Dual regulation of Myc by Abl.

The tyrosine kinase c-Abl (or Abl) and the prolyl-isomerase Pin1 cooperatively activate the transcription factor p73 by enhancing recruitment of the acetyltransferase p300. As the transcription factor c-Myc (or Myc) is a known target of Pin1 and p300, we hypothesized that it might be regulated in a similar manner. Consistent with this hypothesis, overexpression of Pin1 augmented the interaction of Myc with p300 and transcriptional activity. The action of Abl, however, was more complex than predicted. On one hand, Abl indirectly enhanced phosphorylation of Myc on Ser 62 and Thr 58, its association with Pin1 and p300 and its acetylation by p300. These effects of Abl were exerted through phosphorylation of substrate(s) other than Myc itself. On the other hand, Abl interacted with the C-terminal domain of Myc and phosphorylated up to five tyrosine residues in its N-terminus, the principal of which was Y74. Indirect immunofluorescence or immunohistochemical staining suggested that the Y74-phosphorylated form of Myc (Myc-pY74) localized to the cytoplasm and coexisted either with active Abl in a subset of mammary carcinomas or with Bcr-Abl in chronic myeloid leukemia. In all instances, Myc-pY74 constituted a minor fraction of the cellular Myc protein. Thus, our data unravel two potential effects of Abl on Myc: first, Abl signaling can indirectly augment acetylation of Myc by p300, and most likely also its transcriptional activity in the nucleus; second, Abl can directly phosphorylate Myc on tyrosine: the resulting form of Myc appears to be cytoplasmic, and its presence correlates with Abl activation in cancer.

Genomic binding and transcriptional regulation by the Drosophila Myc and Mnt transcription factors.

Deregulated expression of members of the myc oncogene family has been linked to the genesis of a wide range of cancers, whereas their normal expression is associated with growth, proliferation, differentiation, and apoptosis. Myc proteins are transcription factors that function within a network of transcriptional activators (Myc) and repressors (Mxd/Mad and Mnt), all of which heterodimerize with the bHLHZ protein Mad and bind E-box sequences in DNA. These transcription factors recruit coactivator or corepressor complexes that in turn modify histones. Myc, Mxd/Max, and Mnt proteins have been thought to act on a specific subset of genes. However, expression array studies and, most recently, genomic binding studies suggest that these proteins exhibit widespread binding across the genome. Here we demonstrate by immunostaining of Drosophila polytene chromosome that Drosophila Myc (dMyc) is associated with multiple euchromatic chromosomal regions. Furthermore, many dMyc-binding regions overlap with regions containing active RNA polymerase II, although dMyc can also be found in regions lacking active polymerase. We also demonstrate that the pattern of dMyc expression in nuclei overlaps with histone markers of active chromatin but not pericentric heterochromatin. dMyc binding is not detected on the X chromosome rDNA cluster (bobbed locus). This is consistent with recent evidence that in Drosophila cells dMyc regulates rRNA transcription indirectly, in contrast to mammalian cells where direct binding of c-Myc to rDNA has been observed. We further show that the dMyc antagonist dMnt inhibits rRNA transcription in the wing disc. Our results support the view that the Myc/Max/Mad network influences transcription on a global scale.

TGF-beta flips the Myc switch.

Although transforming growth factor-beta (TGF-beta) can affect cell cycle arrest, not much molecular detail is known about how TGF-beta-dependent arrest is mediated. Two recent papers shed some light on how this is accomplished. Orian and Eisenman discuss how Myc interacts with Miz-1 to block the expression of a cell cycle inhibitory protein, p15(INK4b), and how TGF-beta is able to unblock Myc-dependent repression of Miz-1.

The Smad transcriptional corepressor TGIF recruits mSin3.

The homeodomain protein TG-interacting factor (TGIF) represses transcription by histone deacetylase-dependent and -independent means. Heterozygous mutations in human TGIF result in holoprosencephaly, a severe genetic disorder affecting craniofacial development, suggesting that TGIF is critical for normal development. After transforming growth factorbeta (TGFbeta) stimulation, Smad proteins enter the nucleus and form transcriptional activation complexes or interact with TGIF, which functions as a corepressor. The relative levels of Smad corepressors and coactivators present within the cell may determine the outcome of a TGFbeta response. We show that TGIF interacts directly with the paired amphipathic alpha-helix 2 domain of the mSin3 corepressor, and TGIF recruits mSin3 to a TGFbeta-activated Smad complex. The mSin3 interaction domain of TGIF has been shown to be essential for repression of a TGFbeta transcriptional response. Thus, TGIF represents a targeting component of the mSin3 corepressor complex.

