Members of the Myc proto-oncogene family encode transcription factors that function in multiple aspects of cell behavior, including proliferation, differentiation, transformation and apoptosis. Recent studies have shown that MYC activities are modulated by a network of nuclear bHLH-Zip proteins. The MAX protein is at the center of this network in that it associates with MYC as well as with the family of MAD proteins: MAD1, MXI1, MAD3 and MAD4. Whereas MYC-MAX complexes activate transcription, MAD-MAX complexes repress transcription through identical E-box binding sites. MAD proteins therefore act as antagonists of MYC. This study reports the expression patterns of the Mad gene family in the adult and developing mouse. High level of Mad gene expression in the adult is limited to tissues that display constant renewal of differentiated cell populations. In embryos, Mad transcripts are widely distributed with expression peaking during organogenesis at the onset of differentiation. A detailed analysis of their pattern of expression during chrondrocyte and neuronal differentiation in vivo, and during neuronal differentiation of P19 cells in vitro, shows that Mad family genes are sequentially induced. Mad3 transcripts and proteins are detected in proliferating cells prior to differentiation. Mxi1 and Mad4 transcripts are most abundant in cells that have further advanced along the differentiation pathway, whereas Mad1 is primarily expressed late in differentiation. Taken together, these data suggest that the different members of the MAD protein family exert their functions at distinct steps during the transition between proliferation and differentiation (Queva, 1998).
The small constitutively expressed bHLHZip protein Max is known to form sequence-specific DNA binding heterodimers with members of both the Myc and Mad families of bHLHZip proteins. Myc:Max complexes activate transcription, promote proliferation, and block terminal differentiation. In contrast, Mad:Max heterodimers act as transcriptional repressors, have an antiproliferative effect, and are induced upon differentiation in a wide variety of cell types. A novel bHLHZip Max-binding protein, Mnt, has been identified that belongs to neither the Myc nor the Mad families and is coexpressed with Myc in a number of proliferating cell types. Mnt:Max heterodimers act as transcriptional repressors and efficiently suppress Myc-dependent activation from a promoter containing proximal CACGTG sites. Transcription repression by Mnt maps to a 13-amino-acid amino-terminal region related to the Sin3 interaction domain (SID) of Mad proteins. This region of Mnt mediates interaction with mSin3 corepressor proteins and its deletion converts Mnt from a repressor to an activator. Furthermore, wild-type Mnt suppresses Myc+Ras cotransformation of primary cells, whereas Mnt containing a SID deletion cooperates with Ras in the absence of Myc to transform cells. This suggests that Mnt and Myc regulate an overlapping set of target genes in vivo. When mnt is expressed as a transgene under control of the beta-actin promoter in mice the transgenic embryos exhibit a delay in development and die during mid-gestation, when c- and N-Myc functions are critical. It is proposed that Mnt:Max:Sin3 complexes normally function to restrict Myc:Max activities associated with cell proliferation (Hurlin, 1997).
X-ray structures of the basic/helix-loop-helix/leucine zipper (bHLHZ) domains of Myc-Max and Mad-Max heterodimers bound to their common DNA target (Enhancer or E box hexanucleotide, 5'-CACGTG-3') have been determined at 1.9 Å and 2.0 Å resolution, respectively. E box recognition by these two structurally similar transcription factor pairs determines whether a cell will divide and proliferate (Myc-Max) or differentiate and become quiescent (Mad-Max). Deregulation of Myc has been implicated in the development of many human cancers, including Burkitt's lymphoma, neuroblastomas, and small cell lung cancers. Both quasi-symmetric heterodimers resemble the symmetric Max homodimer, albeit with marked structural differences in the coiled-coil leucine zipper regions that explain preferential homo- and hetero-meric dimerization of these three evolutionarily related DNA-binding proteins. The Myc-Max heterodimer, but not its Mad-Max counterpart, dimerizes to form a bivalent heterotetramer, which explains how Myc can upregulate expression of genes with promoters bearing widely separated E boxes (Nair, 2003).
