Project 4’s objective is to characterize the influence of enhancer regions of the genome, which exert crucial control over gene expression, in opioid addiction. Work in several tissues has demonstrated that enhancer regions are “marked” by several specific histone modifications, such as H3K27ac (histone 3 Lys27 acetylation), and are enriched in several chromatin remodeling factors. One example is BRD4, a member of the BET (bromodomain and extra-terminal) subfamily, which binds acetylated histones through its bromodomains and is tightly linked with enhancers in several tissues. We have found elevated levels of both H3K27ac and BRD4 in striatum of humans with heroin use disorders, with such elevations correlating strongly with years of heroin use and expression levels of key gene networks. Similar regulation is seen in nucleus accumbens (NAc) and dorsal striatum (DS) of rats that self-administer heroin. Moreover, we have found that small molecules that disrupt BRD4/BET function—in clinical development for cancer—exert therapeutic-like effects in heroin addiction models in rodents. This Project will further these studies in several ways. We will obtain genome-wide maps of enhancer regions in NAc and DS of humans with heroin use disorders and matched controls, and in rats after heroin self-administration, by use of ChIP-seq for H3K27ac and BRD4, among other marks, in conjunction with the Gene and Chromatin Analysis Core. This will be performed separately on nuclei isolated from neurons and non-neuronal cells of these brain regions. We will work with the Core on advanced bioinformatics approaches to overlay these datasets, which is required for reliable identification of enhancers, along with RNA-seq, ATAC-seq, and Hi-C datasets that we are currently generating and that will provide further insight into the specific target genes affected via enhancer mechanisms. We will test the causal role of enhancer regulation in heroin addiction by studying the effect of bidirectional manipulations of BRD4 in NAc and DS on gene expression and behavioral endpoints in rat models, including screening several BRD4/BET inhibitors for anti-addiction actions. These studies offer a translational opportunity to advance novel therapies for drug addiction.