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Julie Secombe,
John T. Little,
Leni-Sue Carlos,
Cynthia Yost,
Pei-Feng Cheng,
Erin M. Flynn,
Bruce A. Edgar, and
Robert N. Eisenman*
Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington
Received 4 May 2005/ Returned for modification 22 May 2005/ Accepted 27 May 2005
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ABSTRACT |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Much research has focused on mammalian Mad family proteins and Mnt, since they appear to antagonize Myc activity and could function, at least in principle, as tumor suppressors. The mammalian Mad family of transcriptional repressors is encoded by four paralogs: mad1, mxi1, mad3, and mad4 (reviewed in reference 39). The other characterized Max-binding repressor, Mnt (also known as Rox), possesses the two conserved domains common to all Mad family members: the N-terminal Sin-interacting domain (SID), which interacts with the Sin3 corepressor, and the bHLHZ domain required for heterodimerization with Max (26, 42). However, Mnt is considerably larger than any of the Mad family proteins and contains other regions, including proline- and proline/histidine-rich sequences, that are unique to Mnt.
Attempts to understand the physiological roles of mammalian Mad and Mnt proteins have entailed both overexpression and targeted gene deletion studies. In general, this work has provided support for the notion that Mad and Mnt antagonize Myc function and promote cell cycle exit (12, 13, 20, 28, 33, 34, 55, 59, 74).
Surprisingly, given the capacity for overexpressed mad to inhibit proliferation, targeted deletions of mad family genes in mice did not result in dramatic phenotypes relating to differentiation and development. Mice homozygous for a mad1 null mutation displayed no detectable differences in viability, fertility, size, behavior, or incidence of neoplasia compared to controls. However, their granulocyte progenitor cells, when cultured in vitro, exhibited a delay in differentiation due to an inhibition of cell cycle exit (18). mxi1 knockout mice showed generalized hyperplasia in certain tissues and an increased incidence of carcinogen-induced tumors in older animals (62). The only detectable phenotype in mad3 homozygous null mice was an increased sensitivity to gamma irradiation in neural progenitor cells and thymocytes (57). These subtle phenotypes may be the result of redundancy with other Mad family members or with other cell cycle regulatory proteins. In fact, an apparently compensatory increase in Mxi1 and Mad3 expression was observed in the thymi of mad1 knockout mice (18). Therefore, as suggested for Myc family gene deletions, functional redundancy may obscure the developmental roles of mad genes in targeted deletion studies (15, 67). This may be less true for Mnt, whose targeted deletion in mice results in craniofacial abnormalities and perinatal lethality (71). Conditional loss of mnt in murine breast epithelium leads to adenocarcinomas (29). Furthermore, loss of Mnt function in fibroblasts results in enhanced proliferation and upregulation of Myc target genes (29, 46). Thus, mammalian Mnt is a tumor suppressor that presumably functions to antagonize Myc activity. However, because these studies of Mnt have been carried out in settings where wild-type Mad1 to Mad4 proteins are expressed, it is difficult to sort out the extent and consequences of the overlapping functions of these proteins.
The identification of Myc and Max homologs in Drosophila melanogaster has greatly facilitated genetic analysis of candidate functional pathways and targets. The D. melanogaster homologs of Myc and Max (dMyc and dMax) heterodimerize, bind E-box sequences, activate transcription, and in the case of dMyc, recapitulate mammalian Myc functions (19, 48, 63, 72). Furthermore, both loss-of-function and gain-of-function studies with dMyc have demonstrated it to be a positive regulator of cell growth (i.e., cell mass) (31). Here we report the identification and characterization of the third member of the Max network in D. melanogaster, dMnt, the sole homolog of mammalian Mad and Mnt proteins. We show dMnt to be a negative regulator of cell growth that also plays a role in regulating D. melanogaster body size and life span.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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SID and dMnt ZIP splice forms were isolated from an ovary library (68).
Glutathione S-transferase (GST) pull-down assays.
The cDNAs for dMax, dMyc, dMnt, dMnt SID, and dMnt ZIP were cloned into pCite (Novagen, Inc.) and the proteins were expressed in a rabbit reticulocyte lysate (Promega) containing [35S]methionine. dMnt bHLHZ (amino acids [aa] 149 to 386) and dMnt bHLH (aa 149 to 335) were cloned into the pGEX-5X-1 vector (Pharmacia), and fusion proteins were expressed in Escherichia coli. Pull-down assays were performed as previously described (28).
Antibody generation. Monoclonal antibodies (P5D6) to a GST fusion protein containing aa 149 to 248 of dMnt were generated by the Fred Hutchinson Cancer Research Center Antibody Development Facility (Seattle, WA). dMnt-GST fusion proteins were expressed in E. coli and purified on glutathione Sepharose beads (Pharmacia). Antibody specificity was determined by detection of in vitro-translated dMnt isoforms, but not unrelated proteins such as dMyc, in immunoblot assays and immunostaining of dMnt mutant tissues (see Fig. 3B).
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Fly stocks.
All flies were grown at 25°C unless otherwise indicated. w1118, en-GAL4/CyO;UAS-GFP, ey-GAL4/CyO, yw,hs-FLP122, w;;Actin5C>CD2>GAL4, UAS-GFP (45, 52), yw;;[2-3]Sb/TM6B, and EP(X)1559 (58) (kindly provided by Gerald Rubin's lab), dmnt1 (or dmnt2)/Binsinscy flies were used.
