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REVIEW |
a Ontario Cancer Institute/Princess Margaret Hospital, Division of Cell and Molecular Biology, Department of Medical Biophysics, University of Toronto, Toronto, Canada M5G 2M9
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONOVERVIEW OF c-MYC STRUCTURE,...REFERENCES |
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Key Words: c-myc • oncogene • transactivation • repression • transcription • cancer • gene regulation
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONOVERVIEW OF c-MYC STRUCTURE,...REFERENCES |
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OVERVIEW OF c-MYC STRUCTURE, REGULATION, AND FUNCTION |
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TOPABSTRACTINTRODUCTIONOVERVIEW OF c-MYC STRUCTURE,...REFERENCES |
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The product of the c-myc proto-oncogene is a highly conserved nuclear phosphoprotein. P1- and P2-initiated mRNA transcripts both have the capacity to encode the two major species of human c-Myc protein: Myc1, with an apparent molecular mass of 67 kDa; and a 64 kDa protein, Myc2. The former contains an additional 14 amino acids at the amino-terminal end of the Myc molecule. The relative abundance of Myc2 vs. Myc1 protein varies among tissues and cell lines, with the 64 kDa Myc2 species being the major isoform in most cases. Recently, differential transcription regulatory properties and activities have been suggested for these two Myc proteins, with Myc2 being involved in cell growth and Myc1 protein mediating cellular quiescence in COS cells (7). A third isoform of c-Myc protein, termed Myc-S, has been described recently (8). This protein originates from a translation initiation site approximately 100 amino acids downstream of the amino terminus and lacks Myc transcriptional activation activity. Unless otherwise indicated, c-Myc protein refers to the more abundant, growth-promoting 64 kDa Myc2 molecule.
The c-Myc protein is highly conserved among different species. Homologues to human c-Myc have been isolated from mammals, birds, frogs, starfish (as reviewed in refs 9, 10), and most recently, Drosophila (11). However, no homologous gene or protein has yet been isolated from yeast or nematodes. The c-Myc oncoprotein belongs to a larger family of related genes. Members of this family directly involved in the transformation process include the cellular N-myc and L-myc genes (12, 13). Both N-myc and L-myc genes conserve the three exon structure of c-myc, with coding sequences contained exclusively in exons II and III. The N-Myc and L-Myc proteins share several regions of at least 80% amino acid sequence homology to c-Myc, including the Myc box I (MbI) and Myc box II (MbII) domains as well as a basic helix-loop-helix and leucine zipper region (BR-HLH-Zip) (
Fig. 1).
As these domains are also conserved among species, it is believed that they define important functional domains.
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The c-Myc protein structure has not yet been solved. Structural modeling predicts that the c-Myc protein is organized into three domains: a globular end from amino acids 1 to 203, a middle unstructured region between amino acids 204 to 237, and an 
-helical carboxyl end from amino acids 238 to 439. The amino-terminal domain is comprised of putative activation domains, including glutamine-rich and proline-rich regions, from amino acids 1 to 41 and 41 to 103, respectively, and an acidic region from amino acids 104 to 143. Indeed, the first 143 amino acids of c-Myc can function as a transcriptional activator of a GAL4 reporter construct when linked to a GAL4 DNA binding domain (
Fig. 1) (14). The amino terminus also contains the highly conserved MbI (amino acids 45 to 63) and MbII (amino acids 129 to 141) domains. The latter is essential for all known Myc biological activities, yet recent data suggest both of these highly conserved regions may be sites of interaction with cellular proteins (see below). The Myc protein contains two nuclear localization sequences (NLS): a primary motif at amino acids 320 to 328 and a secondary signal from amino acids 364 to 374. The second NLS falls within a basic region (BR) (amino acids 355 to 368) implicated in specific DNA sequence recognition and binding. Immediately downstream of this domain are contiguous helix-loop-helix (amino acids 368 to 410) and leucine zipper (amino acids 411 to 439) (HLH-Zip) motifs responsible for specific heterodimer formation between Myc and its binding partner, Max. Contiguous BR-HLH-Zip motifs are characteristic of transcription factors that bind to specific DNA sequences as multimeric complexes, such as USF, TFE3, and TFEB (15–17). Thus, c-Myc has the potential to undergo protein–'otein interactions, bind to specific DNA sequences, and modulate gene transcription.
Regulation of c-myc expression and function
In general, c-myc expression correlates tightly with the proliferative potential of the cell (reviewed in refs 4, 18). In quiescent cells, c-myc expression is virtually undetectable. Upon mitogen or serum stimulation, there is a rapid and transient burst in both c-myc mRNA and c-Myc protein expression as cells enter the G1 phase, followed by a gradual decline to low but detectable steady-state levels in proliferating cells (19–21). This induction of c-myc transcription occurs in the absence of de novo protein synthesis, indicating that c-myc is an immediate early gene and is directly downstream of mitogenic signaling cascades. In the continual presence of growth factors, basal c-myc expression remains invariant throughout the cell cycle (22–24). However, upon growth factor withdrawal or terminal differentiation signals, c-myc mRNA and c-Myc protein levels decline and are virtually undetectable in terminally differentiated cells, although exceptions have been noted (25–27). Whether c-myc down-regulation is the trigger for differentiation or a consequence of this cell fate is unclear.
A variety of overlapping control mechanisms cooperate to regulate c-myc, allowing Myc expression and function to be rapidly and efficiently modulated in response to internal and external signals. c-myc RNA production is modulated transcriptionally at the level of initiation through mechanisms such as the homeostatic Myc negative autoregulation mechanism (28, 29) as well as by transcriptional attenuation. c-Myc expression is also regulated posttranscriptionally at the levels of protein translation and mRNA stability. Indeed, c-myc mRNA and Myc protein are both short-lived molecules with half-lives of less than 30 min (23, 30, 31). Thus, to achieve functional Myc activity within the cell, active and continuous synthesis of both c-myc RNA and protein is required.
