This research seeks to establish the molecular basis for the regulation of the vascular smooth muscle (VSM) alpha-actin gene during fibroproliferative responses characteristic of vascular injury and disease. Previous studies in our laboratories point to a model in which transcriptional repression of the mouse VSM alpha-actin gene in fibroblasts and undifferentiated myoblasts results from the interaction of sequence-specific single-stranded DNA (ssDNA) binding proteins with opposite strands of an essential TEF-1 enhancer element. Binding site screening of cDNA expression libraries has now resulted in the cloning of these proteins and their subsequent identification as MSY1, a member of the Y-box family of nucleic acid binding proteins, Puralpha, a retinoblastoma (Rb)-binding protein initially identified in Hela cells, and Purbeta, a related protein of unknown properties. Preliminary studies of human atherosclerotic coronary arteries suggest a functional role for these proteins in cardiovascular disease. Experiments proposed in this application will test a central hypothesis that transcriptional repression results from a ssDNA-binding protein-dependent disruption of base pairing within the TEF-1 enhancer and that regulation of this process depends, in part, upon highly specific protein-protein interactions. This model will be tested by delineating functional domains within the ssDNA-binding proteins essential for protein-protein interactions, by determining the effects of mutations within these domains on the ability of these proteins to modulate enhancer topology and function, and by in vivo footprinting of enhancer structure in VSM alpha-actin expressing cell types. Lastly, the involvement of these proteins in human atherogenesis will be studied using a repertoire of anti-Pur and anti-MSYl peptide-specific antibodies. These studies will extend our understanding of a novel mechanism for the regulation of smooth muscle actin synthesis which may be important to the pathogenesis of coronary artery disease.
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