Overexpression of Id protein inhibits the muscle differentiation program: in vivo association of Id with E2A proteins.
The helix-loop-helix (HLH) protein Id lacks the basic DNA-binding domain common to this class of proteins. In vitro experiments suggested that Id could associate tightly with two other HLH proteins encoded by the E2A gene, E12 and E47 (referred to here collectively as E proteins) and prevent their binding to a sequence present in the muscle creatine kinase (MCK) enhancer either as homo-oligomers or hetero-oligomers with MyoD. In this report we present evidence for the in vivo roles of Id and E proteins: (1) Id and E proteins co-fractionate and co-immunoprecipitate in whole-cell extracts prepared from myoblasts; (2) the loss of Id protein observed during the conversion of proliferating myoblasts into mature myotubes correlates with the formation of MyoD/E hetero-oligomeric complexes in whole-cell extracts (these complexes do not form when purified Id protein is added to the extracts); and (3) stable overexpression of Id mRNA and protein in the C2C12 muscle cell line inhibits differentiation in these cells 16 hr post-induction. The myotubes that do eventually form 48 hr post-induction have no detectable Id protein in the nucleus despite the persistence of exogenous Id mRNA. These data support a model in which Id can inhibit muscle cell differentiation by associating with E proteins and preventing them from forming active hetero-oligomeric complexes with the muscle determination gene products.
Jen
Y
Y
Department of Cell Biology, Memorial Sloan Kettering Cancer Center, New York, New York 10021.
Weintraub
H
H
Benezra
R
R
D000818:Animals; D015152:Blotting, Northern; D002454:Cell Differentiation / Q000502:physiology*; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D051798:Inhibitor of Differentiation Protein 1; D009132:Muscles / Q000166:cytology / Q000378:metabolism*; D011233:Precipitin Tests; D012097:Repressor Proteins; D051820:TCF Transcription Factors; D058125:Transcription Factor 7-Like 1 Protein; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
D004268:DNA-Binding Proteins; D051798:Inhibitor of Differentiation Protein 1; D012097:Repressor Proteins; D051820:TCF Transcription Factors; D058125:Transcription Factor 7-Like 1 Protein; D014157:Transcription Factors