3/11 Epigenetic Regulation of Neuroimmune Pathways

项目总结 酒精使用障碍(AUD)是一种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。众所周知，长期接触乙醇会改变整个过程中的多个分子通路 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侧重于 关于长非编码RNA(LncRNAs)在神经免疫系统分子适应中的作用 和澳元的发展。非编码转录组的数量明显多于编码蛋白质的转录组 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 提示lncRNAs是基因组结构和长期基因的重要表观遗传调节因子。 表情。我们假设lncRNAs是对酒精反应的基因表达的表观遗传调节因子 积极协调细胞功能和动物行为的持续变化。我们已经提出了三个建议 具体目的是从实验上检验这一假设，并帮助实现INIA的总体目标 财团。目的1利用扰动序列对神经免疫基因中涉及的lncRNAs进行功能检测 使用建立的乙醇暴露的体外模型进行表达。定期组合使用群集化 高通量RNA片段的CRISPR基因组编辑方法 对这一目标进行测序将识别由特定的lncRNAs调控的神经免疫途径。AIM 2将使用 染色体构象捕获测序显示细胞空间取向的物理变化 染色质(例如，启动子-增强子相互作用)是由于过量的乙醇暴露造成的。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谱，该图谱将确定参与 酒精反应的神经免疫通路。AIM 3将在体内使用CRISPR介导的基因组编辑 确定单个lncRNAs在调节神经免疫激活和酒精相关方面的作用的策略 行为表型。这个特定的目标将创造几个新的基因工程小鼠品系，用于 多个合作项目，以测试与酒精相关的行为和与 过量接触酒精。拟议研究的完成将扩大我们对 AUD神经免疫系统的表观遗传调节及其潜在的分子适应 过量接触酒精。

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.