Repression of muscle-specific gene activation by the murine Twist protein.
Inhibition of myogenic differentiation can be achieved by various mechanisms. The murine bHLH protein Twist has been shown to inhibit muscle differentiation in mammalian cells. Here we demonstrate that this inhibition is cell autonomous and does not alter cell proliferation. By overexpression of E12, we can distinguish the inhibitory mechanisms of Twist and the dominant negative HLH factor Id. A difference is seen both for the native muscle-specific enhancers of myogenin and myosin light chain 1/3 and for an enhancer consisting only of four E-boxes. Mutagenesis experiments revealed that both the basic region and an evolutionarily conserved carboxy-terminal domain are required for the Twist-specific type of inhibition. Loss of either of these regions renders Twist less efficient and more similar to Id. Gel mobility shift assays demonstrate that Twist can bind to the muscle creatine kinase E-box and inhibit DNA binding of heterodimers of E12 with myogenic bHLH transcription factors like MyoD. However, a fourfold excess of Twist compared to MyoD is required for both effects. Our results suggest that Twist inhibits muscle-specific gene activation by formation of actively inhibitory complexes rather than by sequestering E-proteins.
Hebrok
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M
Max-Planck-Institut für Immunbiologie, Freiburg, Germany.
Füchtbauer
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A
Füchtbauer
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EM
D000818:Animals; D002454:Cell Differentiation / Q000235:genetics; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology; D018507:Gene Expression Regulation, Developmental / Q000502:physiology*; D018257:Helix-Loop-Helix Motifs / Q000235:genetics / Q000502:physiology; D051798:Inhibitor of Differentiation Protein 1; D046911:Macromolecular Substances; D051379:Mice; D009124:Muscle Proteins / Q000096:biosynthesis / Q000235:genetics*; D009132:Muscles / Q000166:cytology / Q000196:embryology*; D016297:Mutagenesis, Site-Directed; D017570:MyoD Protein / Q000037:antagonists & inhibitors / Q000502:physiology; D009687:Nuclear Proteins / Q000235:genetics / Q000502:physiology*; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012097:Repressor Proteins; D017384:Sequence Deletion; D051820:TCF Transcription Factors; D058125:Transcription Factor 7-Like 1 Protein; D014157:Transcription Factors / Q000502:physiology; D015533:Transcriptional Activation; D014162:Transfection; D051879:Twist-Related Protein 1
D004268:DNA-Binding Proteins; C497455:Idb1 protein, mouse; D051798:Inhibitor of Differentiation Protein 1; D046911:Macromolecular Substances; D009124:Muscle Proteins; D017570:MyoD Protein; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D051820:TCF Transcription Factors; C544913:Tcf7l1 protein, mouse; D058125:Transcription Factor 7-Like 1 Protein; D014157:Transcription Factors; D051879:Twist-Related Protein 1