Targeted deletion of the S-phase-specific Myc antagonist Mad3 sensitizes neuronal and lymphoid cells to radiation-induced apoptosis.

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. We show here that 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, we disrupted the mad3 gene 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.

Solution structure of the interacting domains of the Mad-Sin3 complex: implications for recruitment of a chromatin-modifying complex.

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 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.

The Myc/Max/Mad network and the transcriptional control of cell behavior.

The Myc/Max/Mad network comprises a group of transcription factors whose distinct interactions result in gene-specific transcriptional activation or repression. A great deal of research indicates that the functions of the network play roles in cell proliferation, differentiation, and death. In this review we focus on the Myc and Mad protein families and attempt to relate their biological functions to their transcriptional activities and gene targets. Both Myc and Mad, as well as the more recently described Mnt and Mga proteins, form heterodimers with Max, permitting binding to specific DNA sequences. These DNA-bound heterodimers recruit coactivator or corepressor complexes that generate alterations in chromatin structure, which in turn modulate transcription. Initial identification of target genes suggests that the network regulates genes involved in the cell cycle, growth, life span, and morphology. Because Myc and Mad proteins are expressed in response to diverse signaling pathways, the network can be viewed as a functional module which acts to convert environmental signals into specific gene-regulatory programs.

The interferon- and differentiation-inducible p202a protein inhibits the transcriptional activity of c-Myc by blocking its association with Max.

p202a is a murine protein that is induced during the fusion of myoblasts to myotubes and can also be induced by interferon. Even 2-3-fold overexpression of p202a in cells retards proliferation. p202a was shown to modulate transcription by binding, and inhibiting the activity of several transcription factors including c-Fos, c-Jun, AP-2, E2F1, E2F4, NF-kappaB, MyoD, and myogenin. Here we report that p202a also bound the c-Myc protein in vitro and in vivo; the C-terminal p202a b segment bound the C-terminal basic region helix-loop-helix-leucine zipper (bHLHLZ) region of c-Myc. The transfection of a p202a expression plasmid inhibited the c-Myc-dependent expression of reporter plasmids in transient assays; moreover, overexpression of p202a in stable cell lines decreased the endogenous levels of mRNAs whose expression is driven by c-Myc. These effects of p202a are consistent with our finding that the binding of p202a to c-Myc inhibited the binding of c-Myc to Max in vitro and in vivo. p202a also inhibited the c-Myc-induced anchorage-independent growth and apoptosis of Rat-1 cells. The inhibition of c-Myc-dependent transcription, proliferation, and apoptosis by p202a is in line with the involvement of p202a in differentiation.

Analysis of Myc/Max/Mad network members in adipogenesis: inhibition of the proliferative burst and differentiation by ectopically expressed Mad1.

Transcription factors of the Myc/Max/Mad network affect multiple aspects of cellular behavior, including proliferation, differentiation, and apoptosis. Recent studies have shown that Mad proteins can inhibit cellular growth and transformation and thus antagonize the function of Myc proteins. To define further the contribution of these proteins to cellular growth control, we have studied the expression of the respective genes and proteins in 3T3-L1 cells, both upon serum stimulation of quiescent cells and during adipocytic differentiation in response to insulin, dexamethasone, and isobutylmethylxanthine. We found distinct expression patterns for the mad genes. Mad4 was induced when cells exit the cell cycle and, together with mad1, during the late phase of differentiation. In contrast, mad3 expression was associated with progression through S phase and the proliferative burst of differentiating preadipocytes, overlapping in part c-myc expression. DNA binding analyses revealed that the most prominent network complex both in cycling and in differentiating cells was Mnt/Max, whereas c-Myc/Max complexes were detectable only during peak c-Myc expression periods. Ectopic expression of Mad1 in preadipocytes resulted in the inhibition of S phase and the proliferation associated with the proliferative burst; as a consequence, adipocytic differentiation was significantly inhibited. Our findings suggest that the precise temporal regulation of Myc/Max/Mad network proteins is critical for determining cellular behavior.

Expression analysis with oligonucleotide microarrays reveals that MYC regulates genes involved in growth, cell cycle, signaling, and adhesion.

