Identification of MRF4, myogenin, and E12 oligomer complexes by chemical cross-linking and two-dimensional gel electrophoresis.
The muscle-specific regulatory factors MRF4 and myogenin, as well as the ubiquitous factor E12, belong to a protein family which shares a common structural motif referred to as the basic helix-loop-helix domain. Recent studies have demonstrated that MRF4 and myogenin, in the presence of E12, bind efficiently to enhancer regions of muscle-specific genes, thereby activating their transcription. Although several lines of evidence suggest that MRF4 and E12 or myogenin and E12 hetero-oligomers exist, direct studies revealing the composition of these protein complexes have not been reported. Here, we demonstrate that MRF4 and myogenin preferentially form heterodimers with E12 in solution and that heterodimer formation in vitro is greatly enhanced when the two proteins are co-synthesized. Utilizing a novel two-dimensional gel electrophoresis system, we have found that MRF4 and myogenin, when complexed with E12, bind as heterodimers to the E-box consensus sequences associated with the troponin I, M-creatine kinase, and myosin light chain gene enhancers. In all cases, higher order oligomer structures were not detected, demonstrating that in vitro DNA binding abilities of these basic helix-loop-helix proteins require heterodimer formation.
Lin
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Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907.
Konieczny
S F
SF
D001483:Base Sequence; D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional; D004742:Enhancer Elements, Genetic; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000235:genetics / Q000378:metabolism*; D018006:Myogenic Regulatory Factors; D018008:Myogenin; D011233:Precipitin Tests; D014176:Protein Biosynthesis; D051820:TCF Transcription Factors; D058125:Transcription Factor 7-Like 1 Protein; D014157:Transcription Factors; D014158:Transcription, Genetic
D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins; D009124:Muscle Proteins; D018006:Myogenic Regulatory Factors; D018008:Myogenin; D051820:TCF Transcription Factors; D058125:Transcription Factor 7-Like 1 Protein; D014157:Transcription Factors; C063403:myogenic factor 6