UAS-mediated ectopic expression and FACS analysis.
pUAS-dMnt, pUAS-dMnt SID, and pUAS-dMnt ZIP were constructed by cloning the corresponding cDNAs into the pUAST vector (9). Standard P-element transformation was used to introduce the constructs into the Drosophila germ line (60). The Flp/Gal4 method (44, 52, 69) was used to generate random clones overexpressing upstream activation sequence (UAS)-regulated transgenes. Flp/Gal4 clones were induced by heat shock for 1 h at 37°C (in an air incubator) 48 h after egg deposition. Clones for fluorescence-activated cell sorting (FACS) analysis or confocal microscopy were analyzed 120 h after egg deposition. FACS analysis was performed as previously described (44). For confocal analysis, wing disks from third-instar larvae were fixed in 4% paraformaldehyde in phosphate-buffered saline for 45 min, washed, and stained with propidium iodide for nuclear staining. A Leica TCS SP confocal microscope was used.
P-element excision screen.
dmnt mutants were generated in an imprecise P-element excision screen using a stock EP(X)1559 (58) containing a P-element insertion between the second and third exons of dMnt. Briefly, homozygous EP(X)1559 females were crossed with Binsinscy;;[2-3]Sb/+ males, then EP(X)1559/Binsinscy;;[2-3]Sb/+ females were crossed with Binsinscy males or EP(X)1559;;[2-3]Sb/+ males were crossed with Df/Binsinscy females. Viable white-eyed male and female offspring were analyzed by PCR analysis using primer pairs consisting of a P-element-specific primer and a dMnt genomic primer flanking the left or right of the P-element insertion site. The lines which appeared to have a P-element excision event were further analyzed by PCR analysis using primers specific to individual exons and a primer set that amplifies the closest exon of the neighboring gene, ralA. The regions of deletion for the two mutant lines were also mapped by Southern blotting (data not shown).
RT-PCR analysis.
Reverse transcription-PCR (RT-PCR) analysis was performed to detect the presence of dMnt, dMnt SID, and dMnt ZIP mRNA. Total RNA from adult males was isolated with TRIzol (Invitrogen) according to the manufacturer's instructions. Sensiscript reverse transcriptase (QIAGEN) was used in the RT reaction according to the manufacturer. dMnt and dMnt ZIP cDNAs (detection was based on size differences of the PCR products) were then PCR amplified (one cycle of 94°C for 3 min; 35 cycles of 94°C for 1 min, 56°C for 1 min, and 72°C for 1 min 30 s; one cycle of 72°C 7 min) with a 5' primer (5'-GGTGCTCCTCGAGGCGGCTC-3') and a 3' primer (5'-CGGGTGCCCGCGCCATTAC-3'), primers 1 and 3, respectively (see Fig. 5A). The dMnt SID cDNA was PCR amplified using the same conditions mentioned above with a 5' primer (5'-TCGGAGGACGATAATTCCTC-3') and the same 3' primer mentioned above, primers 2 and 3, respectively (see Fig. 5A). Rp49 was used as the loading control. PCR products were run on a 1.5% agarose gel containing ethidium bromide.
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Longevity assays. dmnt1, dmnt2, and P-element precise excision allele (wild-type control) virgins were collected and allowed to age in the absence of males at a density of 20 flies per vial. Flies were transferred to fresh food every 3 days, and scores for dead flies were determined at this time. Longevity studies were repeated three times with independent sets of parents. The average percent survivorship from the three independent experiments was then calculated for each collection point.
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RESULTS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ZIP contains SID and the basic helix-loop-helix domains but lacks the zipper domain. Lastly, dMnt SID has a novel amino terminus lacking SID but retains the bHLHZ domain. Sequence comparisons indicate that the predicted protein sequence of SID for dMnt is 56% identical to the SID domains of mouse Mad1 and Mnt, and the bHLHZ domain is 55% and 33% identical to the bHLHZ domains of mouse Mnt and Mad1, respectively. RT-PCR analysis indicates that all three forms are expressed in adult male flies (Fig. 1A).
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250 aa). Also, the overall organization of dMnt is more similar to that of mammalian Mnt. The SID and bHLHZ domains of Mnt proteins are approximately 200 aa apart, whereas the Mad proteins have an average of 30 aa that separate these two domains. To determine relatedness among mnt and mad genes in various organisms, we constructed a phylogenetic tree based on sequence homology within the bHLHZ domain (81 aa) (Fig. 1C). This domain contained the largest block of amino acids common to all Max network proteins analyzed. The tree indicates that the dMnt bHLHZ and the predicted bHLHZ regions for the Anopheles gambiae mosquito and T. rubripes are more closely related to mammalian Mnt than to other members of the Mad family of proteins. These analyses indicate that the gene products we identified are overall more homologous to Mnt. A search of the D. melanogaster genome sequence has failed to identify any other Mad-related protein. Thus, dMnt and its splice forms may be considered to be the sole D. melanogaster homolog of both Mnt and Mad.
dMnt is biochemically and functionally similar to vertebrate Mad and Mnt proteins.
