Myosin is a family of proteins that demonstrates force-generating ATPase activity when it interacts with actin. Whereas myosins from vertebrate muscle and nonmuscle cells share common subunit composition and native structure, sarcomeric and nonsarcomeric myosin heavy chains (MHCs) consist of a distinct subfamily of isoforms. Individual MHC isoforms are encoded by different genes, and expression of various MHC genes is tightly associated with cell growth and differentiation. Using a mouse myogenic cell line (C2Cl2) as a model, we analyzed changes of nonmuscle MHC gene expression by RNA blot analysis. Proliferating C2 myoblasts express nonmuscle MHCs abundantly, similar to NIH 3T3 fibroblasts. When C2 cells differentiate to form multinucleated myotubes, nonmuscle MHC expression is dramatically decreased. Concomitantly, the sarcomeric MHC begins to be expressed. To understand the mechanism for the MHC isoform switch during skeletal muscle differentiation, we have been searching for cis-regulatory element(s) which may be involved in upregulating nonmuscle MHC gene expression in C2 myoblasts and fibroblasts and/or downregulating it in C2 myotubes. The region between 20 kb upstream to transcriptional start sites and 40 kb downstream, which includes the 37 kb 1st intron of human nonmuscle MHC-A gene, was segmented by restriction enzymes or PCR amplification and introduced into the reporter gene construct which contains the nonmuscle MHC-A gene promoter and luciferase CDNA. Following transfection of various luciferase constructs into NIH 3T3, C2 myoblasts and C2 myotubes, three segments, 200 bp, 500 bp and 5.5 Kbp in size, are found to possess activities modulating gene expression in a cell-specific manner. The 200 bp segment, located in the 1st exon and the beginning of 1st intron, shows a 10-15-fold increase in luciferase activity in NIH 3T3 and a 5- fold increase in C2 myoblasts. The 500 bp and 5.5 Kbp segments, both located in the 1st intron, increase gene expression 3-fold in NIH 3T3 and C2 myoblasts. None of these three segments, however, cause an increase in luciferase activity in C2 myotubes. The 200 bp segment functions in a position- and orientation-dependent manner. Quantitation of luciferase MRNA content by competitive PCR and Rnase protection assay revealed that the increase seen in luciferase MRNA, due to the 200 bp segment, is 3-5- fold in NIH 3T3 cells whereas changes seen in luciferase activity (protein amount) is 10-15-fold. Thus, the 200 bp 1st exon-intron segment seems to affect both pretranslational (transcription or RNA stability) and translational steps.
