The long-term goal of this research is to determine the molecular basis for regulation of gene expression and cell differentiation by the c-myc oncogene. This research will determine which of the known activities of c-myc, if any, is required for its ability to inhibit cell differentiation and to repress gene expression. These activities are the ability to immortalize primary cells, to transform established cell lines and to bind to DNA. This will be determined by examining various mutant c-myc genes that lack these activities for their ability to inhibit 3T3-L1 cell differentiation and to repress pro alpha 2(1) collagen mRNA expression. The possibility that c-myc exerts its repressing action by modulating enhancer activity will be examined. Stable cell lines that contain chloramphenicol acetyltransferase (CAT) minigenes under the transcriptional control of the pro alpha 2(I) collagen gene promoter or enhancer will be constructed. CAT activity will be examined in these cell lines before and after introduction of a recombinant c-myc gene. We will test the hypothesis that c-myc affects gene expression by controlling the concentration or activity of specific DNA-binding proteins. The activity of specific DNA-binding proteins will be assayed by gel mobility-shift and DNase I footprinting analysis using nuclear extracts from cells having either high or low amounts of c-myc. The CAT fusion genes will be used to determine whether the decrease in collagen gene expression during 3T3-L1 cell differentiation is controlled at the level of transcription and if it is due to decreased activity of the collagen promoter, enhancer, or both. DNase I footprinting analysis will be used to determine whether this regulation involves a change in the concentration or activity of a DNA-binding protein. In addition, this proposal will test the hypothesis that c-myc represses pro alpha 2(1) collagen gene expression by altering the concentration (activity) of a specific DNA-binding protein that is expressed in a differentiation- dependent manner. This proposal is very relevant to cancer since it investigates the mechanism of action of an oncogene that is known to have a central role in neoplastic development.
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