Expression of mammalian Mad proteins is generally associated with differentiation and organogenesis, while Mnt appears to be more constitutively expressed in both actively dividing cells and those that are exiting the cell cycle (Hurlin, 1997; Hurlin, 1995; Meroni, 1997; Queva, 1998). To examine the expression pattern of dMnt, a monoclonal antibody, P5D6, was raised to the region common to all three dMnt splice forms. To verify the specificity of the dMnt antibody, the larvae of a dmnt null mutant (dmnt1) and a mutant that retains the dMnt DeltaSID transcript (dmnt2) were stained with P5D6. Nuclear staining was clearly seen in wild-type cells and, to a lesser degree in dmnt2 mutant cells, but is not detectable in dmnt1 mutant cells in larval salivary gland cells. These results support the observation that P5D6 is a dMnt-specific antibody (Loo, 2005).
Although dmnt mRNA is maternally deposited and is present until cellularization, dMnt protein is first detected in cells of the epidermis at embryonic cell cycle 17 (stage 11), coincident with these cells ceasing proliferation and beginning differentiation. At this and subsequent stages, dMnt is also expressed in peripheral and central nervous system cells, as evidenced by the colocalization of dMnt and the neural antigen ELAV. dMnt expression is not, however, limited to cells that are differentiating during embryogenesis; dMnt is also expressed in the embryonic salivary gland primordium at stage 11 when these cells are actively endocycling (Loo, 2005).
dMnt is also expressed in both differentiating and actively dividing cells during the third larval instar of Drosophila development. During eye imaginal disc development, cells are recruited to become peripheral nervous system cells and express the neural antigen ELAV posterior to a morphological marker called the morphogenetic furrow (MF). dMnt is predominantly expressed in differentiated cells posterior to the MF. However, a group of dMnt-positive, ELAV-negative cells is detectable at the MF, indicating that dMnt is expressed as the neural fate of these cells is being determined. In addition, dMnt is expressed in actively dividing peripodial membrane cells of the wing and eye-antennal imaginal disks in third-instar larvae. dMnt is also present in endocycling salivary gland and fat body cells. dMnt is therefore expressed in a temporally and spatially dynamic pattern in both actively replicating mitotic and endoreplicating tissue and differentiating cells (Loo, 2005).
Members of the Myc family of proto-oncogenes have long been implicated in regulating proliferation, apoptosis and oncogenesis. Recently, transcriptional and biological studies have suggested a direct role for Myc in regulating growth. dm4, a new null allele of the Drosophila diminutive (dm) gene, which encodes dMyc on the X chromosome, has been used to investigate a role for dMyc in larval endoreplicating tissues, where cellular growth and DNA replication occur in the absence of cell division. Hemizygous dm4/Y mutants arrest as second instar larvae, and fat body nuclei of dm4/Y mutants fail to attain normal size and normal levels of DNA, resulting from a reduced frequency of S-phase. Thus, dMyc is required for endoreplication and larval growth. In support of this, dMyc, as well as its antagonist dMnt, are expressed in larval tissues in a pattern consistent with their involvement in regulating endoreplication. Overexpression of dMyc in endoreplicating cells results in dramatic increases in nuclear DNA content and cell and nucleolar size, whereas dMnt overexpression has the opposite effect. BrdU incorporation and Cyclin E protein levels continue to oscillate in dMyc-overexpressing cells, indicating that the normal cell cycle control mechanisms are not disrupted. dMyc driven growth and endoreplication are strongly attenuated when the endocycle is blocked with Cyclin E or the cdk inhibitor p21. By contrast, the ability of dMyc to promote growth and endoreplication is only partly reduced when PI3K activity is blocked, suggesting that they influence distinct growth pathways. These results indicate that larval growth and endoreplication are coupled processes that, although linked to cell cycle control mechanisms, are regulated by dMyc and dMnt (Pierce, 2004).
To determine whether there is a requirement for dMyc in driving endoreplication, dm4, a null allele of dm, was isolated. The failure of dm4 mutants to grow beyond the second instar indicates that dMyc is required for growth at the organismal level. It is presumed that maternally deposited dm gene products, or other maternal products, are sufficient for development of the embryo. The fact that maternal dm transcripts and protein are undetectable by the time of hatching suggests that dMyc is not required for the initiation of larval growth and may not be required for completion of embryogenesis, although it is possible that a small amount of residual maternal dMyc supports the growth of dm4 mutant larvae prior to their arrest. The massive growth that takes place during larval development is tightly coupled to the endoreplication that takes place in all larval tissues except the imaginal discs and nervous system. The finding that dMyc and dMnt are expressed in distinct groups of cells in these tissues is suggestive of roles in promoting (dMyc) and limiting (dMnt) endoreplication and suggests that the failure of dm4 mutants to grow is the result of loss of dMyc in endocycling tissues (Pierce, 2004).
