Myosin is found in all eukaryotic cells and appears to be involved in diverse cellular motile processes such as cytokinesis. This laboratory has isolated two different cDNAs for nonmuscle myosin heavy chains (MHCs) which are encoded by two different genes in both human and chicken. We have demonstrated the tissue and cell type-dependent expression for the two MHC mRNAs as well as changes in mRNA expression associated with cell growth and differentiation using RNA blot analysis. To understand the mechanisms responsible for regulating the expression of nonmuscle MHC genes, we cloned and have begun to characterize the 5' portion of a human nonmuscle MHC gene which includes the promoter region. A major and a minor transcriptional start site were identified by primer extension and RNase protection analyses using RNA from human leukemia (Jurkat) and epidermoid carcinoma (A431) cells. The region both upstream and downstream from the transcriptional start site is rich in GC sequence, but lacks an apparent TATA box. This is consistent with nonmuscle MHC genes belonging to the family of housekeeping genes. Promoter activity has been monitored using luciferase as a reporter. Following transfection of various luciferase constructs into NIH 3T3 fibroblasts, a fragment of approximately 175 bp (115 bp upstream and 60 bp downstream from the major transcriptional start site) was found to possess core promoter activity. Including the DNA fragment approximately 200 bp further upstream results in a 2-fold increase in transcriptional activity, while including the fragment approximately 150 bp further downstream results in an approximately 10-fold increase in transcriptional activity in fibroblasts. In constructs, this downstream sequence does not activate transcription in the differentiated skeletal muscle cells (C2C12 myotubes), in which endogenous mRNA for this gene is also down-regulated. Gel shift assay using nuclear extracts from NIH 3T3 cells and C2C12 myotubes demonstrated the downstream 150 bp DNA fragment forms a different type of complex with NIH 3T3 nuclear protein compared to C2C12 myotube nuclear protein.
