Identifying and Characterizing Readers of the Neural Histone Code

神经组蛋白密码阅读器的识别和表征

• 批准号: 7294684
• 负责人: MARK T. BEDFORD
• 金额: $ 15.4万
• 依托单位: UNIVERSITY OF TX MD ANDERSON CAN CTR
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-09-20 至 2009-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7294684
• 关键词: Aging; Antibodies; Appendix; Arginine; Behavioral; Binding; Binding Proteins; Brain; Brain Diseases; Cells; Chimeric Proteins; Chromatin; Chromatin Structure; Code; DNA Methylation; Data; Deposition; Drug Addiction; Enzymes; Epigenetic Process; Epitopes; Event; Genetic; Glass; Glutathione S-Transferase; Histone Code; Histones; In Vitro; Knockout Mice; Laboratories; Learning; Libraries; Lysine; Mediating; Memory; Mental Retardation; Methylation; Modification; Monitor; Mono-S; Mutate; Nature; Numbers; PC12 Cells; PRTN3 gene; Peptides; Post-Translational Protein Processing; Prevalence; Process; Property; Protein Array; Protein Microchips; Protein-Arginine N-Methyltransferase; Protein-arginine deiminase; Proteins; Reader; Reading; Recruitment Activity; Regulation; Reporting; Side; Site; Slide; Specificity; Surface; Syndrome; Tail; Tertiary Protein Structure; Testing; addiction; arginyllysine; coactivator-associated arginine methyltransferase 1; combinatorial; gain of function; histone acetyltransferase; histone methyltransferase; histone-binding proteins; in vivo; knock-down; methyl group; relating to nervous system; research study; small hairpin RNA; theories

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.

描述(申请人提供)：学习、记忆和成瘾领域的一种新兴理论，即通过DNA甲基化和组蛋白修饰调节染色质结构，介导长期的行为变化。组蛋白上的大量翻译后修饰组合是可能的。这种密码被称为组蛋白代码。该密码子的甲基化部分与含有Chromo、Tudor、WD40、MBT、PhD结构域的蛋白质结合，被称为“读码蛋白”。在这里，我们关注在大脑中表达的包含这些甲基结合模块的蛋白质，以便识别那些能够在组蛋白尾部引入表观遗传变化后被招募到染色质的蛋白质。这项研究将首次让我们一窥组蛋白密码在大脑中的作用。我们假设，含有PHD结构域的蛋白质在大脑中形成一组突出的甲基依赖组蛋白结合蛋白，精氨酸和赖氨酸甲基化都调节这些相互作用。目的1.利用蛋白质芯片鉴定甲基依赖的脑结合蛋白。在大脑中表达并含有潜在的甲基结合结构域的蛋白质已经被识别出来。这些甲基结合模块(PHD、Tudor、MBT和Chromo结构域)将被克隆为GST融合蛋白并排列到玻片上。最终得到的微阵列将用代表核心组蛋白上所有已知甲基化位点的生物素化多肽进行探测，以确定这些结构域的结合特异性。6个含有PHD结构域的蛋白在不同的智力低下综合征中发生突变。这些遗传数据，加上新发现的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取器。目的2.确定R2me/K4me3双标记物在体外和细胞内均可产生。组蛋白H3K4甲基标记结合了BPTF和ING2的PhD结构域。附近精氨酸2(R2)残基的甲基化可能调节这些相互作用。为了证实R2me2和K4me3在细胞中共存，我们将提高针对H3R2me2K4me3组合表位的甲基特异性抗体。使用该抗体，我们将通过对从PC12细胞中分离出的核心组蛋白的Western分析来评估双重甲基化修饰的流行率。这种抗体也将被用来鉴定在体外可以沉积这些标记的酶。目的3.建立R2me/K4me3双标记功能相关性。在体外导致H3R2甲基化的PRMT将在PC12细胞中单独和联合被敲除(ShRNA)，以确定哪个PRMT是细胞中这一标记的主要贡献者。GFP-PhD结构域蛋白的亚细胞定位将在功能丧失和功能获得的细胞中进行监测。我们还将研究双重H3R2me2K4me3修饰是否与活性或非活性染色质相关。关于我们如何学习，以及记忆如何被大脑保留的理解，在很大程度上仍然没有答案。这些机制显然不是遗传的。学习过程可能是通过修改大脑中的蛋白质来实现的，这些不同的修改可能组合在一起形成一个密码。在这项研究中，我们计划研究这种记忆代码是如何被读取的。这项基础科学研究将明显影响药物成瘾、智力低下和脑老化疾病等领域。