Regulation of c-Myc protein activity also occurs posttranslationally through direct and indirect mechanisms. These can potentiate or antagonize Myc activity, and include protein phosphorylation as well as interaction with other cellular proteins. c-Myc can be phosphorylated at more than a dozen serine and threonine sites, including Thr58, Ser62, Ser71, Ser82, and Ser164 at the amino terminus, a cluster of five sites within residues 240 to 262, as well as Ser293, Thr343, Ser344, Ser347, and Ser348 at the carboxyl terminus ( Fig. 1) (32–34). Of these sites, Thr58 and Ser62 appear to be the most functionally relevant. Both mitogen-activated protein kinase and p34cdc2 can phosphorylate Ser62, whereas Thr58 is a target of glycogen synthase kinase 3 (35–38). Hierarchical phosphorylation of these two residues is cell cycle regulated and appears to be linked to Myc transformation activity (33, 36, 37). Mutation of Thr58 can enhance focus formation in a cotransformation assay with activated Ras oncoprotein, whereas a Ser62 substitution can severely inhibit transforming activity (37). Conflicting data surround the role of Thr58/Ser62 phosphorylation in Myc transactivation activity. One study concluded that Thr58/Ser62 phosphorylation is required for maximal transactivation by Myc (39), whereas another demonstrated that mutations at these sites do not affect Myc transactivation through promoter E-box elements (33). The role of phosphorylation in Myc function at these and other sites clearly requires further investigation.
Biological activities of c-Myc protein
Regulated c-myc gene expression is critical for controlled cell proliferation whereas deregulated, often constitutive, expression of c-myc characterizes oncogenic activation and is a frequent hallmark of tumor-derived cells. Myc deregulation can be defined as an inappropriate, nonphysiological increase in Myc activity. Gross genetic alterations resulting in oncogenic activation of c-myc include translocation, proviral insertion, and gene amplification (2, 3). Myc activation may also be achieved by more subtle mutations that disrupt any one of the many regulatory mechanisms controlling c-myc expression (40). Deregulation may also occur by altering the concentration of functional Myc protein within the cell through point mutations in Myc protein coding sequences (41). These mutations may disrupt binding to a negative regulator or alter protein secondary structure and thereby increase the specific activity of the Myc polypeptide. For example, Myc activation in BL involves a chromosomal translocation that places Myc coding sequences under the control of constitutively active immunoglobulin enhancer elements. In many BL tumors, the activated c-Myc protein contains amino-terminal point mutations thought to further affect phosphorylation patterns and/or biological potency (41). Thus, although numerous gross genetic alterations affect c-myc deregulation, subtle activation mechanisms also exist and have yet to be fully defined.
Activated c-myc expression has been shown to contribute to the stepwise progression of tumorigenesis in both in vitro and in vivo systems. Constitutive overexpression of ectopic c-Myc can immortalize fibroblasts grown in culture and prevent withdrawal from the cell cycle (42, 43). Accompanying this effect are a decreased requirement for serum growth factors (44–46) and a shortening of the G1 phase of the cell cycle (47). In primary rat embryo fibroblasts and hematopoietic cells, constitutive expression of c-myc, along with a cooperating oncogene such as ras or v-abl, results in malignant transformation (48, 49). Domains of the c-Myc protein required for this activity localize to MbII in the amino terminus and the carboxyl-terminal BR-HLH-Zip motifs (50). Furthermore, Myc oncogenic activity is wholly dependent on Myc-Max association, suggesting that Myc regulation of gene transcription is a component of the Myc transformation mechanism (51).
In cell cycle regulation, Myc is required for G1 to S phase progression of primary cells, and induction of c-Myc expression stimulates quiescent nontransformed cells to traverse G1 phase and enter S phase (52, 53). Enormous effort has been devoted to identify the critical G1 checkpoint regulated by Myc. It has recently been shown that introduction of exogenous Myc expression will bypass G1 phase growth arrest imposed by ectopic expression of the p16INK4a cyclin-dependent kinase inhibitor in rodent fibroblasts (54, 55). Under these conditions, Myc drives the cell cycle by up-regulating cyclin E-cdk2 activity in the presence of hypophosphorylated retinoblastoma susceptibility protein (pRb) family. Independent experiments further suggest that Myc acts upstream of cyclin E and in parallel with pRb to regulate the cell cycle. The mechanism for Myc activation of cyclin E activity is thought to involve release of the p27Kip1 cyclin-dependent kinase inhibitor from the cyclin E-cdk2 complex (54–58). Myc expression contributed to only a part of the induction of cdk2 activity, and full activity was achieved by expression of Myc along with other growth factors (58). This observation is in keeping with reports placing Myc downstream of Src, but separate from Ras signaling pathways (59), and the observation that Ras cooperates with Myc to induce cyclin E-dependent kinase activity in REF52 cells (60). Whereas c-Myc plays a key role during the G0/G1 to S phase transition, continuous low-level c-myc expression throughout the cell cycle suggests that it is required at other phases. Indeed, it has been shown that disruption of both c-myc alleles in Rat-1 cells leads to a prolonged G2 as well as G1 phase of the cell cycle (61). In these distinct phases, Myc may regulate a variety of similar or distinct functions. The best experimental evidence to support this concept comes from elegant studies of Xenopus oocytes, which suggests that Myc may be directly involved in regulating DNA replication in the absence of gene transcription [see Lemaitre et al. (27) for a comprehensive review of Myc function during embryogenesis].
Since expression of c-Myc is a strong potentiator of cellular proliferation, down-regulation of c-Myc expression accompanies terminal differentiation and permanent withdrawal from the cell cycle. Simply blocking c-Myc activity by using antisense oligonucleotides, ectopic expression of Max, or dominant interfering Myc molecules can induce differentiation of HL-60, F9, K562, and MEL cells (62–65). Similarly, several independent studies report that ectopic c-Myc or v-Myc expression is sufficient to block the induction of differentiation in MEL, 3T3-L1 preadipocyte, F9 teratocarcinoma, PC12 neuronal, and U-937 monoblastic cell lines (66–72). Some exceptions show that Myc expression is not always uncoupled from differentiation. For example, ectopic Myc expression can drive (73) as well as prevent (74) full myoblast differentiation. Indeed, it has recently been shown that Myc activation in keratinocyte stem cells can stimulate execution of the differentiation program (75). In addition, endogenous Myc expression has been noted in differentiating cells of neuronal as well as other lineages (25, 26, 76–79); for additional references, see ref 27). The significance of these unique associations of Myc expression with cell differentiation remains unclear. The domains of the c-Myc protein required to inhibit cellular differentiation closely overlap with transforming regions of the protein, including BR-HLH-Zip domains and transcription regulatory regions (80). Despite this overlap, the ability of Myc to block cell differentiation can be uncoupled from its ability to drive cell cycle progression (81, 82). Identifying the pathways downstream of Myc involved in each of these activities will ultimately reveal their interdependence. In recent years it has consistently been observed that Myc can antagonize CCAAT/enhancer binding protein (C/EBP) activity, a transcription factor whose expression strongly promotes adipocyte differentiation in 3T3-L1 cells (83–85). Indeed, Myc can repress transcription of the C/EBP gene in NIH 3T3 fibroblasts and murine hibernoma cells through core promoter elements, although it is not known whether this occurs directly or indirectly (86, 87). Moreover, Myc expression can inhibit C/EBP-dependent transcription activation of myelomonocytic-specific genes as well as the growth arrest and DNA damage-inducible gene 45 (gadd45) (83, 85). The full biological role of Myc in differentiation requires further investigation; however, the ability of Myc to prevent growth arrest and differentiation likely involves Myc-mediated modulation of gene transcription.
