Is symmetrical dimethylation of Arginine 2 of Histone H3 used as an epigenetic si

组蛋白 H3 的精氨酸 2 的对称二甲基化是否用作表观遗传 si

基本信息

批准号：
7911689
负责人：
MARJORIE A OETTINGER
金额：
$ 43.81万
依托单位：
MASSACHUSETTS GENERAL HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2008
资助国家：
美国
起止时间：
2008-09-30 至 2011-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7911689
关键词：
Address Affect Animal Model Antibodies Antigen Receptors Arginine Binding Bioinformatics Biological Process Cell Cycle Cell Line Cell model Cell physiology Cells Chromatin Chromatin Structure DNA DNA Damage DNA Sequence Rearrangement Deposition Development Drosophila melanogaster Epigenetic Process Euchromatin Event Exhibits Fingers Fission Yeast Generations Genes Genetic Recombination Genome Goals Heterochromatin Histone H3 Histones Human Immune system Immunofluorescence Immunologic Immunoprecipitation In Vitro Individual Lead Link Lymphoid Lymphoid Cell Lysine Mammalian Cell Mediating Modification Mus Nuclear Extract Pattern Peptides Plants Play Proteins Regulation Role Saccharomyces cerevisiae Series Site Staging Structure System T-Cell Receptor Genes Testing Tissues V(D)J Recombination Western Blotting abstracting cell type chromatin immunoprecipitation epigenomics eta-kappa antigen T cell receptor genetic analysis genome-wide histone modification homeodomain human disease in vivo insight novel preference programs recombinase research study response

项目摘要

DESCRIPTION (provided by applicant): The highly dynamic structure of chromatin governs access of many proteins to the underlying DNA, affecting the regulation of numerous fundamental biological processes and developmental programs. The generation of a competent immune system requires the assembly of antibody and T cell receptor genes by a tightly regulated series of site-specific DNA recombination events that occur during lymphoid development. All these V(D)J rearrangement events are mediated by the same RAG1/RAG2 recombinase, yet the rearrangements themselves are lineage-specific and occur in a preferred temporal order, implying that the recombinase can only access a correctly opened locus. We recently showed that RAG2 contains a plant homeodomain (PHD) finger that specifically recognizes histone H3 trimethylated on lysine 4, and exhibits an even stronger preference for binding H3 that is concurrently trimethylated on lysine 4 and symmetrically dimethylated at arginine 2 (H3R2me2s/K4me3). Symmetrical dimethylation of histone H3 R2 has not been described in mammalian cells or model organisms. The goal of this proposal is to test the hypothesis that H3R2me2s alone or in combination with H3K4me3 is a novel epigenetic mark in mammalian cells, with a role in governing lymphoid development and perhaps many other cellular processes. First, we will develop and characterize antibodies to the individual histone H3R2me2s modification, and to the dual modification H3R2me2s/K4me3. We will also make a third antibody that recognizes H3R2me2s only in the absence of K4me3 (H3R2me2s/K4me3). We will use these antibodies to determine if these modifications are present at a bulk level in mammalian cells. Second, we will perform ChIP and quantitative PCR on chromatin from lymphoid cells to determine if there are locus, stage or lineage-specific distribution patterns of H3R2me2s and H3R2me2s/K4me3. We will also perform immunoprecipitation followed by Western blot analysis to determine if RAG2 PHD finger binds H3R2me2s/K4me3 in chromatin both in vitro and in vivo. Third, we will analyze the epigenomic landscape of H3R2me2s, H3R2me2s/K4me3, and H3R2me2s/K4me3 in mammalian cells in depth, using a combination of immunofluorescence and genome-wide chromatin immunoprecipitation on a range of cell types under various conditions. This analysis should enable us to determine functional correlates of these modifications. Fourth, we will analyze these modifications in model organisms (S. cerevisiae, S. pombe and Drosophila melanogaster), determining both the genome-wide localization pattern of the modifications and whether they are enriched in particular developmental stages, tissues, stages in the cell cycle, or in response to DNA damage. In summary, the proposed experiments should establish whether H3R2me2s is a novel epigenetic modification in mammalian cells and elucidate its possible functional roles. PUBLIC HEALTH RELEVANCE: DNA in human cells is packaged with proteins that can be modified to regulate how genes are expressed. We are studying a novel modification that we believe may play a role in generating a functional immune system and other aspects of cell development. The experiments proposed here should define the roles of this modification and lead to insights into its possible role in both normal development and human disease.

描述（由申请人提供）：染色质的高度动态结构控制着许多蛋白质对潜在的DNA的访问，从而影响了许多基本生物学过程和发育计划的调节。合理的免疫系统的产生需要通过在淋巴样发育过程中发生的一系列位点特异性DNA重组事件组装抗体和T细胞受体基因。所有这些V（d）J重排事件均由相同的RAG1/RAG2重组酶介导，但是重排本身是谱系特异性的，并且以优选的时间顺序发生，这意味着重点酶只能访问正确打开的基因座。我们最近表明，RAG2含有一种植物同源域（PHD）手指，该手指专门识别赖氨酸4上的组蛋白H3三甲基化，并且表现出更强的对结合H3的偏爱，在赖氨酸4上同时将甲基化在赖氨酸4上，并在精氨酸2（H3R2ME2S/K4ME3）上对称地甲基化。在哺乳动物细胞或模型生物中，尚未描述组蛋白H3 R2的对称二甲基化。该提案的目的是检验单独或与H3K4ME3联合使用H3R2ME2S的假设是哺乳动物细胞中的一种新型表观遗传标记，在管理淋巴样发育以及许多其他细胞过程中起作用。首先，我们将开发并表征对单个组蛋白H3R2ME2S修饰的抗体，以及对双重修饰H3R2ME2S/K4ME3的抗体。我们还将制造仅在没有K4ME3（H3R2ME2S/K4ME3）的情况下才能识别H3R2ME2S的第三抗。我们将使用这些抗体来确定这些修饰是否存在于哺乳动物细胞中。其次，我们将对来自淋巴样细胞的染色质进行芯片和定量PCR，以确定H3R2ME2S和H3R2ME2S/K4ME3是否存在基因座，阶段或谱系特异性分布模式。我们还将进行免疫沉淀，然后进行蛋白质印迹分析，以确定RAG2 PHD手指是否在体外和体内结合了染色质和体内染色质中H3R2ME2S/K4ME3。第三，我们将在哺乳动物细胞中分析H3R2ME2S，H3R2ME2S/K4ME3和H3R2ME2S/K4ME3的表观遗传组景观，以在各种条件下在各种条件下的范围内，使用免疫荧光和基因组免疫蛋白免疫蛋白免疫原料的组合。该分析应该使我们能够确定这些修饰的功能相关性。第四，我们将分析模型生物中的这些修饰（S. cerevisiae，S。Pombe和果蝇Melanogaster），确定了修饰的全基因组定位模式，以及它们是否在特定的发育阶段，组织，细胞周期的组织，还是对DNA损害的响应。总而言之，提出的实验应确定H3R2ME2S是否是哺乳动物细胞中一种新型的表观遗传修饰，并阐明其可能的功能作用。公共卫生相关性：人类细胞中的DNA与可以修饰以调节基因表达的蛋白质包装。我们正在研究一种新颖的修饰，我们认为，我们认为可能在产生功能免疫系统和细胞发育的其他方面发挥作用。这里提出的实验应定义这种修饰的作用，并导致对其在正常发育和人类疾病中可能作用的见解。