Genetic characterization of the promoters of putative myc regulated genes has provided further evidence for a physiological role for Myc-Max heterotetramerization. Oligonucleotide microarray analysis has identified several Myc target genes that contain multiple E boxes within promoters, typically separated by at least 100 nucleotides. Given the persistence length of DNA, this separation of Myc-Max binding sites is compatible with DNA looping stabilized by bivalent Myc-Max heterotetramers bound to two cognate sequences. Moreover, in vitro binding studies of Myc-Max heterodimers recognizing the lactate dehydrogenase gene, a natural high-affinity target containing two consensus E boxes, demonstrates that Myc-Max complexes can engage multiple cognate sites. Functional studies with adjacent E boxes found within the lactate dehydrogenase promoter have shown that mutations in either of the two E boxes severely affect Myc-dependent activation of transcription. These results suggest that Myc-Max complexes can form higher order structures that are consistent with the heterotetrameric assembly observed in the cocrystal structure (Nair, 2003).
Proteins of the Myc and Mad family are involved in transcriptional regulation and mediate cell differentiation and proliferation. These molecules share a basic-helix-loop-helix leucine zipper domain (bHLHZip) and bind DNA at the E box (CANNTG) consensus by forming heterodimers with Max. A human gene and its mouse homolog have been isolated, characterized and mapped that encodes a new member of this family of proteins, named Rox. Through interaction mating and immunoprecipitation techniques, it has been demonstrate that Rox heterodimerizes with Max and weakly homodimerizes. Interestingly, bandshift assays demonstrate that the Rox-Max heterodimer shows a novel DNA binding specificity, having a higher affinity for the CACGCG site compared with the canonical E box CACGTG site. Transcriptional studies indicate that Rox represses transcription in both human HEK293 cells and yeast. Repression in yeast is through interaction between the N-terminus of the protein and the Sin3 co-repressor, as previously shown for the other Mad family members. ROX is highly expressed in quiescent fibroblasts and expression markedly decreases when cells enter the cell cycle. Moreover, ROX expression appears to be induced in U937 myeloid leukemia cells stimulated to differentiate with 12-O-tetradecanoylphorbol-13-acetate. The identification of a novel Max-interacting protein adds an important piece to the puzzle of Myc/Max/Mad coordinated action and function in normal and pathological situations. Furthermore, mapping of the human gene to chromosome 17p13.3 in a region that frequently undergoes loss of heterozygosity in a number of malignancies, together with the biochemical and expression features, suggest involvement of ROX in human neoplasia (Meroni, 1997).
Myc and Mad family proteins play opposing roles in the control of cell growth and proliferation. The subcellular locations of complexes formed by Myc/Max/Mad family proteins were visualized using bimolecular fluorescence complementation (BiFC) analysis. Max is recruited to different subnuclear locations by interactions with Myc versus Mad family members. Complexes formed by Max with Mxi1, Mad3, or Mad4 are enriched in nuclear foci, whereas complexes formed with Myc are more uniformly distributed in the nucleoplasm. Mad4 is localized to the cytoplasm when it is expressed separately, and Mad4 is recruited to the nucleus through dimerization with Max. The cytoplasmic localization of Mad4 is determined by a CRM1-dependent nuclear export signal located near the amino terminus. The relative efficiencies of complex formation among Myc, Max, and Mad family proteins in living cells were compared using multicolor BiFC analysis. Max forms heterodimers with the basic helix-loop-helix leucine zipper (bHLHZIP) domain of Myc (bMyc) more efficiently than it forms homodimers. Replacement of two amino acid residues in the leucine zipper of Max reverses the relative efficiencies of homo- and hetero-dimerization in cells. Surprisingly, Mad3 forms complexes with Max less efficiently than bMyc, whereas Mad4 forms complexes with Max more efficiently than bMyc. The distinct subcellular locations and the differences between the efficiencies of dimerization with Max indicate that Mad3 and Mad4 are likely to modulate transcription activation by Myc at least in part through distinct mechanisms (Grinberg, 2004).
