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The proto-oncogene c-mycis transcribed from a dual promoter P1/P2, with transcription initiation sites 160 base pairs apart. Here we have studied the transcriptional activation of both promoters on chromatin templates. c-mycchromatin was reconstituted on stably transfected, episomal, Epstein-Barr virus-derived vectors in a B cell line. Episomal P1 and P2 promoters showed only basal activity but were strongly inducible by histone deacetylase inhibitors. The effect of promoter mutations on c-mycactivity, chromatin structure, and E2F binding was studied. The ME1a1 binding site between P1 and P2 was required for the maintenance of an open chromatin configuration of the dual c-mycpromoters. Mutation of this site strongly reduced the sensitivity of the core promoter region of P1/P2 to micrococcal nuclease and prevented binding of polymerase II (pol II) at the P2 promoter. In contrast, mutation of the P2 TATA box also abolished binding of pol II at the P2 promoter but did not affect the chromatin structure of the P1/P2 core promoter region. The E2F binding site adjacent to ME1a1 is required for repression of the P2 promoter but not the P1 promoter, likely by recruitment of histone deacetylase activity. Chromatin precipitation experiments with E2F-specific antibodies revealed binding of E2F-1, E2F-2, and E2F-4 to the E2F site of the c-mycpromoter in vivoif the E2F site was intact. Taken together, the analyses support a model with a functional hierarchy for regulatory elements in the c-mycpromoter region; binding of proteins to the ME1a1 site provides a nucleosome-free region of chromatin near the P2 start site, binding of E2F results in transcriptional repression without affecting polymerase recruitment, and the TATA box is required for polymerase recruitment.