Myc Family Proteins Research Articles

N-myc functions in transcription and development

N-Myc is a member of the Myc family of proteins, which are best known for their potent oncogenic activities and association with a large proportion of human cancers. Intense scrutiny of the oncogenic properties of Myc family proteins over the last several decades has revealed a great deal about their transcriptional and oncogenic activities. Myc proteins have broad effects on transcription and can stimulate a variety of cell behaviors that contribute to the malignant phenotype. N-Myc and c-Myc also play essential functions during embryonic development, and loss of these proteins has deleterious effects in most, if not all, tissues and organ systems. What remains to be fully unraveled is the relationship between the diverse activities associated with deregulated and overexpressed Myc and their normal roles during embryonic development and tissue homeostasis. In this review I summarize our understanding of the transcriptional activities of Myc family proteins and the roles of N-myc in morphogenesis, particularly as they relate to cellular proliferation and apoptosis.

Growth inhibition of murine neuroblastoma cells by C-Myc with cell cycle arrest in G2/M

c-myc and N-myc belong to the myc family of proteins that plays an important role in cell proliferation, differentiation and apoptosis. The N-myc gene is amplified in aggressive neuroblastoma and c-myc is overexpressed in many lymphomas and cancers. However, c-myc has not been implied in tumorigenesis or progression of neuroblastoma. We therefore investigated the so far unknown effects of c-myc overexpression on the aggressiveness of neuroblastoma cells with single copy N-myc. c-myc overexpression in serum-deprived murine NXS2 neuroblastoma cells led to cell cycle progression and massive apoptosis, causing a net decrease of viable cells. In serum-replete medium c-myc caused NXS2 cells to arrest in G2/M. Furthermore, c-myc decreased clonogenic growth of neuroblastoma cells. Taken together, these data suggest that c-myc attenuates the malignant phenotype of NXS2 neuroblastoma cells. Thus, although c-myc increased NXS2 tumor mass in vivo, c-myc appears to have decreased malignant potency in neuroblastoma cells compared to N-myc. This may be one reason why c-myc does not play a role in neuroblastomagenesis.

Evidence for a cancer-specific switch at the CDK4 promoter with loss of control by both USF and c-Myc.

USF and c-Myc are basic helix-loop-helix transcription factors with similar DNA-binding specificities, but antagonistic effects on cellular transformation. In order to determine how these opposite functions correlate with the transcriptional activities of the two factors on particular downstream targets, we investigated the roles of USF and c-Myc in expression of CDK4, a known direct target of c-Myc. Overexpression of either c-Myc or USF2, but not USF1, stimulated the expression of CDK4 promoter-driven reporter genes in the non-tumorigenic mammary epithelial MCF-10A cells. Dominant-negative mutants specific to either Myc or USF family proteins inhibited reporter gene activity as well as endogenous CDK4 expression, demonstrating involvement of both USF and Myc in CDK4 transcriptional control. In contrast, in two different breast cancer cell lines where USF is transcriptionally inactive and c-Myc is overexpressed, CDK4 promoter activity was no longer responsive to either transcription factor. Accordingly, chromatin immunoprecipitation revealed significantly lower levels of both USF and c-Myc bound to the endogenous CDK4 promoter in breast cancer cells than in MCF-10A cells, with a concomitant decrease in associated histone H3 acetylation. These results suggest that a major switch in the transcriptional control of CDK4 occurs during breast carcinogenesis, with likely alteration of cell cycle regulation.

Visualization of Myc/Max/Mad family dimers and the competition for dimerization in living cells.

Myc and Mad family proteins play opposing roles in the control of cell growth and proliferation. We have visualized the subcellular locations of complexes formed by Myc/Max/Mad family proteins using bimolecular fluorescence complementation (BiFC) analysis. Max was recruited to different subnuclear locations by interactions with Myc versus Mad family members. Complexes formed by Max with Mxi1, Mad3, or Mad4 were enriched in nuclear foci, whereas complexes formed with Myc were more uniformly distributed in the nucleoplasm. Mad4 was localized to the cytoplasm when it was expressed separately, and Mad4 was recruited to the nucleus through dimerization with Max. The cytoplasmic localization of Mad4 was determined by a CRM1-dependent nuclear export signal located near the amino terminus. We compared the relative efficiencies of complex formation among Myc, Max, and Mad family proteins in living cells using multicolor BiFC analysis. Max formed heterodimers with the basic helix-loop-helix leucine zipper (bHLHZIP) domain of Myc (bMyc) more efficiently than it formed homodimers. Replacement of two amino acid residues in the leucine zipper of Max reversed the relative efficiencies of homo- and heterodimerization in cells. Surprisingly, Mad3 formed complexes with Max less efficiently than bMyc, whereas Mad4 formed 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.