Larval growth involves both cytoplasmic growth and DNA endoreplication. The reduced rate of BrdU incorporation in early larval tissues and the failure of dm4 mutant nuclei to grow suggests that these mutants undergo reduced DNA replication. The fact that mutant cells and larvae are smaller than age-matched controls indicates that that there is also a growth defect. Thus, directly or indirectly, dMyc is required for both growth and DNA replication during endoreplication (Pierce, 2004).
Consistent with findings that dMyc loss of function negatively affects endoreplication and growth, overexpression of dMyc drives both cellular growth and DNA replication. By contrast, overexpression of the Drosophila ortholog of Mad, dMnt, blocks cellular growth and DNA replication. These results are consistent with a model in which dMyc and dMnt act antagonistically, with dMnt binding and repressing the genes required for endoreplication that dMyc activates. dMnt is normally highly expressed in the third instar salivary gland and other tissues that have exited the endocycle, indicating that dMnt-mediated gene repression may be necessary for this transition. However, dMnt, the only Mad family ortholog in Drosophila, is non-essential. dMnt null mutants develop normally into adults with modestly increased body weights and shorter lifespans. Although the increased body weight of dMnt mutants is consistent with negative regulation of growth by dMnt, no altered endoreplication was observed in mutants, suggesting that negative regulators of endoreplication other than dMnt must exist (Pierce, 2004).
In endoreplicating cells ectopic expression of dMyc results in increased cytoplasmic and nuclear volume, as well as in enlarged nucleoli, as detected by increased anti-fibrillarin staining. Fibrillarin has been implicated as a Myc target gene in both vertebrate and Drosophila cells and its augmented expression is consistent with the notion that dMyc/Myc promotes ribosome biogenesis. The pitchoune gene, which encodes a putative RNA-helicase localized to the nucleolus, is also induced by ectopic dMyc, and pit null mutants have a severe larval growth defect similar to dm4 mutants. The mammalian ortholog of pit, MrDB (DDX18), has been identified as a direct target of c-Myc. In addition, many other known and suspected targets of the Myc family are involved in this process (Pierce, 2004).
S-phase of the endocycle is initiated by the activity of Cyclin E/cdk2 but endoreplication can be blocked by continuous ectopic expression of Cyclin E or the human cdk inhibitor p21. It is thought that Cyclin E levels must drop after each S-phase and then increase again prior to the next S-phase to allow reinitiation of DNA replication. In mitotic cells, this prevents more than one round of DNA replication from occurring during each cell cycle. In endoreplicating cells it results in discrete S-phases separated by a gap phase. Ectopic p21 is likely to inhibit the activity of cdk2 even in the presence of Cyclin E. The extra endocycles driven by ectopic dMyc appear to be normal, in that there are discrete periods of DNA replication and Cyclin E appears to oscillate. As ectopic dMyc induces cells to accumulate high levels of DNA earlier in development, it is presumed that S-phases and Cyclin E oscillations occur more frequently than normal. It is also possible that the S-phases are shorter and that Cyclin E peaks at higher levels when ectopic dMyc is present. When co-expressed with ectopic unregulated Cyclin E or p21, dMyc drives very little endoreplication, suggesting that the cell cycle control exerted by oscillating Cyclin E/cdk2 activity is downstream of dMyc function. Consistent with this, ectopic dMyc can post-transcriptionally increase Cyclin E levels in wing discs and studies in mammalian cells suggest that Myc can indirectly induce Cyclin E expression. Microarray analysis did not identify Cyclin E as a transcriptional target of dMyc, suggesting that the transcriptional oscillation of Cyclin E is not directly regulated by dMyc. Thus, dMyc is unable to drive endoreplication in the absence of normal cdk activity. Although the level of fibrillarin staining was not quantified, dMyc appears to drive somewhat more nucleolar growth than DNA accumulation when co-expressed with Cyclin E or p21, indicating that dMyc may be able to drive a limited amount of nucleolar growth in the absence of DNA replication (Pierce, 2004).
The Drosophila insulin signaling pathway is also essential for growth. Mutations in the receptor InR and downstream components of the pathway, including Dp110, a PI3 kinase homolog, cause larval growth defects. When PI3 kinase signaling is blocked by ectopic expression of p60, dMyc is still able to induce a significant amount of cellular growth and DNA replication. This suggests either that dMyc is downstream of PI3 kinase signaling or that dMyc and dDp110 represent independent pathways that are both essential for growth. Recent studies have found that Dp110 or InR overexpression do not result in increased dMyc transcription and that activated Ras increases dMyc levels and PI3 kinase activity via independent effector pathways, suggesting that dMyc transcription is not downstream of the insulin signaling pathway. In addition, although ectopic expression of either dMyc or Dp110 leads to increased cell growth, the increase in nuclear size is more pronounced in response to dMyc whereas the increase in cytoplasmic volume is more pronounced in response to Dp110, further supporting the idea that dMyc and Dp110 regulate growth and endoreplication independently (Pierce, 2004).
