Chromatin remodeling mechanisms of gene transcription in memory

描述（由申请人提供）：本提案旨在了解记忆中染色质生物学的机制。组蛋白甲基化依赖的表观遗传机制在成人神经系统的成熟神经元中起调节基因转录的作用。在这里，我们专注于SET结构域/PHD结构域的蛋白质赖氨酸甲基转移酶，G9 a/GLP，催化组蛋白H3赖氨酸9二甲基化（H3 K9 me 2）标记和组蛋白甲基化，染色质重塑和转录调控之间的分子连接器的功能。然而，关于G9 a/GLP-H3 K9 me 2相互作用在神经系统或记忆背景中的作用知之甚少。AIM 1.使用神经科学领域中两种最具创新性的方法，因为它涉及表观遗传学，我们将首先选择性地对神经元染色质进行排序，然后进行免疫沉淀物的大规模平行测序（ChIP-seq），以深入了解海马，内嗅皮层和杏仁核成熟神经元中的H3 K9 me 2景观。然后，我们将定义在成熟神经元中以基因启动子特异性方式招募到H3 K9 me 2甲基化标记的表观遗传读取器。还将在基因启动子区域确定遗传操纵G9 a/GLP活性的影响，并评估行为结果。这些遗传数据，以及在H3 K9 me 2景观上收集的信息，将强烈暗示G9 a/GLP是记忆巩固期间成年大脑中基因转录的主要调节因子。AIM 2.目前，对于任何细胞类型，与这些分子过程耦合的信号传导机制还一无所知。因此，我们将使用药理学方法和激光捕获显微切割技术，在记忆巩固过程中通过NMDA受体激活招募的神经元细胞类型中，确定与G9 a/GLP-H3 K9 me 2相互作用相关的信号通路。AIM 3. NF-B（p65）是蛋白质赖氨酸甲基转移酶的非组蛋白靶标，并且一旦甲基化NF-B可以与蛋白质如G9 a/GLP缔合。在这里，我们将确定这种转录因子如何在记忆巩固过程中作为成熟神经元中G9 a/GLP-H3 K9 me 2相互作用的表观遗传启动子。通过基因敲除p65、阻断肽和赖氨酸脱甲基酶抑制剂，我们将操纵p65-G9 a/GLP相互作用并评估行为结果。总之，提出的研究将提供对成人大脑中H3 K9 me 2甲基化标记的表观遗传发起者和作者的第一次一瞥。有趣的是，人类9号染色体（9 q34）的亚端粒缺失（包括G9 a/GLP）与人类智力迟钝或智力残疾障碍相关，其特征在于学习和认知方面的重大缺陷。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的新治疗方法来改善与衰老，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，从而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.