Chromatin-mediated alternative splicing in reward pathophysiology

项目摘要 表观基因组是指核组蛋白蛋白及其衍生物的共价修饰。 相关的DNA，其功能是调节基因表达。有大量的数据 暗示神经生物学功能和行为的表观遗传重塑，特别是在 奖励病理学的背景，如成瘾和抑郁。但由于 混杂的因素，以前的研究未能区分单纯的 特定基因处给定染色质修饰的存在和功能相关性。 这限制了对神经表观遗传的精确分子机制的阐明， 重塑调节转录。为了解决这个问题，我们采用多学科方法， 涉及（1）染色质免疫印迹（ChIP）和RNA测序（seq）分析 意志奖励行为后大脑奖励区域的数据集和（2）直接 操纵这种修改，使用基因靶向表观遗传的创新策略， 使用工程化转录因子（ETF）进行编辑。 目前的建议测试的假设，组蛋白翻译后修饰 HPTMs，特别是组蛋白H3赖氨酸36甲基化（H3K36me3），直接作用于 奖励介导的前mRNA选择性剪接。这一假设的基本原理包括 最近发表的数据表明，选择性剪接和H3K36me3富集都是高度相关的。 我们的初步发现， 全基因组H3K36me3富集与表达的H3K36me3的剪接复杂性之间的关系， 替代同种型。虽然染色质介导的选择性剪接在细胞中已经很好地建立， 文化系统，它还没有被描述为一种机制，在大脑中，尽管流行 神经元中广泛的染色质重塑和选择性剪接。这项建议 概述了一种新的策略，以证明神经元H3K36me3介导的选择性剪接， 这种转录机制对动机行为的功能意义。特别是， 我们将分析药物或天然药物给药后小鼠大脑奖励区域的神经元变化， 奖励自我管理。这项工作将建立一个战略，通过它我们可以检查 不同脑区中染色质介导的可变剪接的其它机制， 超出了目前提案的最初假设。

PROJECT SUMMARY The epigenome refers to covalent modifications of nuclear histone proteins and their associated DNA, which function to regulate gene expression. There is a wealth of data implicating epigenetic remodeling in neurobiological function and behavior, especially in the context of reward pathologies, such as addiction and depression. However, due to the promiscuity of the factors involved, previous studies have failed to distinguish between the mere presence and the functional relevance of a given chromatin modification at a specific gene. This limits the elucidation of the precise molecular mechanisms by which neuroepigenetic remodeling regulates transcription. To address this, we utilize a multidisciplinary approach that involves (1) analysis of chromatin immunoprecipiation (ChIP)- and RNA-sequencing (seq) datasets from brain reward regions following volitional reward behavior and (2) direct manipulation of such modifications, using an innovative strategy of gene-targeted epigenetic editing using engineered transcription factors (ETFs). The current proposal tests the hypothesis that histone posttranslational modifications (HPTMs), specifically histone H3 lysine 36 methylation (H3K36me3), directly functions in reward-mediated pre-mRNA alternative splicing. The rationale for this hypothesis includes recently published data that both alternative splicing and H3K36me3 enrichment are highly regulated by cocaine exposure, and our preliminary finding that there is a significant correlation between genome-wide H3K36me3 enrichment and the splicing complexity of expressed alternative isoforms. While chromatin-mediated alternative splicing is well established in cell- culture systems, it has not yet been described as a mechanism in brain, despite the prevalence of both widespread chromatin remodeling and alternative splicing in neurons. This proposal outlines a novel strategy to demonstrate neuronal H3K36me3-mediated alternative splicing, and the functional significance of this transcriptional mechanism to motivated behavior. In particular, we will analyze neuronal changes in mouse brain reward regions following drug or natural reward self-administration. This work will establish a strategy through which we can examine additional mechanisms of chromatin-mediated alternative splicing in various brain regions, expanding beyond the initial hypotheses of the current proposal.