dMyc overexpression augments cell growth in mitotic wing disc cells by shortening the mass doubling time. Such cells display a decrease in the length of G1 and a compensatory increase in the length of G2/M, resulting in a division time equal to that of control cells. They retain their normal ploidy and show little effect on the length of S phase. In endoreplicating cells, dMyc drives both cellular growth and DNA replication. What is the relationship of dMyc function to these processes? dMyc transcriptionally activates a wide range of genes involved in ribosome biogenesis, translation and metabolism, suggesting that the relationship of dMyc to growth is likely to be very direct. The absence of an effect of dMyc on S phase length and cell division rate in mitotic cells argues that perhaps the only role of dMyc is to regulate cell growth. Interestingly, the division rate of dMyc-overexpressing mitotic cells is increased by introduction of String, which accelerates G2/M, resulting in the generation of a larger number of cells. Perhaps in endoreplicating cells, which lack G2/M entirely, dMyc simply increases the growth rate thereby shortening the G1-S transition and leading to a higher rate of S phase entry. Because such cells are incapable of division, the net effect observed is larger cells with increased ploidy. In this model, dMyc is thought to augment endoreplication indirectly, through its promotion of growth. dMyc is also required during oogenesis for somatic and germ cell growth and endoreplication, but not for proliferation prior to the onset of endoreplication. The finding that dMyc mutant follicle cells exhibit reduced growth prior to endoreplication suggests that the defect in endoreplication may be secondary to the defect in cellular growth (Pierce, 2004).
Alternatively, dMyc might affect endoreplication more directly. Although both mammalian and Drosophila Myc target genes are predominantly growth related, a smaller number of gene targets are involved in cell cycle control and DNA replication. Importantly, dMyc does not increase transcript levels of Cyclin E or the Drosophila E2F1 transcription factor, the only known limiting factors for endocycles in endoreplicating tissues. The finding that the effect of dMyc on growth is attenuated when the cell cycle is blocked by continuous expression of Cyclin E or p21 indicates that dMyc-induced growth is tightly coupled to DNA replication, at least in endoreplicating cells. The large number and diversity of the genes identified as likely targets of Myc genes indicates that Myc activity impinges on a broad range of cellular functions that must be highly coordinated for proper cell behavior. Interestingly Myc overexpression has been reported to lead to endoreplication and polyploidy in human kertinocytes. Furthermore, in murine fibroblasts treated with colcemid, Myc overexpression leads to abrogation of the G2/M checkpoint and marked polyploidy. These results suggest that Myc function is involved in controlling S-phase entry and G2/M in diverse vertebrate cell types. The Drosophila endoreplicating cell system should provide a good model for better defining the precise role of Myc in coordinating growth and cell cycle (Pierce, 2004).
To determine whether expression of dMnt splice variants can influence cell behavior in Drosophila, the UAS/GAL4 expression system was used to express dMnt in endocycling and mitotically dividing cells during development. dMnt proteins were ectopically expressed in the developing eye (employing an eyeless-GAL4 driver) and posterior compartments (engrailed-GAL4). In all tissues ectopically expressing the dMnt cDNA, a marked reduction was observed in the size of the respective adult structure. These phenotypic changes were more severe when dMnt and dMax were ectopically coexpressed in these tissues, suggesting that the observed dMnt effects were mediated by the transcriptional repression activity of dMnt-dMax heterodimers. No obvious adult phenotypes were observed dMax, dMnt DeltaSID, or dMnt DeltaZIP was expressed using these drivers (Loo, 2005).
Proliferation was measured in cell clones ectopically expressing the dMnt cDNA to determine whether the observed dMnt ectopic expression phenotypes were the result of dMnt's ability to inhibit proliferation utilizing the Flp/Gal4 method. The sizes of clones ectopically expressing dMnt were compared with GFP to clones expressing GFP alone in both the imaginal wing disc and the fat body. Clones ectopically expressing dMnt were significantly smaller than clones expressing GFP alone. To determine whether the reduced clone size was due to an inhibition of proliferation and/or growth or apoptosis, dMnt was coexpressed with the baculovirus apoptosis inhibitor p35. There was no significant change in the size of clones coexpressing dMnt and p35 compared to clones expressing dMnt without p35. These results support the observation that dMnt expression is associated with an inhibition of cellular proliferation. Surprisingly, in contrast to what was observed with the eyeless-GAL4 and engrailed-GAL4 drivers, expression of the dMnt DeltaSID or dMnt DeltaZIP splice form in the wing discs of random clones appeared to have an intermediate effect on proliferation relative to dMnt. Because only the latter experiments were carried out under conditions where cell competition is known to occur, it is likely that even relatively weak effects of dMnt DeltaSID or dMnt DeltaZIP compared to dMnt would be detected. To determine whether the smaller clone size caused by dMnt expression was due to effects on cell cycle or cell size, wing disc cells were dissected and examined using flow cytometry. GFP-positive cells, which express the transgene, were compared to GFP-negative cells, which represent the wild-type cell profile. When dMnt was expressed, an increase in the population of cells was observed in the G0/G1 phase of the cell cycle and a decrease in the population of cells in S and G2/M. When the relative sizes of the cells ectopically expressing dMnt and the nonexpressing population were compared by forward scatter, it was found that dMnt-expressing cells were significantly smaller than the wild-type cells. This size difference is not due to the increased fraction of G0/G1 cells in the dMnt-expressing population because when individual cell cycle phases were gated and compared, it was found that the size difference was observed in all phases. Expression of the dMnt DeltaSID or dMnt DeltaZIP proteins resulted in no detectable effects on cell cycle phasing or cell size, suggesting that these splice forms are likely to act through a mechanism distinct from that of dMnt. Overall, these results indicate that expression of dMnt has effects on cellular proliferation, cell cycle progression, and cell growth (Loo, 2005).