Id2 is dispensable for Myc-induced epidermal neoplasia.

We have previously described a transgenic mouse model of epidermal neoplasia wherein expression of a switchable form of c-Myc, MycER(TAM), is targeted to the postmitotic suprabasal keratinocytes of murine epidermis via the involucrin promoter. Sustained activation of c-MycER(TAM) results in a progressive neoplastic phenotype characterized by aberrant ectopic proliferation and delayed differentiation of suprabasal keratinocytes, culminating in papillomatosis. Transcription of the Id2 gene is regulated by Myc family proteins. Moreover, Id2 is implicated as a pivotal determinant of cell fate in multiple lineages and has a demonstrated role in mediating Myc-dependent cell proliferation in vitro through its interaction with retinoblastoma protein. Using Id2 nullizygous mice, we assessed in vivo the requirement for Id2 in mediating Myc-induced papilloma formation in skin. We show that absence of Id2 has no discernible impact on any measurable attribute of Myc function or on the timing or extent of eventual tumor formation. Thus, our data argue against any essential role for Id2 in mediating Myc action in vivo.

Functions of Myc:Max in the Control of Cell Proliferation and Tumorigenesis

Deregulation and elevated expression of members of the Myc family of bHLHZip transcription factors are observed in a high percentage of tumors. This close association with human cancers has led to a tremendous effort to define their biological and biochemical activities. Although Myc family proteins have the capacity to elicit a wide range of cell behaviors, their principal function appears to be to drive cells into the cell cycle and to keep them there. However, forced expression of Myc profoundly sensitizes normal cells to apoptosis. Therefore, tumor formation caused by deregulated Myc expression requires cooperating events that disrupt pathways that mediate apoptosis. Myc-dependent tumor formation may also be impeded by a set of related bHLHZip proteins with the demonstrated potential to act as Myc antagonists in cell culture experiments. In this review, we examine the complex activities of Myc family proteins and how their actions might be regulated in the context of a network of bHLHZip proteins.

Mad4 is regulated by a transcriptional repressor complex that contains Miz-1 and c-Myc.

Myc and Mad family proteins are central regulators of cellular proliferation and differentiation. We show that various Mad family genes have distinct patterns of expression during the chemically induced differentiation of mouse erythroleukaemia (MEL) cells, suggesting that they each serve a different function. Mad4 RNA is highly induced and persists in terminally differentiated cells, in agreement with observations in other systems. Using reporter gene assays in stably transfected MEL cells, we show that induction of Mad4 is mediated by a 49 nt core promoter region. We demonstrate that the initiator element is required for Mad4 activation, and show that induction is associated with the loss from the initiator of a complex that contains Miz-1 and c-Myc. Miz-1 activates the Mad4 promoter in transient transfection assays, and this effect is antagonized by c-Myc. We therefore identify Mad4 as a novel target of transcriptional repression by c-Myc. These data suggest that the expression of Mad4 in proliferating undifferentiated cells is suppressed by the binding of a c-Myc-Miz-1 repressor complex at the initiator, and that the activation of Mad4 during differentiation results, at least in part, from a decrease in c-Myc-mediated repression.

Gene-target recognition among members of the myc superfamily and implications for oncogenesis.

Myc and Mad family proteins regulate multiple biological processes through their capacity to influence gene expression directly. Here we show that the basic regions of Myc and Mad proteins are not functionally equivalent in oncogenesis, have separable E-box-binding activities and engage both common and distinct gene targets. Our data support the view that the opposing biological actions of Myc and Mxi1 extend beyond reciprocal regulation of common gene targets. Identification of differentially regulated gene targets provides a framework for understanding the mechanism through which the Myc superfamily governs the growth, proliferation and survival of normal and neoplastic cells.

Nmi protein interacts with regions that differ between MycN and Myc and is localized in the cytoplasm of neuroblastoma cells in contrast to nuclear MycN.

