Physical and functional interactions between the transcriptional inhibitors Id3 and ITF-2b. Evidence toward a novel mechanism regulating muscle-specific gene expression.
We have used an interaction cloning strategy to identify an inhibitory isoform of the ITF-2 transcription factor, ITF-2b, that interacts with the transcriptional inhibitor Id3/HLH462. The interaction was confirmed in vitro, and inside intact myogenic C2C12 cells. As expected, overexpression of either Id3/HLH462 or ITF-2b effectively inhibited the activation of the muscle-specific creatine kinase promoter by the myogenic transcription factor MyoD. However, when overexpressed simultaneously, ITF-2b and Id3/HLH462 counteracted each other's inhibitory effect to produce a reduced overall inhibition. Moreover, while ITF-2b inhibited the creatine kinase promoter, it acted as a weak transactivator on an artificial promoter consisting of three tandem copies of the consensus myogenic factor DNA binding site. Further investigation indicated that the ITF-2b/MyoD heterodimer bound to its specific DNA binding site in vitro, and the DNA binding was effectively blocked by Id3/HLH462. Additional analysis revealed the presence of transcripts for both the activating (ITF-2a) and inhibitory (ITF-2b) isoforms in differentiating C2C12 cultures, suggesting that both isoforms might participate in regulating the differentiation process. Taken together, this study reveals a more complex pattern of regulatory interactions involving the helix-loop-helix proteins than was previously anticipated.
Chen
B
B
Department of Pharmacology, University of Missouri, School of Medicine, Columbia, Missouri 65212, USA.
Lim
R W
RW
D000199:Actins / Q000235:genetics / Q000378:metabolism; D000818:Animals; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D002478:Cells, Cultured; D018076:DNA, Complementary / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005786:Gene Expression Regulation; D018257:Helix-Loop-Helix Motifs; D051796:Inhibitor of Differentiation Proteins; D051379:Mice; D009132:Muscles / Q000378:metabolism*; D018008:Myogenin / Q000235:genetics / Q000378:metabolism; D009363:Neoplasm Proteins; D009419:Nerve Tissue Proteins; D016133:Polymerase Chain Reaction; D051820:TCF Transcription Factors; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*; D000073940:Transcription Factor 4; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
D000199:Actins; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D051796:Inhibitor of Differentiation Proteins; C503801:Myog protein, mouse; D018008:Myogenin; D009363:Neoplasm Proteins; D009419:Nerve Tissue Proteins; D051820:TCF Transcription Factors; C090743:Tcf4 protein, mouse; D015534:Trans-Activators; D000073940:Transcription Factor 4; D014157:Transcription Factors; C075640:ID3 protein, human