Accurate identification of specific groups of proteins by their amino acid sequence is an important goal in genome research. This study combines information theory with fuzzy logic search procedures to identify sequence signatures or predictive motifs for members of the Myc-Max-Mad transcription factor network. Myc is a well known oncoprotein, and this family is involved in cell proliferation, apoptosis, and differentiation. A small set of amino acid sites from the N-terminal portion of the basic helix-loop-helix (bHLH) domain is described that provide very accurate sequence signatures for the Myc-Max-Mad transcription factor network and three of its member proteins. A predictive motif involving 28 contiguous bHLH sequence elements found 337 network proteins in the GenBank NR database with no mismatches or misidentifications. This motif also identifies at least one previously unknown fungal protein with strong affinity to the Myc-Max-Mad network. Another motif found 96% of known Myc protein sequences with only a single mismatch, including sequences from genomes previously not thought to contain Myc proteins. The predictive motif for Myc is very similar to the ancestral sequence for the Myc group estimated from phylogenetic analyses. Based on available crystal structure studies, this motif is discussed in terms of its functional consequences. The results provide insight into evolutionary diversification of DNA binding and dimerization in a well characterized family of regulatory proteins and provide a method of identifying signature motifs in protein families (Atchley, 2005).
Mad1 is a member of the Mad family. This family is part of the larger Myc/Max/Mad b-HLH-LZ eukaryotic transcription-factor network. Mad1 forms a specific heterodimer with Max and acts as a transcriptional repressor when bound to an E-box sequence (CACGTG) found in the promoter of c-Myc target genes. Mad1 cannot form a complex with DNA by itself under physiological conditions. A global model for the molecular recognition has emerged in which the Mad1 b-HLH-LZ homodimer is destabilized and the Mad/Max b-HLH-LZ heterodimer is favored. The detailed structural determinants responsible for the molecular recognition remain largely unknown. In this study, focus was placed on the elucidation of the structural determinants responsible for the destabilization of the Mad1 b-HLH-LZ homodimer. Conserved acidic residues at the dimerization interface (position a) of the LZ of all Max-interacting proteins have been hypothesized to be involved in the destabilization of the homodimeric states. In Mad1, this position corresponds to residue Asp 112. As reported for the complete gene product of Mad1, it was shown that wild-type b-HLH-LZ does not homodimerize or bind DNA under physiological conditions. In contrast, the single mutation of Asp 112 to an Asn enables the b-HLH-LZ to dimerize and bind DNA. These results suggest that Asp 112 is implicated in the destabilization of Mad1 b-HLH-LZ homodimer. Interestingly, this side chain is observed to form a salt bridge at the interface of the LZ domain in the crystal structure of Mad1/Max heterodimeric b-HLH-LZ bound to DNA. This clearly suggests that Asp 112 plays a crucial role in the molecular recognition between Max and Mad1 (Montagne, 2005).
The switch from transcriptionally activating MYC-MAX to transcriptionally repressing MAD1-MAX protein heterodimers has been correlated with the initiation of terminal differentiation in many cell types. To investigate the function of MAD1-MAX dimers during differentiation, the Mad1 gene was disrupted by homologous recombination in mice. Analysis of hematopoietic differentiation in homozygous mutant animals reveals that cell cycle exit of granulocytic precursors is inhibited following the colony-forming cell stage, resulting in increased proliferation and delayed terminal differentiation of low proliferative potential cluster-forming cells. Surprisingly, the numbers of terminally differentiated bone marrow and peripheral blood granulocytes are essentially unchanged in Mad1 null mice. This imbalance between the frequencies of precursor and mature granulocytes is correlated with a compensatory decrease in granulocytic cluster-forming cell survival under apoptosis-inducing conditions. Recovery of the peripheral granulocyte compartment following bone marrow ablation is significantly enhanced in Mad1 knockout mice. Two Mad1-related genes, Mxi1 and Mad3, are expressed ectopically in adult spleen, indicating that functional redundancy and cross-regulation between MAD family members may allow for apparently normal differentiation in the absence of MAD1. These findings demonstrate that MAD1 regulates cell cycle withdrawal during a late stage of granulocyte differentiation, and suggest that the relative levels of MYC versus MAD1 mediate a balance between cell proliferation and terminal differentiation (Foley, 1998).
