The nuclear receptor (NR) superfamily of ligand regulated transcription factors has proven to be a rich source of targets for the development of therapeutics for a wide range of human diseases. Endogenous small molecule regulation of these allosteric proteins control processes central to most aspects of mammalian physiology. Our lab has focused on synthetic ligand development and structure-function analysis of the NR1F subfamily of NRs known as the retinoic acid receptor-related orphan receptors or the RORs. This subfamily contains three genes that are involved in but not limited to regulation of glucose and lipid metabolism, bone growth, and immune functions. In this proposal we seek to expand our understanding of ligand-dependent regulation of NR1F3 (RORy; gene name RORC), in the context of the intact full-length receptor. There are two isoforms of RORy, RORy1 and RORy2, that differ in only their N-terminal sequence. RORy1 is broadly expressed, and in the liver it plays an important role in circadian rhythms and glucose and lipid metabolism. The expression of RORy2 or RORyt, is T cell specific and has been shown to be the key lineage-defining transcription factor to initiate the differentiation program of TH17 cells making RORyt an essential regulator for TH17 and T,17 differentiation. Importantly, these cells that have demonstrated anti-tumor efficacy and RORyt controls gene programs that enhance immunity and decrease immune suppression. We have reported sterols and oxygenated sterols as high affinity endogenous ligands and others have confirmed these findings and provided key evidence that they are indeed physiological RORy ligands. Although in certain experimental paradigms RORy can recruit coactivators without addition of exogenous ligand, suggesting the receptor may be constitutively active. Recent evidence clearly demonstrates that RORy is dependent on ligand binding for activation. While extensive structural studies on isolated domains of the receptor have provided important insight into high affinity ligand binding for both agonists and antagonists, there is a lack of information on how the modular domains of RORy act together in the context of the intact full-length receptor. Given the importance of RORy as a therapeutic target, it is surprising that we have an incomplete understanding of how small molecules modulate its activity. The mechanism for "turning off' RORy activity appears straightforward; however. we have an incomplete understanding on how the receptor is "turned on." We hypothesize that ligand-dependent structural perturbations manipulate the localization and PTM status of the receptor influencing its coregulator and DNA interactions to modulate of the RORy transcriptome. To provide the groundwork to test this hypothesis, we propose to develop and validate an integrated structural model of intact full-length RORy/DNA complex to expand our understanding of ligand-dependent regulation of RORy. Illuminating RORy activation m...