Transcription from one of the two initiation sites, P1 and P2, of the dual human MYC promoter seems to be essential in all proliferating cells. To identify proteins and target structures for MYC regulation, a DNA region was analyzed that is critical for P2 promoter activity. Here, we show that a nuclear factor binds to a DNA element within P2, which is conserved perfectly between mouse and man and displays a striking homology to the Ela-inducible E2 promoter of adenovirus type 5 (AdS}. We demonstrate that the same transcription factor, defined recently as E2F, which plays an essential role in the activation of adenovirus early promoters and enhancers, also interacts as a dominant nuclear factor with the MYC promoter. The presence of an intact E2F binding site is required for basic expression and for trans-activation of the P2 promoter by Ela proteins. The human MYC promoter is the first cellular target described for E2F. The results suggest that expression of MYC might be regulated via modulation of E2F by cellular 'Ela-like' factors.

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The paper describes a rapid and sensitive assay for DNA binding proteins which interact with specific and defined binding sites. It exploits the observation that complexes of proteins and small synthetic DNA fragments (40 bp) containing the protein/DNA binding site can enter native polyacrylamide gels and remain stably associated during electrophoresis under non-denaturing conditions. The assay was applied to nuclear factor I, to its identification and purification from porcine liver, to an analysis of its binding site on adenovirus type 5 DNA and to an exploration of other potential binding sites for DNA binding proteins within the inverted terminal repetition of adenovirus DNA. The extreme sensitivity of the assay which surpasses that of conventional footprint assays by at least two orders of magnitude permitted the identification of nuclear factor I-like activities in Saccharomyces cerevisiae.

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The decanucleotide sequence (dc) TNATTTGCAT is an upstream regulatory sequence of immunoglobulin genes and occurs also upstream of certain other eukaryotic and prokaryotic genes (compiled in the accompanying paper). We now investigated the binding of proteins from nuclear extracts of a number of cell types and organisms to the dc sequence using a sensitive gel electrophoretic DNA binding assay. Binding studies with specifically designed oligonucleotides led to the following conclusions: the central T of the dc sequence can be altered with only a slight decrease of protein binding activity: the sequences in the neighborhood of dc have a positive or negative effect on the efficiency of protein binding; C-rich sequences which occur in many K chain promoters have a protein binding activity independent of dc; the dc binding protein(s) of human lymphoid cells elute from a Sephadex column in the 30.000-60.000 molecular weight range; dc binding proteins were found in nuclear extracts of lymphoid as well as non-lymphoid human and murine cell lines, of Xenopus oocytes, and of yeast cells. The finding of dc binding proteins in a wide variety of different organisms and the occurrence of dc-related sequences in the regulatory regions of several gene families point to a general role in the transcriptional regulation of the respective genes.