The long-term goal of this proposal is to explore how the nuclear receptors of thyroid hormones regulate the expression of specific cellular genes. There are two major types of thyroid hormone receptors, TRalpha and TRbeta. Hormone-free TRs actively silence transcription of genes bearing TR-response elements. Ligand binding to the receptor triggers the release of silencing and leads to the activation of target gene expression. Recent studies indicate that a corepressor, NCoR, associates with unliganded TR to critically regulate its repression function.
The specific aims of this proposal are to: 1. Define the polypeptide constituents of a native corepressor complex containing NCoR. TRBETA binds to a multiprotein complex containing the corepressor NCoR, which is essential for receptor-mediated silencing in HeLa nuclear extracts. The NCoR complex that interacts with TRBETA will be isolated by protein affinity or immunoaffinity methods and its polypeptide constituents will be identified. 2. Identify the silencing domains of NCoR and mSin3A that participate in TR-mediated repression in an in vitro transcription system and investigate their mechanisms of action. NCoR is associated with another corepressor, mSin3A, in HeLa extracts, raising the possibility that mSin3A is also involved in the repression process. Site-directed mutagenesis will be performed to identify the amino acid residues in NCoR that mediate its interaction with mSin3A. These mutant NCoRs will be used to determine the role of mSin3A in TR-mediated silencing. 3. Analyze the mechanism by which the NCoR-mSin3A complex inhibits the initiation of basal transcription by the RNA polymerase II transcription machinery. Recent studies suggested that the NCoR-mSin3 corepressor complex silences transcription by recruiting a histone deacetylase (HDAC) that creates a repressive chromatin structure. However, trichostatin A, an inhibitor of HDAC, failed to inhibit TR-mediated silencing in HeLa nuclear extracts. Transcriptional repression by the corepressor complex may therefore proceed through an alternative, HDAC-independent pathway. Interestingly, the NCoR-mSin3 complex interacts with the basal initiation factor TFIIB. The hypothesis that a component(s) of the transcription initiation complex could be a direct target of repression by the corepressors will be tested by analyzing the functional consequences of interactions between the NCoR complex and the components of the basal transcription machinery. The proposed research will develop a better understanding of the mechanisms that determine the gene regulatory activity of TR in normal cellular physiology and the molecular basis of abnormalities in the TR signaling pathway in human disease states such as GRTH.
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