Transcriptional regulation of the human nonmuscle myosin II heavy chain-A gene. Identification of three clustered cis-elements in intron-1 which modulate transcription in a cell type- and differentiation state-dependent manner.
In an attempt to identify cis-acting elements for transcriptional regulation 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 reporter constructs, a 100-base pair (bp) region (N2d) in intron 1, located 23 kb downstream from the transcriptional start sites, has been found to activate transcription in a cell type- and differentiation state-dependent manner. Maximum activity (approximately 20-fold) is seen in NIH 3T3 fibroblasts and intermediate activity (7-fold) in proliferating and undifferentiated C2C12 myoblasts. In contrast, this region is almost inactive in terminally differentiated C2C12 myotubes, in which endogenous nonmuscle MHC-A expression is down-regulated. Gel mobility shift assays and methylation interference analyses were performed using NIH 3T3 nuclear extracts to determine the protein-binding elements for transcription factors. Three binding elements have been identified within the N2d region. Antibody-supershift experiments, as well as competition experiments using consensus binding sequences for specific transcription factors, revealed that the most 5'-element, C (GGGAGGGGCC) is recognized specifically and exclusively by Sp1 and Sp3 transcriptional factors. Element C is immediately followed by a novel element, A (GTGACCC). A third element, F (GTGTCAGGTG), which contains an E-box, is located 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 three elements in various combinations demonstrate that the A and C binding factors cooperatively activate transcriptional activity in NIH 3T3 cells. The F binding factor shows an additive effect on transcription.
Beohar
N
N
Laboratory of Molecular Cardiology, NHLBI, National Institutes of Health, Bethesda, Maryland 20892, USA.
Kawamoto
S
S
D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002454:Cell Differentiation; D019175:DNA Methylation; D004268:DNA-Binding Proteins / Q000378:metabolism; D004742:Enhancer Elements, Genetic; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D006801:Humans; D007438:Introns; D008156:Luciferases / Q000096:biosynthesis; D051379:Mice; D008969:Molecular Sequence Data; D018995:Myosin Heavy Chains / Q000096:biosynthesis* / Q000235:genetics*; D009928:Organ Specificity; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012045:Regulatory Sequences, Nucleic Acid; D014158:Transcription, Genetic; D014162:Transfection
D004268:DNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D008156:Luciferases; D018995:Myosin Heavy Chains