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Quantitative PCR-based Assay to Measure Sonic Hedgehog Signaling in Cellular Model of Ciliogenesis
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Mediator modulates Gli3-dependent Sonic hedgehog signaling
Haiying Zhou1, Seokjoong Kim, Shunsuke Ishii
1Department of Molecular Medicine, Institute of Biotechnology, The University of Texas Health Science Center at San Antonio, 15355 Lambda Drive, San Antonio, TX 78245-3207, USA.
Molecular and Cellular Biology
|September 27, 2006
Summary
Researchers discovered that Gli3 protein interacts with MED12 in the Mediator complex, which is crucial for regulating Sonic hedgehog (Shh) signaling pathways and target gene transcription.
Area of Science:
- Molecular Biology
- Developmental Biology
- Gene Regulation
Background:
- Sonic hedgehog (Shh) signaling is vital for development and disease.
- The precise nuclear mechanisms of Gli transcription factors remain unclear.
- The RNA polymerase II Mediator complex plays a role in gene transcription.
Purpose of the Study:
- To elucidate the interaction between Gli3 and the Mediator complex.
- To understand how this interaction affects Shh target gene transcription.
- To identify the specific domain of Gli3 involved in Mediator binding.
Main Methods:
- In vitro and in vivo binding assays to test Gli3-MED12 interaction.
- Analysis of Gli3 transactivation domain (MBD) mutants.
- Dominant-negative interference and RNA interference to disrupt the interaction.
- Assays to measure target gene induction in response to Shh signaling.
Main Results:
- Gli3 physically interacts with MED12 and the intact Mediator complex.
- This interaction is mediated by the Gli3 MBD, requiring functional cooperation with CBP.
- Disrupting the Gli3-MED12 interaction impairs Gli3 transactivation and Shh target gene induction.
- MED12 depletion enhances Gli3 activity and target gene expression.
Conclusions:
- Activated Gli3 targets the MED12 interface of the Mediator complex.
- This interaction reverses Mediator-dependent suppression of Shh target gene transcription.
- MED12 modulates Gli3-dependent Shh signaling, implicating Mediator in Shh-driven processes.
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