Project Summary The United States is facing an unprecedented opioid epidemic caused by the misuse and abuse of both prescription pain relievers and illegal opioids. This issue has devastating consequences for public health, including a significant increase in overdoses related fatalities, in neonatal withdrawal syndrome and spread of infectious diseases such as HIV and hepatitis. Numerous studies indicate that opioid use disorder (OUD) has a strong genetic component. However, the genes networks implicated in opioid addiction remain poorly understood. The primary goal of this proposal is to study the transcriptional regulatory mechanisms that underlie the development of distinct stages of oxycodone abuse disorder. We will leverage the power of quantitative epigenomic methods that will provide a comprehensive map of regulatory elements, transcription factors and downstream target genes that are dysregulated in specific stages along the OUD trajectories. Our major innovation is the use of capped small (cs)RNA-seq, a method that we developed to quantify newly initiated transcripts with high sensitivity and high spatial resolution directly from total RNA. This approach enables the unbiased annotation of Transcriptional Start Sites (TSS) of both activated genes and transcribed regulatory elements at single nucleotide resolution. Compared with other epigenomic profiling, csRNA-seq is highly sensitive to changes in transcription, and it can capture the dynamic regulation of both stable genes and unstable transcripts, such as enhancer RNA. To study regulatory changes in distinct stages of OUD, we will use a rat model of oxycodone self-administration under extended access conditions. This model recapitulates several aspects of the human addiction-like behaviors, including tolerance, dependence, and motivation. Thus, it enhances the translational relevance of our results. This proposal will use two inbred strains that exhibit large differences in their motivation to seek oxycodone during abstinence while having similar pharmacokinetic for oxycodone and similar exposure to oxycodone. Using transcriptional initiation profiling, in combination with other sensitive profiling techniques, we will investigate the transcriptional regulatory networks underlying different stages of the OUD, including initial exposure, escalation of use, acute and sustained abstinence. Together, our proposed studies will have a broad impact in the field by defining regulatory networks that underlie phenotypes associated with vulnerability to distinct stages along the OUD trajectory in rats, and it may lead to novel therapeutic targets to treat OUD.