Muscle-specific regulation of a transfected rabbit myosin heavy chain beta gene promoter

We have examined the transcriptional regulation of the rabbit myosin heavy chain (HC) beta gene by using DNA-mediated transfection experiments. To analyze the activity of the myosin HC beta promoter in a myogenic background, cultured myoblasts from 12-day-old chick embryonic breast muscle were transfected with a chimeric gene containing 781 base pairs of the promoter region fused to the gene for chloramphenicol acetyltransferase (CAT). As indicated by the transient expression of chloramphenicol acetyltransferase, the activity of the promoter in myoblast cultures increased at least 32-fold following differentiation and was selectively inhibited when myogenesis was blocked with 5-bromodeoxyuridine. Furthermore, RNase protection experiments showed that the in vivo myosin HC beta transcriptional initiation (or cap) site was utilized in the transfected skeletal muscle cells and also that the regulation of the exogenous promoter was similar to the induction of the endogenous skeletal alpha-actin gene. The results indicated that the exogenous promoter is regulated in a tissue- and stage-specific manner. By creating progressive 5' deletions of the promoter, we showed that only the region extending -294 base pairs upstream from the cap site is necessary for the muscle-specific expression. Linker-scanner mutagenesis of this region indicated that the positive regulation in differentiated skeletal muscle is mediated by at least two distinct elements within the 5'-flanking region of the myosin HC beta gene.