Project Summary Opioid use disorder (OUD) has reached epidemic levels in the Unites States, associated with increased rates of hospitalization and drug overdose death, the latter showing a significant steep rise of up to 29.4% in 2020. While genetic risk factors have been identified in recent large-scale genome-wide association studies (GWAS) of OUD, these explain only part of the variance and often map to noncoding regions. Epigenetic modifications have been implicated in the etiology of opioid use disorders (OUD) underlying the gene and environment interplay. We and others have found that alterations of DNA methylation (5mC), one of the most studied epigenetic mechanisms, is associated with OUD in both human peripheral and postmortem brain. However, most of this work has been done in bulk tissues, which obscures the functional role of the cellular diversity in human cells. Further, research is needed to assess additional and novel epigenetic regulatory layers to gain a better understanding of its contribution to gene regulation and its ability to interpret the functionality of GWAS genetic variants in the context of OUD. Here, I offer a novel framework to tackle these gaps and challenges: 1) conduct a simultaneous profiling of DNA methylation, DNA hydroxymethylation, and 3D genome structure in single human nuclei, 2) identify OUD-dependent regulatory signatures within cell types and brain regions, 3) evaluate the crosstalk between the different epigenomic regulatory layers, and 4) construct gene programs to finely map OUD GWAS variants and polygenic signals to function. This comprehensive single-cell multiomics mapping of OUD will examine the dorsolateral prefrontal cortex (DLPFC), amygdala (BLA), and nucleus accumbens (NAcc), part of the addiction circuitry, of human postmortem brain samples collected from the UTHealth Brain Collection datasets and using the VA Brain Bank (NPBB) as a validation cohort. This work is highly innovative and will open new lines of research the genetics and epigenetics of OUD by providing novel mechanistic insights on its gene regulatory structure in the human brain. This proposed study will identify and help inform molecular targets to be used as prevention and treatment efforts for individuals suffering from OUD.