MYC affects normal and neoplastic cell proliferation by altering gene expression, but the precise pathways remain unclear. We used oligonucleotide microarray analysis of 6,416 genes and expressed sequence tags to determine changes in gene expression caused by activation of c-MYC in primary human fibroblasts. In these experiments, 27 genes were consistently induced, and 9 genes were repressed. The identity of the genes revealed that MYC may affect many aspects of cell physiology altered in transformed cells: cell growth, cell cycle, adhesion, and cytoskeletal organization. Identified targets possibly linked to MYC's effects on cell growth include the nucleolar proteins nucleolin and fibrillarin, as well as the eukaryotic initiation factor 5A. Among the cell cycle genes identified as targets, the G1 cyclin D2 and the cyclin-dependent kinase binding protein CksHs2 were induced whereas the cyclin-dependent kinase inhibitor p21(Cip1) was repressed. A role for MYC in regulating cell adhesion and structure is suggested by repression of genes encoding the extracellular matrix proteins fibronectin and collagen, and the cytoskeletal protein tropomyosin. A possible mechanism for MYC-mediated apoptosis was revealed by identification of the tumor necrosis factor receptor associated protein TRAP1 as a MYC target. Finally, two immunophilins, peptidyl-prolyl cis-trans isomerase F and FKBP52, the latter of which plays a role in cell division in Arabidopsis, were up-regulated by MYC. We also explored pattern-matching methods as an alternative approach for identifying MYC target genes. The genes that displayed an expression profile most similar to endogenous Myc in microarray-based expression profiling of myeloid differentiation models were highly enriched for MYC target genes.

Mga, a dual-specificity transcription factor that interacts with Max and contains a T-domain DNA-binding motif.

The basic-helix-loop-helix-leucine zipper (bHLHZip) proteins Myc, Mad and Mnt are part of a transcription activation/repression system involved in the regulation of cell proliferation. The function of these proteins as transcription factors is mediated by heterodimerization with the small bHLHZip protein Max, which is required for their specific DNA binding to E-box sequences. We have identified a novel Max-interacting protein, Mga, which contains a Myc-like bHLHZip motif, but otherwise shows no relationship with Myc or other Max-interacting proteins. Like Myc, Mad and Mnt proteins, Mga requires heterodimerization with Max for binding to the preferred Myc-Max-binding site CACGTG. In addition to the bHLHZip domain, Mga contains a second DNA-binding domain: the T-box or T-domain. The T-domain is a highly conserved DNA-binding motif originally defined in Brachyury and characteristic of the Tbx family of transcription factors. Mga binds the preferred Brachyury-binding sequence and represses transcription of reporter genes containing promoter-proximal Brachyury-binding sites. Surprisingly, Mga is converted to a transcription activator of both Myc-Max and Brachyury site-containing reporters in a Max-dependent manner. Our results suggest that Mga functions as a dual-specificity transcription factor that regulates the expression of both Max-network and T-box family target genes.

c-Myc enhances protein synthesis and cell size during B lymphocyte development.

Members of the myc family of nuclear protooncogenes play roles in cell proliferation, differentiation, and apoptosis. Moreover, inappropriate expression of c-myc genes contributes to the development of many types of cancers, including B cell lymphomas in humans. Although Myc proteins have been shown to function as transcription factors, their immediate effects on the cell have not been well defined. Here we have utilized a murine model of lymphomagenesis (Emu-myc mice) to show that constitutive expression of a c-myc transgene under control of the Ig heavy-chain enhancer (Emu) results in an increase in cell size of normal pretransformed B lymphocytes at all stages of B cell development. Furthermore, we show that c-Myc-induced growth occurs independently of cell cycle phase and correlates with an increase in protein synthesis. These results suggest that Myc may normally function by coordinating expression of growth-related genes in response to mitogenic signals. Deregulated c-myc expression may predispose to cancer by enhancing cell growth to levels required for unrestrained cell division.

Drosophila myc regulates cellular growth during development.

Transcription factors of the Myc proto-oncogene family promote cell division, but how they do this is poorly understood. Here we address the functions of Drosophila Myc (dMyc) during development. Using mosaic analysis in the fly wing, we show that loss of dMyc retards cellular growth (accumulation of cell mass) and reduces cell size, whereas dMyc overproduction increases growth rates and cell size. dMyc-induced growth promotes G1/S progression but fails to accelerate cell division because G2/M progression is independently controlled by Cdc25/String. We also show that the secreted signal Wingless patterns growth in the wing primordium by modulating dMyc expression. Our results indicate that dMyc links patterning signals to cell division by regulating primary targets involved in cellular growth and metabolism.

Analysis of the Max-binding protein MNT in human medulloblastomas.

