Functional activity of myogenic HLH proteins requires hetero-oligomerization with E12/E47-like proteins in vivo.
In this report we provide four lines of evidence indicating that E12/E47-like proteins interact in vivo with the myogenic HLH proteins MyoD and myogenin. First, cotransfection of MyoD and E47 in COS cells indicates that these factors synergistically enhance transcription of a reporter gene containing an oligomerized MyoD-binding site. Second, mobility-shift assays of muscle cell nuclear extracts, "double shifted" with specific antisera, have identified complexes binding to the MEF1 site that contain either MyoD or myogenin in association with E12/E47-like proteins. Third, association with E47 alters the phosphorylation state of MyoD. Fourth, C3H10T1/2 cells expressing antisense E2A transcripts contain low levels of E2A gene products and display less terminal muscle differentiation when infected with retroviral MyoD or when challenged to differentiate with 5-azacytidine treatment. In addition we demonstrate that MyoD, in conjunction with E12/E47-like proteins, is functioning as a regulatory nodal point for activation of several other downstream muscle regulators.
Lassar
A B
AB
Department of Genetics, Fred Hutchinson Cancer Research Center, Seattle, Washington 98104.
Davis
R L
RL
Wright
W E
WE
Kadesch
T
T
Murre
C
C
Voronova
A
A
Baltimore
D
D
Weintraub
H
H
D017456:Adenovirus Early Proteins; D000818:Animals; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D007106:Immune Sera; D007118:Immunoassay; D046911:Macromolecular Substances; D009124:Muscle Proteins / Q000235:genetics / Q000378:metabolism*; D009132:Muscles / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D017570:MyoD Protein; D018008:Myogenin; D015345:Oligonucleotide Probes; D009856:Oncogene Proteins, Viral / Q000235:genetics; D010766:Phosphorylation; D051820:TCF Transcription Factors; D058125:Transcription Factor 7-Like 1 Protein; D014157:Transcription Factors / Q000235:genetics; D014158:Transcription, Genetic; D014162:Transfection
D017456:Adenovirus Early Proteins; D004268:DNA-Binding Proteins; D007106:Immune Sera; D046911:Macromolecular Substances; D009124:Muscle Proteins; D017570:MyoD Protein; D018008:Myogenin; D015345:Oligonucleotide Probes; D009856:Oncogene Proteins, Viral; D051820:TCF Transcription Factors; D058125:Transcription Factor 7-Like 1 Protein; D014157:Transcription Factors