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: 8005000
• 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
• 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/8005000
• 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; 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与底层 DNA 的接触，影响着许多基本生物过程的调节。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。 。此外，我们表明，RAG2 对 H3K4me3 的识别是体内质粒底物有效重组和细胞系中 Ig D 至 J 重链重排所必需的。 本资助中描述的实验的目标是从机械角度理解 RAG2 PHD 手指对 H3K4me3（和/或 H3R2me2s/K4me3）的识别对于 V(D)J 重排的重要性以及如何进行。将采用补充的体外和体内实验。首先，我们将在转化的 Pro B 细胞系中使用诱导重组测定来询问 RAG2 PHD 指中的突变是否会导致切割前或切割后缺陷。其次，我们将采用生化方法，使用裸露 DNA 和核小体底物来剖析 H3K4me3 结合对观察到的重组缺陷的贡献。在这里，我们测试了 H3K4me3 结合除了通常设想的组蛋白尾识别在转录中的作用之外还具有其他作用的可能性。第三，我们将测试在 RAG1（序列特异性 DNA 识别所需的结合配偶体）存在或不存在的情况下，RAG2 的 PHD 指是否足以靶向或富集内源抗原受体基因座（和/或所有具有 H3K4me3 的基因座）处的 RAG2 蛋白。 。第四，我们询问除 IgH 基因座外的其他抗原受体基因座的重排是否也需要 RAG2 识别 H3K4me3，以及 RAG2 未能结合 H3K4me3 对小鼠免疫系统发育有何影响。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免疫缺陷。总之，所提出的实验应该揭示 H3K4me3 识别在 V(D)J 重组酶促过程中的机制作用，并可能揭示不同于转录、复制或 DNA 修复的组蛋白修饰的新功能。