-487

Schreiber-Agus, N., Stein, D., Chen, K., Goltz, J. S., Stevens, L. and DePinho, R. A. (1997). Drosophila Myc is oncogenic in mammalian cells and plays a role in the diminutive phenotype. Proc. Natl. Acad. Sci. 94: 1235-1240

Schwamborn, J. C., Berezikov, E. and Knoblich, J. A. (2009). The TRIM-NHL protein TRIM32 activates microRNAs and prevents self-renewal in mouse neural progenitors. Cell 136: 913-925. PubMed Citation: 19269368

Sears, R., Ohtani, K. and Nevins, J. R. (1997). Identification of positively and negatively acting elements regulating expression of the E2F2 gene in response to cell growth signals. Mol. Cell. Biol. 17(9): 5227-5235

Sears, R., et al. (2000). Multiple Ras-dependent phosphorylation pathways regulate Myc protein stability. Genes Dev. 14: 2501-2514

Secombe, J., Li, L., Carlos, L., and Eisenman, R. N. (2007). The Trithorax group protein Lid is a trimethyl histone H3K4 demethylase required for dMyc-induced cell growth. Genes Dev. 21: 537-551. Medline abstract: 17311883

Seoane, J., Le, J. V. and Massagué, J. (2002). Myc suppression of the p21Cip1 Cdk inhibitor influences the outcome of the p53 response to DNA damage. Nature 419: 729-734. 12384701

Shcherbata, H. R., et al. (2004). The mitotic-to-endocycle switch in Drosophila follicle cells is executed by Notch-dependent regulation of G1/S, G2/M and M/G1 cell-cycle transitions. Development 131: 3169-3181. 15175253

Shi, J., et al. (2013). Role of SWI/SNF in acute leukemia maintenance and enhancer-mediated Myc regulation. Genes Dev 27: 2648-2662. PubMed ID: 24285714

Shim, H., et al. (1998). A unique glucose-dependent apoptotic pathway induced by c-Myc. Proc. Natl. Acad. Sci. 95(4): 1511-1516. PubMed ID: 9465046

Shimono, A., Okuda, T. and Kondoh, H. (1999). N-myc-dependent repression of ndr1, a gene identified by direct subtraction of whole mouse embryo cDNAs between wild type and N-myc mutant. Mech Dev 83(1-2): 39-52

Shiio, Y., et al. (2002). Quantitative proteomic analysis of Myc oncoprotein function. EMBO J. 21: 5088-5096. 12356725

Sierra, J., Yoshida, T., Joazeiro, C. A. and Jones, K. A. (2006). The APC tumor suppressor counteracts beta-catenin activation and H3K4 methylation at Wnt target genes. Genes Dev. 20(5): 586-600. 16510874

Sitcheran, R., et al. (2005). Positive and negative regulation of EAAT2 by NF-kappaB: a role for N-myc in TNFalpha-controlled repression. EMBO J. 24: 510-520. 15660126

Sjostrom, S. K., et al. (2005). The Cdk1 complex plays a prime role in regulating N-Myc phosphorylation and turnover in neural precursors. Dev. Cell 9: 327-338. 16139224

Sodir, N. M., et al. (2011). Endogenous Myc maintains the tumor microenvironment. Genes Dev. 25(9): 907-16. PubMed Citation: 21478273

Sommer, A., et al. (1997). Cell growth inhibition by the Mad/Max complex through recruitment of histone deacetylase activity. Curr. Biol. 7(6): 357-65. PubMed Citation: 9197243

Song, Y. and Lu, B. (2011). Regulation of cell growth by Notch signaling and its differential requirement in normal vs. tumor-forming stem cells in Drosophila. Genes Dev. 25(24): 2644-58. PubMed Citation: 22190460

Staller, P., et al. (2001). Repression of p15INK4b expression by Myc through association with Miz-1. Nat Cell Biol. 3(4): 392-9. 11283613

Stanton, B. R., et al. (1992). Loss of N-myc function results in embryonic lethality and failure of the epithelial component of the embryo to develop. Genes Development 6: 2235-47

Steiger, D., Furrer, M., Schwinkendorf, D. and Gallant, P. (2008). Max-independent functions of Myc in Drosophila melanogaster. Nat. Genet. 40(9): 1084-1091. PubMed Citation: 19165923

