PROJECT SUMMARY Non-alcoholic fatty liver disease (NAFLD) contributes strongly to the development of insulin resistance, and the two mutually regulate each other in type 2 diabetes. In the liver of NAFLD and insulin resistance, hundreds to thousands of genes are either upregulated or downregulated. Epigenetic modifications such as histone methylation and acetylation modulate homochromatin or heterochromatin states to enhance or suppress gene expression in a context-dependent manner. However, significant knowledge gaps exist in mapping the epigenetic landscape and identifying the major epigenetic factors regulating the development of NAFLD and insulin resistance. In the current proposal, we take advantage of the newly developed CUT&TAG technology and create an epigenetic landscape of histone modifications, including H3K4me1 (poised marker), H3K4me2, and H3K27ac (active marker), and H3K9me2 (suppressive marker) in the liver of mouse and human NAFLD. A comprehensive analysis of the histone modification landscape identifies RE1 Silencing Transcription Factor (REST) as an epigenetic modulator that coordinates the activity of these histone markers. REST recruits histone deacetylases (HDAC1&2), which deacetylate H3K27ac, and lysine-specific demethylase 1 (LSD1), which demethylates H3K4me1 and H3K9me2, to regulate gene expression. Our preliminary data show that nuclear REST protein levels are increased in the liver of mouse and human NAFLD. Insulin and glucose treatment of cultured hepatocytes mimicking insulin resistance drives REST nuclear translocation. Knocking down REST using REST antisense oligonucleotides (ASO) in the liver of adult obese mice (REST-LKD) alleviates fatty liver and improves glucose and insulin tolerance. Hyperinsulinemic-euglycemic clamp studies show that REST knockdown in the liver increases glucose uptake in adipose tissue and muscle, indicating crosstalk between the liver and adipose tissue/muscle. These data show that hepatic REST is activated in insulin resistance, and the activated REST promotes the development of NAFLD and insulin resistance, forming a vicious cycle. Our central hypothesis is that REST is the key epigenetic factor orchestrating histone methylation and acetylation to regulate lipid and glucose metabolism in NAFLD and insulin resistance. We propose three aims to investigate what causes REST activation in NAFLD and insulin resistance, why the activated REST promotes NAFLD and insulin resistance, and how the activated REST induces systemic insulin resistance. Aim 1: To determine the mechanisms for the increased REST activity in the liver of NAFLD. Aim 2: To elucidate the mechanisms by which hepatic REST regulates lipid and glucose metabolism. Aim 3: To investigate the mechanisms for improved systemic insulin sensitivity in REST-LKD mice. Successful execution of the proposal will fill the knowledge gap by mapping the epigenetic landscape and identifying REST as a key epigenetic factor that reprograms metabolic ge...