Chromatin remodeling mechanisms of gene transcription in memory

描述(申请人提供)：本提案旨在了解染色质生物学在记忆中的作用机制。组蛋白甲基化依赖的表观遗传机制用于调节成年神经系统成熟神经元中的基因转录。在这里，我们关注的是含有SET结构域/PHD结构域的蛋白赖氨酸甲基转移酶G9a/GLP，它催化组蛋白H3赖氨酸9二甲基化(H3K9me2)标记，并在组蛋白甲基化、染色质重塑和转录调控之间发挥分子连接物的作用。然而，人们对G9a/GLP-H3K9me2相互作用在神经系统或记忆方面的作用知之甚少。目的1.使用神经科学领域中最具创新性的两种方法，因为它与表观遗传学有关，我们将首先选择性地对神经元染色质进行分类，然后进行大规模并行免疫沉淀测序(CHIP-SEQ)，以深入了解H3K9me2在来自海马、内嗅觉皮质和杏仁核的成熟神经元中的情况。然后，我们将定义在成熟神经元中以基因启动子特异的方式招募到H3K9me2甲基化标记的表观遗传读取器。基因操纵G9a/GLP活性的效果也将在基因启动子区域确定，并将评估行为结果。这些遗传数据，加上在H3K9me2环境中收集的信息，将有力地暗示G9a/GLP在记忆巩固过程中是成人大脑中基因转录的主要调节因子。目的2.目前，对于任何类型的细胞，与这些分子过程相耦合的信号机制还知之甚少。因此，我们将利用药理学方法和激光捕获显微切割技术，确定在记忆巩固过程中NMDA受体激活所招募的神经细胞类型中与G9a/GLP-H3K9me2相互作用耦合的信号通路。目的3.核因子-B(P65)是蛋白质赖氨酸甲基转移酶的非组蛋白靶标，一旦甲基化，核因子-B可与G9a/GLP等蛋白质结合。在这里，我们将确定该转录因子如何在记忆巩固过程中作为G9a/GLP-H3K9me2相互作用的表观遗传启动子在成熟神经元中发挥作用。通过基因敲除p65、封闭肽和赖氨酸去甲基酶抑制剂，我们将操纵p65-G9a/GLP相互作用并评估行为结果。总之，提出的研究将提供第一次一瞥成人大脑中H3K9me2甲基化标记的表观遗传启动者和编写者。有趣的是，人类染色体9(9q34)亚端粒缺失，包括G9a/GLP，与人类智力低下或智力残疾障碍有关，其特征是学习和认知方面的主要缺陷。因此，这项基础科学研究将通过帮助开发基于操纵表观基因组来改善与衰老、精神分裂症、抑郁症和创伤后应激障碍相关的学习和记忆缺陷的新治疗方法，明显影响认知功能障碍。

DESCRIPTION (provided by applicant): This proposal seeks to understand mechanisms of chromatin biology in memory. Histone methylation- dependent epigenetic mechanisms serve to regulate gene transcription in mature neurons of the adult nervous system. Here, we focus on the SET-domain/PHD-domain-containing protein lysine methyltransferase, G9a/GLP, that catalyses the histone H3 lysine 9 dimethylation (H3K9me2) mark and functions as a molecular linker between histone methylation, chromatin remodeling, and transcription regulation. However, very little is known about the role of G9a/GLP-H3K9me2 interactions in the nervous system or in the context of memory. AIM 1. Using two of the most innovative approaches in the field of neuroscience as it pertains to epigenetics, we will first selectively sort neuronal chromatin followed by massively parallel sequencing of immunoprecipitates (ChIP-seq) to obtain insight into the H3K9me2 landscape in mature neurons from the hippocampus, entorhinal cortex, and amygdala. We will then define the epigenetic readers recruited to the H3K9me2 methylation marks in a gene promoter-specific manner in mature neurons. The effect of genetically manipulating G9a/GLP activity will also be determined at gene promoter regions and behavioral outcomes will be assessed. This genetic data, together with information gathered on the H3K9me2 landscape, will strongly implicate G9a/GLP as a major regulator of gene transcription in the adult brain during memory consolidation. AIM 2. Currently, nothing is known about the signaling mechanisms coupled to these molecular processes for any cell-type. Thus, we will determine the signaling pathways coupled to G9a/GLP-H3K9me2 interactions in neuronal cell types recruited by NMDA receptor activation during memory consolidation using pharmacological approaches and laser-capture microdissection technology. AIM 3. NF-¿B (p65) is a non-histone target of protein lysine methyltransferases, and once methylated NF¿B can associate with proteins such as G9a/GLP. Here, we will determine how this transcription factor serves as an epigenetic initiator of the G9a/GLP- H3K9me2 interaction in mature neurons during memory consolidation. Through genetic knockdown of p65, blocking peptides, and lysine demethylase inhibitors, we will manipulate the p65-G9a/GLP interaction and assess behavioral outcomes. Together, the research studies proposed will provide the first glimpse into the epigenetic initiators and writers of the H3K9me2 methylation mark in the adult brain. Interestingly, subtelomeric deletion of the human chromosome 9 (9q34), which includes G9a/GLP, is associated with human mental retardation or intellectual disability disorders characterized by major defects in learning and cognition. Thus, this basic scientific study will clearly impact cognitive dysfunction by helping to develop new therapeutic approaches based on manipulating the epigenome to improve learning and memory deficits associated with aging, schizophrenia, depression, and post-traumatic stress disorder.