Identifying and Characterizing Readers of the Neural Histone Code

描述（由申请人提供）：学习、记忆和成瘾领域的一个新兴理论是，通过DNA甲基化和组蛋白修饰调节染色质结构介导持久的行为变化。组蛋白上的翻译后修饰的大量组合是可能的。这种密码被称为组蛋白密码。该密码子的甲基化部分与具有染色体、Tudor、WD 40、MBT、PHD结构域的蛋白质结合，并被称为“密码子阅读蛋白”。在这里，我们专注于在大脑中表达的蛋白质，并含有这些甲基结合模块，以确定那些能够被招募到染色质后，表观遗传变化已被引入组蛋白尾部的蛋白质。这项研究将提供对大脑中组蛋白密码效应器的第一次一瞥。我们假设PHD结构域蛋白在大脑中形成一组显著的甲基依赖性组蛋白结合蛋白，精氨酸和赖氨酸甲基化调节这些相互作用。AIM 1.使用蛋白质微阵列来识别甲基依赖性结合脑蛋白。在大脑中表达的蛋白质，并具有潜在的甲基结合域，已被确定。将这些甲基结合模块（PHD、Tudor、MBT和染色体结构域）克隆为GST融合蛋白并排列在载玻片上。将用代表核心组蛋白上所有已知甲基化位点的生物素化肽探测所得微阵列，以确定这些结构域的结合特异性。六个PHD结构域蛋白在不同的精神发育迟滞综合征中发生突变。这些遗传数据，以及新发现的PHD结构域的甲基结合特性，强烈暗示携带这些结构域的蛋白质是大脑中表观遗传标记的读者。AIM 2.确定双重R2 me/K4 me 3标记可以在体外和细胞中产生。组蛋白H3 K4甲基标记结合BPTF和ING 2的PHD结构域。附近的精氨酸2（R2）残基的甲基化可以调节这些相互作用。为了证实R2 me 2和K4 me 3在细胞中共存，我们将产生针对组合的H3 R2 me 2K 4 me 3表位的甲基特异性抗体。使用这种抗体，我们将通过从PC 12细胞分离的核心组蛋白的Western分析来评估双重甲基修饰的流行率。该抗体也将用于鉴定能够在体外存款这些标记的酶。AIM 3.建立双重R2 me/K4 me 3标记功能相关性。在体外促进H3 R2甲基化的PRMT将在PC 12细胞中单独和组合地被敲低（shRNA），以确定哪种PRMT是细胞中该标记的主要贡献者。将在功能丧失和获得细胞中监测GFP-PHD结构域蛋白的亚细胞定位。我们还将研究双重H3 R2 me 2K 4 me 3修饰是否与活性或非活性染色质相关。关于我们如何学习，以及大脑如何保留记忆的理解在很大程度上仍然没有答案。这些机制在本质上显然不是遗传的。学习过程可以通过大脑中蛋白质的修饰来实现，这些不同的修饰可以联合收割机形成一个代码。在这项研究中，我们计划研究如何读取这种记忆代码。这项基础科学研究将明显影响吸毒成瘾、智力迟钝和大脑衰老疾病等领域。

DESCRIPTION (provided by applicant): An emerging theory in the fields of learning, memory and addiction, is that the regulation of chromatin structure through DNA methylation and histone modification mediate long-lasting behavioral changes. A large number of combinations of posttranslational modifications on histones are possible. This cipher is referred to as the histone code. The methylated component of this code is bound by proteins with Chromo, Tudor, WD40, MBT, PHD domains and have been termed "code-reading proteins". Here we focus on proteins that are expressed in the brain, and that contain these methyl-binding modules, in order to identify those proteins that are capable of being recruited to chromatin after epigenetic changes have been introduced on the histone tails. This study will provide the first glimpse at the effectors of the histone code in the brain. We hypothesize that PHD domain- containing proteins form a prominent group of methyl-dependent histone binding proteins in the brain, and that both arginine and lysine methylation regulate these interactions. AIM 1. Use protein microarrays to identify methyl-dependent binding brain proteins. Proteins that are expressed in the brain, and harbor potential methyl-binding domains, have been identified. These methyl- binding modules (PHD, Tudor, MBT and Chromo domains) will be cloned as GST fusion proteins and arrayed onto glass slides. The resulting microarrays will be probed with biotinylated peptides that represent all the known methylation sites on the core histones, to determine the binding specificity of these domains. Six PHD domain-containing proteins are mutated in different mental retardation syndromes. This genetic data, together with the newly discovered methyl-binding properties of PHD domains, strongly implicate proteins carrying these domains as readers of epigenetic marks in the brain. AIM 2. Determine that the dual R2me/K4me3 mark can be generated in vitro and in cells. The histone H3K4 methyl-mark binds the PHD domains of BPTF and ING2. The methylation of a nearby arginine 2 (R2) residue may regulate these interactions. To confirm that the R2me2 and K4me3 co-exist in cells, we will raise methyl-specific antibodies to the combined H3R2me2K4me3 epitope. Using this antibody, we will assess the prevalence of the duel methyl-modification by Western analysis of core histone isolated from PC12 cells. This antibody will also be used to identify the enzymes that can deposit these marks in vitro. AIM 3. Establish that functional relevance of the dual R2me/K4me3 mark. PRMTs that contribute to H3R2 methylation in vitro will be knocked-down (shRNA) in PC12 cells, individually and in combination, to determine which PRMT is the major contributor to this mark in cells. The subcellular localization of GFP-PHD domain proteins will be monitored in loss- and gain-of-function cells. We will also investigate whether the dual H3R2me2K4me3 modification associates with active or inactive chromatin. The understanding of how we learn, and how memories are retained by the brain remains largely unanswered. These mechanisms are clearly not genetic in nature. The learning process may be achieved through the modification of proteins in the brain, and these different modifications may combine to form a code. In this study we plan to investigate how this memory code is read. This basic scientific study will clearly impact the fields of drug addiction, mental retardation and aging diseases of the brain.