Analysis of the inhibition of MyoD activity by ITF-2B and full-length E12/E47.

MyoD heterodimerizes with E type factors (E12/E47 and ITF-2A/ITF-2B) and binds E box sequences within promoters of muscle-specific genes. In transient transfection assays, MyoD activates transcription in the presence of ITF-2A but not ITF-2B, which contains a 182-amino acid N-terminal extension. The first 83 amino acids of the inhibitory N terminus of ITF-2B show high sequence homology to the N terminus of full-length E12/E47. Previous studies that showed activation of MyoD by E12 used an artificially N-terminally truncated form. Here we show that the full-length form of E12 inhibits MyoD function. A conserved alpha-helix motif, capable of interacting with the transcriptional machinery, was not essential for inhibition. Furthermore, the fusion of N-terminal ITF-2B sequences or non-inhibiting ITF-2A sequences to truncated E12 was sufficient in converting the activator into an inhibitor. Overexpression of ITF-2B did not inhibit C2C12 myogenesis or affect levels of endogenous muscle gene expression, consistent with the finding that inhibitory E type proteins are present in muscle. Furthermore, we found that MyoD co-transfected with either ITF-2B or ITF-2A converted fibroblasts into myoblasts with the same frequency. Our findings suggest that the ability of E type proteins to inhibit MyoD activity is dependent on the context of the E box.