Identification of a contractile-responsive element in the cardiac alpha-myosin heavy chain gene.
The mechanisms by which the cardiac-specific alpha-myosin heavy chain (alpha-MHC) gene responds to contractile activity was studied in cultured cardiomyocytes and in vivo. Deletion analysis of the alpha-MHC promoter transiently transfected into neonatal rat cardiomyocytes localized the contractile-responsive element within -80 to -40 base pairs of the transcriptional start site. Mutational analysis of an E-box motif at position -47 showed that it was necessary for the contractile response both in cultured cardiomyocytes and in the intact heart. Competition gel mobility shift experiments indicated that the protein-DNA complex formed within the -39 to -59 base pair region could be competed by the E-box element at -309 of the alpha-MHC gene and that base substitutions within an E-box motif at -47 eliminated the protein-DNA complex. To identify the contractile-responsive nuclear protein, antibodies specific for E12/E47, an E-box binding basic-helix-loop-helix (bHLH) protein, and antibodies recognizing upstream stimulatory factor (USF), a widely expressed bHLH-leucine zipper transcription factor, were studied for their ability to inhibit cardiomyocyte nuclear protein binding to the E-box motif at -47. Anti-USF antibody abolished formation of the protein-DNA complex, thus identifying the protein as antigenically related to USF and demonstrating that bHLH-leucine zipper proteins are involved in the contractile-induced expression of the cardiac alpha-MHC gene.
Department of Medicine, North Shore University Hospital/Cornell University Medical College, Manhasset, New York 11030, USA.
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Ojamaa
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Samarel
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Klein
D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D008969:Molecular Sequence Data; D009200:Myocardial Contraction / Q000235:genetics*; D009206:Myocardium / Q000378:metabolism*; D018995:Myosin Heavy Chains / Q000235:genetics*; D011401:Promoter Regions, Genetic; D051381:Rats; D012045:Regulatory Sequences, Nucleic Acid; D014157:Transcription Factors / Q000378:metabolism; D014162:Transfection
D014157:Transcription Factors; D018995:Myosin Heavy Chains