This proposal will focus on biochemical, molecular and cellular aspects of endocrinology, especially as it relates to Vitamin A signaling. The major hypothesis of this proposal is that the transcriptional regulation by retinoids is mediated through distinct co-activator and co-repressor proteins that interact with nuclear receptors in a hormone-dependent fashion. Consistent with the notion that enzymatic activities may be required to regulate target gene expression, the nuclear receptor co-repressors SMRT and NCoR, by association with Class 1 and Class II HDACs, Sin-3A, SHARP, and a collection of other represser related proteins, enable the formation of a multimeric deacetylase complex. The goal of Specific Aim I is to define the molecular basis for co-repressor recognition of non-liganded receptors by detailed functional and physiological analysis of the receptor interaction domains (RIDs).
Specific Aim II recognizes that SMRT as a large platform protein interacts with more than a dozen signaling pathways. To zero in on receptor specific function, we have created a series of targeting constructs, which disable RID 1, RID 2, or RID 1 +2, function. Germline transmission has been obtained for each of these vectors, which should prove to be an effective strategy to selectively understand the role of repressers in hormone signaling.
In Specific Aim III, we will characterize co-repressor associated proteins by the creation of stable cell lines harboring Flag-tagged SMRT or HDACS expression vectors .We will also establish an in vitro transcription system reconstituted with chromatin templates to mechanistically dissect the role of represser and represser-associated proteins in the transcription cascade. Finally, in Specific Aim IV, we will continue to characterize the role of RAR and RXR and retinoid target genes in proliferation, differentiation, and function, utilizing gene array cataloguing. Together, these studies will provide important new insights in to the biochemical and molecular mechanisms that underlie broad aspects of endocrine physiology in human disease including Vitamin A deficiency, metabolic physiology, stem cell function and embryonic development. ? ?
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