Myc so actively prevents cells from exiting the cell cycle that Myc activation and growth arrest appear to be mutually exclusive and incompatible with cell viability (88, 89). Myc plays a key role in regulating apoptosis in different cell types and under a wide variety of physiological conditions (88–92). For example, anti-sense c-myc RNA inhibits activation-induced programmed cell death in CD3 cross-linked T cell hybridomas (93) and apoptosis induced by tumor necrosis factor is dependent on c-Myc expression (94, 95). An exception can be found in immature B cell lymphoma cell lines, where induction of apoptosis by transforming growth factor ß or immunoglobulin is preceded by a decline in c-myc expression that can be prevented by stabilization of c-Myc protein (96, 97). The molecular events associated with Myc-induced apoptosis are not well defined but may involve p53-dependent and -independent pathways, cyclin D3, and the Fas/Fas ligand signaling cascade (92, 98–101). Inhibition of Myc-induced apoptosis appears critical in order to allow the full transformation potential of Myc to be realized. Inhibitors include Bcl-2 as well as specific growth and survival factors such as platelet-derived growth factor and insulin-like growth factor-1 (102–104). The latter is thought to function through activation of phosphatidylinositol-3 kinase, protein kinase B/Akt, and downstream effector substrates (105, 106). Identification of inhibitors and potentiators of Myc-induced apoptosis will reveal both the molecular mechanisms of this pathway and the nature of cooperating mutations that can abrogate this mechanism of programmed cell death and thus contribute to the potent transforming properties of Myc.
The ability of Myc to promote proliferation and apoptosis as well as block differentiation is strongly supported by animal models. Transgenic mice expressing a c-myc transgene under the control of the immunoglobulin heavy-chain enhancer promoter (Eµ-myc) accumulate large pools of polyclonal undifferentiated pre-B lymphocytes (107, 108). One hypothesis for the potent transforming properties of c-Myc suggests that the main effect of Myc is to inhibit terminal differentiation by preventing cells from exiting the cell cycle. This results in an enlarged population of undifferentiated precursor cells, thereby increasing the probability that a significant number of cells will acquire further transforming mutations (109). Indeed, the Eµ-myc mice do show evidence of tumor formation (110, 107). However, the onset of tumor formation is greatly accelerated in double transgenic mice expressing Eµ-myc and other cooperating oncogenes such as Eµ-abl, Eµ-bcl-2, and Eµ-pim-1, as well as in MMTVD/myc mice overexpressing Notch1 (111–114). When ES cells harboring a targeted gene disruption of both c-myc alleles are tested for developmental potential, the resultant knockout mutation is lethal at embryonic day 9.5 to 10.5 (115), which further underscores the essential biological role of Myc in growth control. Thus, c-myc is required for embryonic development; although oncogenic activation of c-myc alone is a strong potentiator of cellular proliferation, transformation to the malignant phenotype requires additional cooperating oncogenic lesions.
MOLECULAR MECHANISMS OF MYC ACTIVITIES
Myc-Max interaction is key to Myc function
Experimental evidence clearly defines the critical role of c-Myc in various biological activities, including growth potentiation and transformation. The next challenge lies in understanding the molecular mechanisms that form the basis of Myc function. Because Myc protein contains two highly conserved dimerization motifs, binds to a specific DNA element (116), and Myc homodimers do not form at physiological Myc protein concentrations, it was hypothesized that Myc function may be dependent on at least one cellular binding partner. Indeed, another cellular BR-HLH-Zip protein with high binding affinity for Myc was identified (117, 118) ( Fig. 1). Like Myc, Max is a well-conserved, predominantly nuclear phosphoprotein. Myc-Max interaction is required for key biological functions of Myc, including G1 to S phase cell cycle progression, Myc-induced apoptosis, Myc-negative autoregulation, cellular transformation (51, 118–121), and Myc-mediated transcriptional activation (122). Alternatively spliced mRNA transcripts yield two major isoforms of Max protein: the p21 Max (short) and p22 Max (long) protein containing a nine amino acid insert within the amino terminus of the molecule. Structurally, Max contains a nuclear localization sequence at the 23 carboxyl-terminal amino acids and a BR-HLH-Zip domain from amino acids 15 to 99, but lacks transcription regulatory motifs (reviewed in refs 5, 123).
In contrast to c-myc, Max protein expression is abundant, remains constant in quiescent and proliferating cells, and is not responsive to mitogenic signaling (124, 125), whereas max RNA levels may be up-regulated by serum growth factors (118, 126). The expression of max in differentiated cells appears to vary with cell type. A suppression of max was observed during erythroid and myelomonocytic differentiation (127, 128) but not upon differentiation of U-937, HL-60, or ML-1 myeloid cells (129). Growth-regulated max expression has been noted in one epithelial cell line (126), and a unique report describes the complete absence of wild-type Max protein (due to the synthesis of mutant max mRNA transcripts) in the PC12 pheochromocytoma cell line (130). The biological implications of these exceptions are unclear.
Myc family proteins form high-affinity heterodimers with Max both in vitro and in vivo (131) that are capable of recognizing and binding to specific DNA E-box elements to activate transcription (117, 118, 122, 131, 132). Analysis of binding sites for Myc-Max heterodimers by in vitro-selected and amplified binding sequence assays identified CACGTG and CACATG as the preferred binding sites, as well as several low-affinity, noncanonical sites (116, 133, 134). However, both CACGTG and CACATG are recognized with high affinity by other BR-HLH-Zip transcription factors, including the highly abundant USF and TFE3 family. To determine high-affinity in vivo binding sites specific for Myc-Max heterodimers, Myc- and Max-specific antibodies were used to immunoprecipitate genomic DNA fragments from Myc- and Max-transformed CB33 lymphocytes (135). The critical determinants of the binding sites isolated by this method included 5' and 3' flanking dinucleotides as well as the inner hexamer core. The majority of these sites corresponded to previously identified noncanonical sites, indicating that the preferred binding sites in vivo may be distinct from those described in vitro.
