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In Vitro Transcription Assays and Their Application in Drug Discovery
Published on: September 20, 2016
OUT, a novel basic helix-loop-helix transcription factor with an Id-like inhibitory activity
1Department of Molecular Genetics, Graduate School of Medicine, Kyoto University, Shogoin Kawahara-cho 53, Sakyo-ku, 606-8507 Kyoto, Japan.
The Journal of Biological Chemistry
|February 1, 2000
Summary
A novel basic helix-loop-helix (bHLH) protein, OUT, was identified and characterized. OUT acts as a negative regulator, inhibiting bHLH factor DNA binding and cell differentiation.
Area of Science:
- Molecular Biology
- Developmental Biology
- Genetics
Background:
- Basic helix-loop-helix (bHLH) transcription factors regulate cell differentiation.
- The novel bHLH factor OUT was isolated and characterized.
Purpose of the Study:
- To investigate the function and regulatory role of the novel bHLH factor OUT.
- To determine OUT's mechanism of action in relation to other bHLH proteins.
Main Methods:
- Co-immunoprecipitation assays to confirm protein interactions.
- Gel mobility shift assays to assess DNA binding inhibition.
- Luciferase assays to evaluate transactivation inhibition.
- Deletion studies to identify functional domains.
- Exogenous expression in C2C12 myoblasts to study differentiation effects.
Main Results:
- OUT associates with E proteins but does not bind DNA itself.
- OUT inhibits DNA binding and transactivation by other bHLH factors (e.g., MyoD-E12).
- The inhibitory function is localized to OUT's bHLH and C-terminal regions.
- Exogenous OUT inhibits terminal differentiation of C2C12 myoblasts.
Conclusions:
- OUT functions as a negative regulator of bHLH transcription factors.
- OUT likely inhibits bHLH activity by forming inactive heterodimers, similar to Id proteins.
- OUT plays a role in regulating cell differentiation, particularly in reproductive organs.
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