Decoding the grammar of transcriptional enhancers regulating different stages of opioid use disorder

项目摘要 美国正面临着前所未有的阿片类药物流行病，这是由滥用和滥用两者造成的。 处方止痛药和非法阿片类药物这一问题对公共卫生造成了毁灭性的后果， 包括与过量用药有关的死亡、新生儿戒断综合征和 感染性疾病，如艾滋病毒和肝炎。许多研究表明，阿片类药物使用障碍（OUD）具有 强大的遗传成分。然而，与阿片类药物成瘾有关的基因网络仍然很差 明白该提案的主要目标是研究转录调节机制， 是羟考酮滥用障碍不同阶段发展的基础。我们将利用 定量表观基因组学方法，将提供一个全面的地图的调控元件，转录 在沿着OUD轨迹的特定阶段中失调的因子和下游靶基因。我们 一个主要的创新是使用加帽的小（cs）RNA-seq，我们开发的一种方法，以量化新的 以高灵敏度和高空间分辨率直接从总RNA启动转录物。这种方法 能够无偏地注释激活基因和转录的转录起始位点（TSS）， 调节元件在单核苷酸分辨率。与其他表观基因组分析相比，csRNA-seq 它对转录的变化高度敏感，可以捕获稳定基因的动态调节， 不稳定的转录物，如增强子RNA。为了研究OUD不同阶段的监管变化，我们将 使用大鼠模型的羟考酮自我给药在扩展的访问条件。该模型概括了 人类成瘾行为的几个方面，包括耐受性，依赖性和动机。因此，在本发明中， 它增强了我们结果的翻译相关性。该提案将使用两个近交系， 他们在禁欲期间寻求羟考酮的动机存在差异， 羟考酮和类似的羟考酮暴露。使用转录起始分析，结合 其他敏感的分析技术，我们将调查转录调控网络的基础 OUD的不同阶段，包括初始接触、使用升级、急性和持续戒断。 总之，我们提出的研究将通过定义监管网络在该领域产生广泛影响， 是与大鼠OUD轨迹沿着不同阶段易感性相关的表型的基础，它可能 从而产生治疗OUD新的治疗靶点。

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.