Cyclic amplification and selection of targets for multicomponent complexes: myogenin interacts with factors recognizing binding sites for basic helix-loop-helix, nuclear factor 1, myocyte-specific enhancer-binding factor 2, and COMP1 factor.
Myogenin is one of four muscle-specific basic helix-loop-helix regulatory factors involved in controlling myogenesis. We here describe various protein complexes that increase the affinity of myogenin for DNA. We mixed an oligonucleotide containing a degenerate center large enough to accommodate multiple binding sites with crude myotube nuclear extracts and used cyclic amplification and selection of targets with an antimyogenin monoclonal antibody to isolate protein-DNA complexes. Since each cycle of selection results in the enrichment for the sequences with the highest affinity, we isolated multicomponent sites in which myogenin binding was increased by its interaction with other DNA binding proteins. Myogenin interacts with members of the nuclear factor 1 family, the muscle-specific factor myocyte-specific enhancer-binding factor 2, and another factor, COMP1 (cooperates with myogenic proteins 1), that binds to the sequence TGATTGAC. Myogenin also exhibits cooperative binding with other proteins that recognize CANNTG motifs, and various constraints on spacing and orientation were observed. The application of this approach to other transcription factors should not only help identify the different functions of myogenin versus other members of the muscle basic helix-loop-helix regulatory family but also help define the general combinatorial mechanisms involved in eukaryotic gene regulation.
Funk
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WD
Department of Cell Biology and Neuroscience, University of Texas Southwestern Medical Center, Dallas 75235.
Wright
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WE
D001483:Base Sequence; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000378:metabolism*; D066298:In Vitro Techniques; D064326:MEF2 Transcription Factors; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000378:metabolism*; D009132:Muscles / Q000502:physiology*; D018006:Myogenic Regulatory Factors; D018008:Myogenin; D009687:Nuclear Proteins / Q000378:metabolism*; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D012045:Regulatory Sequences, Nucleic Acid; D016415:Sequence Alignment; D014157:Transcription Factors / Q000378:metabolism*
D004268:DNA-Binding Proteins; D064326:MEF2 Transcription Factors; D046911:Macromolecular Substances; D009124:Muscle Proteins; D018006:Myogenic Regulatory Factors; D018008:Myogenin; D009687:Nuclear Proteins; D009838:Oligodeoxyribonucleotides; D014157:Transcription Factors