A major dilemma facing the Myc researcher is understanding how c-Myc regulation of gene transcription translates into the proliferative and oncogenic activities mediated by c-Myc protein. Indeed, much effort has focused on attempting to link c-Myc activation of gene transcription with both cell cycle progression and transformation mechanisms. Considerable progress has been made in recent years, with the identification of new Myc binding proteins as well as novel cellular targets of Myc-Max complexes. These discoveries have yielded more than a few surprises and challenged those working in the field to rethink traditional paradigms. It is now evident that c-Myc can also repress the transcription of specific genes, and Myc-mediated repression appears to be linked to Myc-dependent transformation. We summarize the evidence on Myc biological and molecular functions with regard to Myc-Max transcriptional regulation. In addition, we reevaluate current models of Myc transcriptional modulation in light of the discovery of new Myc binding partners and novel downstream target genes. Finally, we explore whether direct transactivation of cellular genes by Myc-Max heterodimers is sufficient for the growth-promoting and transforming activities of Myc or whether other molecular activities of Myc, such as Myc-mediated repression, may play a key role.

Increasing evidence supports an important biological role for Myc in the downregulation of specific gene transcription. Recent studies suggest that c-Myc may suppress promoter activity through proteins of the basal transcription machinery. We have previously reported that Myc protein, in combination with additional cellular factors, suppresses transcription initiation from the c-myc promoter. To characterize the cis components of this Myc negative autoregulation pathway, fragments of the human c-myc promoter were inserted upstream of luciferase reporter genes and assayed for responsiveness to inducible MycER activation in Rat-1 fibroblasts. We found four-to fivefold suppression of a c-myc P2 minimal promoter fragment upon induction of wild-type MycER protein activity, while induction of a mutant MycER protein lacking amino acids 106 to 143 required for Myc autosuppression failed to elicit this response. This assay is physiologically significant, as it reflects Myc autosuppression of the endogenous c-myc gene with regard to kinetics, dose dependency, cell type specificity, and c-Myc functional domains. Analysis of mutations within the P2 minimal promoter indicated that the cis components of Myc autosuppression could not be ascribed to any known protein-binding motifs. In addition, to address the trans factors required for Myc negative autoregulation, we expressed MycEG and MaxEG leucine zipper dimerization mutants in Rat-1 cells and found that Myc-Max heterodimerization is obligatory for Myc autosuppression. Two models for the Myc autosuppression mechanism are discussed.

The c-Myc protein strongly stimulates cellular proliferation, inducing cells to exit G0/G1 and enter the cell cycle. At a molecular level, Myc prevents growth arrest and drives cell cycle progression through the transcriptional regulation of Myc-target genes. Expression of the growth arrest and DNA damage inducible gene 45 (gadd45) is elevated in response to DNA damaging agents, such as ionizing radiation via a p53-dependent mechanism, upon nutrient deprivation, or during di erentiation. Gadd45 holds a vital role in growth arrest as ectopic expression confers a strong block to proliferation. Exposure of quiescent cells to mitogen stimulates a rapid increase in c-Myc expression which is followed by the subsequent reduction in gadd45 expression. The kinetics of these two regulatory events suggest that Myc suppresses the expression of gadd45, contributing to G0/ G1 phase exit of the cell cycle. Indeed, ectopic Myc expression in primary and immortalized ®broblasts results in the suppression of gadd45 mRNA levels, by a mechanism which is independent of cell cycle progression. Using an inducible MycER TM system, rapid suppression of gadd45 mRNA is ®rst evident approximately 0.5 h following Myc activation. The reduction in gadd45 mRNA expression occurs at the transcriptional level and is mediated by a p53-independent pathway. Moreover, Myc suppression and p53 induction of gadd45 following exposure to ionizing radiation are noncompetitive co-regulatory events. Myc suppression of gadd45 de®nes a novel pathway through which Myc promotes cell cycle entry and prevents growth arrest of transformed cells.

Platelet-derived growth factor BB (PDGF BB) is a potent mitogen for fibroblasts as well as many other cell types. Interaction of PDGF BB with the PDGF ␤ receptor (PDGF-␤R) activates numerous signaling pathways and leads to a decrease in receptor expression on the cell surface. PDGF-␤R downregulation is effected at two levels, the immediate internalization of ligand-receptor complexes and the reduction in pdgf-␤r mRNA expression. Our studies show that pdgf-␤r mRNA suppression is regulated by the c-myc proto-oncogene. Both constitutive and inducible ectopic Myc protein can suppress pdgf-␤r mRNA and protein. Suppression of pdgf-␤r mRNA in response to Myc is specific, since expression of the related receptor pdgf-␣r is not affected. We further show that Myc suppresses pdgf-␤r mRNA expression by a mechanism which is distinguishable from Myc autosuppression. Analysis of c-Myc-null fibroblasts demonstrates that Myc is required for the repression of pdgf-␤r mRNA expression in quiescent fibroblasts following mitogen stimulation. In addition, it is evident that the Myc-mediated repression of pdgf-␤r mRNA levels plays an important role in the regulation of basal pdgf-␤r expression in proliferating cells. Thus, our studies suggest an essential role for Myc in a negative-feedback loop regulating the expression of the PDGF-␤R.

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