A variant form of Max protein, dMax, was isolated in detergent nuclear extracts of NIH 3T3 fibroblasts (136). This mutant Max protein of 16 to 17 kDa apparently results from alternative splicing of max mRNA. This splice variant encodes the 9 amino acid insert but possesses an internal deletion of 108 bp, resulting in the removal of the basic, helix 1, and loop regions of the Max protein. The dMax protein binds c-Myc in vivo with high affinity, presumably through the intact Zip motif, but these c-Myc-dMax complexes cannot bind the CACGTG DNA element. Moreover, cotransfection of dMax into NIH 3T3 cells interferes with c-Myc transactivation of E-box-containing promoter constructs, indicating that dMax may function as a potential dominant-negative regulator of c-Myc activity.
In addition to heterodimer formation with Myc, Max also forms homodimers and can heterodimerize with members of a network of related BR-HLH-Zip proteins: Mad1, Mad3, Mad4, Mxi1, and Mnt1 (137–140). Max homodimers and Mad-Max, Mxi1-Max, or Mnt1-Max heterodimers can compete for occupation of E-box elements recognized by Myc-Max complexes. Unlike Myc-Max heterodimers, these other Max complexes repress transcription when bound to E-box sites (137, 138, 141). Max-Max homodimers can antagonize the activity of Myc-Max by competitively binding to the same elements. Mad-Max heterodimers repress transcription through an active mechanism. This repression depends on interaction of Mad with repressor complexes consisting of (at least) mSin3, N-Cor, and histone deacetylases (142; reviewed in ref 143). Mad and Mxi1 proteins are expressed in differentiated, nonproliferating cells and may shut off target genes that are activated by Myc under growth-promoting conditions (for a review, see refs 5, 123). The expression of Mnt1, on the other hand, coincides with Myc expression; Mnt1-Max complexes thus may antagonize Myc transcription activation activity.
Other Myc binding proteins
Myc also interacts with a diverse collection of cellular proteins through amino and carboxyl end motifs ( Fig. 1). Some of these interactions can occur in the presence of Max, but others preclude Myc-Max association. In vitro and in vivo interactions have been reported between the amino-terminal transcription regulatory domain of c-Myc and the transformation-associated protein (TR-AP), p107 tumor suppressor, BIN-1, the TATA binding protein (TBP), and -tubulin proteins (144–148; M. Cole, personal communication). Furthermore, c-Myc can form complexes through BR-HLH-Zip domains with the transcription factors Yin Yang-1 (YY-1), AP-2, Miz-1 (Myc interacting zinc finger protein), and the Nmi-1 protein (149–152). In addition, c-Myc protein binds in vitro with the initiator binding protein TFII-I and with pRb (153, 154).
Several of these Myc interacting proteins bind to functionally significant regions of the Myc protein to enhance or abrogate various Myc activities. For example, a novel Myc binding complex was identified whose activity is dependent on interaction with the highly conserved and functionally critical MbII domain (155). More recently, biochemical and molecular analyses have led to the identification of a key component of this complex, TR-AP, a very large, highly conserved novel protein that is ubiquitously expressed and binds to c-Myc, N-Myc, and L-Myc proteins (M. Cole, personal communication). Deletion of MbII in c-Myc disrupts complex formation with this nuclear factor and links Myc-TR-AP interaction to Myc cotransformation activity ( Fig. 1). Indeed, TR-AP participates in the positive growth regulatory functions of Myc, since titration of this molecule by dominant-negative Myc mutants blocks transformation by wild-type Myc protein. It is important that ectopic expression of truncated forms of TR-AP protein can abolish Myc, but not E1A, cotransformation with Ras. Antisense TR-AP RNA blocks Myc-Ras cotransformation, providing further evidence that TR-AP is required for Myc function. Disruption of the yeast homologue of TR-AP, TRA1, leads to growth arrest, underlining the essential function of this protein in the yeast cell cycle. The molecular function of TR-AP is currently unknown. Although the carboxyl terminus demonstrates significant homology to a domain found among members of the PI-3 kinase family, TR-AP is not predicted to exhibit kinase activity due to the absence of critical analytic residues. It is tempting to speculate that TR-AP may function as a transcriptional adapter protein to promote Myc-Max transcription regulatory activities; however, additional models of TR-AP function can be postulated, and further investigation of this novel Myc binding protein is required.
A second Myc interacting protein, p107, is a member of the pRb family and, like pRb, arrests cells at the G1 phase of the cell cycle when in a hypophosphorylated state (145, 146). Coimmunoprecipitation experiments identified p107 protein binding to Myc through a portion of the Myc transactivation domain from amino acids 41 to 103, including phosphorylated Thr58 and Ser62 residues ( Fig. 1). Binding of p107 to Myc inhibits c-Myc transactivation activity on a reporter construct containing Myc-Max CACGTG E-box sites; however, the physiological consequence of such interaction is unclear (145, 146). For example, in some lymphomas, p107 binds to the amino terminus of a mutant Myc protein expressing an altered phosphorylation pattern but no longer inhibits c-Myc transactivation activity (156). As Myc has also been reported to bind in vitro to the product of the retinoblastoma (RB1) gene (154), it will be interesting to determine whether Myc can also interact with other pRb family members and to further explore the biological consequences of Myc interaction with these important cell cycle modulators.
A yeast two-hybrid screen of an E10.5 murine embryo cDNA library, with a portion of the MbI domain as bait, isolated a novel Myc binding protein, BIN-1 (148). BIN-1 requires intact MbI and MbII domains for binding and antagonizes the oncogenic activity of Myc ( Fig. 1) (G. Prendergast, personal communication). Expression of BIN-1 inhibits cotransformation of primary rat embryo fibroblasts by Ras and Myc through a Myc-dependent mechanism, and can also inhibit E1A- and p53-mediated transformation via Myc-independent mechanisms. Consistent with its role as a putative tumor suppressor molecule, BIN-1 expression is either lacking or altered in tumor cells. Furthermore, expression of BIN1 protein is up-regulated and structurally altered during differentiation of C2C12 myoblasts. These changes in BIN-1 do not appear sufficient to initiate differentiation, but rather enhance the differentiation program once the differentiation signals have been triggered. BIN-1 will inhibit transactivation activity of Myc in a manner that depends on promoter context: specifically, BIN-1 expression in NIH 3T3 and HeLa cells inhibited Myc transactivation of the ornithine decarboxylase (ODC) promoter, but not of a reporter construct containing four Myc E-box elements upstream of a CAT gene. This repression activity is not intrinsic to BIN-1; rather, BIN-1 may recruit a repression activity to the Myc-responsive promoter. It is possible that BIN-1 antagonizes Myc's oncogenic activity by interfering with transactivation of Myc target genes.