The Mad family comprises four basic-helix-loop-helix/leucine zipper proteins, Mad1, Mxi1, Mad3, and Mad4, which heterodimerize with Max and function as transcriptional repressors. The balance between Myc-Max and Mad-Max complexes has been postulated to influence cell proliferation and differentiation. The expression patterns of Mad family genes are complex, but in general, the induction of most family members is linked to cell cycle exit and differentiation. The expression pattern of mad3 is unusual in that mad3 mRNA and protein were found to be restricted to proliferating cells prior to differentiation. During murine development mad3 is specifically expressed in the S phase of the cell cycle in neuronal progenitor cells that are committed to differentiation. To investigate mad3 function, the mad3 gene was disrupted by homologous recombination in mice. No defect in cell cycle exit and differentiation could be detected in mad3 homozygous mutant mice. However, upon gamma irradiation, increased cell death of thymocytes and neural progenitor cells was observed, implicating mad3 in the regulation of the cellular response to DNA damage (Queva, 2001).
Mnt is a Max-interacting transcriptional repressor that has been hypothesized to function as a Myc antagonist. To investigate Mnt function the Mnt gene was deleted in mice. Since mice lacking Mnt are born severely runted and typically die within several days of birth, mouse embryo fibroblasts (MEFs) derived from these mice and conditional Mnt knockout mice were used in this study. In the absence of Mnt, MEFs prematurely enter the S phase of the cell cycle and proliferated more rapidly than Mnt+/+ MEFs. Defective cell cycle control in the absence of Mnt is linked to upregulation of Cdk4 and cyclin E and the Cdk4 gene appears to be a direct target of Mnt-Myc antagonism. Like MEFs that overexpress Myc, Mnt-/- MEFs are prone to apoptosis, efficiently escape senescence and can be transformed with oncogenic Ras alone. Consistent with Mnt functioning as a tumor suppressor, conditional inactivation of Mnt in breast epithelium leads to adenocarinomas. These results demonstrate a unique negative regulatory role for Mnt in governing key Myc functions associated with cell proliferation and tumorigenesis (Hurlin, 2003).
The Mnt gene encodes a Mad-family bHLH transcription factor located on human 17p13.3. Mnt is one of 20 genes deleted in a heterozygous fashion in Miller-Dieker syndrome (MDS), a contiguous gene syndrome that consists of severe neuronal migration defects and craniofacial dysmorphic features. Mnt can inhibit Myc-dependent cell transformation and is hypothesized to counterbalance the effects of c-Myc on growth and proliferation in vivo by competing with Myc for binding to Max and by repressing target genes activated by Myc/Max heterodimers. Unlike the related Mad family members, Mnt is expressed ubiquitously and Mnt/Max heterodimers are found in proliferating cells that contain Myc/Max heterodimers, suggesting a unique role for Mnt during proliferation. To examine the role of Mnt in vivo, mice with null (MntKO) and loxP-flanked conditional knock-out (MntCKO) alleles of Mnt were produced. Virtually all MntKO/KO mutants in a mixed (129S6 x NIH Black Swiss) or inbred (129S6) genetic background died perinatally. Mnt-deficient embryos exhibit small size throughout development and show reduced levels of c-Myc and N-Myc. In addition, 37% of the mixed background mutants displayed cleft palate as well as retardation of skull development, a phenotype not observed in the inbred mutants. These results demonstrate an important role for Mnt in embryonic development and survival, and suggest that Mnt may play a role in the craniofacial defects displayed by MDS patients (Toyo-oka, 2004).
Members of the Mad family of bHLHZip proteins heterodimerize with Max and function to repress the transcriptional and transforming activities of the Myc proto-oncogene. Mad:Max heterodimers repress transcription by recruiting a large multi-protein complex containing the histone deacetylases, HDAC1 and HDAC2, to DNA. The interaction between Mad proteins and HDAC1/2 is mediated by the corepressor mSin3A (see Drosophila Sin3A) and requires sequences at the amino terminus of the Mad proteins, termed the SID, for Sin3 interaction domain, and the second of four paired amphipathic alpha-helices (PAH2) in mSin3A. To better understand the requirements for the interaction between the SID and PAH2, mutagenesis and structural studies on the SID have been performed. These studies show that amino acids 8-20 of Mad1 are sufficient for SID:PAH2 interaction. Further, this minimal 13-residue SID peptide forms an amphipathic alpha-helix in solution, and residues on the hydrophobic face of the SID helix are required for interaction with PAH2. Finally, the minimal SID can function as an autonomous and portable repression domain, demonstrating that it is sufficient to target a functional mSin3A/HDAC corepressor complex (Eilers, 1999).
