Multiple muscle-specific regulatory elements are associated with a DNase I hypersensitive site of the cardiac beta-myosin heavy-chain gene.
Using nuclei isolated from neonatal cardiomyocytes, we have mapped the DNase I hypersensitive sites (DHSs) residing within the 5'-upstream regions of the hamster cardiac myosin heavy-chain (MyHC) gene. Two cardiac-specific DHSs within the 5 kb upstream region of the cardiac MyHC gene were identified. One of the DHSs was mapped to the -2.3 kb (beta-2.3 kb) region and the other to the proximal promoter region. We further localized the beta-2.3 kb site to a range of 250 bp. Multiple, conserved, muscle regulatory motifs were found within the beta-2.3 kb site, consisting of three E-boxes, one AP-2 site, one CArG motif, one CT/ACCC box and one myocyte-specific enhancer factor-2 site. This cluster of regulatory elements is strikingly similar to a cluster found in the enhancer of the mouse muscle creatine kinase gene (-1256 to -1050). The specific interaction of the motifs within the beta-2.3 kb site and the cardiac nuclear proteins was demonstrated using gel mobility-shift assays and footprinting analysis. In addition, transfection analysis revealed a significant increase in chloramphenicol acetyltransferase activity when the beta-2.3 kb site was linked to a heterologous promoter. These results suggest that previously undefined regulatory elements of the beta-MyHC gene may be associated with the beta-2.3 kb site.
Huang
W Y
WY
Laboratory for Molecular Cardiology, Departments of Clinical Biochemistry and Medicine, University of Toronto, Toronto, Ontario, Canada.
Chen
J J
JJ
Shih
N
N
Liew
C C
CC
D000818:Animals; D000831:Animals, Newborn; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis; D003402:Creatine Kinase / Q000096:biosynthesis / Q000235:genetics; D006224:Cricetinae; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000378:metabolism*; D003850:Deoxyribonuclease I; D005347:Fibroblasts; D007668:Kidney; D051379:Mice; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000378:metabolism*; D009206:Myocardium / Q000378:metabolism*; D018995:Myosin Heavy Chains / Q000096:biosynthesis* / Q000235:genetics*; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012045:Regulatory Sequences, Nucleic Acid; D016415:Sequence Alignment; D012689:Sequence Homology, Nucleic Acid; D014162:Transfection
D004268:DNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D015500:Chloramphenicol O-Acetyltransferase; D003402:Creatine Kinase; D003850:Deoxyribonuclease I; D018995:Myosin Heavy Chains