YY-1 is a multifunctional, zinc finger DNA binding protein that acts as an activator, repressor, and/or initiator of transcription. YY-1 associates in vitro and in vivo with the carboxyl terminal of the c-Myc protein (152, 157) ( Fig. 1). This interaction is independent of Max, as Myc and YY-1 fail to form a ternary complex with GST-Max fusion protein. The presence of Max will abolish Myc binding to GST-YY-1 (152), and overexpression of YY-1 in COS-7 cells cannot disrupt Myc-Max heterodimer formation (B. Luscher, personal communication). Nevertheless, Myc overexpression can antagonize YY-1 activity and alter the levels of free YY-1 within the nucleus, possibly through a Max-independent mechanism. Cotransfection of YY-1 and Myc in P3X plasmacytoma cells strongly inhibits YY-1-mediated activation of a c-myc promoter-luciferase reporter construct. As Myc-YY-1 association does not block the ability of YY-1 to bind DNA, it has been suggested that this effect of Myc on YY-1 activity may involve interference of TBP and/or TFIIB binding to YY-1 (157). The Myc-YY-1 interaction also has ramifications for Myc function, as YY-1 can antagonize the growth-promoting activities of c-Myc (B. Luscher, personal communication). It will be interesting to examine this interaction in greater detail, since Myc binding to YY-1 is one of the few Max-independent Myc properties.
The AP-2 transcription factor is another cellular protein that binds c-Myc through the BR-HLH-Zip domains (150) ( Fig. 1). In contrast to the Myc-YY-1 interaction, association of AP-2 and the c-Myc carboxyl terminal can occur concurrent with Myc-Max heterodimer formation. This interaction interferes with binding of the Myc-Max-AP-2 complex to DNA E-box elements and consequently abrogates Myc-Max transcriptional activation. The -prothymosin gene contains AP-2 sites adjacent to and overlapping with Myc E-box elements. Furthermore, binding of AP-2 to the AP-2 element within the -prothymosin gene can quench Myc-Max transcription activation of this gene at the E-box sites (150). Thus, AP-2 acts as a negative regulator of Myc transactivation activity by two distinct mechanisms.
Another protein that interacts with the carboxyl terminus of c-Myc, Miz-1 may participate in at least one mechanism by which Myc negatively regulates transcription (see below) (151). Identified by a yeast two-hybrid screen, Miz-1 is a novel POZ domain protein that interacts specifically with the HLH domain of Myc but not of Max or USF ( Fig. 1). In the absence of Myc, Miz-1 binds to the transcription initiator regions of the adenovirus major late (AdML) and cyclin D1 promoters and activates transcription. Expression of Myc inhibits Miz-1 activation, suggesting a mechanism whereby Myc represses transcription of these two promoters. Additional experiments are required to elucidate the molecular role of Miz-1 in Myc-mediated repression and cellular transformation, as ectopic expression of Miz-1 results in growth arrest.
In addition to Max, the other Myc binding proteins that are likely to play an integral or accessory role to promoting Myc function include TR-AP, TBP, and Miz-1. Conversely, Myc interacting proteins such as AP-2, BIN-1, p107, and YY-1 function primarily as antagonists of Myc activity. The molecular and biological interplay among these diverse proteins with respect to Myc transformation, proliferative, and apoptotic activities has yet to be explored. As the family of Myc binding proteins grows, it may become evident that certain factors influence specific Myc activities whereas others, like Max, affect all pathways of Myc function.
Transcriptional activation by Myc
One approach to understanding how Myc activity at the molecular level translates into growth promotion and transformation is to examine the nature, function, and regulation of direct molecular targets of Myc and work up from the level of the gene. Several genes have been identified whose expression is up-regulated in response to c-myc expression, but it remains unclear whether increased expression is a direct result of Myc-Max transactivation or an indirect consequence of Myc-induced cell cycle progression. For example, cyclin A mRNA and protein levels are up-regulated with delayed kinetics by inducible, ectopic Myc expression, but conflicting opinions exist as to whether this induction reflects a direct or indirect mechanism (57, 158–160). Despite these complexities, a growing number of cellular genes that represent direct targets of Myc-Max transactivation have been isolated ( Table 1),
including the -prothymosin, carbamoyl-phosphate synthase/aspartate carbamoyltransferase/dihydroorotase (cad), cdc25a, ECA39, eukaryotic translation initiation factor eIF-4E, interferon-stimulated gene factor 3 gamma (ISGF3
), lactate dehydrogenase A (LDH-A), MrDb RNA helicase, ODC, p53, and the novel rcl gene (135, 161–170). These genetic targets of Myc activation can be placed into four general categories on the basis of function. ODC, cad, and LDH-A are rate-limiting enzymes for the biosynthesis and metabolic production of polyamines, pyrimidines, and lactate, respectively, essential molecules required for cell division and growth. Similarly, MrDb and eIF-4E play critical roles in governing RNA structure and metabolism, thereby participating in global regulation of gene expression. Myc transactivation of cdc25a, ISGF3, and p53 can play an important role in cell cycle control. Finally, the molecular function of -prothymosin, ECA39, and rcl are as yet unknown. It thus appears that many target genes whose expression is directly up-regulated by Myc belong to components of key cellular pathways.
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The target genes effecting the strong role of Myc in transformation might be those directly modulating cell cycle progression, including ODC, cad, eIF-4E, and cdc25a. When overexpressed, ODC displays properties of an oncogene and can transform NIH 3T3 fibroblasts in culture (178, 179). Moreover, expression of the translation initiation factor eIF-4E is elevated in transformed cells and can promote cellular transformation, possibly by up-regulating levels of cyclin D1 protein (180, 181–183). Finally, cdc25a is an attractive control point for Myc because this phosphatase activates cyclin-dependent kinases (cdk) through the dephosphorylation of cdk molecules, allowing cyclin-cdk complexes to phosphorylate key cell cycle regulatory molecules such as pRb and p107. Together, these data suggest a direct role for Myc in the control of cell cycle progression; however, the connection between these particular downstream genes and the promotion of cell growth has yet to be firmly established. Indeed, evidence suggests that key target genes of Myc transactivation have yet to be uncovered, since a primary control point of Myc in cell cycle progression seems to point to the regulation of cyclin E kinase activity through the inhibition of the cyclin kinase inhibitor p27Kip1 (54–58). Clearly, a large, diverse subset of genes must be regulated downstream of Myc to fully effect the many biological activities attributed to Myc.
At a mechanistic level, an unexpected discovery was localization of the Myc-specific E-box elements in most of these target genes to sequences downstream of the transcription initiation site either within the first (-prothymosin, ODC, p53) or second intron (cdc25a), in 5' untranslated regions as for cad and ECA39, or within protein coding sequences, as for MrDb ( Fig. 2a, b).
