Mechanisms of transcriptional control of adipocyte formation and function through nuclear receptor coactivators - PROJECT SUMMARY Dysregulation of energy storage in white adipocytes and of energy expenditure in brown or beige adipocytes, respectively, cause lipodystrophy and obesity, which lead to metabolic disorders that include insulin resistance, diabetes, and cardiovascular disease. A favorable metabolic state is achieved through storage of excess energy as lipids by white adipose and dissipation of energy as heat by brown adipose, as well as by conversion of white adipose tissue to brown-like beige tissue. Studies, including our own, have established that adipose tissue physiology is largely regulated at the level of transcription by a web of interactions between nuclear receptors (NRs) that include PPARs and ERRs and their associated coactivators, including the multi-subunit Mediator complex, that are recruited to enhancers of adipocyte-specific genes. Remarkably, our biochemical and genetic studies in mice collectively demonstrate conditional requirements for the two NR-interacting NR boxes of the MED1 subunit of the Mediator and consequent dramatic beneficial effects of attenuating NR-Mediator interactions via the NR boxes. However, the detailed mechanisms underlying these observations remain completely unknown. We hypothesize that differential recruitment of cofactors by unique combinations of NRs and other transcription factors bound to target gene enhancers determines their adipocyte-specific spatiotemporal expression patterns. Our hypothesis further posits that Mediator recruited by these factors is the critical node in establishing the locus-specific higher-order complexes that ultimately determine the amplitude of the transcriptional output. Here, we propose a holistic approach that merges biochemical and cell- and animal- based methods to investigate how adipocyte identity arises from combinatorial effects of enhancer-bound NRs and other transcription factors as channeled through the Mediator. Our specific aims therefore are (i) to uncover using in vitro transcription, cross-link mass spectroscopy, multiomics, and rapid degradation approaches how cooperative interactions of PPARg, C/EBP pioneer factors, Mediator, and other coactivators at adipocyte lineage- specific enhancers give rise to a transcriptional program that establishes white and brown adipocyte identity, as well as how additional interactions among ERRg, NCOA3, and Mediator are specifically responsible for function of brown/beige adipocytes; and (ii) to analyze how transcriptional activation proceeds in mature adipocytes, both when Mediator recruitment to the enhancer is mediated by MED1 NR boxes and when alternate pathways are operative. The underlying cellular and molecular mechanisms of the metabolically favorable phenotypes of mice harboring MED1 NR box mutants under obesogenic conditions will also be investigated. Projected research outcomes thus include both a detailed understanding of enhancer-mediated transcriptional mechanisms that regulate the formation, interconversion, and function of different adipocyte cell types, as well as insights into rationally developing therapeutic modalities for treating metabolic diseases.
