The steroid receptor coactivator, GRIP-1, is necessary for MEF-2C-dependent gene expression and skeletal muscle differentiation.
Nuclear receptor-mediated activation of transcription involves coactivation by cofactors collectively denoted the steroid receptor coactivators (SRCs). The process also involves the subsequent recruitment of p300/CBP and PCAF to a complex that synergistically regulates transcription and remodels the chromatin. PCAF and p300 have also been demonstrated to function as critical coactivators for the muscle-specific basic helix-loop-helix (bHLH) protein MyoD during myogenic commitment. Skeletal muscle differentiation and the activation of muscle-specific gene expression is dependent on the concerted action of another bHLH factor, myogenin, and the MADS protein, MEF-2, which function in a cooperative manner. We examined the functional role of one SRC, GRIP-1, in muscle differentiation, an ideal paradigm for the analysis of the determinative events that govern the cell's decision to divide or differentiate. We observed that the mRNA encoding GRIP-1 is expressed in proliferating myoblasts and post-mitotic differentiated myotubes, and that protein levels increase during differentiation. Exogenous/ectopic expression studies with GRIP-1 sense and antisense vectors in myogenic C2C12 cells demonstrated that this SRC is necessary for (1) induction/activation of myogenin, MEF-2, and the crucial cell cycle regulator, p21, and (2) contractile protein expression and myotube formation. Furthermore, we demonstrate that the SRC GRIP-1 coactivates MEF-2C-mediated transcription. GRIP-1 also coactivates the synergistic transactivation of E box-dependent transcription by myogenin and MEF-2C. GST-pulldowns, mammalian two-hybrid analysis, and immunoprecipitation demonstrate that the mechanism involves direct interactions between MEF-2C and GRIP-1 and is associated with the ability of the SRC to interact with the MADS domain of MEF-2C. The HLH region of myogenin mediates the direct interaction of myogenin and GRIP-1. Interestingly, interaction with myogenic factors is mediated by two regions of GRIP-1, an amino-terminal bHLH-PAS region and the carboxy-terminal region between amino acids 1158 and 1423 (which encodes an activation domain, has HAT activity, and interacts with the coactivator-associated arginine methyltransferase). This work demonstrates that GRIP-1 potentiates skeletal muscle differentiation by acting as a critical coactivator for MEF-2C-mediated transactivation and is the first study to ascribe a function to the amino-terminal bHLH-PAS region of SRCs.
Chen
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University of Queensland, Institute for Molecular Biosciences, Centre for Molecular and Cellular Biology, Ritchie Research Laboratories, B402A, St. Lucia, 4072, Queensland, Australia.
Dowhan
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DH
Hosking
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BM
Muscat
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GE
D000818:Animals; D001665:Binding Sites; D002454:Cell Differentiation; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000378:metabolism; D018507:Gene Expression Regulation, Developmental; D018257:Helix-Loop-Helix Motifs; D006801:Humans; D026161:MADS Domain Proteins; D064326:MEF2 Transcription Factors; D051379:Mice; D018482:Muscle, Skeletal / Q000166:cytology* / Q000378:metabolism; D018006:Myogenic Regulatory Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018008:Myogenin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D051958:Nuclear Receptor Coactivator 2; D009928:Organ Specificity; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D016372:RNA, Antisense / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020218:Response Elements / Q000235:genetics; D017384:Sequence Deletion / Q000235:genetics; D051820:TCF Transcription Factors; D058125:Transcription Factor 7-Like 1 Protein; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015533:Transcriptional Activation; D020798:Two-Hybrid System Techniques
D004268:DNA-Binding Proteins; D026161:MADS Domain Proteins; D064326:MEF2 Transcription Factors; C578040:MEF2C protein, human; C503799:MYOG protein, human; C578041:Mef2c protein, mouse; C503801:Myog protein, mouse; D018006:Myogenic Regulatory Factors; D018008:Myogenin; C498001:NCOA2 protein, human; C498002:Ncoa2 protein, mouse; D051958:Nuclear Receptor Coactivator 2; D016372:RNA, Antisense; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D051820:TCF Transcription Factors; C544912:TCF7L1 protein, human; C544913:Tcf7l1 protein, mouse; D058125:Transcription Factor 7-Like 1 Protein; D014157:Transcription Factors