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...

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Bibliographic Details
Published in:The Journal of biological chemistry 2000-08, Vol.275 (33), p.25095-25101
Main Authors: Petropoulos, Helen, Skerjanc, Ilona S.
Format: Article
Language:English
Subjects:
Actins - antagonists & inhibitors
Actins - genetics
Amino Acid Sequence
Animals
Basic Helix-Loop-Helix Leucine Zipper Transcription Factors
Blotting, Northern
Blotting, Western
Cell Differentiation
Cell Line
DNA, Complementary - metabolism
DNA-Binding Proteins - chemistry
DNA-Binding Proteins - metabolism
Fibroblasts - metabolism
Humans
In Situ Hybridization
Mice
Molecular Sequence Data
Muscles - metabolism
Myocardium - chemistry
MyoD Protein - antagonists & inhibitors
Nerve Tissue Proteins
Plasmids - metabolism
Promoter Regions, Genetic
Protein Isoforms
Protein Structure, Tertiary
Recombinant Fusion Proteins - metabolism
Sequence Homology, Amino Acid
TCF Transcription Factors
Trans-Activators - chemistry
Trans-Activators - metabolism
Transcription Factor 4
Transcription Factor 7-Like 1 Protein
Transcription Factor 7-Like 2 Protein
Transcription Factors
Transcription, Genetic
Transcriptional Activation
Transfection
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Summary: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 α-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.
ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.M004251200