Vertebrate Myc, Max, and Mad/Mnt are all members of the bHLHZ class of transcription factors in which the bHLHZ domains mediate highly specific heterodimerization of Myc and Mad with Max as well as sequence-specific DNA binding (3, 6). To determine whether dMnt heterodimerizes with dMax, we carried out in vitro binding assays. Chimeric proteins in which GST was fused to the dMnt bHLHZ domain or the dMnt ZIP bHLH domain were bacterially expressed and purified. GST alone or GST fusion proteins were incubated with in vitro-transcribed and in vitro-translated [35S]methionine-labeled dMax, dMyc, or each of the three dMnt splice forms (Fig. 2A). GST alone did not specifically associate with any of the in vitro-translated proteins. However, the GST-dMnt bHLHZ fusion protein interacted specifically with dMax but did not interact with dMyc or any of the dMnt splice forms. The GST-dMnt ZIP bHLH fusion protein failed to interact with dMax, dMyc, or any of the dMnt splice forms. These results demonstrate that dMnt is able to heterodimerize with dMax through the bHLHZ domain but is unable to homodimerize or interact with dMyc. In addition, the leucine zipper domain is required for dMnt heterodimerization with dMax.
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SID form. No interaction of the dMnt splice forms with GST alone was observed.
Vertebrate Myc-Max and Mad-Max heterodimers have been demonstrated to bind specifically to the consensus E-box sequence CACGTG (4, 6, 61), and D. melanogaster Myc-Max dimers bind the same E-box sequence (19, 63). We tested in vitro-translated dMnt, dMnt ZIP, or dMnt SID together with dMax in electrophoretic mobility shift assays and determined that dMnt-dMax and dMnt SID-dMax heterodimers bind specifically to a labeled CACGTG oligonucleotide (see Fig. S1 in the supplemental material). As expected, given that dMnt ZIP is unable to heterodimerize with dMax (Fig. 2A), no specific binding to the labeled oligonucleotide was observed in the sample containing dMnt ZIP and dMax.
The DNA binding activity of these proteins is related to their transcriptional function. Synthetic reporter genes, as well as cellular target genes, are transcriptionally activated by both vertebrate and D. melanogaster Myc-Max heterodimers and are transcriptionally repressed by mammalian Mad-Max and Mnt-Max. Activation and repression by these groups of heterodimers are dependent on the presence of the E-box sequence (for reviews, see references 21, 37, 49, and 77). We found that expression of dMnt and dMax repressed transcription of a reporter construct containing an E-box (CACGTG) twofold compared to empty vector control (see Fig. S2 in the supplemental material). In contrast, the dMnt SID and dMnt ZIP splice forms failed to influence transcriptional activity relative to empty vector. Taken together, these results indicate that dMnt, like vertebrate Mad and Mnt proteins, dimerizes with dMax, binds E-box sequences, and represses transcription of an E-box-containing promoter in a SID-dependent manner.
Expression of dMnt during development.
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 (26, 27, 42, 56). To examine the expression pattern of dMnt, we raised a monoclonal antibody, P5D6, to the region common to all three dMnt splice forms (see Materials and Methods for details). To verify the specificity of the dMnt antibody, the larvae of a dmnt null mutant (dmnt1) and a mutant that retains the dMnt SID transcript (dmnt2) were stained with P5D6 (see below for derivation and analysis of mutants). As seen in Fig. 3Ba to c, nuclear staining is 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.
Although dmnt mRNA is maternally deposited and is present until cellularization (data not shown), 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 (Fig. 3A). 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 (47) (Fig. 3Aa to f). 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 (data not shown).
dMnt is also expressed in both differentiating and actively dividing cells during the third larval instar of D. melanogaster development. During eye imaginal disk 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 (Fig. 3Ag to i). In addition, dMnt is expressed in actively dividing peripodial membrane cells of the wing and eye-antennal imaginal disks in third-instar larvae (K. McClure, personal communication; also data not shown). dMnt is also present in endocycling salivary gland (51) and fat body cells (Fig. 3B; data not shown). dMnt is therefore expressed in a temporally and spatially dynamic pattern in both actively replicating mitotic and endoreplicating tissue and differentiating cells.
Ectopic expression of dMnt inhibits cellular growth and proliferation.
To determine whether expression of dMnt splice variants can influence cell behavior in D. melanogaster, we used the UAS/GAL4 expression system (9) 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, we observed a marked reduction in the size of the respective adult structure (see Fig. S3 in the supplemental material). 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. We did not observe any obvious adult phenotypes when dMax, dMnt SID, or dMnt ZIP was expressed using these drivers (see Fig. S3A in the supplemental material; also data not shown).
We measured proliferation 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 (see Materials and Methods for details). We compared the sizes of clones ectopically expressing dMnt with GFP to clones expressing GFP alone in both the imaginal wing disk (Fig. 4A) and the fat body (51). 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 (data not shown). 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 SID or dMnt ZIP splice form in the wing disks of random clones appeared to have an intermediate effect on proliferation relative to dMnt (Fig. 4A). 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 SID or dMnt ZIP 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, we dissociated wing disk cells and examined them using flow cytometry (Fig. 4B). We compared GFP-positive cells, which express the transgene, to GFP-negative cells, which represent the wild-type cell profile. When dMnt was expressed, we observed an increase in the population of cells in the G0/G1 phase of the cell cycle and a decrease in the population of cells in S and G2/M. When we compared the relative sizes of the cells ectopically expressing dMnt and the nonexpressing population by forward scatter (Fig. 4B), we 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 we gated and compared individual cell cycle phases, we found that the size difference was observed in all phases (data not shown). Expression of the dMnt SID or dMnt ZIP 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.
