Expression of the alpha-myosin heavy chain gene in the heart is regulated in part by an E-box-dependent mechanism.
Proximal regulatory element B (PRE-B), located from positions -318 to -284 in the alpha-myosin heavy chain (MHC) promoter, stimulated expression from an otherwise weak alpha-MHC promoter fragment in primary rat neonatal cardiomyocytes but not in the C2C12 myogenic cell line. PRE-B interacted with alpha-MHC binding factor 2 (BF-2), a protein found in nuclear extracts from several neonatal rat tissues and cell types including cardiomyocytes. BF-2 DNA binding activity was greatly reduced in adult versus neonatal tissues. Methylation interference footprints indicated that BF-2 bound to an element that included an E-box consensus sequence. Site-directed mutations in the BF-2-binding site, that abolish BF-2 binding, reduced expression from the full-length alpha-MHC promoter by 70%. A BF-2-like protein interacts within the HF-1a element of the myosin light chain-2 (MLC-2) promoter suggesting that one of the proteins that regulates the alpha-MHC and MLC-2 genes is identical or closely related. Analysis of binding by competition gel shift experiments indicated that both BF-2 and HF-1a are E-box-binding proteins. The alpha-MHC and MLC-2 genes encode contractile proteins which are precursors of myosin. Regulation by the same transcription factor might indicate that the expression of alpha-MHC and MLC-2 is coordinately controlled.
Molkentin
J D
JD
Department of Physiology, Medical College of Wisconsin, Milwaukee 53226.
Brogan
R S
RS
Jobe
S M
SM
Markham
B E
BE
D000818:Animals; D001483:Base Sequence; D004268:DNA-Binding Proteins / Q000378:metabolism; D005786:Gene Expression Regulation; D066298:In Vitro Techniques; D008745:Methylation; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009206:Myocardium / Q000378:metabolism*; D009218:Myosins / Q000235:genetics*; D009687:Nuclear Proteins / Q000378:metabolism; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D012045:Regulatory Sequences, Nucleic Acid; D013329:Structure-Activity Relationship; D014158:Transcription, Genetic
D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D009838:Oligodeoxyribonucleotides; D012333:RNA, Messenger; D009218:Myosins