Gene-specific targeting of the Sin3 corepressor complex by DNA-bound repressors is an important mechanism of gene silencing in eukaryotes. The Sin3 corepressor specifically associates with a diverse group of transcriptional repressors, including members of the Mad family, that play crucial roles in development. The NMR structure of the complex formed by the PAH2 domain of mammalian Sin3A with the transrepression domain (SID) of human bHLHZip protein Mad1 reveals that both domains undergo mutual folding transitions upon complex formation generating an unusual left-handed four-helix bundle structure and an amphipathic alpha helix, respectively. The SID helix is wedged within a deep hydrophobic pocket defined by two PAH2 helices. Structure-function analyses of the Mad-Sin3 complex provide a basis for understanding the underlying mechanism(s) that lead to gene silencing (Brubaker, 2000).
Sin3 appears to function as a large protein scaffold capable of multiple protein-protein interactions. While Sin3 interacts with class I histone deacetylases (HDAC1 and HDAC2) and presumed accessory proteins such as RbAp48, SAP30, and SAP18, it also associates with a surprisingly wide range of DNA binding transcription factors, including the nuclear hormone receptors (through the N-CoR and SMRT corepressors), MeCP2, Ski, p53, Ikaros and Aiolos, REST/NRSF, MNF-beta, and the Mad family of Max binding bHLH-Zip transcriptional repressors. The activities of these proteins and their ability to interact with Sin3 are thought to be crucial for cell proliferation and differentiation (Brubaker, 2000 and references therein).
The nature and possible regulation of the specific interaction between transcription factors and Sin3 is of great interest. For nuclear hormone receptors, the interaction with N-CoR/SMRT is hormone regulated, while for 'dedicated' repressors such as the Mad protein family, the association appears to be constitutive. In the case of the Mad proteins, all four family members (Mad1, Mxi1, Mad3, and Mad4) and the related repressor, Mnt (or Rox) contain an ~30-residue, N-terminally located segment known as the Sin3 interaction domain, or SID, which is both necessary and sufficient for Sin3 association and for transcriptional repression. Deletion or specific mutation of the SID abrogates Mad repression as well as its growth inhibitory functions. Furthermore, the Mad SID is capable of conferring repression activity when fused to a heterologous DNA binding domain. Helical wheel analysis and circular dichroism (CD) studies of the Mad SID suggest that it has the potential to form an amphipathic alpha helix. Mutational analyses further demonstrate that a cluster of residues on the apolar face of the helix is essential for interaction with mammalian Sin3A (mSin3A) (Brubaker, 2000 and references therein).
Sin3 interacts with many proteins in the complex through four imperfect repeats of ~100 residues known as paired amphipathic helix (PAH) domains. The PAH domains, which were each suggested to be organized into two alpha helices separated by a flexible spacer region, are among the most evolutionarily conserved regions of the large Sin3 proteins (100-170 kDa). Indeed, these domains are important for Sin3 function as a corepressor, most likely through their independent associations with various repressors and other associated proteins. For example, PAH2 is both necessary and sufficient for interaction with the Mad proteins as well as with a newly discovered Sin3-interacting protein, Pf1. However, PAH1 associates with N-CoR and PLZF, while PAH3 binds the SAP30 protein (Brubaker, 2000 and references therein).
While previous work on repressor-Sin3 corepressor interactions has localized functionally important regions and provides hints regarding their structure, details of these important interactions have remained largely unknown. In this study, a high-resolution structure is described for the Mad1 SID bound to the PAH2 domain of mSin3A determined by NMR (nuclear magnetic resonance) methods. Mutational studies of mSin3A are presented that confirm many of the specific interactions predicted from the NMR structure. Finally, it is shown that an unrelated Sin3-interacting protein, Pf1, with an interaction domain distinct from the Mad family SID, is likely to interact with PAH2 in a manner closely resembling Mad1 SID (Brubaker, 2000).
