A dominant-negative form of transcription factor MEF2 inhibits myogenesis.
A biological role for MEF2 (myocyte enhancer factor 2) activity during mammalian myogenesis has been inferred but not directly proven because of its role in the transcriptional activation of many muscle-specific genes. Therefore, our purpose was to determine whether MEF2 activity is absolutely required for mammalian myogenesis. Using a dominant-negative approach to address this question, we constructed a mutated MEF2A protein comprised of the amino-terminal DNA binding/dimerization domain of MEF2A without its trans-activation domain as a bacterial fusion protein (GST-131) or in a eukaryotic expression vector (pcDNA-131). GST-131 and the protein encoded by pcDNA-131 bind specifically to the MEF2 cis element and abrogate trans-activation of a MEF2-responsive luciferase reporter gene by wild type MEF2A, thus serving a role as trans-dominant inhibitors of MEF2 function. In congruence with their ability to interfere with wild type MEF2 function, microinjection of GST-131 or pcDNA-131 into L6E9 or C2C12 myoblasts inhibited myotube formation. Immunofluorescence analysis showed that the expression of myogenin, myosin heavy chain, and MEF2A were inhibited in the GST-131 or pcDNA-131-injected cells compared with GST or pcDNA-injected controls. We also document that this trans-dominant MEF2 inhibitor impairs the myogenic conversion of C3H10T1/2 fibroblasts by MyoD. Thus, these data provide evidence that the trans-activation function of the MEF2 proteins during mammalian myogenesis is required for muscle-specific gene expression and differentiation.
Ornatsky
O I
OI
Departments of Biology and Kinesiology, Faculty of Pure and Applied Science, York University, Toronto M3J 1P3, Canada.
Andreucci
J J
JJ
McDermott
J C
JC
D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors*; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D026161:MADS Domain Proteins; D064326:MEF2 Transcription Factors; D008845:Microinjections; D024510:Muscle Development; D017570:MyoD Protein / Q000037:antagonists & inhibitors; D018006:Myogenic Regulatory Factors; D018008:Myogenin / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000008:administration & dosage / Q000494:pharmacology; D014157:Transcription Factors / Q000037:antagonists & inhibitors*; D015533:Transcriptional Activation
D004268:DNA-Binding Proteins; D026161:MADS Domain Proteins; D064326:MEF2 Transcription Factors; C578035:MEF2A protein, human; C503799:MYOG protein, human; D017570:MyoD Protein; D018006:Myogenic Regulatory Factors; D018008:Myogenin; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D004247:DNA; D005982:Glutathione Transferase