Medulloblastomas (MBs) are the most frequent malignant brain tumors in children. The molecular pathogenesis of these tumors is still poorly understood. Microsatellite and restriction-fragment-length polymorphism studies have revealed allelic loss of genetic material on the short arm of chromosome 17 in the region 17p13 in approximately 50% of MBs, suggesting the presence of a tumor-suppressor gene in this region. A candidate for this putative tumor-suppressor is the MNT gene, located at 17p13.3 and encoding a Max-interacting nuclear protein with transcriptional-repressor activity. In this study, we analyzed MNT mRNA and protein expression in 44 MB samples, including 32 primary tumors, 3 recurrent tumors and 9 MB cell lines. Allelic loss at 17p13.3 was found in 49% of informative cases. RT-PCR showed MNT mRNA expression in all cases analyzed. Endogenous Mnt protein with an apparent molecular weight of 72 to 74 kDa was detected in lysates from MB cell lines. The presence and functional integrity of Mnt in MBs were tested in electrophoretic mobility-shift assays. These experiments demonstrated that Mnt interacts with Max, and that this heterodimer binds DNA specifically, suggesting a functional bHLHZip domain of MB-derived Mnt. In support, single-strand conformation-polymorphism (SSCP) analyses revealed no mutation in the bHLHZip region. Deletion of the Mnt Sin3 interaction domain was shown to convert Mnt from an inhibitor of myc/ras-co-transformation into a molecule capable of cooperating with Ras in transformation. This region therefore was screened for mutation by SSCP: again, no alterations were found. These findings indicate that the MNT gene located at 17p13.3 is not likely to be involved in the molecular pathogenesis of MBs.

Two MAD tails: what the recent knockouts of Mad1 and Mxi1 tell us about the MYC/MAX/MAD network.

Members of the MAD/MXI protein family heterodimerize with MAX and repress transcription by recruiting a chromatin-modifying co-repressor complex to specific DNA target genes. Repression mediated by MAD is thought to antagonize the transcriptional activation and proliferation-promoting functions of MYC-MAX heterodimers. Because they are induced during differentiation, it has been suggested that MAD proteins act to limit cell proliferation during terminal differentiation. There is also controversial evidence that these proteins may function as tumor suppressors. Recently, targeted gene deletions of two members of this gene family, Mad1 and Mxi1, have been carried out in mice. Although these animals display what appear to be quite different phenotypes, further analysis supports the view that both these proteins function in cell-cycle exit during terminal differentiation, and that at least MXI1 can act as a tumor suppressor.

Dwarfism and dysregulated proliferation in mice overexpressing the MYC antagonist MAD1.

The four members of the MAD family are bHLHZip proteins that heterodimerize with MAX and act as transcriptional repressors. The switch from MYC-MAX complexes to MAD-MAX complexes has been postulated to couple cell-cycle arrest with differentiation. The ectopic expression of Mad1 in transgenic mice led to early postnatal lethality and dwarfism and had a profound inhibitory effect on the proliferation of the hematopoietic cells and embryonic fibroblasts derived from these animals. Compared to wild-type cells, Mad1 transgenic fibroblasts arrested with altered morphology and reduced density at confluence, cycled more slowly, and were delayed in their progression from G0 to the S phase. These changes were accompanied by accumulation of hypophosphorylated retinoblastoma protein and p130. Cyclin D1-associated kinase activity was dramatically reduced in MAD1-overexpressing fibroblasts. However, wild-type cell-cycle distribution and morphology could be rescued in the Mad1 transgenic cells by the introduction of HPV-E7, but not an E7 mutant incapable of binding to pocket proteins. This indicates that the activities of the retinoblastoma family members, via the cyclin D pathway, are likely to be the major targets for MAD1-mediated inhibition of proliferation in primary mouse fibroblasts.

Pim-1 kinase and p100 cooperate to enhance c-Myb activity.

The pim-1 oncogene is regulated by hematopoietic cytokine receptors, encodes a serine/threonine protein kinase, and cooperates with c-myc in lymphoid cell transformation. Using a yeast two-hybrid screen, we found that Pim-1 protein binds to p100, a transcriptional coactivator that interacts with the c-Myb transcription factor. Pim-1 phosphorylated p100 in vitro, formed a stable complex with p100 in animal cells, and functioned downstream of Ras to stimulate c-Myb transcriptional activity in a p100-dependent manner. Thus, Pim-1 and p100 appear to be components of a novel signal transduction pathway affecting c-Myb activity, linking all three to the cytokine-regulated control of hematopoietic cell growth, differentiation, and apoptosis.