Only the eIF-4E, LDH-A, and ISGF3 genes contain regulatory, Myc-responsive E-box sites within the 5' promoter region (c). The presence of E-box elements within the rcl gene has not yet been determined. It is becoming apparent that both the nature of the E-box element and its context within the promoter are critical to Myc-Max transactivation. Whereas in vitro binding assays identified CACGTG as the optimal sequence for Myc-Max binding, the isolation of the MrDb gene by antibodies specific to Myc-Max complexes bound to fragmented genomic DNA suggests that Myc-Max heterodimers may prefer other sites in vivo. Myc E-box elements have the potential to be recognized by at least two other members of the BR-HLH-Zip family of transcription factors: USF and TFE-3. Specificity of Myc-Max vs. USF or TFE-3 binding to these elements may involve several distinct mechanisms, depending on the promoter context. One of these mechanisms involves distance of the E-box from the promoter and may explain the unusual location of the sites. For the -prothymosin gene, CACGTG E-box elements proximal to the transcription start site are recognized by USF and TFE-3 as well as Myc, whereas the distal elements are recognized specifically by Myc-Max alone (184). Indeed, Myc-Max transcription activation through the CACGTG motif can operate over distances of 1.7 kb in a manner independent of element position and orientation (185). However, on the cad promoter, selectivity between USF vs. Myc-Max binding is not determined by the position of the E-box element, but rather by the sequence of nucleotides flanking the core CACGTG hexamer (186). Finally, whereas a synthetic reporter construct consisting of four tandem E-box elements upstream of a minimal promoter has been used extensively to demonstrate Myc-Max transactivation, a recent finding suggests this artificial promoter may not be representative of all naturally occurring Myc-responsive promoters. In particular, the recently characterized BIN-1 protein (see previous section) can antagonize Myc transactivation of the ODC promoter, but has no effect on Myc-Max transactivation of the artificial Myc E-box reporter construct, underlining the importance of promoter context to Myc molecular function.
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Another surprising finding arising from the identification of endogenous Myc target genes is the modest level of induction of these genes by Myc, averaging between 2- and 10-fold depending on the conditions of the assay and cell line used. This poses the question of how such modest and cell-specific changes in transcription can contribute to the global and dramatic effects of Myc on the cell cycle. One possible explanation is that the genetic background or transformation status of the cell can influence the outcome of Myc transcriptional activity. Indeed, Myc expression can lead to induction of cdc2 only in the presence of activated Ras, and both Myc and Ras are required to induce cyclin E-cdk2 activity (60, 187). Another potential variable is the contribution of endogenous c-myc expression among cell lines and assay conditions. Basal Myc protein levels are significantly higher in proliferating vs. quiescent cells, and this basal Myc protein may occupy Myc-Max DNA binding sites and activate gene transcription. Thus, the contribution of endogenous Myc expression could significantly compromise the sensitivity of assays designed to identify Myc target genes. This technical problem may be overcome by the recent construction of rat fibroblasts in which both c-myc alleles have been rendered nonfunctional by targeted gene disruption (61). These Myc null cells should prove valuable in understanding the mechanisms of Myc in gene regulation and should demonstrate the essential role of Myc in the regulation of known as well as novel target genes.
Apart from cellular Myc concentration, the degree of Myc transactivation may be affected indirectly by the expression of antagonists of Myc function, including certain Myc binding proteins or members of the Max network. An example of the former includes the BIN-1 protein, which interacts with MbI and MbII domains of Myc, inhibits Myc transactivation of the ODC gene in NIH 3T3 and HeLa cells, and suppresses Myc and Ras cotransformation in rat embryo fibroblasts (148). Another functional antagonist of Myc activity may include the novel member of the Max interacting family of proteins, Mnt-1 (138). Coexpression of Mnt-1 will repress Myc activation of the CACGTG reporter construct as well as suppress Myc and Ras cotransformation of primary rat embryo fibroblasts. Moreover, deletion of the mSin3 interaction domain allows Mnt-1 to activate E-box-dependent transcription and to substitute for Myc in promoting cotransformation with the Ras oncogene, again suggesting that Myc transcriptional transactivation and transformation activities are tightly linked. The authors also suggest that the overlapping expression patterns of Myc and Mnt-1 in mammalian cells limit Myc activity and may account for the modest levels of Myc transactivation in mammalian cells vs. the dramatic transactivation of E-box-dependent promoters in yeast systems. This hypothesis will be interesting to test once the critical cellular targets of Mnt-1-Max repression have been identified.
Transcriptional repression by Myc
The identification of nearly a dozen target genes of Myc transactivation has hinted at a few pathways by which Myc may promote the cell cycle, yet direct support for these mechanisms has not been forthcoming. Indeed, there is growing evidence to suggest that Myc transactivation of specific gene transcription may not be the sole contributor to the transforming properties of the c-myc oncogene and that other activities of Myc, such as Myc-mediated repression, also play an important role. To this end, we may make note of lessons learned from the adenovirus E1a viral oncoprotein. Like Myc, E1a is a strong potentiator of both cellular proliferation and programmed cell death, cooperates with activated Ras to transform cells in culture, and inhibits terminal differentiation of a number of cell types (70, 188–191). Furthermore, Myc and E1a can activate and repress transcription from numerous viral and cellular promoters. Despite low sequence homology between the E1a and c-Myc oncoproteins, domains of the E1a protein that mediate transformation can functionally complement c-Myc transformation domains, and vice versa (192). More important, the shorter 12S species of E1a, which can repress but not activate transcription, still retains full transforming ability, demonstrating that E1a-mediated transformation is tightly linked to transcriptional repression and is separable from transactivation.
By comparison, Myc suppression of gene transcription may also be an important component of the ability of c-Myc to promote cell growth and induce cellular transformation, similar to the transforming mechanism of the E1a viral oncoprotein. Substitution of the weak transactivation domain of Myc with the VP16 transactivator does not reconstitute the transforming properties of Myc (155), indicating that transactivation is insufficient to account for Myc transformation activity. In addition, Myc protein mutants that are defective for Myc-mediated repression of the AdML promoter do not cooperate with Ras to transform rat embryo fibroblasts but retain the ability to activate transcription of this promoter through E-box elements, demonstrating a clear separation between transactivation and transformation (87). Likewise, a lymphoma-derived mutation at Phe115 was found to be more potent for rodent fibroblast transformation and transcriptional suppression of the AdML promoter (193). Conversely, a mutation at Trp136 abrogated both the transforming and transcriptional suppression activities of c-Myc (155, 193). Furthermore, the domains of the human c-Myc protein required to suppress the transcription of several Myc-regulated genes overlap with those required for cotransformation, underlining the strong association between Myc-mediated repression and Myc tumorigenic functions (87, 194).