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ZIP splice forms but does not extend to the exon containing the translational start codon of the dMnt SID splice form. RT-PCR analysis demonstrated that dmnt1 lacks mRNA for the dMnt, dMnt ZIP, and dMnt SID splice forms (Fig. 5B), while dmnt2 lacks detectable mRNA for the dMnt and dMnt ZIP splice forms but retains the mRNA for dMnt SID (Fig. 5A and B). In addition, we performed RT-PCR analyses on the neighboring gene, ralA, to determine whether expression of this gene was affected in the dmnt mutant alleles. No difference in ralA mRNA expression was detectable for either of the dmnt mutant alleles compared to the wild-type control, indicating that our deletion has not extended beyond the dmnt locus (data not shown). 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, we tested hatching rates on the two homozygous mutant lines and compared them to a wild-type (precise excision) control and the parental EP(X)1559 allele. We observed no significant differences 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. We also analyzed larval development and did not detect any delays in larval molting, pupation, or eclosion. These results suggest that dMnt is not essential for the normal developmental program.
dMnt mutants have a growth phenotype and are short-lived. Because ectopic expression of dMnt inhibits cellular proliferation and growth, we determined whether loss of dMnt function influences weight and cell size in adult flies. We compared the average individual weights of dmnt mutant adult males and wild-type control males and observed that both dmnt1 and dmnt2 mutant males are 20% and 12% heavier than wild-type controls (Fig. 6A). 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 (Fig. 6B), indicative of increased cell size. These results demonstrate that dMnt proteins normally play a role in limiting cellular growth.
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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dmnt is the sole homolog of mnt and mad genes in D. melanogaster. Sequence comparison and phylogenetic tree construction based on the bHLHZ domain indicate that the protein product of this gene is most similar to vertebrate Mnt (Fig. 1). In addition to the bHLHZ domain, dMnt possesses the SID domain characteristic of mammalian Mnt and the four Mad family proteins. We show that both these domains are functional—the dMnt bHLHZ mediates association with Max and DNA binding, while the dMnt SID interacts directly with the Sin3 corepressor (Fig. 2). On the other hand, dMnt lacks the proline-rich and proline/histidine-rich domains characteristic of mammalian Mnt. Because at this time there is no data indicating that these domains are required for Mnt function, their absence in D. melanogaster Mnt cannot be taken as evidence of functional divergence. We surmise that dMnt represents a progenitor of the vertebrate Mad family and Mnt proteins. This notion is consistent with the developmental patterns of expression we have observed for dMnt (Fig. 3A) which appear similar to those for mammalian Mad proteins (expressed during cell cycle exit) as well as Mnt (expressed in proliferating cells). The numerous vertebrate paralogs are likely a reflection of the more complex and diverse regulation imposed on these genes during vertebrate development.
It is probably not a coincidence that the alternatively spliced forms of dMnt that we have observed involve functionally significant alterations in the SID and bHLHZ domains (Fig. 1A, 2, and 5A). In contrast to dMnt, which functions as a transcriptional repressor, neither the dMnt Zip protein nor the dMnt SID protein displayed positive or negative transcriptional activity on a synthetic reporter gene containing E-box binding sites (see Fig. S2 in the supplemental material). These forms could potentially act as dMnt dominant interfering proteins. However, both the dMnt Zip and dMnt SID forms contain unique sequences introduced by alternative splicing and not found in dMnt (Fig. 1A and 5A). We speculate that it is the polypeptide regions that are unique to the Zip and SID isoforms that confer distinct E-box- and Sin3-independent functions on these proteins. For example, overexpression of dMnt, but not dMnt Zip or dMnt SID, had strong effects on wing and eye formation (see Fig. S3 in the supplemental material). However, while all three isoforms reduced the size of random clones in the wing disk, dMnt had the most dramatic effects and also influenced cell size and cell cycle phasing (see below). By contrast, the Zip and SID isoforms had no detectable effects on cell size or cell cycle (Fig. 4). It will be of interest to further explore potential protein interactions mediated by the alternatively spliced sequences in the Zip and SID isoforms and to delineate in greater detail their expression patterns and biological roles.
Our imprecise excision screen of an existing allele containing a P-element insertion in the dmnt gene generated dmnt1, a null allele, and dmnt2, an allele that expresses only the dMnt SID form (Fig. 5). To our surprise, both mutant alleles are viable with no obvious pattern of defects or effects on male or female fertility. We did, however, observe that dmnt1 and dmnt2 mutants were 20% and 12% heavier than their wild-type controls, respectively. Furthermore, dmnt1 and dmnt2 alleles had an average of 18% and 10% fewer cells per unit area, respectively, than the wild-type controls, indicative of increased cell size (Fig. 6). The intermediate effect observed with dmnt2, which expresses only dMnt SID, again suggests that this alternatively spliced form functions in a pathway related to the dMnt pathway. The augmentation of cell and organismal size in the dMnt mutant flies is generally consistent with dMnt acting as an antagonist of dMyc. Flies with dMyc loss-of-function alleles are smaller and have increased trichome density (19, 31, 63), while dMyc overexpression generates larger cells and flies (16, 31). Mice with single deletions of murine mad family genes mad1, mxi1, and mad3 were viable, although hyperplasia, sensitivity to radiation damage, and a delay in differentiation were observed. The relatively mild phenotypes were generally ascribed to redundancy between mad family members. Our data derived from the dmnt null mutant suggest that mad genes are not essential for survival. However, a strong synthetic effect in mice bearing both mad1 and p27KIP1 null alleles indicates that mad genes may be redundant with other genes that act to limit cell growth and proliferation (38). Interestingly, we did not observe any obvious genetic interaction or developmental or adult phenotypes in mutant flies which were hemizygous for dmnt1 and heterozygous for dacapo, the sole Drosophila p21CIP1/p27KIP1 ortholog (data not shown).
