diminutive

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).

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 dmyc^P0. 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 dmyc^PL35 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 dmyc^PL35 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 dmyc^PG45 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 dmyc^PG45 or dmyc^PL35 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).

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 (Ras^V12) promotes these processes in the developing wing, but the effector pathways involved are unclear. Evidence is presented indicating that Ras^V12 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 Ras^V12-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 (Ras^V12) is capable of increasing dMyc protein levels as well as levels of PI3K signaling, suggesting that Ras^V12 drives growth and G₁/S progression via both of these mechanisms. Ras^V12 effector loop mutants were used to show that Ras^V12 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 dmyc^P0 or dmyc^P1 homozygotes than for dmyc^P0 heterozygotes in regions of the wing disc that normally contain high dMyc levels (i.e., wing pouch and notum. Because dmyc^P0/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. Ras^V12 increases dMyc levels post-transcriptionally, and studies in mammalian cell culture has shown that Ras^V12 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 (Tkv^Q238D) 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 (hfp^EP) harbors a recessive lethal P element insertion in the 5' UTR of hfp, 94 bp upstream of the initiating methionine codon. Homozygous hfp^EP larvae were of similar size to age-matched wild type third instar larvae. However, the pupariation of hfp^EP 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 hfp^EP mutant animals; however, they failed to eclose and died as pharate adults that were larger than wild type. The hfp^EP/hfp^EP 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 hfp^EP/hfp^EP and hfp^EP/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 hfp^EP 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 hfp^EP/hfp^EP larvae (Quinn, 2004).

In order to investigate whether Hfp regulates cell proliferation during Drosophila development, BrdU incorporation was measured in wing discs from wandering hfp^EP/hfp^EP 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 hfp^EP 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 hfp^EP 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).

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 SCF^Ago 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 SCF^Ago 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 SCF^Ago 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 SCF^Ago 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 SCF^Ago 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).

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 Lidís 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 proteinís 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 dm^P0. 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 proteinís 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).

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