Our long-term goal is to utilize the telokin promoter as a model system to determine the mechanisms regulating smooth muscle-specific gene expression. Unraveling these mechanisms is crucial to our understanding of smooth muscle development and of the pathology of many different vascular, pulmonary, intestinal and urogenital diseases that are associated with altered contractile protein expression and altered contractility. Experiments are proposed to test the hypothesis that modular transcription elements control gene expression in different smooth muscle tissues. Transgenic mice will be utilized to identify the minimal cell-specific telokin promoter. Important cis-acting regulatory elements identified in vitro, will be characterized by mutational analysis, of telokin promoter- beta-galactosidase transgenes. To determine if regulatory elements are important for basal expression or smooth muscle specificity we will employ a novel approach using chimeric telokin/SM22a promoters. Several transcription factors that regulate the telokin promoter activity have been identified, including SRF. HFH-l, HFH-8, CDP, TEF and HMGI. A major goal of this proposal is to determine the role played by these factors in regulating expression of telokin and other smooth muscle contractile proteins in normal and injured, phenotypically modified, smooth muscle cells. Specifically we will test the hypothesis that HFH-l and CDP repress and HFH-8 and TEF stimulate telokin gene expression. The role of architectural transcription factors HMGI(Y) in regulating telokin promoter activity and contractile protein expression will also be evaluated. SRF is known to be a key regulator of all smooth muscle genes thus far examined, although it is not known if it is required for basal expression of smooth muscle genes or if it is involved in mediating their tissue specific expression. It is likely that the tissue specific functions of SRF result from its interaction with other cell-type specific factors, hence, modified yeast-2 hybrid screens are proposed to identify novel smooth muscle restricted binding partners. Preliminary results have yielded two interesting proteins, UBC9 and Duplin, which although widely expressed, may have fundamental effects on SRF's activity through Sumo modification and interaction with beta--catenin, respectively. The function of SRFs' interaction with these proteins will be elucidated.
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