To analyze the effects of loss of dmnt function, the Drosophila database was sought for any mutations in the dmnt genomic region (cytological region 3E5). Two P-element insertions were identified in the dmnt gene and one insertion immediately upstream of the putative transcription start site. An imprecise P-element excision screen was initiated to obtain stronger dmnt mutant alleles. By mobilizing the P-element [EP(X)1559], two independent dmnt mutant alleles were generated, dmnt1 and dmnt2; a precise excision served as a wild-type control allele. On the basis of genomic PCR and Southern blotting analysis, dmnt1 is a null allele. All exons contributing to the open reading frame of dMnt are deleted in this allele. dmnt2 contains a smaller deletion that removes the exon containing the translational start codons of the dMnt and dMnt DeltaZIP splice forms but does not extend to the exon containing the translational start codon of the dMnt DeltaSID splice form. RT-PCR analysis demonstrated that dmnt1 lacks mRNA for the dMnt, dMnt DeltaZIP, and dMnt DeltaSID splice forms, while dmnt2 lacks detectable mRNA for the dMnt and dMnt DeltaZIP splice forms but retains the mRNA for dMnt DeltaSID (Loo, 2005).
dmnt1 and dmnt2 mutants are homozygous viable and fertile with no detectable decrease in fitness. To determine whether there are any delays or developmental defects during embryogenesis, hatching rates were tested on the two homozygous mutant lines and they were compared to a wild-type (precise excision) control and the parental EP(X)1559 allele. No significant differences were observed in the hatching percentage or rate in the mutants compared to the wild-type control, suggesting that the loss of dMnt has no significant effects on developmental timing during embryogenesis. Larval development was analyzed and no delays in larval molting, pupation, or eclosion were detected. These results suggest that dMnt is not essential for the normal developmental program (Loo, 2005).
Because ectopic expression of dMnt inhibits cellular proliferation and growth, a determination was made whether loss of dMnt function influences weight and cell size in adult flies. The average individual weights of dmnt mutant adult males and wild-type control males were compared and it was observed that both dmnt1 and dmnt2 mutant males are 20% and 12% heavier than wild-type controls. Cell size was measured by determining trichome density (each trichome represents one cell) in a defined area of the adult wing. dmnt1 mutant males had 18% fewer cells in a unit area and dmnt2 mutants had 10% fewer cells than the wild-type controls, indicative of increased cell size. These results demonstrate that dMnt proteins normally play a role in limiting cellular growth (Loo, 2005).
Several mutants that affect adult body size, such as those that compromise insulin signaling in Drosophila, also influence life span. To test whether dMnt mutant adults have an altered life expectancy, dmnt1 and dmnt2 virgins were collected, their ages were determined, and the number of dead flies was scored every 3 days and compared to the death rate of virgins from isogenic wild-type controls. The results are the average of three independent and highly reproducible experiments. The average life spans of dmnt1 and dmnt2 females were 41 and 48 days, respectively, compared with 54 days for wild-type controls. This reduction of 13 days represents a significant decrease (24%) in the life span of the fly. The maximal life expectancy (90% mortality) is also reduced in dMnt mutant animals, with wild-type flies living an average of 71 days compared with 59 and 63 days for dmnt1 and dmnt2 alleles, respectively. The intermediate phenotype observed for dmnt2 in life span is consistent with its less severe effect on adult cell and body size, suggesting that dmnt2 is likely to be a hypomorphic allele. While dmnt mutant females have a shorter life span than an isogenic wild-type control, these flies are generally not unhealthy. dmnt mutant strains can be kept as homozygous stocks and show no developmental delays. Furthermore, dmnt mutant larvae and adults are neither more sensitive to starvation nor more susceptible to bacterial infection. Therefore, it is unlikely that the shortened life span of the mutants is due to developmental defects, infection, or generalized unhealthiness. Taken together, these results suggest that dMnt plays a role in regulating life span (Loo, 2005).
