Nuclear receptors are transcription factors that play key roles in regulating glucose and lipid metabolism and energy homeostasis. Among these, PPARgamma is a lipid sensor and master regulator of adipogenesis with important functions in adaptive thermogenesis and insulin sensitivity, TRs are involved in many aspects of metabolic homeostasis that include adaptive thermogenesis, and ERRalpha is an activator of genes involved in mitochondrial biogenesis and oxidative phosphorylation in muscle. These receptors interact with many coregulatory proteins that include the p1607SRC proteins and interacting chromatin modifying factors (HATs, HMTs), the TRAP220 component of the TRAP/Mediator complex that facilitates receptor communication with the general transcription machinery, and an inducible coactivator (PGC-1alpha) that is elevated in tissues with high energy demands and that interacts both with p160/SRC proteins and chromatin modifying factors and with TRAP220. The hypothesis is that a detailed understanding of the roles of PGC-1alpha, TRAP220 and other interacting cofactors in receptor functions in adipose and muscle tissues will lead to a better appreciation of ways to control receptor-dependent genes related to obesity and diabetes. Relevance to disease is underscored by the recent association of insulin resistance and type 2 diabetes with obesity (dependent upon PPARgamma) and reduced expression of ERRa, PGC-1alpha and mitochondrial and oxidation phosphorylation target genes. Using a combination of mechanistic assays in cell-free transcription systems reconstituted with purified factors and DNA versus recombinant chromatin templates, cell-based transcription and chromatin immunoprecipitation assays, and tissue-specific TRAP220 knockouts and knockins in mice, the aims are (i) to detail biochemical mechanisms involved in receptor-mediated transcription involving p160/SRC's, TRAP/Mediator, PGC-1a, and various histone acetyltransferases and methyltransferases - including cofactors involved in the transition between chromatin remodeling and transcription preinitiation complex formation and function, (ii) to determine the dynamics of nuclear receptor coactivators and histone modifications on key target genes during receptor-mediated gene activation events in cells - in order both to guide and to validate the in vitro studies, (iii) to determine structure-function relationships in TRAP220 and effects of mutations in different nuclear receptor/coactivator interaction sites on adipogenesis and metabolic homeostasis in mice, and (iv) to identify and characterize novel TRAP220-interacting cofactors important for adipogenesis and for tissue-selective functions of the TRAP220 N- and C-terminal domains.
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