Chromatin-mediated alternative splicing in reward pathophysiology

项目总结 表观基因组是指核中组蛋白及其受体的共价修饰 相关的DNA，其功能是调节基因的表达。有大量的数据 在神经生物学功能和行为中涉及表观遗传重塑，特别是在 奖赏病理的背景，如成瘾和抑郁。然而，由于 所涉及的滥交因素，以前的研究都未能区分仅仅 特定基因上特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学的确切分子机制的阐明 重塑调控转录。为了解决这个问题，我们利用了多学科的方法 涉及(1)染色质免疫沉淀(ChIP)分析和rna测序(Seq)。 来自大脑奖赏区域的数据集跟踪自愿奖赏行为和(2)直接奖赏 使用基因靶向表观遗传学的创新策略来操纵这种修饰 使用工程转录因子(ETF)编辑。 目前的建议验证了组蛋白翻译后修饰的假设 (HPTMS)，特别是组蛋白H3赖氨酸36甲基化(H3K36me3)，直接在 奖赏介导的Pre-mRNA选择性剪接。这一假设的基本原理包括 最近公布的数据显示，替代剪接和H3K36me3富集率都很高 受可卡因暴露的调节，我们的初步发现与 全基因组H3K36me3富集度与表达产物剪接复杂性之间的关系 另一种异构体。虽然染色质介导的选择性剪接在细胞中得到了很好的确立- 培养系统，它还没有被描述为大脑中的一种机制，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来展示神经元H3K36me3介导的选择性剪接，以及 这一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后小鼠大脑奖赏区域的神经元变化 奖励自我管理。这项工作将建立一个战略，通过这个战略，我们可以检查 染色质介导的不同脑区选择性剪接的其他机制， 在当前提案的初始假设之外扩展。

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.