Our finding that both the dmnt loss-of-function alleles resulted in a 24% decrease in life span and a diminished life expectancy was surprising (Fig. 7). Although it is possible that dmnt loss of function might have a nonspecific toxic effect, we believe this is unlikely because we fail to observe sensitivity to starvation or bacterial infection in the dmnt1 mutant flies (Fig. 7; see Fig. S4 in the supplemental material). Nonetheless, we have been unable to demonstrate increased longevity using a dMnt transgene and a motor neuron-specific GAL4 driver used previously to overexpress superoxide dismutase and increase life span (50). However, a negative result with the transgene may well be due to a failure to target expression to the appropriate cell type or developmental time. While this work was in progress, the mad/mnt homolog, mdl-1 in Caenorhabditis elegans (76) was identified as a positively regulated target of the DAF-16 transcription factor (43), which is known to influence the rate of aging during early adulthood. In addition, mdl-1 small interfering RNA resulted in a 10% decrease in the life span of C. elegans. Perhaps D. melanogaster Mad/Mnt homologs function as part of a conserved pathway involved in longevity. Clearly, further studies will be required to define dMnt's possible role in aging. Interestingly, mammalian c-Myc has been recently shown to have the ability to attenuate cellular senescence and promote transformation through its direct regulation of the Werner syndrome gene, also known to function in aging (22).
Longevity has been closely linked to cell growth pathways in many organisms including C. elegans, D. melanogaster, and vertebrates (7, 14, 25, 35, 36, 43, 75). We have shown, through dMnt overexpression and loss of function, that this transcriptional repressor has a role in regulating growth. Expression array studies in murine thymocytes containing a Mad1 transgene indicate that Mad downregulates the expression of numerous genes involved in ribosome biogenesis, translation, and metabolism (30). The majority of these repressed genes are activated by Myc. Furthermore, genomic binding studies of dMnt in D. melanogaster indicate direct association of dMnt and dMyc proteins with many of the same growth-related genes (48). These findings, taken together with the data reported here, further extend the notion that Myc and Mad/Mnt proteins are antagonists in terms of their transcriptional activities and effects on cell and organismal growth and longevity. It remains to be determined whether dmnt is regulated by Drosophila FOXO, the only fly homolog of FOXO/DAF-16 (32, 54). If so, dMnt may act to suppress a specific subset of growth-related genes in the absence of insulin and perhaps other growth factors.
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ACKNOWLEDGMENTS |
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This work was supported by grants from NIH/NCI (RO1 CA15738 to R.N.E., R01 GM51186 to B.A.E., and F32 GM18246-02 to L.W.M.L.) and the Lady Tata Memorial Trust to J.S. R.N.E. is an American Cancer Society Research Professor.
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FOOTNOTES |
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Supplemental material for this article may be found at http://mcb.asm.org/.
Both authors contributed equally to this work.
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REFERENCES |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2. Armstrong, J. A., and B. M. Emerson. 1998. Transcription of chromatin: these are complex times. Curr. Opin. Genet. Dev. 8:165-172.[CrossRef][Medline]
3. Ayer, D. E., L. Kretzner, and R. N. Eisenman. 1993. Mad: a heterodimeric partner for Max that antagonizes Myc transcriptional activity. Cell 72:211-222.[CrossRef][Medline]
4. Ayer, D. E., Q. A. Lawrence, and R. N. Eisenman. 1995. Mad-Max transcriptional repression is mediated by ternary complex formation with mammalian homologs of yeast repressor Sin3. Cell 80:767-776.[CrossRef][Medline]
5. Baudino, T. A., and J. L. Cleveland. 2001. The Max network gone Mad. Mol. Cell. Biol. 21:691-702.
6. Blackwood, E. M., and R. N. Eisenman. 1991. Max: a helix-loop-helix zipper protein that forms a sequence-specific DNA binding complex with Myc. Science 251:1211-1217.