Myc oncoproteins are essential regulators of the growth and proliferation of mammalian cells. In Drosophila the single ortholog of Myc (dMyc), encoded by the dm gene, influences organismal size and the growth of both mitotic and endoreplicating cells. A null mutation in dm results in attenuated endoreplication and growth arrest early in larval development. Drosophila also contains a single ortholog of the mammalian Mad/Mnt transcriptional repressor proteins (dMnt), which is thought to antagonize dMyc function. Animals lacking both dMyc and dMnt display increased viability and grow significantly larger and develop further than dMyc single mutants. Increased endoreplication and growth of larval tissues is observed in these double mutants, along with disproportionate growth of the imaginal discs. Gene expression analysis indicates that loss of dMyc leads to decreased expression of genes required for ribosome biogenesis and protein synthesis. The additional loss of dMnt partially rescues expression of a small number of dMyc and dMnt genes that are primarily involved in rRNA synthesis and processing. These results indicate that dMnt repression is normally overridden by dMyc activation during larval development. Therefore the severity of the dm null phenotype is likely due to unopposed repression by dMnt on a subset of genes critical for cell and organismal growth. Surprisingly, considerable growth and development can occur in the absence of both dMyc and dMnt (Pierce, 2008).
These experiments show that Drosophila larvae lacking both dMyc and dMnt display increased viability, grow significantly larger, and advance considerably further in larval development than dMyc single mutants. dm4dmnt1 mutant larvae reached a maximum size that was much larger than the maximum size of dm4 mutants, which are terminally growth arrested at an early larval stage. In addition, a substantial fraction of dm4dmnt1 mutant larvae can continue to develop and form pupae, although they fail to metamorphose (Pierce, 2008).
The most dramatic defect in larvae lacking dMyc is the failure of endoreplicating cells to attain both wild-type size and DNA levels. Similar effects have been observed in germline and somatic follicle cells mutant for dm. Because endoreplicating cells comprise the bulk of the early larva and are responsible for most of the 200-fold increase in mass that occurs between embryogenesis and pupariation, it is likely that the defect in endoreplicating tissues accounts to a large extent for the very limited larval growth and the inviability in dm4 animals. The additional removal of dMnt results in increased cell and nuclear growth, which is accompanied by a modest increase in the fraction of cells undergoing endoreplication in the early larva as well as increased survival. It is surmised that this increase in endoreplication and growth, combined with the extended larval period, is likely to account for the overall growth rescue. These results indicate that the strong larval growth arrest phenotype in the dMyc single mutant (dm4) is due, in part, to the presence of dMnt, whose activity as a repressor would be unopposed by dMyc in dm4. As a result, when dMnt is removed in the dm4 background, a significant amount of growth can occur in the absence of dMyc. A recent study in mammalian cells and Xenopus extracts has demonstrated that c-Myc plays a regulatory role in initiation of DNA replication through direct association, together with Max, with the pre-replication complex (Dominguez-Sola, 2007). It is possible that dMyc could drive endoreplication directly rather than as a secondary consequence of increased growth. Further experiments will be required to determine if dMyc is involved in DNA replication in Drosophila and, if so, whether dMnt plays an antagonistic role (Pierce, 2008).
In Drosophila, dMyc has been demonstrated to drive the growth of mitotic as well as endoreplicating cells. dMyc overexpression results in dramatically larger wing disc cells and analysis of dMyc overexpressing clones has demonstrated that dMyc drives cell growth with little effect on cell division time. Clones of mitotic cells with reduced dMyc activity are smaller than clones of wild-type cells, indicating that the mutant cells grow more slowly. As expected, overexpression of dMnt results in small cells and cell clones while dMnt loss of function produces larger cells in the adult wing as well as heavier animals. In mammals, primary mouse embryo fibroblasts (MEFs) and other cell types lacking c-Myc are unable to grow and proliferate in response to mitogenic signals. However, MEFs lacking both c-Myc and Mnt display accelerated proliferation following selection for cells that have escaped apoptosis. While no reports to date have addressed loss of both Myc and Mnt function in tissues or whole organisms, the MEF studies are generally consistent with the finding that the imaginal discs, which consist of mitotically dividing cells, are rescued in larvae lacking dMyc and dMnt (Pierce, 2008).
It is likely that the increased growth of the endoreplicating tissues plays a significant role in the growth rescue of the whole animal and the discs. In particular, the fat body is a critical tissue for mediating the response to nutritional signals and is required to promote growth and proliferation in mitotic cells and larvae in a cell non-autonomous fashion. However, if larvae are starved after growth begins, mitotic cells continue to proliferate after endoreplication stops. Thus is it likely that the increased growth of the endoreplicating tissues, particularly the fat body, promotes the growth of the entire animal to a point at which the imaginal discs can continue to proliferate despite the limited growth of the endoreplicating tissues. In animals that lack only dMyc, there may be sufficient growth of the endoreplicating tissues to initiate growth of the animal as a whole, but not enough to sustain that growth to a point at which the discs can proliferate more independently (Pierce, 2008).
