The mechanisms which govern cardiogenic differentiation and cardiac-- specific gene expression are unknown. Our previous data have shown the time of cardiogenic commitment and the embryonic period when cardiac genes are activated in the developing heart. Our data suggest that the activation of muscle gene expression in the heart may involve unique mechanisms not seen after the initial conversion of mesoderm to myocyte. Finally, two genes, cardiac troponin I (TnI) and ventricular myosin heavy chain one (VMHC1) Which are activated at the initiation of cardiomyogenic differentiation were identified. Cardiac TnI is expressed in a truly cardiac-specific manner while VMHC1 is expressed in all striated myogenic cells at the initiation of myogenic differentiation but then is immediately restricted to ventricular myocytes. We propose to identify regulatory sequences which direct the expression of these two cardiac genes. We will: 1) isolate and characterize the 5' and proximal sequences of the cardiac TnI and VMHC1 genes, 2) transfect putative regulatory sequences fused to the lacZ reporter gene of pTMG vector into cardiogenic mesoderm, differentiated cardiac myocytes, skeletal myogenic cells and non-myogenic cells, and 3) identify specific regions of DNA/protein interaction in the regulatory elements using DNA footprinting and electrophoretic mobility shift assays. Our hypothesis is that the cardiac TnI gene contains regulatory elements which confer cardiac specific expression, while the VMHC1 gene contains sequences which direct a unique ubiquitous phase of expression followed by a strict cardiac specific phase. Our present goal is to identify the sequences which direct these activities by comparing and contrasting putative regulatory elements of the genes and thereby aid in the determination of the molecular mechanisms which govern cardiac-specific gene expression and cardiogenic differentiation.
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