TRANSCRIPTIONAL REGULATION OF THE HUMAN NONMUSCLE MYOSIN-II HEAVY CHAIN-A GENE - IDENTIFICATION OF 3 CLUSTERED CIS-ELEMENTS IN INTRON-1 WHICH MODULATE TRANSCRIPTION IN A CELL TYPE-DEPENDENT AND DIFFERENTIATIONSTATE-DEPENDENT MANNER

In an attempt to identify cis-acting elements for transcriptional regu lation of the human nonmuscle myosin II heavy chain (MHC)-A gene, the region extending 20 kilobases (kb) upstream and 40 kb downstream from the transcription start sites, which includes the entire 37-kb intron 1, was examined. Using transient transfection analysis of luciferase r eporter constructs, a 100-base pair (bp) region (N2d) in intron 1, loc ated 23 kb downstream from the transcriptional start sites, has been f ound to activate transcription in a cell type-and differentiation stat e-dependent manner, Maximum activity (similar to 20-fold) is seen in N IH 3T3 fibroblasts and intermediate activity (7-fold) in proliferating and undifferentiated C2C12 myoblasts, In contrast, this region is alm ost inactive in terminally differentiated C2C12 myotubes, in which end ogenous nonmuscle MHC-A expression is down-regulated, Gel mobility shi ft assays and methylation interference analyses were performed using N IH 3T3 nuclear extracts to determine the protein-binding elements for transcription factors, Three binding elements have been identified wit hin the N2d region, Antibody-supershift experiments, as well as compet ition experiments using consensus binding sequences for specific trans cription factors, revealed that the most 5'-element, C (GGGAGGGGCC) is recognized specifically and exclusively by Sp1 and Sp3 transcriptiona l factors, Element C is immediately followed by a novel element, A (GT GACCC), A third element, F (GTGTCAGGTG), which contains an E-box, is l ocated 50 bp 3' to element A. Element F can be recognized partially by upstream stimulatory factors, USF1 and/or USF2, Transfection studies with luciferase reporter constructs which include mutations in all thr ee elements in various combinations demonstrate that the A and C bindi ng factors cooperatively activate transcriptional activity in NIH 3T3 cells, The F binding factor shows an additive effect on transcription.