A novel E1A domain mediates skeletal-muscle-specific enhancer repression independently of pRB and p300 binding.

Abstract

The adenovirus E1A oncoprotein completely blocks muscle differentiation and specifically inhibits the transactivating function of myogenic basic helix-loop-helix (bHLH) transcription factors. This inhibition is dependent on the conserved region CR1 of E1A, which also constitutes part of the binding sites for the pocket proteins pRB, p107, and p130 and the transcriptional coactivators p300 and CBP. Here we report a detailed mutational analysis of E1A and the identification of a muscle inhibition motif within CR1. This motif encompasses amino acids 38 to 62 and inhibits Myf-5- or MyoD-mediated activation of myogenin and the muscle creatine kinase gene. Overexpression of this E1A region also inhibits the conversion of 10T1/2 fibroblasts to the myogenic lineage. The sequence motif EPDNEE (amino acids 55 to 60) within CR1 appears to be particularly important, because point mutations of this sequence diminish the E1A inhibitory activity. Interactions of E1A with pRB and with p300 do not seem to be necessary for the muscle-specific enhancer repression, because E1A mutants which lack these interactions still inhibit Myf-5- and MyoD-mediated transactivation. Moreover, overexpression of p300 fails to overcome muscle-specific inhibition by wild-type E1A and mutant E1A protein which lacks pRB binding. Since we have no evidence for direct E1A interaction with bHLH proteins, we propose that E1A may target a necessary cofactor of the muscle-specific bHLH transcription complex.