Mxi1 belongs to the Mad (Mxi1) family of proteins, which function as potent antagonists of Myc oncoproteins. This antagonism relates partly to their ability to compete with Myc for the protein Max and for consensus DNA binding sites and to recruit transcriptional co-repressors. Mad(Mxi1) proteins have been suggested to be essential in cellular growth control and/or in the induction and maintenance of the differentiated state. Consistent with these roles, mxi1 may be the tumour-suppressor gene that resides at region 24-26 of the long arm of chromosome 10. This region is a cancer hotspot, and mutations here may be involved in several cancers, including prostate adenocarcinoma. Mice lacking Mxi1 exhibit progressive, multisystem abnormalities. These mice also show increased susceptibility to tumorigenesis either following carcinogen treatment or when also deficient in Ink4a. This cancer-prone phenotype may correlate with the enhanced ability of several mxi1-deficient cell types, including prostatic epithelium, to proliferate. These results show that Mxi1 is involved in the homeostasis of differentiated organ systems, acts as a tumour suppressor in vivo, and engages the Myc network in a functionally relevant manner (Schreiber-Agus, 1998).
The genes of the myc/max/mad family play an important role in controlling cell proliferation and differentiation. The first homologues of the mad and max genes in the nematode C. elegans, named mdl-1 and mxl-1 respectively, have been identified. Like the vertebrate MAD proteins, MDL-1 binds an E-box DNA sequence (CACGTG) when dimerized with MXL-1. However, unlike vertebrate MAX, MXL-1 can not form homodimers and bind to DNA alone. Promoter fusions to a GFP reporter suggest that these genes are coexpressed in posterior intestinal and post-mitotic neuronal cells during larval development. The coexpression in the posterior intestinal cells occurs before their final division at the end of the L1 stage and persists afterwards, demonstrating that mad and max expression can be correlated directly to the cell cycle state of an individual cell type. These data also show that mxl-1 is an obligate partner for mdl-1 in vivo and in vitro and indicate that these genes may play an important role in post-embryonic development. Finally, MDL-1 can suppress activated c-MYC/RAS-induced focus formation in a rat embryo fibroblast transformation assay. Like the vertebrate MAD protein, MDL-1 activity in suppressing transformation is dependent on a functional SIN3 interaction domain (Yuan, 1998).
Activated lymphocytes must increase in size and duplicate their contents (cell growth) before they can divide. The molecular events that control cell growth in proliferating lymphocytes and other metazoan cells are still unclear. Transgenesis has been utilized to provide evidence suggesting that the basic helix-loop-helix-zipper (bHLHZ) transcriptional repressor Mad1, considered to be an antagonist of Myc function, inhibits lymphocyte expansion, maturation and growth following pre-T-cell receptor (pre-TCR) and TCR stimulation. Furthermore, cDNA microarray technology was used to determine that of the genes repressed by Mad1, the majority (77%) are involved in cell growth, which correlates with a decrease in size of Mad1 transgenic thymocytes. Over 80% of the genes repressed by Mad1 have previously been found to be induced by Myc. These results suggest that a balance between Myc and Mad levels may normally modulate lymphocyte proliferation and development in part by controlling expression of growth-regulating genes (Iritani, 2002).
The growth inhibitory cytokine TGF-beta enforces homeostasis of epithelia by activating processes such as cell cycle arrest and apoptosis. Id2 expression is often highest in proliferating epithelial cells and declines during differentiation. Recently, Id2 expression has been found to depend on Myc-Max transcriptional complexes. TGF-beta signaling inhibits Id2 expression in human and mouse epithelial cell lines from different tissue origins. Furthermore, the observed Id2 down-regulation by TGF-beta in mouse mammary epithelial cells occurs without a concurrent drop in c-Myc levels. However, sustained Id2 repression in these cells and in human keratinocytes coincides with induction of the Myc antagonistic repressors Mad2 and Mad4, decreased formation of Myc-Max heterodimers and the replacement of Myc-Max complexes with Mad-Max complexes on the Id2 promoter. These results argue that induction of Mad expression and Id2 down-regulation are important events during the TGF-beta cytostatic program in epithelial cells (Siegel, 2003).
