c-myc plays a key role in regulating mammalian cell proliferation and apoptosis. The gene codes for a transcription factor, Myc, that belongs to the helix-loop-helix/leucine zipper (HLH/LZ) family of proteins. Myc heterodimerizes with a partner protein termed Max; the heterodimeric complex binds to CAC(G/A)TG (E-box) sequences and activates transcription from these sites. However, several other HLH/LZ proteins, including USF and TFE-3, bind to and trans-activate from the same element, yet have no documented effect on cell proliferation or apoptosis. Therefore, it is likely that mechanisms exist that discriminate between these proteins for activation of natural target genes of Myc. We now show that trans-activation from the E-box in the rat prothymosin-oL intron enhancer is indeed specific for Myc, and identify both the distance from the start site of transcription and a second E-box element adjacent to that recognized by Myc as critical determinants of specificity. Surprisingly, transcription activation domains required for Myc to activate from this distal enhancer position differ from previously mapped domains and closely correlate with those domains essential for transformation. As observed in transformation assays, Myc and Max strongly synergize in activation from a distal enhancer position. Our data suggest that trans-activation from the prothymosin intron enhancer is a faithful reflection of the transforming properties of the Myc protein.

The human papillomavirus type 16 E6 protein exerts a transforming activity through inactivation of tumor suppressor p53. Recently E6 has been shown to have additional transforming activities independent of p53. E6 is able to transactivate or repress several specific viral promoters. However, underlying molecular mechanisms and cellular target genes for the activity are not well understood. Using a differential hybridization technique, we identified the prothymosin alpha gene as a cellular target of E6 transactivation. E6 was able to transactivate the prothymosin alpha promoter in H358 cells lacking p53 and in C33A cells harboring a mutant p53 allele. Disruption of the E-box in intron 1 of the prothymosin alpha promoter abolished the responsiveness to E6. Then we determined if E6 up-regulates the expression of Myc, by which the prothymosin alpha promoter is transactivated through the E-box. We found that E6 is also able to transactivate the c-myc promoter in H358 cells and in C33A cells. These results suggest that E6 is able to transactivate the c-myc promoter independently of p53, and that the prothymosin alpha promoter is subsequently transactivated by Myc.

c-myc was discovered as the cellular homologue of the transduced oncogene of several avian retroviruses. The gene encodes a transcription factor, which forms a heteromeric protein complex with a partner protein termed Max. In mammalian cells, Myc is a central regulator of cell proliferation and links external signals to the cell cycle machinery. Myc also induces cells to undergo apoptosis, unless specific signals provided either by cytokines or by oncogenes block the apoptotic pathway. Recent progress sheds light both on the factors regulating the function and expression of Myc and on the downstream targets in the cell cycle. Together, these findings suggest the existence of a novel signal transduction pathway regulating both apoptosis and proliferation.

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Although USF-1 and -2 are the major proteins that bind to Myc-regulated E-box (CACGTG) elements in many cells, there is no clear role for USF during Mycdependent gene regulation. Using dominant negative alleles of USF-1 we now show that DNA binding by USF at a Myc-regulated E-box limits the ability of another E-box binding factor, TFE-3, to activate a target gene of Myc in vivo and to stimulate S phase entry in resting ®broblasts. Similarly, dominant negative alleles of USF-1 relieve the restriction that prevents activation of the IgH enhancer by TFE-3 in non B-cells. DNA binding activity of USF complexes is abundant in primary human B-cells and is signi®cantly downregulated during B-cell immortalization. Re-expression of USF-1 in immortalized B-cells retards proliferation. Our data establish an essential role for USF in restricting E-box dependent gene activation in vivo and suggest that this control is relaxed during cellular immortalization.