THE MYC NEGATIVE AUTOREGULATION MECHANISM REQUIRES MYC-MAX ASSOCIATION AND INVOLVES THE C-MYC P2 MINIMAL PROMOTER

Increasing evidence supports an important biological role for Myc in t he downregulation of specific gene transcription. Recent studies sugge st that c-Myc may suppress promoter activity through proteins of the b asal transcription machinery. We have previously reported that Myc pro tein, in combination with additional cellular factors, suppresses tran scription initiation from the c-myc promoter. To characterize the cis components of this Myc negative autoregulation pathway, fragments of t he 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 P 2 minimal promoter fragment upon induction of wild-type MycER protein activity, while induction of a mutant MycER protein tacking amino acid s 106 to 143 required for Myc autosuppression failed to elicit this re sponse, 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 th e cis components of Myc autosuppression could not be ascribed to any k nown protein-binding motifs. In addition, to address the trans factors required for Myc negative autoregulation, se expressed MycEG and MaxE G leucine zipper dimerization mutants in Rat-1 cells and found that My c-Max heterodimerization is obligatory for autosuppression, Two models for the Myc autosuppression mechanism are discussed.