THE ROLE OF AN E-BOX-BINDING BASIC HELIX-LOOP-HELIX PROTEIN IN THE CARDIAC MUSCLE-SPECIFIC EXPRESSION OF THE RAT CYTOCHROME-OXIDASE SUBUNIT-VIII GENE

We have characterized the rat gene for muscle-specific cytochrome oxid ase VIII (COX VIII(H)) and mapped the distal promoter region responsib le for transcription activation in C2C12 skeletal myocytes and H9C2 ca rdiomyocytes. In both cell types, the promoter elements responding to the induced differentiation of myocytes map to two E boxes, designated as El and E2 boxes with a core sequence of CAGCTG. Gel mobility shift analysis showed that both El and E2 box motifs form complexes with nu clear extracts from H9C2 cardiomyocytes that were supershifted with mo noclonal antibody to E2A but not with antibody to myo-D. Extracts from induced and uninduced H9C2 cardiomyocytes yielded different gel mobil ity patterns and also different E2A antibody supershifts suggesting a difference in the DNA-bound protein complexes cross-reacting with the E2A antibody. Transcriptional activity of the promoter construct conta ining intact E boxes was inhibited by coexpression with Id in differen tiated H9C2 cardiomyocytes. Our results show the involvement of an E b ox binding basic helix loop helix protein in the cardiac muscle-specif ic regulation of the COX VIII(H) promoter.