Two distinct factor-binding DNA elements in cardiac myosin light chain 2 gene are essential for repression of its expression in skeletal muscle. Isolation of a cDNA clone for repressor protein Nished.
The expression of the cardiac myosin light chain 2 (MLC2) gene is repressed in skeletal muscle as a result of the negative regulation of its transcription. Two regulatory elements, the cardiac specific sequence (CSS) located upstream (-360 base pairs) and a downstream negative modulatory sequence (NMS), which function in concert with each other, are required for repression of the MLC2 promoter activity in skeletal muscle. Individually, CSS and NMS have no effect. Transient transfection analysis with recombinant plasmids indicated that CSS- and NMS-mediated repression of transcription is position- and orientation-dependent and is transferable to heterologous promoters. A minimal conserved motif, GAAG/CTTC, present in both CSS and NMS, is responsible for repression as the mutation in the core CTTC sequence alone was sufficient to abrogate its repressor activity. The DNA binding assay by gel mobility shift analysis revealed that one of the two complexes, CSSBP2, is significantly enriched in embryonic skeletal muscle relative to cardiac muscle. In extracts from adult skeletal muscle, where the cardiac MLC2 expression is suppressed, both complexes, CSSBP1 and CSSBP2, were present, whereas the cardiac muscle extracts contained CSSBP1 alone, suggesting that the protein(s) in the CSSBP2 complex accounts for the negative regulation of cardiac MLC2 in skeletal muscle. A partial cDNA clone (Nished) specific for the candidate repressor factor was isolated by expression screening of the skeletal muscle cDNA library by multimerized CSS-DNA as probe. The recombinant Nished protein binds to the CSS-DNA, but not to DeltaCSS-DNA where the core CTTC sequence was mutated. The amino acid sequence of Nished showed a significant structural similarity to the sequence of transcription factor "runt," a known repressor of gap and pair-rule gene expression in Drosophila.
Dhar
M
M
Center for Cardiovascular and Muscle Research, Department of Anatomy and Cell Biology, State University of New York, Brooklyn, New York 11203, USA.
Mascareno
E M
EM
Siddiqui
M A
MA
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D024722:Cardiac Myosins; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D002642:Chick Embryo; D002645:Chickens; D017124:Conserved Sequence; D018983:DNA Footprinting; D004268:DNA-Binding Proteins / Q000378:metabolism*; D003850:Deoxyribonuclease I; D015723:Gene Library; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009206:Myocardium / Q000378:metabolism; D018994:Myosin Light Chains / Q000096:biosynthesis* / Q000235:genetics; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid; D012097:Repressor Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D014157:Transcription Factors / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D014158:Transcription, Genetic; D014162:Transfection
D004268:DNA-Binding Proteins; D018994:Myosin Light Chains; C107185:Nished protein, Gallus gallus; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D012097:Repressor Proteins; D014157:Transcription Factors; C107184:myosin light chain 2; D003850:Deoxyribonuclease I; D024722:Cardiac Myosins