The list of Myc-repressed genes continues to grow ( Table 1). Overexpression of c-Myc in 3T3-L1 cells indirectly suppresses CCAAT transcription factor/nuclear factor-1 (CTF/NF-1) -dependent promoters via Myc-induced phosphorylation of the CTF/NF-1 transcription factor (195). c-Myc expression can also lead to suppression of the lymphocyte function-associated antigen-1 via a posttranscriptional mechanism (196). For many genes whose expression is down-regulated in response to Myc, it remains unclear whether a transcriptional or posttranscriptional mechanism is involved. These include gadd153/CHOP (a transcription factor of the C/EBP family), lysozyme, major histocompatability complex I (MHC I), myb-induced myeloid protein-1 (mim-1), and thrombospondin-1 (tsp-1) (85, 173, 176, 177). For the remaining Myc-repressed genes identified, repression occurs at the transcriptional level; however, it is not known whether Myc down-regulation of promoter activity involves a direct or indirect mechanism. Elevated expression of Myc protein appears to down-regulate transcription from several genes through core promoter regions, including the AdML promoter as well as the c-myc, C/EBP, albumin, cyclin D1, terminal deoxynucleotidyl transferase (TdT), and 
5 promoters (86, 87, 120, 171, 175). Myc-mediated repression of gene transcription also targets the gadd45 gene, immunoglobulin heavy-chain enhancer (IgEµ), immunoglobulin light-chain kappa (Ig
), and the platelet-derived growth factor ß receptor (PDGF-ßR); although the respective promoters can mediate Myc-repression of these targets, the precise regulatory regions have not been fully defined (83, 172, 174; W. Marhin, S. Chen, L. Facchini, P. Dion, M. Post, A. Ishisaki, K. Funa, and L. and Penn, unpublished results).
The function and expression pattern of several of these genes suggest that their down-regulation may contribute to Myc proliferative and tumorigenic activities. In particular, the gadd45 gene is expressed during G0, promotes growth arrest when overexpressed, and must be suppressed in order to allow entry into the cell cycle. Likewise, one function of the C/EBP transcription factor is to induce a subset of genes expressed during differentiation; moreover, its expression is required to initiate differentiation of preadipocytes. Thus, down-regulation of C/EBP by Myc may contribute to the Myc-induced differentiation block in at least some cell lines. The Tsp-1 protein can function as an angiogenesis inhibitor, suggesting that repression of the tsp-1 gene may be one component of the ability of Myc to promote tumor formation. Other genes repressed by Myc, such as PDGF-ßR and c-myc itself, are suppressed as part of negative feedback pathways contributing to regulated control of cell cycle progression. Finally, several Myc-repressed genes, including IgEµ, Ig, lysozyme, MHC I, mim-1, and TdT, are expressed only in terminally differentiated cells. Down-regulation of these genes by Myc may ensure they are not improperly expressed in actively proliferating cells. By contrast, Myc suppression of cyclin D1 is uncoupled from transformation (171) and is dependent on cell type as well as the transformation state of the cell (197). The exact mechanism (or mechanisms) of Myc transcriptional repression are unknown and the role of Myc-Max interactions in most of these mechanisms is unclear or has not been addressed.
Once again, repression activities of the E1a protein may provide insight into potential mechanisms of Myc-mediated transcriptional repression. Several mechanisms have been described to account for E1a transcriptional repression. E1a down-regulates enhancer-driven transactivation of several cellular genes including AP-1-dependent transactivation of collagenase and ferritin H genes, enhancer-driven transactivation of SV40, metallothionein, MHC I genes, and CREB- and MyoD-dependent transactivation (198–202). This repression results from E1a binding to and sequestering CBP or p300 coactivators from the transcription activation complex, and may be a component of E1a transformation activity. In addition, a direct role for E1a in repressing transcription from the HIV-1 LTR promoter has been described and appears to involve E1a interaction with components of the basal transcription complex, including TBP (203).
Evidence suggests Myc may repress transcription by several mechanisms both common to and distinct from E1a-mediated repression. One model proposes that c-Myc down-regulates transcription initiation at the AdML promoter by binding to and sequestering the general transcription factor and initiator binding protein TFII-I, thereby preventing the formation of a functional RNA polymerase II preinitiation complex ( Fig. 3a)
(153). Indeed, a role for the Inr element has been described for Myc repression of C/EBP, cyclin D1, albumin, TdT, and 5 promoters (87, 171, 175). However, other experiments have demonstrated that the C/EBP Inr element is dispensable for Myc-mediated repression and suggest, instead, that Myc may act through other components of the basal transcription machinery (86). Alternatively, another Myc interacting Inr binding protein, Miz-1, may be involved because Myc expression can abrogate Miz-1 activation of the cyclin D1 and AdML promoters (151). To date, only one group has reported Myc in complexes at Inr elements (175). Conflicting data also surround the regions of Myc protein that are required for down-regulation through core promoter elements ( Fig. 1). Cyclin D1 promoter suppression involves a distinct region of the Myc amino terminus that does not correlate with Myc cotransformation activity, similar to cyclin D1 repression by the E1a protein (171, 204). However, the amino-terminal domain of Myc required for suppression of the AdML and c-myc promoters coincides with the cotransformation domain and is uncoupled from those required for transcription activation, reminiscent of the E1a transformation mechanism (87, 194).
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Like E1a, Myc can also repress enhancer-driven transcription from specific promoters ( Fig. 3b). In particular, Myc inhibits C/EBP-dependent transactivation of the mim-1, lysozyme, and gadd45 promoters, although it has not been determined whether this repression operates directly through C/EBP or indirectly through a p300-like coactivator (83, 85, 172). The promoter region required for suppression of gadd45 includes octamer/CCAAT motifs as well as the core promoter region (172). Myc repression of Ig and IgEµ also requires an octamer binding promoter element; however, complexes containing Myc-Max or Oct proteins were not detectable on either Ig promoter (174).
Myc binding to TBP through the Myc amino-terminal domain suggests that Myc may regulate transcription through direct interaction with the basal transcription machinery ( Fig. 3c). Myc in conjunction with Max may repress transcription directly by binding to TBP and interfering with either the formation or function of the preinitiation complex via an active repression mechanism. Examples of this type of repression are becoming increasingly evident and have been described for transcription repression mediated by E1a, p53, the Dr1 repressor, the Drosophila even-skipped protein, and unliganded thyroid hormone receptor alpha (205–209).
