Project Summary The goal of the Epigenetics Core is to provide a robust common platform for the identification and analysis of genes affected by withdrawal from chronic ethanol exposure in the brain. During the current funding period, the Epigenetics Core identified gene networks both induced and repressed by alcohol exposure or withdrawal in the amygdala, hippocampus, ventral tegmental area and limbic cortex and specific target genes that showed changes in RNA levels, as well as, in H3K9/14ac levels were functionally validated. In the current submission, the proposed Aims will allow preclinical CARE Research Projects (Projects 1-3) to obtain novel information regarding genome-wide changes in chromatin accessibility (ATAC-seq) and histone methylation (H3K27me3 ChIP-seq). These findings will be integrated with data obtained during the current funding period (RNA-seq and H3K9/14ac ChIP-seq). We will also perform ATAC-seq and RNA-seq in the prefrontal cortex (PFC) of the alcohol use disorder (AUD)/control cohort obtained from the New South Wales Tissue Resource Center (Project 4). In addition, we will investigate the across genome levels of 5- hydroxymethylcytosine (5hmC) in the same tissue using Illumina’s EPIC Methylation Array. Results from these studies will reveal brain region-selective changes in genes that are likely to contribute to alcohol use disorders. The above studies constitute Aim 1 of the Core. Aim 2 proposes bioinformatics pipelines for the analyses of the genome-wide datasets and the 5hmC array and integrating the large datasets for both preclinical and post- mortem studies. The integration of datasets from multiple non-biased approaches is innovative and will provide context for interpreting changes in the gene regulatory landscape in response to withdrawal from alcohol. In Aim 3, we propose to employ a novel dCas9:P300 acetyl transferase fusion protein to alter acetylation levels at specific genomic loci. This will allow us to activate expression of key genes in specific brain regions by altering acetylation selectively at their corresponding promoters. Similarly, we will use a dCas9:Tet1 fusion protein to alter 5mC and 5hmC levels at glucocorticoid receptor (Nr3c1) to modulate mRNA expression. The Epigenetics Core is using state-of-the-art technology to obtain genome-wide information to provide additional resources for each CARE component to address their specific hypotheses. The Core will perform quality control measures at many of the key steps of the protocols described above. In addition, key findings in the genome-wide measures will be validated by Core personnel in consult with Project leaders. The Epigenetics Core is an important platform, not only for individual CARE Projects, but also for University-wide NIH-funded epigenetics investigators.