Steiner, P., et al. (1995). Identification of a Myc-dependent step during formation of active G1 cyclin-cdk complexes. EMBO J. 14: 4814-26

Strom, A., et al. (2007). Unique mechanisms of growth regulation and tumor suppression upon Apc inactivation in the pancreas. Development 134: 2719-2725. Medline abstract: 17596282

Takahashi, K. and Yamanaka, S. (2006). Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126: 1-14. Medline abstract: 16904174

Takayama, M. A., Taira, T., Tamai, K., Iguchi-Ariga, S. M., Ariga, H. (2000). ORC1 interacts with c-Myc to inhibit E-box-dependent transcription by abrogating c-Myc-SNF5/INI1 interaction. Genes Cells 5: 481- 490. 10886373

Tan, K., Shaw, A. L., Madsen, B., Jensen, K., Taylor-Papadimitriou, J., and Freemont, P. S. (2003). Human PLU-1 has transcriptional repression properties and interacts with the developmental transcription factors BF-1 and PAX9. J. Biol. Chem. 278: 20507-20513. Medline abstract: 12657635

Tanaka, H., et al. (2002). E2F1 and c-Myc potentiate apoptosis through inhibition of NF-kappaB activity that facilitates MnSOD-mediated ROS elimination. Molec. Cell, Vol 9: 1017-1029. 12049738

Tapon, N., et al. (2001). The Drosophila Tuberous sclerosis complex gene homologs restrict cell growth and cell proliferation. Cell 105: 345-355. 11348591

Teleman, A. A., Hietakangas, V., Sayadian, A. C. and Cohen, S. M. (2008). Nutritional control of protein biosynthetic capacity by insulin via Myc in Drosophila. Cell Metab 7: 21-32. PubMed ID: 18177722

Tevosian, S. G., et al. (1997). HBP1: a HMG box transcriptional repressor that is targeted by the retinoblastoma family. Genes Dev. 11:383-396

Tomonaga, T. and Levens, D. (1996). Activating transcription from single stranded DNA. Proc. Natl. Acad. Sci. 93: 5830-5835

Trumpp, A., et al. (2001). c-Myc regulates mammalian body size by controlling cell number but not cell size. Nature 414(6865): 768-73. 11742404

Vafa, O., et al. (2002). c-Myc can induce DNA damage, increase reactive oxygen species, and mitigate p53 function: A mechanism for oncogene-induced genetic instability. Molec. Cell 9: 1031-1044. 12049739

van de Wetering, M., et al. (2002). The ß-Catenin/TCF-4 complex imposes a crypt progenitor phenotype on colorectal cancer cells. Cell 111: 241-250. 12408868

van Riggelen, J., et al. (2010). The interaction between Myc and Miz1 is required to antagonize TGFbeta-dependent autocrine signaling during lymphoma formation and maintenance. Genes Dev. 24(12): 1281-94. PubMed Citation: 20551174

Vendrell, V., Lopez-Hernandez, I., Alonso, M. B., Feijoo-Redondo, A., Abello, G., Galvez, H., Giraldez, F., Lamonerie, T. and Schimmang, T. (2015). Otx2 is a target of N-myc and acts as a suppressor of sensory development in the mammalian cochlea. Development 142(16):2792-800. PubMed ID: 26160903

von der Lehr, N., et al. (2003). The F-Box protein Skp2 participates in c-Myc proteosomal degradation and acts as a cofactor for c-Myc-regulated transcription. Molec. Cell 11: 1189-1200. 12769844

Wakamatsu, Y., et al. (1997). Regulation of the neural crest cell fate by N-myc: promotion of ventral migration and neuronal differentiation. Development 124: 1953-1962

Walker, W., et al. (2005). Mnt-Max to Myc-Max complex switching regulates cell cycle entry. J. Cell Biol. 169(3): 405-13. 15866886

Wang, J., et al. (1998). Myc activates telomerase. Genes Dev. 12(12): 1769-1774

Wartiovaara, K., et al. (2002). N-myc promotes survival and induces S-phase entry of postmitotic sympathetic neurons. J. Neurosci. 22(3): 815-824. 11826111