Myc family proteins play an important role in cellular processes such as proliferation, differentiation, apoptosis and transformation. A number of interaction partners of Myc have been identified, such as Max, p107, TBP, YY1, Miz-1, AP-2 and Nmi. Both Max and Nmi also bind to MycN. In contrast to the well defined binding of Max to Myc family proteins the interaction of Nmi with Myc or MycN is only poorly characterized. By employing the yeast two-hybrid system we have mapped the regions of MycN and Myc responsible for binding to Nmi. For MycN exclusively a central region mediates binding to Nmi. In contrast, for Myc a C-terminal portion of the protein, and possibly also a central part, is involved in Nmi interaction. Nmi does not interact with Max and has no transactivation capabilities in yeast, suggesting that Nmi alone is not a transcriptional activator in mammalian cells. Immunofluorescence demonstrates that both in 293 embryonic kidney cells and in Kelly neuroblastoma cells all detectable ectopically expressed Nmi is localized in the cytoplasm, in part in a punctate, granular pattern. MycN, which is highly expressed in Kelly cells consequent to amplification, appears to be localized exclusively in the nuclei. This directly demonstrates that in the same cell at least the major proportion of MycN and Nmi is localized in different cellular compartments. This result is confirmed by the finding that endogenous Nmi, which is expressed in Kelly cells only after stimulation with interferon gamma, is detected exclusively in the cytoplasm of these cells. Therefore only a very small amount of MycN and Nmi is likely to be involved in MycN/Nmi interaction in vivo.

Multiple androgen response elements and a Myc consensus site in the androgen receptor (AR) coding region are involved in androgen-mediated up-regulation of AR messenger RNA.

The androgen receptor (AR) gene is transcriptionally regulated by AR (autoregulation); however, the androgen response elements (AREs) required for this process have not been found in the AR promoter or in the 5'-flanking region. We previously showed that the AR cDNA contains AREs involved in AR mRNA autoregulation and that auto(up)regulation is reproduced in PC3 cells (a human prostate cancer cell line) expressing the human AR cDNA driven by a heterologous promoter. A 350-bp fragment of the AR cDNA contains the requisite AREs (ARE-1 and ARE-2) and, when linked upstream of a reporter gene, confers androgen inducibility in a cell-specific manner. Here we report that, although an AR cDNA harboring silent mutations of ARE-1 and ARE-2 produces a transcriptionally active AR, AR mRNA encoded by this mutant cDNA is not up-regulated in androgen-treated PC3 cells. Thus, ARE-1 and ARE-2 are essential for androgen-mediated up-regulation of AR mRNA in this model. Since ARE-1 and ARE-2 are located on separate exons (exons D and E) in the AR gene, we evaluated these AREs in their native context, a 6.5-kb AR genomic fragment. Androgen regulated the 6.5-kb AR genomic fragment and the 350-bp region of the AR cDNA at comparable levels, suggesting that sequences in exons D and E are likely to be involved in androgen-mediated up-regulation of the native AR gene. Furthermore, androgen regulated both responsive regions in U2OS cells, a human osteoblastic cell line that exhibits androgen-mediated up-regulation of native AR mRNA. DNAse I footprinting of the 350-bp region with recombinant AR (DNA- and ligand-binding domains) suggested the presence of additional AREs. Gel shift analyses and mutational studies showed that maximal androgen regulation and AR binding were dependent on the integrity of four AREs (ARE-1, ARE-1A, IVSARE, and ARE-2). While the presence of multiple, nonconsensus AREs is common among other androgen-regulated enhancers, the androgen-responsive region of the AR gene is unique because it contains exonic AREs. DNA binding studies with nuclear extracts were performed to determine whether non-AR transcription factors contribute to androgen regulation of the 350-bp region. These studies, in conjunction with mutational analysis and reporter gene assays with dominant negative Myc and Max expression vectors, showed that Myc and Max interaction with a Myc consensus site is required for androgen regulation of the 350-bp fragment. These results represent a novel interaction between AR and the Myc family of proteins and support a model of androgenic control of AR mRNA via AR and Myc family interaction with a unique internal androgen-responsive region harboring multiple exonic regulatory sequences.