7. Bohni, R., J. Riesgo-Escovar, S. Oldham, W. Brogiolo, H. Stocker, B. F. Andruss, K. Beckingham, and E. Hafen. 1999. Autonomous control of cell and organ size by CHICO, a Drosophila homolog of vertebrate IRS1-4. Cell 97:865-875.[CrossRef][Medline]
8. Bourbon, H. M., G. Gonzy-Treboul, F. Peronnet, M. F. Alin, C. Ardourel, C. Benassayag, D. Cribbs, J. Deutsch, P. Ferrer, M. Haenlin, J. A. Lepesant, S. Noselli, and A. Vincent. 2002. A P-insertion screen identifying novel X-linked essential genes in Drosophila. Mech. Dev. 110:71-83.[CrossRef][Medline]
9. Brand, A., and N. Perrimon. 1993. Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development 118:401-415.[Abstract]
10. Brown, N. H., and F. C. Kafatos. 1988. Functional cDNA libraries from Drosophila embryos. J. Mol. Biol. 203:425-437.[CrossRef][Medline]
11. Brubaker, K., S. M. Cowley, K. Huang, L. Loo, G. S. Yochum, D. E. Ayer, R. N. Eisenman, and I. Radhakrishnan. 2000. Solution structure of the interacting domains of the Mad-Sin3 complex: implications for recruitment of a chromatin-modifying complex. Cell 103:655-665.[CrossRef][Medline]
12. Cerni, C., K. Bousset, C. Seelos, H. Burkhardt, M. Henriksson, and B. Luscher. 1995. Differential effects by Mad and Max on transformation by cellular and viral oncoproteins. Oncogene 11:587-596.[Medline]
13. Chen, J., T. Willingham, L. R. Margraf, N. Schreiber-Agus, R. A. DePinho, and P. D. Nisen. 1995. Effects of the MYC oncogene antagonist, MAD, on proliferation, cell cycling and the malignant phenotype of human brain tumor cells. Nat. Med. 1:638-643.[CrossRef][Medline]
14. Clancy, D. J., D. Gems, L. G. Harshman, S. Oldham, H. Stocker, E. Hafen, S. J. Leevers, and L. Partridge. 2001. Extension of life-span by loss of CHICO, a Drosophila insulin receptor substrate protein. Science 292:104-106.
15. Davis, A. C., M. Wims, G. D. Spotts, S. R. Hann, and A. Bradley. 1993. A null c-myc mutation causes lethality before 10.5 days of gestation in homozygous and reduced fertility in heterozygous female mice. Genes Dev. 7:671-682.
16. de la Cova, C., M. Abril, P. Bellosta, P. Gallant, and L. A. Johnston. 2004. Drosophila myc regulates organ size by inducing cell competition. Cell 117:107-116.[CrossRef][Medline]
17. Eisenman, R. N. 2001. Deconstructing myc. Genes Dev. 15:2023-2030.
18. Foley, K. P., G. A. McArthur, C. Quéva, P. J. Hurlin, P. Soriano, and R. N. Eisenman. 1998. Targeted disruption of Mad1 inhibits cell cycle exit during granulocyte differentiation. EMBO J. 17:774-785.[CrossRef][Medline]
19. Gallant, P., Y. Shiio, P. F. Cheng, S. Parkhurst, and R. N. Eisenman. 1996. Myc and Max homologs in Drosophila. Science 274:1523-1527.
20. Gehring, S., S. Rottmann, A. R. Menkel, J. Mertsching, A. Krippner-Heidenreich, and B. Luscher. 2000. Inhibition of proliferation and apoptosis by the transcriptional repressor mad1. Repression of fas-induced caspase-8 activation. J. Biol. Chem. 275:10413-10420.
21. Grandori, C., S. M. Cowley, L. P. James, and R. N. Eisenman. 2000. The MYC/MAX/MAD network and the transcriptional control of cell behavior. Annu. Rev. Cell Dev. Biol. 16:653-699.[CrossRef][Medline]
22. Grandori, C., K. J. Wu, P. Fernandez, C. Ngouenet, J. Grim, B. E. Clurman, M. J. Moser, J. Oshima, D. W. Russell, K. Swisshelm, S. Frank, B. Amati, R. Dalla-Favera, and R. J. Monnat, Jr. 2003. Werner syndrome protein limits MYC-induced cellular senescence. Genes Dev. 17:1569-1574.
23. Grunstein, M. 1997. Histone acetylation in chromatin structure and transcription. Nature 389:349-352.[CrossRef][Medline]
24. Hartwell, L. H., J. J. Hopfield, S. Leibler, and A. W. Murray. 1999. From molecular to modular cell biology. Nature 402(Suppl.):C47-C52.[CrossRef][Medline]
25. Hsu, A. L., C. T. Murphy, and C. Kenyon. 2003. Regulation of aging and age-related disease by DAF-16 and heat-shock factor. Science 300:1142-1145.
