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与底层 DNA 的接触，影响着许多基本生物过程和发育程序的调节。有效免疫系统的产生需要通过淋巴发育过程中发生的一系列严格调控的位点特异性 DNA 重组事件来组装抗体和 T 细胞受体基因。所有这些 V(D)J 重排事件都是由相同的 RAG1/RAG2 重组酶介导的，但重排本身是谱系特异性的，并且以首选的时间顺序发生，这意味着重组酶只能访问正确打开的基因座。我们最近表明，RAG2 包含一个植物同源结构域 (PHD) 指，可特异性识别在赖氨酸 4 上三甲基化的组蛋白 H3，并且对结合同时在赖氨酸 4 上三甲基化和在精氨酸 2 上对称二甲基化的 H3 (H3R2me2s/K4me3) 表现出更强的偏好。。组蛋白 H3 R2 的对称二甲基化尚未在哺乳动物细胞或模型生物体中得到描述。该提案的目的是检验以下假设：单独的 H3R2me2 或与 H3K4me3 组合是哺乳动物细胞中的一种新型表观遗传标记，在控制淋巴发育以及可能的许多其他细胞过程中发挥作用。首先，我们将开发并表征针对单个组蛋白 H3R2me2s 修饰和双重修饰 H3R2me2s/K4me3 的抗体。我们还将制作第三种抗体，仅在 K4me3 不存在的情况下识别 H3R2me2s (H3R2me2s/K4me3)。我们将使用这些抗体来确定这些修饰是否大量存在于哺乳动物细胞中。其次，我们将对淋巴细胞染色质进行 ChIP 和定量 PCR，以确定 H3R2me2s 和 H3R2me2s/K4me3 是否存在位点、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 Finger 是否在体外和体内结合染色质中的 H3R2me2s/K4me3。第三，我们将结合免疫荧光和全基因组染色质免疫沉淀对各种条件下的一系列细胞类型，深入分析哺乳动物细胞中 H3R2me2s、H3R2me2s/K4me3 和 H3R2me2s/K4me3 的表观基因组景观。这种分析应该使我们能够确定这些修改的功能相关性。第四，我们将分析模式生物（酿酒酵母、粟酒裂殖酵母和黑腹果蝇）中的这些修饰，确定这些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、细胞阶段中富集循环，或响应 DNA 损伤。总之，拟议的实验应确定 H3R2me2s 是否是哺乳动物细胞中的一种新型表观遗传修饰，并阐明其可能的功能作用。公共健康相关性：人类细胞中的 DNA 与蛋白质包装在一起，可以对其进行修改以调节基因的表达方式。我们正在研究一种新的修饰，我们相信它可能在产生功能性免疫系统和细胞发育的其他方面发挥作用。这里提出的实验应该定义这种修饰的作用，并深入了解它在正常发育和人类疾病中可能的作用。