Multiple Androgen Response Elements and a Myc Consensus Site in the Androgen Receptor (AR) Coding Region Are Involved in Androgen-Mediated Up-Regulation of AR Messenger RNA

The androgen receptor (AR) gene is transcriptionally regulated by AR (autoregulation); however, the androgen response elements (AREs) required for this process have not been found in the AR promoter or in the 5′-flanking region. We previously showed that the AR cDNA contains AREs involved in AR mRNA autoregulation and that auto(up)regulation is reproduced in PC3 cells (a human prostate cancer cell line) expressing the human AR cDNA driven by a heterologous promoter. A 350-bp fragment of the AR cDNA contains the requisite AREs (ARE-1 and ARE-2) and, when linked upstream of a reporter gene, confers androgen inducibility in a cell-specific manner. Here we report that, although an AR cDNA harboring silent mutations of ARE-1 and ARE-2 produces a transcriptionally active AR, AR mRNA encoded by this mutant cDNA is not up-regulated in androgen-treated PC3 cells. Thus, ARE-1 and ARE-2 are essential for androgen-mediated up-regulation of AR mRNA in this model. Since ARE-1 and ARE-2 are located on separate exons (exons D and E) in the AR gene, we evaluated these AREs in their native context, a 6.5-kb AR genomic fragment. Androgen regulated the 6.5-kb AR genomic fragment and the 350-bp region of the AR cDNA at comparable levels, suggesting that sequences in exons D and E are likely to be involved in androgen-mediated up-regulation of the native AR gene. Furthermore, androgen regulated both responsive regions in U2OS cells, a human osteoblastic cell line that exhibits androgen-mediated up-regulation of native AR mRNA. DNAse I footprinting of the 350-bp region with recombinant AR (DNA- and ligand-binding domains) suggested the presence of additional AREs. Gel shift analyses and mutational studies showed that maximal androgen regulation and AR binding were dependent on the integrity of four AREs (ARE-1, ARE-1A, IVSARE, and ARE-2). While the presence of multiple, nonconsensus AREs is common among other androgen-regulated enhancers, the androgen-responsive region of the AR gene is unique because it contains exonic AREs. DNA binding studies with nuclear extracts were performed to determine whether non-AR transcription factors contribute to androgen regulation of the 350-bp region. These studies, in conjunction with mutational analysis and reporter gene assays with dominant negative Myc and Max expression vectors, showed that Myc and Max interaction with a Myc consensus site is required for androgen regulation of the 350-bp fragment. These results represent a novel interaction between AR and the Myc family of proteins and support a model of androgenic control of AR mRNA via AR and Myc family interaction with a unique internal androgen-responsive region harboring multiple exonic regulatory sequences.

Identification of differentially regulated gene targets in the Myc and Mad family proteins

Identification of differentially regulated gene targets in the Myc and Mad family proteins

Regulation of c-Myc through Phosphorylation at Ser-62 and Ser-71 by c-Jun N-Terminal Kinase

The expression of c-myc promotes cell proliferation and also sensitizes cells to various extracellular apoptotic stimuli. However, signal pathways regulating the function of Myc proteins during apoptosis are unknown. c-Jun N-terminal kinase (JNK) is activated by various apoptotic stimuli, but neither the target molecule(s) or the action of JNK has been identified in Myc-mediated apoptosis. Here, we found that JNK selectively interacted with, and phosphorylated, c-Myc at Ser-62 and Ser-71 as confirmed with phospho-c-Myc-specific antibodies. Interestingly, dominant negative mutant JNK(APF) impaired the c-Myc-dependent apoptosis, but not mutated c-Myc (S62A/S71A)-dependent apoptosis triggered by UV irradiation. Furthermore, c-Myc (S62A/S71A)-expressing NIH3T3 cells were not sensitized like wild type c-Myc-expressing NIH3T3 cells to JNK-activating apoptotic stimuli, such as UV and Taxol. These results indicate that the JNK pathway is selectively involved in the c-Myc-mediated apoptosis and that the apoptotic function of c-Myc is directly regulated by JNK pathway through phosphorylation at Ser-62 and Ser-71.

