E1A-mediated inhibition of myogenesis correlates with a direct physical interaction of E1A12S and basic helix-loop-helix proteins.
The observation that adenovirus E1A gene products can inhibit differentiation of skeletal myocytes suggested that E1A may interfere with the activity of myogenic basic helix-loop-helix (bHLH) transcription factors. We have examined the ability of E1A to mediate repression of the muscle-specific creatine kinase (MCK) gene. Both the E1A12S and E1A13S products repressed MCK transcription in a concentration-dependent fashion. In contrast, amino-terminal deletion mutants (d2-36 and d15-35) of E1A12S were defective for repression. E1A12S also repressed expression of a promoter containing a multimer of the MCK high-affinity E box (the consensus site for myogenic bHLH protein binding) that was dependent, in C3H10T1/2 cells, on coexpression of a myogenin bHLH-VP16 fusion protein. A series of coprecipitation experiments with glutathione S-transferase fusion and in vitro-translated proteins demonstrated that E1A12S, but not amino-terminal E1A deletion mutants, could bind to full-length myogenin and E12 and to deletion mutants of myogenin and E12 that spare the bHLH domains. Thus, the bHLH domains of myogenin and E12, and the high-affinity E box, are targets for E1A-mediated repression of the MCK enhancer, and domains of E1A required for repression of muscle-specific gene transcription also mediate binding to bHLH proteins. We conclude that E1A mediates repression of muscle-specific gene transcription through its amino-terminal domain and propose that this may involve a direct physical interaction between E1A and the bHLH region of myogenic determination proteins.
Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710.
D A
DA
Taylor
V B
VB
Kraus
J J
JJ
Schwarz
E N
EN
Olson
W E
WE
Kraus
D017500:Adenovirus E1A Proteins / Q000378:metabolism*; D001483:Base Sequence; D002454:Cell Differentiation; D003402:Creatine Kinase; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004742:Enhancer Elements, Genetic; D005786:Gene Expression Regulation; D006801:Humans; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000235:genetics*; D009132:Muscles / Q000166:cytology*; D018008:Myogenin; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D011485:Protein Binding; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D013329:Structure-Activity Relationship; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
D017500:Adenovirus E1A Proteins; D004268:DNA-Binding Proteins; C503799:MYOG protein, human; D009124:Muscle Proteins; D018008:Myogenin; D009838:Oligodeoxyribonucleotides; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D014157:Transcription Factors; D003402:Creatine Kinase