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 oxidase VIII (COX VIII(H)) and mapped the distal promoter region responsible for transcription activation in C2C12 skeletal myocytes and H9C2 cardiomyocytes. In both cell types, the promoter elements responding to the induced differentiation of myocytes map to two E boxes, designated as E1 and E2 boxes with a core sequence of CAGCTG. Gel mobility shift analysis showed that both E1 and E2 box motifs form complexes with nuclear extracts from H9C2 cardiomyocytes that were supershifted with monoclonal antibody to E2A but not with antibody to myo-D. Extracts from induced and uninduced H9C2 cardiomyocytes yielded different gel mobility 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 containing intact E boxes was inhibited by coexpression with Id in differentiated H9C2 cardiomyocytes. Our results show the involvement of an E box binding basic helix loop helix protein in the cardiac muscle-specific regulation of the COX VIII(H) promoter.