Of the downstream targets repressed by Myc protein, the mechanism of c-myc autosuppression has been characterized the most extensively. This negative feedback mechanism is operative in primary and immortalized, but not all tumor-derived or transformed, cell lines (28, 29, 120, 210). Myc negative autoregulation is a homeostatic control mechanism whereby Myc protein suppresses transcription initiation from the c-myc promoter in a concentration-dependent manner. The levels of Myc protein required for this response fall within physiological Myc concentrations found in nontransformed proliferating cells (29). In addition, both v-Myc and N-Myc proteins can elicit this suppressive response on the c-myc promoter, indicating that conserved domains within the Myc protein are important. Indeed, the c-Myc protein domains required for autosuppression overlap with those necessary for Myc oncogenic activity and include amino acids 106 to 143, which contain the highly conserved MbII sequence, as well as carboxyl-terminal BR-HLH-Zip motifs (50, 194). Somatic cell hybridization between a variant NIH 3T3 cell line, which does not demonstrate Myc autosuppression, and feedback-competent Rat-1 cells indicates that the defect in the NIH 3T3 cells could be complemented by rat cell factors (29). These results suggest that the Myc autosuppression pathway involves trans-acting cellular factors in addition to Myc protein. One of these factors is the Max protein, since Myc-Max heterodimerization is obligatory for Myc autosuppression (120). Myc autosuppression involves the P2 core promoter, but no Myc-Max-specific E-box elements are found within this region. Indeed, affinity capture assays failed to detect Myc-Max complexes on Xenopus c-myc genomic DNA (211). Thus, this mechanism clearly does not involve Myc-Max binding to a CACGTG DNA element within the c-myc promoter and may involve Myc interaction with the basal transcription machinery.
PERSPECTIVES
Discoveries of the last few years have advanced our understanding of c-Myc function in growth and transformation. All experimental evidence gathered to date supports an essential role for Myc-Max interaction in Myc biological activity. Yet within this functional heterodimer, Myc remains the critically regulated component. Myc regulation is accomplished by modulation of Myc expression through intricate and overlapping mechanisms, including transcriptional, posttranscriptional, translational, and posttranslational mechanisms. More recently we have come to realize that molecular agonists and antagonists of Myc function can contribute further to the complexity of Myc regulation by interaction or competition with Myc protein. These molecules may be Myc binding proteins such as TR-AP, BIN-1, p107 and YY-1, which directly modulate Myc activity, or they may be competitors that affect the ability of the Myc-Max functional unit to recognize and act at DNA E-box elements. Representatives of this latter category may include Max-Max homodimers, Mnt-1-Max heterodimers, or Myc interaction with dMax.
Myc molecular activity clearly involves, but is not necessarily limited to, modulation of gene transcription. Identification of numerous target genes that are activated or repressed by c-Myc provides us with the challenge of deciphering how these transcriptional changes translate into potentiation of the cell cycle and transformation. So far, these pathways remain hidden. Activation of genes such as cdc25a, ODC, cad, and eIF-4E may directly or indirectly drive or allow cell cycle progression. Similarly, Myc down-regulation of gadd45 and C/EBP may play a role in the induction of quiescent cells into the cell cycle and the inhibition of differentiation pathways, respectively. These transcriptional changes evoked by Myc are subtle rather than dramatic. Furthermore, activation or repression of any one Myc target gene is not sufficient to account for the essential role of Myc in cell cycle progression or its potent transforming activity, leaving the Myc transformation mechanism unexplained. Indeed, accumulating structure/function studies suggest that the transformation activity of Myc is separable from its transactivation potential and, instead, correlates more closely with Myc-mediated transcriptional repression. Clearly, more investigation into these highly provocative correlations is required to identify the nature of the Myc transformation mechanism.
For most other Myc target genes, the involvement in growth and transformation is not instinctively clear. One general model for Myc activity proposes that in a proliferating cell, c-Myc allows the expression of a vast number of genes, each required (but not necessarily sufficient) for a different aspect of cell cycle progression. Such categories of Myc-regulated genes might then include direct and indirect cell cycle regulators, genes involved in the synthesis and manipulation of DNA, and essential structural components. Similarly, Myc would also prevent expression of genes specific to the differentiation phenotype whose expression may not be compatible with cell growth. The role of Myc-Max in such a scenario would be similar to a master switch for the transcription machinery, identifying all cell components that must be produced and those that must be suppressed to enable efficient, regulated entry into and passage through the cell cycle.
The past few years have witnessed several exciting advances in the Myc field, and we can anticipate this trend to continue in the near future. Detailed exploration of the mechanisms of Myc-mediated repression will likely provide new insights for understanding the mechanisms of Myc transformation and modulation of gene transcription. New and powerful tools such as Myc null fibroblasts are available whereby the effects of Myc at molecular and biological levels can be tested in the absence of endogenous Myc expression. Finally, the identification of novel Myc binding proteins will undoubtedly take the field in productive and potentially unexpected directions, which may hold the key to unlocking the Myc puzzle.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Correspondence: Ontario Cancer Institute/Princess Margaret Hospital, Division of Cell and Molecular Biology, 610 University Ave., Toronto, Ontario Canada M5G 2M9. E-mail: lpenn{at}oci.utoronto.ca
3 Abbreviations: Inr, initiator element; Max, Myc-associated factor x; NLS, nuclear localization sequences; BR, basic region; HLH-Zip, helix-loop-helix and leucine zipper region; TR-AP, transformation-associated protein; TBP, TATA binding protein; MIZ-1, Myc interacting zinc finger protein; YY-1, Yin Yang-1; ISGF3, interferon-stimulated gene factor 3 gamma; LDH-A, lactate dehydrogenase A; ODC, ornithine decarboxylase; cdk, cyclin-dependent kinase (or kinases); AdML, adenovirus major late promotor; CTF/NF-1, CCAAT transcription factor/nuclear factor-1; MHC, major histocompatibility complex; mim-1, myb-induced myeloid protein-1; tsp-1, thrombospondin-1; TdT, terminal deoxynucleotidyl transferase; IgEµ, immunoglobulin heavy-chain enhancer; Ig, immunoglobulin light-chain kappa; PDGF-ßR, platelet-derived growth factor ß receptor; gadd45, growth arrest and DNA damage-inducible gene 45; C/EBP, CCAAT/enhancer binding protein ; MbI, Myc box I; BL, Burkitt's lymphoma; pRb, retinoblastoma susceptibility protein.
4 Search of abstracts containing `c-myc' or `cMyc' in the Medline database from 1981 to 1997.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONOVERVIEW OF c-MYC STRUCTURE,...REFERENCES |
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