SUMMARY This R01 grant has focused on long-lasting changes induced by stimulant and opioid drugs of abuse in the brain's reward circuitry in rodent models. Our aims have shifted over the years based on an expanding knowl- edge of addiction mechanisms and development of more powerful experimental methods. When the grant was last renewed competitively, it focused on transcription factors that mediate drug regulation of gene expression. Recently, we have shifted increasingly to a host of chromatin-based mechanisms—so-called epigenetics—that work in concert with transcription factors to alter expression levels of target genes. Our central hypothesis is that drug exposure induces stable “chromatin scars” that drive particularly long-lived changes in gene expression, which in turn mediate many downstream changes in cell and circuit function and behavior. Here, we concentrate on the nucleus accumbens (NAc) based on its central role in brain reward and our empirical finding that it is most dramatically affected by drugs of abuse in our RNA-seq datasets across numerous brain reward regions. In Aim 1, we utilize cocaine or heroin self-administration in mice, combined with unbiased RNA-seq, ATAC-seq, and proteomic methods, to identify candidate chromatin-scar mechanisms in each of the two major subtypes of NAc medium spiny projection neurons, D1- and D2-type MSNs. We then use ChIP-seq to map those candidate mechanisms genome-wide in the affected cell type. In Aim 2, we use viral-mediated gene transfer to bidirectionally manipulate the most highly implicated chromatin-scar mechanisms in a cell- type-specific manner to causally establish their role in mediating the lasting transcriptional and behavioral consequences of drug self-administration. We have already identified two prominent chromatin scars. After prolonged (30 days) withdrawal from cocaine or heroin self-administration, there is profound depletion of H2A.Z (a variant of the core histone subunit H2A) and its acetylated form (H2A.Zac) in both D1 and D2 MSNs, and such depletion associates extensively with those RNAs that show primed or desensitized expression at this time point, an effect particularly prominent in D1 MSNs. Moreover, several of the proteins that control H2A.Z deposition or eviction from nucleosomes or its acetylation are prominently regulated selectively in D1 NAc MSNs. We also have evidence for a role played by H3K79me2 (dimethylation of histone 3 Lys 79) which is induced in NAc after prolonged withdrawal. The histone methyltransferase (DOT1L) and demethylase (KDM2B) that catalyze and remove this mark, respectively, are regulated in D1 and in D2 MSNs. We will now manipulate each of these proteins in a cell-type-selective manner and study downstream behavioral and transcriptional effects. We already have shown that DOT1L knockdown in D1 NAc MSNs profoundly reduces rewarding responses to cocaine. It is important to note that neither H2A.Z nor H3K79me2 have been studied previously in add...