The Myc oncoprotein: a critical evaluation of transactivation and target gene regulation

Mutations which disrupt the regulation or expression level of the c-myc gene are among the most common found in human and animal cancers (reviewed in ref. Cole, 1986; Henriksson and Luscher, 1996; Marcu et al., 1992). Ectopic expression studies define numerous biological activities of the c-myc gene, including transformation, immortalization, blockage of cell differentiation and induction of apoptosis (Askew et al., 1991; Cole, 1986; Evan and Littlewood, 1993; Freytag et al., 1990; Henriksson and Luscher, 1996; Marcu et al., 1992). Furthermore, c-myc is required for efficient progression through the cell cycle (Goruppi et al., 1994; Prochownik et al., 1988; Yokoyama and Imamoto, 1987), although recent studies indicate that it is not absolutely essential (Mateyak et al., 1997). This fascinating array of biological activities makes the c-myc gene one of the most intriguing oncogenes and presents the challenging question of how a single gene can manifest so many different effects. The c-Myc protein exhibits sequence-specific DNA binding when dimerized with its partner Max, and DNA binding is mediated through the basic region, which recognizes the core sequence CACGTG (Berberich et al., 1992; Blackwell et al., 1993; Blackwood and Eisenman, 1991; Prendergast and Ziff, 1991; Prendergast et al., 1991), but exhibits somewhat higher affinity for the more extended sequence ACCACGTGGT (Berberich et al., 1992; Blackwell et al., 1993; Halazonetis and Kandil, 1991). There are three closely related Myc family proteins (c-Myc, N-Myc and L-Myc), each with documented oncogenic potential (Birrer et al., 1988; Schwab et al., 1985; Yancopoulos et al., 1985) and similar DNA binding properties (Mukherjee et al., 1992). For simplicity, we will use the term Myc to refer to all three proteins, but delineate any distinct activities where they apply. The goal of this review is to discuss Myc as a transcriptional activator and critically evaluate the evidence for the transactivation of specific target genes as direct downstream effectors. Since excellent comprehensive reviews on Myc have been published recently (Facchini and Penn, 1998; Henriksson and Luscher, 1996), we will focus on the latest observations that offer mechanistic insight into transactivation and oncogenic transformation.

Myc boxes, which are conserved in myc family proteins, are signals for protein degradation via the proteasome.

Cellular levels of the rapidly degraded c-myc protein play an important role in determining the proliferation status of cells. Increased levels of c-myc are frequently associated with rapidly proliferating tumor cells. We show here that myc boxes I and II, found in the N termini of all members of the myc protein family, function to direct the degradation of the c-myc protein. Both myc boxes I and II contain sufficient information to independently direct the degradation of otherwise stably expressed proteins to which they are fused. At least part of the myc box-directed degradation occurs via the proteasome. The mechanism of myc box-directed degradation appears to be conserved between yeast and mammalian cells. Our results suggest that the myc boxes may play an important role in regulating the level and activity of the c-myc protein.

Dimerization and DNA binding facilitate alpha-helix formation of Max in solution.

Max is a basic region/helix-loop-helix/leucine zipper (b/HLH/Z) protein that forms a hetero-complex with the Myc family proteins Myc, Mad, and Mxi1, and a homo-complex with itself. These complexes specifically bind to double-stranded DNA containing CACGTG sequences. Here, we report on the structural properties in aqueous solution of a 109-amino-acid protein, Max110, corresponding to the N-terminal domain of Max containing the b/HLH/Z motif (residues 2-110), as characterized by combined use of circular dichroism (CD) and sedimentation equilibrium experiments. The results showed that the alpha-helical content of Max110 increases with increasing protein concentration. The sedimentation equilibrium data indicated that Max110 exists as a monomer at low protein concentration, and forms a dimer at high protein concentration. Further increases in the alpha-helical content of Max110 occur upon addition of DNA with the CACGTG recognition sequence. Thus, dimerization and binding to DNA of Max both favor an increase of the alpha-helical content.

Analysis of the DNA-binding activities of Myc/Max/Mad network complexes during induced differentiation of U-937 monoblasts and F9 teratocarcinoma cells.