Myc oncoproteins are overexpressed in most cancers and are sufficient to accelerate cell proliferation and provoke transformation. However, in normal cells Myc also triggers apoptosis. All of the effects of Myc require its function as a transcription factor that dimerizes with Max. This complex induces genes containing CACGTG E-boxes, such as Ornithine decarboxylase (Odc), which harbors two of these elements. In quiescent cells the Odc E-boxes are occupied by Max and Mnt, a putative Myc antagonist, and this complex is displaced by Myc-Max complexes in proliferating cells. Knockdown of Mnt expression by stable retroviral RNA interference triggers many targets typical of the 'Myc' response and provokes accelerated proliferation and apoptosis. Strikingly, these effects of Mnt knockdown are even manifest in cells lacking c-myc. Moreover, Mnt knockdown is sufficient to transform primary fibroblasts in conjunction with Ras. Therefore, Mnt behaves as a tumor suppressor. These findings support a model where Mnt represses Myc target genes and Myc functions as an oncogene by relieving Mnt-mediated repression (Nilsson, 2004).
The Myc/Max/Mad network of transcription factors regulates cell proliferation, differentiation, and transformation. Similar to other proteins of the network, Mnt forms heterodimers with Max and binds CACGTG E-Box elements. Transcriptional repression by Mnt is mediated through association with mSin3, and deletion of the mSin3-interacting domain (SID) converts Mnt to a transcriptional activator. Mnt is coexpressed with Myc in proliferating cells and has been suggested to be a modulator of Myc function. Mnt is expressed both in growth-arrested and proliferating mouse fibroblasts and is phosphorylated when resting cells are induced to re-enter the cell cycle. Importantly, the interaction between Mnt and mSin3 is disrupted upon serum stimulation resulting in decreased Mnt-associated HDAC activity. Furthermore, Mnt binds and recruits mSin3 to the Myc target gene cyclin D2 in quiescent mouse fibroblasts. Interference with Mnt expression by RNAi results in upregulation of cyclin D2 expression in growth-arrested fibroblasts, supporting the view that Mnt represses cyclin D2 transcription in quiescent cells. These data suggest a model in which phosphorylation of Mnt at cell cycle entry results in disruption of Mnt-mSin3-HDAC1 interaction, which allows induction of Myc target genes by release of Mnt-mediated transcriptional repression (Popov, 2005).
The transcription factors of the Myc/Max/Mad network play essential roles in the regulation of cellular behavior. Mad1 inhibits cell proliferation by recruiting an mSin3-corepressor complex that contains histone deacetylase activity. Mad1 is a potent inhibitor of the G(1) to S phase transition, a function that requires Mad1 to heterodimerize with Max and to bind to the corepressor complex. Cyclin E/CDK2, but not cyclin D and cyclin A complexes, fully restored S phase progression. In addition inhibition of colony formation and gene repression by Mad1 are also efficiently antagonized by cyclin E/CDK2. This was the result of cyclin E/CDK2 interfering with the interaction of Mad1 with HDAC1 and reducing HDAC activity. These findings define a novel interplay between the cell cycle regulator cyclin E/CDK2 and Mad1 and its associated repressor complex and suggests an additional mechanism how cyclin E/CDK2 affects the G(1) to S phase transition (Rottmann, 2005).
The c-Myc oncoprotein is strongly induced during the G0 to S-phase transition and is an important regulator of cell cycle entry. In contrast to c-Myc, the putative Myc antagonist Mnt is maintained at a constant level during cell cycle entry. Mnt and Myc require interaction with Max for specific DNA binding at E-box sites, but have opposing transcriptional activities. c-Myc induction during cell cycle entry leads to a transient decrease in Mnt-Max complexes and a transient switch in the ratio of Mnt-Max to c-Myc-Max on shared target genes. Mnt overexpression suppresses cell cycle entry and cell proliferation, suggesting that the ratio of Mnt-Max to c-Myc-Max is critical for cell cycle entry. Furthermore, simultaneous Cre-Lox mediated deletion of Mnt and c-Myc in mouse embryo fibroblasts rescues the cell cycle entry and proliferative block caused by c-Myc ablation alone. These results demonstrate that Mnt-Myc antagonism plays a fundamental role in regulating cell cycle entry and proliferation (Walker, 2005).
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