title: Analysis of the inhibition of MyoD activity by ITF-2B and full-length E12/E47.
abstract: 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.
authors.affiliation: Department of Biochemistry, University of Western Ontario, London, Canada.
authors.forename: H
authors.initials: H
authors.lastname: Petropoulos
authors.forename: I S
authors.initials: IS
authors.lastname: Skerjanc
mesh_terms: D000199:Actins / Q000037:antagonists & inhibitors / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D015152:Blotting, Northern; D015153:Blotting, Western; D002454:Cell Differentiation; D002460:Cell Line; D018076:DNA, Complementary / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D005347:Fibroblasts / Q000378:metabolism; D006801:Humans; D017403:In Situ Hybridization; D051379:Mice; D008969:Molecular Sequence Data; D009132:Muscles / Q000378:metabolism; D017570:MyoD Protein / Q000037:antagonists & inhibitors*; D009206:Myocardium / Q000737:chemistry; D009419:Nerve Tissue Proteins; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D051820:TCF Transcription Factors; D015534:Trans-Activators / Q000737:chemistry / Q000378:metabolism*; D000073940:Transcription Factor 4; D058125:Transcription Factor 7-Like 1 Protein; D058118:Transcription Factor 7-Like 2 Protein; D014157:Transcription Factors; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014162:Transfection
chemical_list: D000199:Actins; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D017570:MyoD Protein; D009419:Nerve Tissue Proteins; D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins; D051820:TCF Transcription Factors; C544912:TCF7L1 protein, human; C544894:TCF7L2 protein, human; C090743:Tcf4 protein, mouse; C544913:Tcf7l1 protein, mouse; C544895:Tcf7l2 protein, mouse; D015534:Trans-Activators; D000073940:Transcription Factor 4; D058125:Transcription Factor 7-Like 1 Protein; D058118:Transcription Factor 7-Like 2 Protein; D014157:Transcription Factors