26. Hurlin, P., C. Queva, and R. N. Eisenman. 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.
27. Hurlin, P. J., K. P. Foley, D. E. Ayer, R. N. Eisenman, D. Hanahan, and J. M. Arbeit. 1995. Regulation of Myc and Mad during epidermal differentiation and HPV-associated tumorigenesis. Oncogene 11:2487-2501.[Medline]
28. Hurlin, P. J., C. Quéva, P. J. Koskinen, E. Steingrímsson, D. E. Ayer, N. G. Copeland, N. A. Jenkins, and R. N. Eisenman. 1995. Mad3 and Mad4: novel Max-interacting transcriptional repressors that suppress c-Myc-dependent transformation and are expressed during neural and epidermal differentiation. EMBO J. 14:5646-5659.[Medline]
29. Hurlin, P. J., Z. Q. Zhou, K. Toyo-Oka, S. Ota, W. L. Walker, S. Hirotsune, and A. Wynshaw-Boris. 2003. Deletion of Mnt leads to disrupted cell cycle control and tumorigenesis. EMBO J. 22:4584-4596.[CrossRef][Medline]
30. Iritani, B. M., J. Delrow, C. Grandori, I. Gomez, M. Klacking, L. S. Carlos, and R. N. Eisenman. 2002. Modulation of T lymphocyte development, growth, and cell size by the Myc-antagonist Mad1 transcriptional repressor. EMBO J. 21:4820-4830.[CrossRef][Medline]
31. Johnston, L. A., D. A. Prober, B. A. Edgar, R. N. Eisenman, and P. Gallant. 1999. Drosophila myc regulates growth during development. Cell 98:779-790.[CrossRef][Medline]
32. Junger, M. A., F. Rintelen, H. Stocker, J. D. Wasserman, M. Vegh, T. Radimerski, M. E. Greenberg, and E. Hafen. 7 August 2003, posting date. The Drosophila Forkhead transcription factor FOXO mediates the reduction in cell number associated with reduced insulin signaling. J. Biol. 2:20. [Online.] http://jbiol.com/content/2/3/20.
33. Koskinen, P. J., D. E. Ayer, and R. N. Eisenman. 1995. Repression of Myc-Ras co-transformation by Mad is mediated by multiple protein-protein interactions. Cell Growth Differ. 6:623-629.[Abstract]
34. Lahoz, E. G., L. Xu, N. Schreiber-Agus, and R. A. DePinho. 1994. Suppression of Myc, but not E1a, transformation activity by Max-associated proteins, Mad and Mxi1. Proc. Natl. Acad. Sci. USA 91:5503-5507.
35. Lee, S. S., S. Kennedy, A. C. Tolonen, and G. Ruvkun. 2003. DAF-16 target genes that control C. elegans life-span and metabolism. Science 300:644-647.
36. Leevers, S. J., D. Weinkove, L. K. MacDougall, E. Hafen, and M. D. Waterfield. 1996. The Drosophila phosphoinositide 3-kinase Dp110 promotes cell growth. EMBO J. 15:6584-6594.[Medline]
37. Luscher, B. 2001. Function and regulation of the transcription factors of the Myc/Max/Mad network. Gene 277:1-14.[CrossRef][Medline]
38. McArthur, G. A., K. P. Foley, M. L. Fero, C. R. Walkley, A. J. Deans, J. M. Roberts, and R. N. Eisenman. 2002. MAD1 and p27KIP1 cooperate to promote terminal differentiation of granulocytes and to inhibit Myc expression and cyclin E-CDK2 activity. Mol. Cell. Biol. 22:3014-3023.
39. McArthur, G. A., C. D. Laherty, C. Quéva, P. J. Hurlin, L. Loo, L. James, C. Grandori, P. Gallant, Y. Shiio, W. C. Hokanson, A. C. Bush, P. F. Cheng, Q. Lawrence, B. Pulverer, P. J. Koskinen, K. P. Foley, D. E. Ayer, and R. N. Eisenman. 1998. The Mad protein family links transcriptional repression to cell differentiation. Cold Spring Harbor Symp. Quant. Biol. 63:423-433.[CrossRef][Medline]
40. McMahon, S. B., H. A. Van Buskirk, K. A. Dugan, T. D. Copeland, and M. D. Cole. 1998. The novel ATM-related protein TRRAP is an essential cofactor for the c-Myc and E2F oncoproteins. Cell 94:363-374.[CrossRef][Medline]
41. McMahon, S. B., M. A. Wood, and M. D. Cole. 2000. The essential cofactor TRRAP recruits the histone acetyltransferase hGCN5 to c-Myc. Mol. Cell. Biol. 20:556-562.
42. Meroni, G., A. Reymond, M. Alcalay, G. Borsani, A. Tanigami, R. Tonlorenzi, C. L. Nigro, S. Messali, M. Zollo, D. H. Ledbetter, R. Brent, A. Ballabio, and R. Carrozzo. 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.[CrossRef][Medline]
43. Murphy, C. T., S. A. McCarroll, C. I. Bargmann, A. Fraser, R. S. Kamath, J. Ahringer, H. Li, and C. Kenyon. 2003. Genes that act downstream of DAF-16 to influence the lifespan of Caenorhabditis elegans. Nature 424:277-283.[CrossRef][Medline]
44. Neufeld, T. P., A. F. A. de la Cruz, L. A. Johnston, and B. A. Edgar. 1998. Coordination of growth and cell division in the Drosophila wing. Cell 93:1183-1193.[CrossRef][Medline]
45. Neufeld, T. P., A. Tang, and G. M. Rubin. 1998. A genetic screen to identify components of the sina signaling pathway in Drosophila eye development. Genetics 148:277-286.
46. Nilsson, J. A., K. H. Maclean, U. B. Keller, H. Pendeville, T. A. Baudino, and J. L. Cleveland. 2004. Mnt loss triggers Myc transcription targets, proliferation, apoptosis, and transformation. Mol. Cell. Biol. 24:1560-1569.
