We have been applying a technique developed by us (Levens, D. and Howley, P., Mol. Cell. Biol. 5: 2307-2316, 1985) for the rapid identification of sequence specific DNA binding proteins to examine transcriptional regulatory factors binding to the c-myc oncogene. By combining this technique with a sensitive exonuclease footprinting assay (Wu, C., Nature 317: 84-87, 1985), DNA-protein complexes can be formed, rapidly separated from the vast majority of protein and competitor nucleic acids, centrifugally concentrated and """"""""footprinted"""""""". This approach has led to the identification of multiple factors binding to a 2.3 kb region upstream from the c-myc promoters P1 and P2. The pattern of proteins binding, reflects the physiological state and particular cell source of the proteins. One of the factors examined has also been shown in our laboratory to bind to the enhancer of the gibbon ape leukemia virus and correlates well with the activity of that enhancer. This protein has been enriched and identified from nuclear extracts with a single step of two cycles. The protein is a 38-40 kd peptide. We are currently devising a purification scheme for this and other c-myc regulatory proteins. The role of these factors in c-myc regulation will be studied by in vitro transcription where we hope to reconstitute some of the properties of the physiological regulation of c-myc.
