Project Summary/Abstract Thyroid hormone receptors (TRs) are nuclear receptors (NRs) that switch from transcriptional repressors to activators in response to thyroid hormone (TH), a powerful regulator of lipid and energy metabolism. Repression is mediated by NR corepressors NCOR1 and NCOR2/SMRT, which function in multiprotein complexes containing histone deacetylase 3 (HDAC3), whose catalytic activity requires interaction with NCOR1/2. This proposal focuses on mechanisms by which TH controls liver metabolism as well as non- canonical metabolic functions of NCOR1/2 and HDAC3. Specific Aim 1 is to determine the molecular mechanisms and role of thyroid hormone action in physiology and in non-alcoholic steatohepatitis (NASH). TRb agonists have great promise in the treatment of NASH, but the mechanisms by which they alleviate fatty liver, inflammation, and fibrosis are unclear. Preliminary data in mouse models demonstrate that many TH-regulated genes are dysregulated in NASH livers, with evidence of both hypo- and hyperthyroidism. We will use novel epitope-tagged TRb mice, functional genomics, and genomic region-specific proximity labelling to determine the mechanisms by which TH-regulated genes are dysregulated in NASH. Our preliminary data has also uncovered regulation of tryptophan (Trp) metabolism by both NASH and TH, and shown that TH upregulates anti-inflammatory kynurenine (Kyn) metabolites downstream of Trp. We will use metabolic flux analysis to understand these changes, and will determine the importance of Kyn metabolites in the improvement of NASH by TH. Specific Aim 2 is to elucidate the physiological functions of nuclear receptor corepressors in the liver. Preliminary data reveal that, in addition to repression of lipogenic genes, NCORs unexpectedly activate gluconeogenic genes via an unrecognized chromatin opening ability. We hypothesize that this is due to recruitment of chromatin remodelers, and will test this by utilizing a functional genomics approach, including ChIP-seq, GRO-seq, and ATAC-seq combined with ChIP-mass spec in novel mice with epitope-tagged NCORs. We will also use genomic region-specific proximity labelling to identify proteins that interact specifically with NCORs at positively regulated genes. In addition we will determine the mechanism underlying the regulation of gluconeogenesis by NCORs in the normal fasting response. Specific Aim 3 is to determine the mechanisms underlying enzyme-independent functions of HDAC3 in the liver. In liver, catalytically inactive HDAC3 can repress de novo lipogenesis and prevent hepatosteatosis, but the mechanisms are not known. We will test the hypothesis that this is due to novel protein interactions by combining functional genomics and proteomics using tagged wild type and mutant HDAC3. These innovative studies address major questions regarding the mechanisms of action of TRs, corepressors, and HDAC3 and will shed new light on the transcriptional and epigenomic control of metabolism ...