The overall goal of this project is to understand the nuclear regulatory components that control cardiac-specific gene transcription. The precise mechanisms that control cardiac specific gene expression are unknown but members of three transcription factor families (MEF-2, GATA and TEF-1) appear to control expression of most cardiac genes characterized to date. In the present proposal, we will analyze the molecular interactions between two distinct regulatory domains that control cardiac-specific transcription of the cardiac troponin T gene (cTNT). Deletion of either regulatory element reduces activity of cTNT promoter constructs transfected into cultured cardiac myocytes to background levels, indicating both are necessary. One regulatory domain (dubbed the """"""""Cardiac Element"""""""") contains binding sites for both GATA and MEF-2 and lies 200 nucleotides upstream of the transcription initiation site. The other regulatory domain lies within the proximal promoter region (less than 100 nucleotides upstream of the transcription initiation site) and contains two MCAT elements which are binding sites for members of the TEF-1 transcription factor family, and a novel co- binding factor, poly (ADP-ribose) polymerase (PARP), recently discovered in this laboratory to play a central and obligatory role in the control of cell-selective transcription by one of the two MCAT elements. The proposed experiments involve detailed analyses of the molecular interactions that occur between transcription factors bound to the cardiac element and those bound to the MCAT elements with the overall goal of elucidating how they collaborate to activate transcription specifically in cardiac myocytes but keep it inactive in other cell types. Because the central factors involved in the regulation of the cTNT gene are also known to be active in the regulation of other cardiac genes we anticipate that our findings regarding their molecular interactions will be generally relevant to the regulation of diverse cardiac genes. In addition, parallel studies comparing the roles of these cardiac gene regulatory domains in transgenic mice versus cultured cardiac myocytes will indicate the degree to which in vitro studies are predictive of mechanisms of gene regulation in vivo. Such direct comparisons are crucial to understand the cardiac gene regulatory mechanisms operating under physiological conditions within the whole animal and, ultimately, in context of cardiac pathologies, including myocardial hypertrophy.
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