Identification of a myocyte nuclear factor that binds to the muscle-specific enhancer of the mouse muscle creatine kinase gene.
The muscle creatine kinase (MCK) gene is transcriptionally induced when skeletal muscle myoblasts differentiate into myocytes. The gene contains two muscle-specific enhancer elements, one located 1,100 nucleotides (nt)5' of the transcriptional start site and one located in the first intron. We have used gel mobility shift assays to characterize the trans-acting factors that interact with a region of the MCK gene containing the 5' enhancer. MM14 and C2C12 myocyte nuclear extracts contain a sequence-specific DNA-binding factor which recognizes a site within a 110-nt fragment of the MCK enhancer region shown to be sufficient for enhancer function. Preparative mobility shift gels were combined with DNase I footprinting to determine the site of binding within the 110-nt fragment. Site-directed mutagenesis within the footprinted region produced a 110-nt fragment which did not bind the myocyte factor in vitro. The mutant fragment had about 25-fold-less activity as a transcriptional enhancer in myocytes than did the wild-type fragment. Complementary oligomers containing 21 base pairs spanning the region protected from DNase degradation were also specifically bound by MM14 and C2C12 myocyte nuclear factors. The oligomer-binding activity was not found in nuclear extracts from the corresponding myoblasts, in nuclear extracts from a variety of nonmuscle cell types (including differentiation-defective MM14-DD1 cells and 10T1/2 mesodermal stem cells), or in cytoplasmic extracts. Both the 5' and intron 1 enhancer-containing fragments competed for factors that bind the oligomer probe, while total mouse genomic DNA and several DNA fragments containing viral and cellular enhancers did not. Interestingly, a 5' MCK proximal promoter fragment that also contains muscle-specific positive regulatory elements did not compete for factor binding to the oligomer. We have designated the factor which interacts with the two MCK enhancers myocyte-specific enhancer-binding nuclear factor 1 (MEF 1). A consensus for binding sites in muscle-specific regulatory regions is proposed.
Department of Biochemistry, University of Washington, Seattle 98195.
J N
JN
Buskin
S D
SD
Hauschka
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002454:Cell Differentiation; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D003402:Creatine Kinase / Q000235:genetics*; D004247:DNA / Q000378:metabolism; D015342:DNA Probes; D003850:Deoxyribonuclease I; D004742:Enhancer Elements, Genetic; D051379:Mice; D009132:Muscles / Q000032:analysis / Q000166:cytology* / Q000201:enzymology; D009154:Mutation; D017570:MyoD Protein; D009687:Nuclear Proteins / Q000032:analysis / Q000378:metabolism; D009841:Oligonucleotides / Q000138:chemical synthesis / Q000378:metabolism; D010750:Phosphoproteins / Q000032:analysis / Q000378:metabolism; D011401:Promoter Regions, Genetic; D015183:Restriction Mapping; D014157:Transcription Factors / Q000032:analysis / Q000378:metabolism*; D014162:Transfection
D015342:DNA Probes; D017570:MyoD Protein; C057363:MyoD1 myogenic differentiation protein; D009687:Nuclear Proteins; D009841:Oligonucleotides; D010750:Phosphoproteins; D014157:Transcription Factors; D004247:DNA; D003402:Creatine Kinase; D003850:Deoxyribonuclease I