3/11 Epigenetic Regulation of Neuroimmune Pathways

项目摘要 酒精使用障碍（AUD）是一种使人衰弱的神经精神障碍，对数百万人的生活产生负面影响 世界各地的个人。已知慢性乙醇暴露会改变多个分子途径， 中枢神经系统，包括异常的神经免疫信号。作为综合神经科学的一部分， 酒精中毒倡议（INIA）神经免疫联盟，拟议的合作研究项目将重点 长链非编码RNA（lncRNA）对神经免疫系统分子适应的贡献 发展AUD。非编码转录组明显多于蛋白质编码转录组， 转录本，但大多数非编码RNA的生物学功能尚未确定。几项研究 已经表明lncRNA是基因组结构和长期基因的关键表观遗传调节因子， 表情我们假设lncRNA是乙醇反应中基因表达的表观遗传调节剂 积极协调细胞功能和动物行为的持续变化。我们提出了三个 具体目标是通过实验检验这一假设，并帮助实现INIA的总体目标 财团目的1利用Perturb-Seq技术对lncRNAs参与神经免疫基因的功能进行检测 使用已建立的乙醇暴露体外模型表达。定期结合使用聚类 间隔短回文重复序列（CRISPR）基因组编辑方法，具有高通量RNA- 测序这一目标将确定由特定lncRNA调控的神经免疫途径。目标2将使用 染色体构象捕获测序，以证明在空间取向的物理变化， 染色质（例如，启动子-增强子相互作用）。这一具体目标将 创建一个新的全基因组染色质相互作用图，将确定参与调控元件， 乙醇反应性神经免疫通路。Aim 3将在体内使用CRISPR介导的基因组编辑 确定单个lncRNA在调节神经免疫激活和乙醇相关性中的作用的策略 行为表型这一特定目标将创造出几种新的基因工程小鼠品系， 多个合作项目，以测试乙醇相关的行为和分子机制， 过量酒精暴露。完成拟议的研究将扩大我们对 AUD中神经免疫系统的表观遗传调节，并确定相关的分子适应 过量酒精暴露。

PROJECT SUMMARY Alcohol use disorder (AUD) is a debilitating neuropsychiatric disorder, negatively affecting the lives of millions of individuals worldwide. Chronic ethanol exposure is known to alter multiple molecular pathways throughout the central nervous system, including aberrant neuroimmune signaling. As part of the Integrative Neuroscience Initiative on Alcoholism (INIA) Neuroimmune consortium, the proposed collaborative research project will focus on the contribution of long non-coding RNAs (lncRNAs) to molecular adaptations in the neuroimmune system and development of AUD. The non-coding transcriptome significantly outnumbers the protein-coding transcripts, but the biological function of most non-coding RNAs has yet to be determined. Several studies have suggested that lncRNAs are critical epigenetic regulators of genomic structure and long-term gene expression. We hypothesis that lncRNAs are epigenetic modulators of gene expression in response to ethanol that actively coordinate persistent alterations in cellular function and animal behavior. We have proposed three specific aims to experimentally test this hypothesis and help accomplish the overall goals of the INIA consortium. Aim 1 will use Perturb-Seq to functionally test the involved of lncRNAs in neuroimmune gene expressing using an established in vitro model of ethanol exposure. Combining use of the clustered regularly interspaced short palindromic repeats (CRISPR) genome editing approach with high-throughput RNA- Sequencing this aim will identify neuroimmune pathways regulated by specific lncRNAs. Aim 2 will use chromosome conformation capture sequencing to demonstrate physical changes in the spatial orientation of chromatin (e.g., promoter-enhancer interactions) due to excessive ethanol exposure. This specific aim will create a new genome-wide chromatin interaction map that will identify regulatory elements involved in the ethanol-responsive neuroimmune pathways. Aim 3 will use CRISPR-mediated genome editing in vivo strategies to determine the role of individual lncRNAs in regulating neuroimmune activation and ethanol-related behavioral phenotypes. This specific aim will create several novel genetically engineered lines of mice, for multiple collaborative projects, to test ethanol-related behaviors and molecular mechanisms associated with excessive ethanol exposure. Completion of the proposed research will broaden our understanding for epigenetic regulation of the neuroimmune system in AUD and determine molecular adaptations underlying excessive ethanol exposure.