Characterization of a dominant inhibitory E47 protein that suppresses C2C12 myogenesis.
Skeletal muscle formation is controlled through the coordinated actions of the muscle regulatory factors (MRFs). The activities of these basic helix-loop-helix proteins is mediated in part through heterodimer formation with a family of ubiquitous bHLH proteins, referred to as E-proteins. The primary E-protein in skeletal muscle is the E2A splice variant, E47. To further address the role of E47 during skeletal myogenesis, we created a chimeric E47 repressor protein by replacing the transcriptional activation domain with the Drosophila Engrailed transcriptional repressor domain. The dominant inhibitory E-protein (EnDeltaE47) formed homodimers capable of binding DNA and abolished E47-directed gene transcription. Stable expression of EnDeltaE47 in mouse C2C12 myoblasts effectively blocked the cells' ability to differentiate into mature myofibers. Closer examination of the molecular basis for the inhibition of myogenesis revealed that EnDeltaE47 preferentially forms heterodimers with myogenin. Interestingly, the chimeric repressor did not form DNA-binding heterodimers with MyoD in C2C12 myocytes. The failure to detect MyoD:EnDeltaE47 heterodimers in myoblasts was not due to protein conformational defects as both wild-type E47 and EnDeltaE47 readily formed DNA binding complexes with MyoD in vitro. These results indicate that E47 plays a crucial role in C2C12 myogenesis by serving as the preferred heterodimer partner of the myogenin protein.
Becker
J R
JR
Department of Poultry Science, The Pennsylvania State University, University Park, Pennsylvania 16802, USA.
Dorman
C M
CM
McClafferty
T M
TM
Johnson
S E
SE
D000818:Animals; D002454:Cell Differentiation; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D019281:Dimerization; D029721:Drosophila Proteins; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D018482:Muscle, Skeletal / Q000166:cytology* / Q000502:physiology; D017570:MyoD Protein / Q000378:metabolism; D018006:Myogenic Regulatory Factors / Q000235:genetics / Q000378:metabolism*; D018008:Myogenin / Q000378:metabolism; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D051820:TCF Transcription Factors; D058125:Transcription Factor 7-Like 1 Protein; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism
D004268:DNA-Binding Proteins; D029721:Drosophila Proteins; C063459:En protein, Drosophila; D018398:Homeodomain Proteins; D017570:MyoD Protein; C503801:Myog protein, mouse; D018006:Myogenic Regulatory Factors; D018008:Myogenin; D011993:Recombinant Fusion Proteins; D051820:TCF Transcription Factors; C544913:Tcf7l1 protein, mouse; D058125:Transcription Factor 7-Like 1 Protein; D014157:Transcription Factors