REGULATION OF THE RAT CARDIAC TROPONIN-I GENE BY THE TRANSCRIPTION FACTOR GATA-4

Troponin I is a thin-filament contractile protein expressed in striate d muscle. There are three known troponin I genes which are expressed i n a muscle-fibre-type-specific manner in mature animals. Although the slow skeletal troponin I isoform is expressed in fetal and neonatal he art, the cardiac isoform is restricted in its expression to the myocar dium at all developmental stages. To study the regulation of this card iac-specific and developmentally regulated gene in vitro, the rat card iac troponin I gene was cloned. Transient transfection assays were per formed with troponin I-luciferase fusion plasmids to characterize the regulatory regions of the gene. Proximal regions of the upstream seque nce were sufficient to support high levels of expression of the report er gene in cardiocytes and relatively low levels in other cell types. The highest luciferase activity in the cardiocytes was noted with a pl asmid that included the region spanning -896 to +45 of the troponin I genomic sequence. Cotransfection of GATA-4 a recently identified cardi ac transcription factor, with troponin I-luciferase constructs permitt ed high levels of luciferase expression in non-cardiac cells. Electrop horetic mobility-shift assays demonstrated specific binding of GATA-4 to oligonucleotides representative of multiple sites of the troponin I sequence. Mutation of a proximal GATA-4 DNA-binding site decreased tr anscriptional activation in transfected cardiocytes. These results ind icate that the proximal cardiac troponin I sequence is sufficient to s upport high levels of cardiac-specific gene expression and that the GA TA-4 transcription factor regulates troponin I-luciferase expression i n vitro.