Evidence for an internal regulatory region in a human nonmuscle myosin heavy chain gene.

We have isolated genomic clones which encode the promoter and flanking region of human nonmuscle myosin heavy chain (MHC)-A. The sequence of this region shows many features typical of a housekeeping gene; there is no TATA element and no functional CAAT box. The GC content is high, having an average GC content of 74% in the 600 base pairs (bp) surrounding the transcriptional start sites, and multiple GC boxes (putative Sp1 binding sites) are present. A number of nucleotide sites are utilized for the initiation of transcription. Promoter activity was monitored using luciferase as a reporter following transient transfection into NIH 3T3 cells. Analysis of 5' and 3' deletion mutants in the promoter region defines the core promoter as extending from nucleotide -112 to +61, where +1 is a major transcriptional start site. An essential sequence for core promoter activity resides in the 36-bp region from -77 to -112 which includes a single potential AP-2 binding site and a single potential Sp1 binding site. The region just downstream from the transcriptional start site (between +62 and +257) was found to be involved in cell type-specific activation of nonmuscle MHC-A gene expression. The increase in luciferase activity due to this proximal downstream region is approximately 15-fold in NIH 3T3 cells, but no increase was observed in C2C12 myotubes and neuroblastoma cells. This 196-bp region, which consists of 100 bp from exon 1 and 96 bp from intron 1, functions in a position- and orientation-dependent manner. Quantitation of luciferase mRNA content driven by the MHC-A promoter, using both competitive polymerase chain reaction and RNase protection assays, revealed that the increase seen in luciferase mRNA due to the 196-bp fragment is approximately 5-fold in NIH 3T3 cells. This only accounts for about one-third of the total increase seen in luciferase activity (protein amounts). Thus, this proximal downstream region appears to activate gene expression in NIH 3T3 cells via both pretranslational (transcription and/or mRNA stability) and translational mechanisms.