Given the strong rescue of overall imaginal disc growth it was surprising to find that clones of imaginal disc cells lacking dMyc alone or lacking both dMyc and dMnt are the same size and significantly smaller than their sibling wild-type clones. This indicates that these cells are at a proliferative disadvantage. Because such mutant clones are surrounded by cells with higher levels of dMyc, their dramatically smaller clone size can be ascribed to cell competition, a process resulting in elimination of more slowly growing cell populations from the disc epithelium. It has been well established that cell clones with lower dMyc levels relative to the surrounding population are at a competitive disadvantage and die through activation of apoptotic pathways. In this regard it is interesting that dMnt loss of function has no discernable effect on the size of dm4 clones in a heterozygous background. One interpretation is that the growth rescue resulting from dMnt loss is simply not sufficient to avoid cell competition induced apoptosis. Alternatively it is possible that dMnt does not influence the pathway through which reduced dMyc levels stimulate apoptosis in cells targeted for elimination (Pierce, 2008).
The signals required for the growth and maturation of multicellular organisms include extrinsic environmental cues, such as nutrition, and intrinsic developmental signals, which may have cell-autonomous and cell non-autonomous effects. The major pathways that regulate growth in response to nutrition in Drosophila are the insulin receptor and TOR signaling pathways. The TOR pathway in particular is responsive to amino acids and, with input from the phosphoinositide 3-kinase (PI3K) branch of the insulin receptor pathway, regulates ribosome biogenesis and translation. In Drosophila dMyc and dMnt have been shown to influence cell and organismal growth primarily through effects on transcription. Yet many Myc- and Mnt-regulated transcriptional targets in flies and vertebrates are themselves involved in the regulation of the translational machinery. For example Myc directly binds to and stimulates RNA PolI transcription of rDNA in mammalian cells and indirectly stimulates RNA PolI transcription in Drosophila. Interestingly, strong mutations in, or inhibition of, components of the insulin and TOR pathways result in larval growth arrest phenotypes that are similar to the effect of amino acid starvation and the phenotype of the dMyc null mutant, dm4. A weaker TOR mutant resembles the dm4dmnt1 mutant in that it grows larger and the wing discs overgrow at the expense of the endoreplicating tissues. Indeed it is also noted that growth of the dm4dmnt1 mutant was sensitive to environmental conditions such as nutrient source and presence of competition. However, clear molecular connections between the insulin and Tor pathways and dMyc/dMnt function have yet to be established (Pierce, 2008).
Although the dm4dmnt1 larvae display delayed pupariation, the pupae are all very uniform in size, suggesting that they have reached a threshold for growth or size. The transition from feeding and growth to pupariation and metamorphosis is normally triggered by induction of the steroid hormone ecdysone. This occurs after larvae reach a minimum viable weight at which they possess sufficient nutritional stores to survive metamorphosis. However, experiments indicate that treatment with ecdysone is unable to rescue the delay in pupariation suggesting that ecdysone is not limiting for dm4dmnt1 pupariation. Since the dm4dmnt1 pupae are abnormal and never result in the formation of adult animals, their extended larval period and other modulatory factors in larvae may interact with ecdysone to trigger the initiation of metamorphosis without the larvae reaching the minimum weight required to sustain metamorphosis. Thus the failure of dm4dmnt1 larvae to survive metamorphosis could be explained by their failure to accumulate sufficient nutritional stores. In addition, there may be a specific requirement for dMyc during pupal development that cannot be rescued by removing dMnt function. This latter possibility would be consistent with earlier work showing that a subset of genomic binding sites for dMyc do not bind dMax or dMnt as well as a study indicating dMax independent functions of dMyc. Because dMax coordinates binding of both dMyc and dMnt to common E-box sites it is unlikely that genes activated by dMyc independent of dMax would be repressed by dMnt (Pierce, 2008).
This is the first analysis of gene expression changes in animals that are null for dMyc, and it was found that the affected genes are primarily involved in protein synthesis. Although it was previously known that dMyc was required for pre-rRNA transcription and that PolI function is required for dMyc-induced cell growth, this study has demonstrated that dMyc is also necessary for the expression of a wide range of genes involved in ribosome biogenesis and translation. This is consistent with what is known about Myc target genes in Drosophila and mammals, in which Myc proteins have been shown to regulate the transcription of genes from all three polymerases thus controlling both ribosome biogenesis and subsequent translation. Because larvae are primarily made up of endoreplicating non-dividing cells, it is not surprising that particularly strong overlap was found between genes for which expression decreased in the absence of dMyc and those for which expression increased when dMyc was overexpressed in larvae and less overlap with dMyc direct binding sites. Note that some of the genes detected are not direct targets of dMyc but are likely to be induced as a secondary response to Myc (Pierce, 2008).