Welcker, M., et al. (2004). The Fbw7 tumor suppressor regulates glycogen synthase kinase 3 phosphorylation-dependent c-Myc protein degradation. Proc. Natl. Acad. Sci. 101(24): 9085-90. 15150404

Weng, A. P., et al. (2006). c-Myc is an important direct target of Notch1 in T-cell acute lymphoblastic leukemia/lymphoma. Genes Dev 20: 2096-2109. Medline abstract: 16847353

Wilson, A., et al. (2004). c-Myc controls the balance between hematopoietic stem cell self-renewal and differentiation. Genes Dev. 18(22): 2747-63. 15545632

Wong, K. K., et al. (1995). v-Abl activates c-myc transcription through the E2F site. Mol. Cell. Biol. 15(12): 6535-6544

Wong, M. M., Liu, M. F. and Chiu, S. K. (2015). Cropped, Drosophila transcription factor AP-4, controls tracheal terminal branching and cell growth. BMC Dev Biol 15: 20. PubMed ID: 25888431

Wood, C. D., Veenstra, H., Khasnis, S., Gunnell, A., Webb, H. M., Shannon-Lowe, C., Andrews, S., Osborne, C. S. and West, M. J. (2016). MYC activation and BCL2L11 silencing by a tumour virus through the large-scale reconfiguration of enhancer-promoter hubs. Elife 5 [Epub ahead of print]. PubMed ID: 27490482

Wood, M. A., McMahon, S. B. and Cole, M. D. (2000). An ATPase/helicase complex is an essential cofactor for oncogenic transformation by c-Myc. Mol. Cell 5(2): 321-30. Medline abstract: 10882073

Wu, D. C. and Johnston, L. A. (2010). Control of wing size and proportions by Drosophila myc. Genetics 184(1): 199-211. PubMed Citation: 19897747

Wu, K. J., Polack, A. and Dalla-Favera, R. (1999a). Coordinated regulation of iron-controlling genes, H-ferritin and IRP2, by c-MYC. Science 283(5402): 676-9.

Wu, K. J., et al. (1999b). Direct activation of TERT transcription by c-MYC. Nat. Genet. 21(2): 220-4

Wu, Y. C., Lee, K. S., Song, Y., Gehrke, S. and Lu, B. (2017). The bantam microRNA acts through Numb to exert cell growth control and feedback regulation of Notch in tumor-forming stem cells in the Drosophila brain. PLoS Genet 13(5): e1006785. PubMed ID: 28520736

Xiao, Q., et al. (1998). Transactivation-defective c-mycS retains the ability to regulate proliferation and apoptosis. Genes Dev. 12(24): 3803-8

Yu, P., et al. (2017). FGF-dependent metabolic control of vascular development. Nature 545(7653): 224-228. PubMed ID: 28467822

Zervos, A. S., Gyuris, J. and Brent, R. (1993). Mxi1, a protein that specifically interacts with Max to bind Myc-Max recognition sites. Cell 72: 223-232

Zaffran, S., et al. (1998). A Drosophila RNA helicase gene, pitchoune, is required for cell growth and proliferation and is a potential target of d-Myc. Development 125(18): 3571-3584. PubMed Citation: 9716523

Zhao, X., et al. (2009). The N-Myc-DLL3 cascade is suppressed by the ubiquitin ligase Huwe1 to inhibit proliferation and promote neurogenesis in the developing brain. Dev. Cell 17(2): 210-21. PubMed Citation: 19686682

Zhang, C., Tinto, S. C., Li, G., Lin, N., Chung, M., Moreno, E., Moberg, K. H. and Zhou, L. (2014). An intergenic regulatory region mediates Drosophila Myc-induced apoptosis and blocks tissue hyperplasia. Oncogene [Epub ahead of print]. PubMed ID: 24931167

Zhang, H., et al. (2007). HIF-1 inhibits mitochondrial biogenesis and cellular respiration in VHL-deficient renal cell carcinoma by repression of C-MYC activity. Cancer Cell 11: 407-420. PubMed Citation: 17482131

Zindy, F., et al. (1998). Myc signaling via the ARF tumor suppressor regulates p53-dependent apoptosis and immortalization. Genes Dev. 12(15): 2424-2433. PubMed Citation: 9694806


Biological Overview

date revised: 18 April 2018

Home page: The Interactive Fly © 2011 Thomas Brody, Ph.D.