Functional role of RAG2 H3K4me3 interaction in V(D)J recombination

RAG2 H3K4me3 相互作用在 V(D)J 重组中的功能作用

• 批准号: 8391252
• 负责人: MARJORIE A OETTINGER
• 金额: $ 41.18万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-12-15 至 2014-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8391252
• 关键词: Address; Affect; Antibodies; Antigen Receptors; Arginine; B-Lymphocytes; Binding; Biochemical; Biological Assay; Biological Process; Cell Line; Cells; Chromatin Structure; Cleaved cell; Complex; Coupled; DNA; DNA Binding; DNA Repair; DNA Sequence Rearrangement; Defect; Development; Disease; Event; Exhibits; Failure; Fingers; Generations; Genes; Genetic Recombination; Genetic Transcription; Goals; Grant; Histone H3; Histones; Human; IGH@ gene cluster; Immune; Immune system; Immunologic Deficiency Syndromes; In Vitro; Infection; Knock-in Mouse; Lead; Light; Link; Lymphoid; Lysine; Measures; Mediating; Modeling; Mus; Mutation; Nucleosomes; Peptide Signal Sequences; Peptides; Physiological; Plants; Plasmids; Positioning Attribute; Process; Proteins; Regulation; Role; Series; Site; Specificity; Synapses; Syndrome; System; Systems Development; T-Cell Receptor; T-Cell Receptor Genes; Tail; Testing; Transformed Cell Line; V(D)J Recombination; base; design; histone modification; homeodomain; in vivo; lymphoid neoplasm; lysine analog; mutant; novel; preference; prevent; public health relevance; receptor; recombinase; research study

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. 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). Moreover, we showed that recognition of H3K4me3 by RAG2 is required in vivo for efficient recombination of plasmid substrates and Ig D to J heavy chain rearrangement in cell lines. The goal of the experiments described in this grant is to understand in mechanistic terms why and how recognition of H3K4me3 (and/or H3R2me2s/K4me3) by the RAG2 PHD finger is important for V(D)J rearrangement. Complementary in vitro and in vivo experiments will be employed. First, we will use an inducible recombination assay in transformed Pro B cell lines to ask whether mutations in the RAG2 PHD finger cause a pre- or post-cleavage defect. Second, we will take a biochemical approach to dissect the contribution of H3K4me3 binding to the observed recombination defect, employing both naked DNA and nucleosomal substrates. Here we test the possibility that H3K4me3 binding could have other roles beyond the recruitment/retention model generally envisioned for the role of histone tail recognition in transcription. Third, we will test whether the PHD finger of RAG2 is sufficient to target or enrich RAG2 protein at endogenous antigen receptor loci (and/or all loci with H3K4me3) in the presence or absence of RAG1, a binding partner required for sequence specific DNA recognition. Fourth, we ask whether rearrangement of other antigen receptor loci in addition to the IgH locus also require RAG2 recognition of H3K4me3 and what the consequences of a failure of RAG2 to bind H3K4me3 are on immune system development in the mouse. In particular, we will test whether the RAG2 W453R mutation associated with Omenn's syndrome in humans causes immunodeficiency in mice. In summary, the proposed experiments should uncover the mechanistic role of H3K4me3 recognition in the enzymatic process of V(D)J recombination and may reveal novel functions for histone modifications distinct from those involved in transcription, replication or DNA repair.

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基础DNA的访问，从而影响了许多基本生物学过程的调节。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。此外，我们表明，需要在体内对H3K4me3的识别，以有效地重组质粒底物，并在细胞系中重组Ig D到J重链重排。 该赠款中描述的实验的目的是从机械术语中理解rag2 phd手指的H3K4ME3（和/或H3R2ME2S/K4ME3）的识别以及如何识别对V（d）J重排的重要性。将采用互补的体外和体内实验。首先，我们将在转化的Pro B细胞系中使用诱导型重组测定法，以询问RAG2 PHD手指中的突变是否引起切割前或后切割后缺陷。其次，我们将采用一种生化方法来剖析H3K4ME3对观察到的重组缺陷的贡献，该缺陷既采用裸体DNA和核小体底物。在这里，我们测试了H3K4ME3结合可能具有除募集/保留模型以外的其他角色的可能性。第三，我们将测试RAG2的PhD手指是否足以在内源性抗原受体基因座（和/或所有带有H3K4ME3的基因座）靶向或富集RAG2蛋白，在存在或不存在RAG1的情况下，RAG1是序列特定DNA识别所需的结合伴侣。第四，我们询问其他抗原受体基因座除了IGH基因座外是否还需要RAG2识别H3K4me3，以及RAG2失败结合H3K4me3的后果是对小鼠免疫系统发育的影响。特别是，我们将测试与人类Omenn综合征相关的RAG2 W453R突变是否会导致小鼠的免疫缺陷。总而言之，提出的实验应揭示H3K4ME3识别在V（d）J重组的酶促过程中的机理作用，并可能揭示了与参与转录，复制或DNA修复的组蛋白修饰的新功能。