Given the growth rescue seen when dMnt function is removed in the context of the dMyc mutant, it was particularly interesting to find that the expression of only a subset of dMyc-responsive genes was altered in the double mutants relative to the dMyc single mutant. There was a modest, but significant, increase in pre-rRNA, which can in part be explained by an increase in PolI components such as Rpl1. There was a more significant increase in the expression of genes that are known or predicted to be involved in rRNA processing. Interestingly, c-Myc has been shown to promote rRNA processing in a human B-cell line. It is surmised that an increase in both rRNA synthesis and processing leads to more efficient ribosome biogenesis and, ultimately, protein synthesis. Although transcription of only one of four ribosomal protein genes that was tested by qPCR was significantly rescued, the availability of ribosomal proteins may be increased through post-transcriptional mechanisms or existing ribosomal proteins may be utilized more efficiently due to increased levels of processed rRNA (Pierce, 2008).
Although it has been shown that dMyc and dMnt are capable of acting antagonistically, in a manner similar to their mammalian counterparts, the lack of a dramatic phenotype in flies lacking dMnt might suggest that dMnt is not normally playing a major regulatory role at dMyc target genes. Nonetheless, a modest increase in expression of growth related genes is seen in dmnt1 larvae indicating that in wild-type flies dMnt is likely functionally required to limit cell growth during normal development. The data showing that dMnt loss produces partial alleviation of the repression of a subset of growth related genes in dm4 larvae is consistent with the notion that dMnt is involved in the downregulation of growth gene expression upon loss of dMyc. In the absence of both dMyc and dMnt, active repression by dMnt is relieved and gene transcription is likely to be dependent on the presence of other transcription factors that may be gene-specific and normally act to regulate expression of specific genes in collaboration with Myc and Mnt. It is also possible that other Drosophila bHLH class activators that do not normally interact with dMyc-dMnt regulated promoters may play a role in the rescue of growth related gene expression when both dMnt and dMyc are absent. It is proposed that it is this rescue of growth related gene expression that leads to increased viability and development (Pierce, 2008).
A similar model has been proposed to explain the effects of deletion of another antagonistic pair of transcription factors dE2F1 and dE2F2. dE2F1 is a transcriptional activator that is essential for normal proliferation and larval growth. dE2F2 is a transcriptional repressor that can inhibit dE2F1-dependent transcription by binding to the same DNA binding sites. The growth defects in larvae lacking dE2F1 can largely be suppressed by also removing dE2F2, indicating that the dE2F1 mutant phenotype is largely due to unchecked repression by dE2F2. Interestingly the E2F proteins are also subunits of the Myb-MuvB/dREAM complex that additionally comprises Drosophila Myb, required for the selective amplification of the chorion genes, and the Mip130, Mip120, and Mip40 repressors of DNA replication (Georlette, 2007). Loss of function mutations of Dm-Myb are lethal, while single Mip mutants and the double Dm-Myb;Mip mutants are viable, indicating that the Mip proteins are primarily responsible for the lethality of the single Dm-Myb mutants (Pierce, 2008).
The results suggest that, in order to activate genes required for growth and development, dMyc must overcome the repressive effect of dMnt. It is therefore likely that dMnt serves to refine or limit the activity of dMyc. Although the weak phenotypes associated with the dMnt single mutant suggest that this role is minor, it is important to note that dMnt mutants as well as mutants in the C. elegans ortholog of dMnt (mdl1) have a reduced lifespan, indicating that the modulation of dMyc activity by dMnt is necessary for wild-type fitness (Pierce, 2008).
Recent evidence indicates that skeletal muscle influences systemic aging, but little is known about the signaling pathways and muscle-released cytokines (myokines) responsible for this intertissue communication. This study shows that muscle-specific overexpression of the transcription factor Mnt decreases age-related climbing defects and extends lifespan in Drosophila. Mnt overexpression in muscle autonomously decreases the expression of nucleolar components and systemically decreases rRNA levels and the size of the nucleolus in adipocytes. This nonautonomous control of the nucleolus, a regulator of ribosome biogenesis and lifespan, relies on Myoglianin, a myokine induced by Mnt and orthologous to human GDF11 and Myostatin. Myoglianin overexpression in muscle extends lifespan and decreases nucleolar size in adipocytes by activating p38 mitogen-activated protein kinase (MAPK), whereas Myoglianin RNAi in muscle has converse effects. Altogether, these findings highlight a key role for myokine signaling in the integration of signaling events in muscle and distant tissues during aging (Demontis, 2014. PubMed ID: 24882005).
Reference names in red indicate recommended papers.
Atchley, W. R. and Fernandes, A. D. (2005). Sequence signatures and the probabilistic identification of proteins in the Myc-Max-Mad network. Proc. Natl. Acad. Sci. 102(18): 6401-6. 15851686
Brubaker, K., et al. (2000). Solution structure of the interacting domains of the Mad-Sin3 complex: Implications for recruitment of a chromatin-modifying complex. Cell 103: 655-665.
Demontis, F., Patel, V. K., Swindell, W. R. and Perrimon, N. (2014). Intertissue control of the nucleolus via a myokine-dependent longevity pathway. Cell Rep 7(5):1481-94. PubMed ID: 24882005
Eilers, A. L., et al. (1999). A 13-amino acid amphipathic alpha-helix is required for the functional interaction between the transcriptional repressor Mad1 and mSin3A. J. Biol. Chem. 274(46): 32750-6.