47. O'Neill, E. M., I. Rebay, R. Tjian, and G. M. Rubin. 1994. The activities of two Ets-related transcription factors required for Drosophila eye development are modulated by the Ras/MAPK pathway. Cell 78:137-147.[CrossRef][Medline]
48. Orian, A., B. van Steensel, J. Delrow, H. J. Bussemaker, L. Li, T. Sawado, E. Williams, L. M. Loo, S. M. Cowley, C. Yost, S. Pierce, B. A. Edgar, S. M. Parkhurst, and R. N. Eisenman. 2003. Genomic binding by the Drosophila Myc, Max, Mad/Mnt transcription factor network. Genes Dev. 17:1101-1114.
49. Oster, S. K., C. S. Ho, E. L. Soucie, and L. Z. Penn. 2002. The myc oncogene: MarvelouslY Complex. Adv. Cancer Res. 84:81-154.[Medline]
50. Parkes, T. L., A. J. Elia, D. Dickinson, A. J. Hilliker, J. P. Phillips, and G. L. Boulianne. 1998. Extension of Drosophila lifespan by overexpression of human SOD1 in motorneurons. Nat. Genet. 19:171-174.[CrossRef][Medline]
51. Pierce, S. B., C. Yost, J. S. Britton, L. W. Loo, E. M. Flynn, B. A. Edgar, and R. N. Eisenman. 2004. dMyc is required for larval growth and endoreplication in Drosophila. Development 131:2317-2327.
52. Pignoni, F., and S. L. Zipursky. 1997. Induction of Drosophila eye development by Decapentaplegic. Development 124:271-278.[Abstract]
53. Poortinga, G., M. Watanabe, and S. M. Parkhurst. 1998. Drosophila CtBP: a Hairy-interacting protein required for embryonic segmentation and hairy-mediated transcriptional repression. EMBO J. 17:2067-2078.[CrossRef][Medline]
54. Puig, O., M. T. Marr, M. L. Ruhf, and R. Tjian. 2003. Control of cell number by Drosophila FOXO: downstream and feedback regulation of the insulin receptor pathway. Genes Dev. 17:2006-2020.
55. Pulverer, B., A. Sommer, G. A. McArthur, R. N. Eisenman, and B. Lüscher. 2000. Analysis of Myc/Max/Mad network members in adipogenesis: inhibition of the proliferative burst and differentiation by ectopically expressed Mad1. J. Cell. Physiol. 183:399-410.[CrossRef][Medline]
56. Queva, C., P. J. Hurlin, K. P. Foley, and R. N. Eisenman. 1998. Sequential expression of the MAD family of transcriptional repressors during differentiation. Oncogene 16:967-977.[CrossRef][Medline]
57. Queva, C., G. A. McArthur, B. M. Iritani, and R. N. Eisenman. 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.
58. Rorth, P. 1996. A modular misexpression screen in Drosophila detecting tissue-specific phenotypes. Proc. Natl. Acad. Sci. USA 93:12418-12422.
59. Roussel, M. F., R. A. Ashmun, C. J. Sherr, R. N. Eisenman, and D. E. Ayer. 1996. Inhibition of cell proliferation by the Mad1 transcriptional repressor. Mol. Cell. Biol. 16:2796-2801.[Abstract]
60. Rubin, G. M., and A. C. Spradling. 1982. Genetic transformation of Drosophila with transposable element vectors. Science 218:348-353.
61. Schreiber-Agus, N., L. Chin, K. Chen, R. Torres, G. Rao, P. Guida, A. I. Skoultchi, and R. A. DePinho. 1995. An amino-terminal domain of Mxi1 mediates anti-Myc oncogenic activity and interacts with a homolog of the yeast repressor SIN3. Cell 80:777-786.[CrossRef][Medline]
62. Schreiber-Agus, N., Y. Meng, T. Hoang, H. Hou, Jr., K. Chen, R. Greenberg, C. Cordon-Cardo, H.-W. Lee, and R. A. DePinho. 1998. Role of Mxi1 in ageing organ systems and the regulation of normal and neoplastic growth. Nature 393:483-487.[CrossRef][Medline]
63. Schreiber-Agus, N., D. Stein, K. Chen, J. S. Goltz, L. Stevens, and R. A. DePinho. 1997. Drosophila Myc is oncogenic in mammalian cells and plays a role in the diminutive phenotype. Proc. Natl. Acad. Sci. USA 94:1235-1240.
64. Seoane, J., H. V. Le, and J. Massague. 2002. Myc suppression of the p21Cip1 Cdk inhibitor influences the outcome of the p53 response to DNA damage. Nature 419:729-734.[CrossRef][Medline]
65. Seoane, J., C. Pouponnot, P. Staller, M. Schader, M. Eilers, and J. Massague. 2001. TGFß influences Myc, Miz-1 and Smad to control the CDK inhibitor p15INK4b. Nat. Cell Biol. 3:400-408.[CrossRef][Medline]
66. Staller, P., K. Peukert, A. Kiermaier, J. Seoane, J. Lukas, H. Karsunky, T. Möröy, J. Bartek, J. Massague, F. Hänel, and M. Eilers. 2001. Repression of p15INK4b expression by Myc through association with Miz-1. Nat. Cell Biol. 3:392-399.[CrossRef][Medline]
67. Stanton, B. R., A. S. Perkins, L. Tessarollo, D. A. Sassoon, and L. F. Parada. 1992. Loss of N-myc function results in embryonic lethality and failure of the epithelial component of the embryo to develop. Genes Dev. 6:2235-2247.
-ELAV) identify differentiated cells of the central and peripheral nervous systems (b, e, and h). 
