ABSTRACT Chronic alcohol misuse is a major risk factor for alcohol-associated liver disease, a serious health condition that represents approximately 50% of deaths in the U.S. related to chronic liver disease. Known pathophysiological processes associated with alcohol-induced liver injury (ALI) include oxidative stress, alteration of cell signaling, and inflammation; however, little is known about the effects of alcohol on the hepatic epigenome, an emerging area with significant implications in the pathogenesis and progression of ALI. Specifically, changes in histone acetylation, which includes the alcohol-induced H3K9Ac chromatin mark, have been observed in several studies utilizing various cell culture and animal models of acute and chronic alcohol exposure. A key feature of alcohol-induced histone acetylation changes is that ethanol-derived metabolites can be incorporated directly into the hepatic epigenome (i.e., acetate to Acetyl-CoA used for histone acetylation), which could have a significant impact on transcriptional processes and subsequent pathophysiological outcomes related to chronic alcohol use. Interestingly, ACSS2, a nucleocytosolic enzyme that catalyzes the conversion of acetate to Acetyl-CoA, has recently been shown to play a role in regulation of lipid metabolism genes where deletion of Acss2 protects against high fat diet-induced hepatic steatosis. However, in the context of alcohol-induced hepatic steatosis and associated histone acetylation changes, the role of ACSS2 is essentially unknown. We propose a novel metabolic tracing approach using stable isotope (non- radioactive)-labeled ethanol and high-resolution mass spectrometry to accurately quantify the contribution of ethanol-derived Acetyl-CoA and ACSS2 to hepatic histone acetylation at site-specific levels in a chronic plus binge alcohol exposure model. We hypothesize that ethanol metabolism as well as ACSS2 activity will enhance alcohol-induced hepatic histone acetylation and that enrichment of these histone acetylation sites occurs at gene/gene promoter regions associated with markers and/or pathways relevant to alcohol-induced liver injury phenotype. To test this hypothesis, we will 1) quantify the hepatocyte-specific contribution of Acetyl-CoA derived from ethanol metabolism and ACSS2 activity to histone acetylation in a mouse model of chronic ethanol exposure and 2) determine the impact of site-specific histone acetylation increases induced by ethanol metabolism and ACSS2 activity on ALI phenotype. The results from this project could reveal a novel link between alcohol-induced changes in hepatic histone acetylation and liver injury phenotype, ultimately providing a foundation to guide future mechanistic studies related to the specific role of ACSS2 in the progression of severe liver injury brought about by excessive alcohol misuse.