ALPHA-MYOSIN HEAVY-CHAIN GENE-REGULATION - DELINEATION AND CHARACTERIZATION OF THE CARDIAC MUSCLE-SPECIFIC ENHANCER AND MUSCLE-SPECIFIC PROMOTER

The alpha-myosin heavy chain (alpha-MHC) gene encodes a cardiac muscle -specific protein involved in active force generation. The mechanism r esponsible for restricting expression of this gene to the heart should provide clues for the identification of transcriptional regulatory ev ents involved in the induction and maintenance of the cardiac cell lin eage. In this report we dissect the alpha-MHC regulatory region to ide ntify the components necessary for directing high levels of cardiac mu scle-restricted expression. Deletion, site-specific mutant and heterol ogous promoter constructs were assayed for expression after injection into adult rat heart and skeletal muscle or transfection into non-musc le cells. These studies indicated that sequences from -344 to -156 dir ected high levels of cardiac-muscle specific expression from a heterol ogous promoter that was independent of position and orientation. This region includes a previously uncharacterized CArG box. alpha-MHC seque nces from -86 to +16 promoted activity from two heterologous enhancers in a muscle-specific fashion. Mutational analysis of an E-box and a C ArG box within the promoter revealed that they act as negative and pos itive regulatory elements, respectively. Based on competitive binding and supershift electrophoretic mobility shift assays, serum response f actor was shown interact with the CArG boxes found in the promoter and enhancer. Similar experiments demonstrated that the E-box bound to a factor immunologically related to upstream stimulatory factor. Togethe r, these results identify two distinct regions with different regulato ry function that are critical for the tissue restricted expression of the alpha-MHC gene. (C) 1996 Academic Press Limited