Foley, K. P., et al. (1998). Targeted disruption of Mad1 inhibits cell cycle exit during granulocyte differentiation. EMBO J. 17: 774-785. 9451002
Grinberg, A. V., Hu, C. D. and Kerppola, T. K. (2004). Visualization of Myc/Max/Mad family dimers and the competition for dimerization in living cells. Mol. Cell. Biol. 24(10): 4294-308. 15121849
Hurlin, P., Queva, C. and Eisenman, R. N. (1997). Mnt, a novel Max-interacting protein is coexpressed with Myc in proliferating cells and mediates repression at Myc binding sites. Genes Dev. 11: 44-58. 9000049
Hurlin, P. J., et al. (1995). Regulation of Myc and Mad during epidermal differentiation and HPV-associated tumorigenesis. Oncogene 11: 2487-2501. 8545105
Hurlin, P. J., et al. (2003). Deletion of Mnt leads to disrupted cell cycle control and tumorigenesis. EMBO J. 22: 4584-4596. 12970171
Iritani, B. M., et al. (2002). Modulation of T-lymphocyte development, growth and cell size by the Myc antagonist and transcriptional repressor Mad1. EMBO J. 21: 4820-4830. 12234922
Loo, L. W., et al. (2005). The transcriptional repressor dMnt is a regulator of growth in Drosophila melanogaster. Mol. Cell. Biol. 25(16): 7078-91. 16055719
Meroni, G., et al. (1997). Rox, a novel bHLHZip protein expressed in quiescent cells that heterodimerizes with Max, binds a non-canonical E box and acts as a transcriptional repressor. EMBO J. 16: 2892-2906. 9184233
Montagne, M., Naud, J. F., McDuff, F. O. and Lavigne, P. (2005). Toward the elucidation of the structural determinants responsible for the molecular recognition between Mad1 and Max. Biochemistry 44(38): 12860-9. 16171401
Nair, S. K. and Burley, S. K. (2003). X-Ray structures of Myc-Max and Mad-Max recognizing DNA: Molecular bases of regulation by proto-oncogenic transcription factors. Cell 112: 193-205. 12553908
Nilsson, J. A., et al. (2004). Mnt loss triggers Myc transcription targets, proliferation, apoptosis, and transformation. Mol. Cell. Biol. 24: 1560-1569. 14749372
Orian, A., et al. (2003). Genomic binding by the Drosophila Myc, Max, Mad/Mnt transcription factor network. Genes Dev. 17(9): 1101-14. 12695332
Pierce, S. B., et al. (2004). dMyc is required for larval growth and endoreplication in Drosophila. Development 131: 2317-2327. 15128666
Pierce, S. B., Yost, C., Anderson, S. A., Flynn, E. M., Delrow, J. and Eisenman, R. N. (2008). Drosophila growth and development in the absence of dMyc and dMnt. Dev. Biol. 315(2): 303-16. PubMed Citation: 18241851
Popov, N., Wahlstrom, T., Hurlin, P. J. and Henriksson, M. (2005). Mnt transcriptional repressor is functionally regulated during cell cycle progression. Oncogene 24(56): 8326-37. 16103876
Queva, C., Hurlin, P. J., Foley, K. P. and Eisenman, R. N. (1998). Sequential expression of the MAD family of transcriptional repressors during differentiation. Oncogene 16: 967-977. 9519870
Queva, C., McArthur, G. A., Iritani, B. M. and Eisenman, R. N. (2001). Targeted deletion of the S-phase-specific Myc antagonist Mad3 sensitizes neural and lymphoid cells to radiation-induced apoptosis. Mol. Cell. Biol. 21: 703-712. 11154258
Rottmann, S., et al. (2005). Mad1 function in cell proliferation and transcriptional repression is antagonized by cyclin E/CDK2. J. Biol. Chem. 280(16): 15489-92. 15722557
Schreiber-Agus, N., et al. (1998). Role of Mxi1 in ageing organ systems and the regulation of normal and neoplastic growth. Nature 393: 483-487. 9624006
Siegel, P. M., Shu, W. and Massague, J. (2003). Mad upregulation and Id2 repression accompany transforming growth factor (TGF)-beta-mediated epithelial cell growth suppression. J. Biol. Chem. 278(37): 35444-50. 12824180
Toyo-oka, K., et al. (2004). Loss of the Max-interacting protein Mnt in mice results in decreased viability, defective embryonic growth and craniofacial defects: relevance to Miller-Dieker syndrome. Hum. Mol. Genet. 13: 1057-1067. 15028671
Walker, W., et al. (2005). Mnt-Max to Myc-Max complex switching regulates cell cycle entry. J. Cell Biol. 169(3): 405-13. 15866886
Yuan, J., et al. (1998). The C. elegans MDL-1 and MXL-1 proteins can functionally substitute for vertebrate MAD and MAX. Oncogene 17: 1109-1118. 9764821
date revised: 25 October 2014
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