The bHLHZip protein Max interacts with both the Myc and Mad family proteins forming heterodimers which specifically bind certain E-box DNA recognition sequences, thereby regulating transcription. Whereas Myc proteins actively promote cell proliferation, Mad complexes have the opposite function. Although the main regulation of this network seems to be the control of myc- and mad family gene expression, regulation at the level of DNA-binding and transactivation may also be in operation. Few studies on the DNA-binding activity of native Myc:Max or Max:Mad complexes have been reported mainly due to technical difficulties. To overcome these problems we have developed a specific and sensitive solid phase DNA-binding assay based on partial purification of native Myc, Max and Mad1 complexes by immunological methods. Using this technique we report that the DNA-binding activity of c-Myc-containing complexes is reduced during induced differentiation of U-937 monoblasts and F9 embryonic teratocarcinoma cells. In contrast, the DNA-binding of Mad1-containing complexes increases during monocytic differentiation. In general, the DNA-binding activity of c-Myc and Mad1 correlate with their expression. However, our studies of early kinetics of TPA-induced differentiation of U-937 cells as well as of late events during F9 differentiation suggest that post-translational regulation of Myc and Max DNA-binding may also occur. The solid phase DNA-binding assay may thus provide a tool to study the regulation of DNA-binding in more detail.

Regulation of cell growth by the Myc-Max-Mad network: role of Mad proteins and YY1.

Since the discovery of the c-Myc dimerization partner Max (Blackwood and Eisenman 1991; Prendergast et al. 1991) a startling number of proteins capable to interact directly or indirectly with c-Myc and/or Max have been described (for review see Marcu et al. 1992; Henriksson and Luscher 1996). It has become evident that at the center of this network lies Max which on one hand forms heterodimers with the Myc family of proteins, including c-, N-, and L-Myc and on the other hand heterodimerizes with proteins of the Mad family, including Mad1, Mxi1 (or Mad2), Mad3, and Mad4. In addition Max has also the ability to form homodimers. Thus Max is the true center of this array of proteins which we will refer to as the Myc/Max/Mad network.

A biochemical and biological analysis of Myc superfamily interactions.

Members of the myc family of nuclear proto-oncogenes (c-, N- and L-myc) play central roles in the control of normal growth and development and in genetic pathways linked to cellular transformation and apoptotic cell death (for reviews see [19, 40]). Accumulating structural, biochemical, and genetic evidence affords the view that the function of Myc family oncoproteins in these diverse processes relates in part to their roles as sequence-specific transcription factors (for reviews see [31, 58]). Myc family proteins possess a multi-functional amino-terminal domain with transactivation potential [32], a region rich in basic amino acid residues responsible for sequence-specific DNA binding activity to the E-box consensus CACGTG [8], and a carboxy-terminal α-helical domain required for dimerization with another basic region helix-loop-helix/leucine zipper (bHLH/LZ) protein, Max [9, 43]. Many of the biochemical and biological activities of Myc appear to be highly dependent upon its association with Max [1, 2, 9, 35, 43). In addition to its key role as an obligate partner in transactivation-competent Myc/Max complexes, Max may also repress Myc-responsive genes through the formation of transactivation-inert complexes that are capable of binding the Myc/Max recognition sequence [5, 10, 33, 35, 39, 41, 44, 65]. These complexes include Max/Max homodimers, and the heterodimers Mad/Max [5, 29] and Mxil/Max [65].

Mad3 and Mad4: novel Max-interacting transcriptional repressors that suppress c-myc dependent transformation and are expressed during neural and epidermal differentiation.

The basic helix-loop-helix-leucine zipper (bHLHZip) protein Max associates with members of the Myc family, as well as with the related proteins Mad (Mad1) and Mxi1. Whereas both Myc:Max and Mad:Max heterodimers bind related E-box sequences, Myc:Max activates transcription and promotes proliferation while Mad:Max represses transcription and suppresses Myc dependent transformation. Here we report the identification and characterization of two novel Mad1- and Mxi1-related proteins, Mad3 and Mad4. Mad3 and Mad4 interact with both Max and mSin3 and repress transcription from a promoter containing CACGTG binding sites. Using a rat embryo fibroblast transformation assay, we show that both Mad3 and Mad4 inhibit c-Myc dependent cell transformation. An examination of the expression patterns of all mad genes during murine embryogenesis reveals that mad1, mad3 and mad4 are expressed primarily in growth-arrested differentiating cells. mxi1 is also expressed in differentiating cells, but is co-expressed with either c-myc, N-myc, or both in proliferating cells of the developing central nervous system and the epidermis. In the developing central nervous system and epidermis, downregulation of myc genes occurs concomitant with upregulation of mad family genes. These expression patterns, together with the demonstrated ability of Mad family proteins to interfere with the proliferation promoting activities of Myc, suggest that the regulated expression of Myc and Mad family proteins function in a concerted fashion to regulate cell growth in differentiating tissues.