Regulation of the VDJ recombinase during genotoxic stress

描述(由申请人提供)：本项目的目标是确定V(D)J重组酶的细胞定位在基因毒性应激过程中是如何调节的，以及这是否与DNA切割活性降低有关。淋巴细胞发育的中心是免疫球蛋白和T细胞受体基因从组成基因片段组装而成。在这个过程中，DNA双链断裂(DSB)是由RAG1和RAG2组成的V(D)J重组酶在中间步骤中产生的。这是一个特别危险的步骤，因为DSB是最具遗传毒性的DNA损伤形式。与此相一致，许多淋巴瘤和白血病含有癌基因和抗原受体基因座之间特有的染色体易位，后者的断裂点通常被归因于RAG介导的切割。承受遗传毒性应激的淋巴细胞将有更大的风险形成这种有害的染色体易位，因为有更多的DNA末端可以异常地连接到由碎布产生的断裂。此外，DNA损伤导致细胞周期停滞于G1期。然而，由于V(D)J重组酶稳定在G1期，延长这一细胞周期阶段预计会增加发生异常重组事件的可能性。在我们使用荧光标记的RAG蛋白的初步研究中，我们确定了电离辐射(IR)引起的DNA损伤促使V(D)J重组酶在细胞中重新定位，这实际上将酶复合体从基因组中隔离出来。这是一种瞬时效应，因为V(D)J重组酶的DNA损伤前共定位特性在DNA修复后重新建立。此外，在DNA损伤反应(DDR)激酶中加入抑制剂可阻断IR诱导的V(D)J重组酶的重分布。鉴于这些结果，我们假设在遗传毒性应激期间，DDR系统通过破坏RAG2和赖氨酸4上三甲基化的组蛋白H3(H3K4me3)之间的相互作用，将V(D)J重组酶从基因组中隔离出来。在这个项目中，我们将使用荧光显微镜和分析相结合的方法来监测细胞蛋白质-蛋白质和蛋白质-DNA相互作用。提出了三个具体目标。首先，我们将确定V(D)J重组酶的空间调控是否是一个与细胞类型无关的普遍过程。其次，我们将确定RAG2和H3K4me3结合的染色质之间的相互作用是否在IR暴露时中断，以及这种作用是否独立于RAG1发生。此外，还将确定调节这一影响的特定DDR因素。第三，我们将确定抗原受体基因座上RAG识别位点的序列特异性结合和切割活性是否受到IR诱导的DNA损伤的影响。该项目将为进一步研究遗传毒性应激过程中V(D)J重组酶的调控机制提供重要的基础。加强这一调控过程的能力可能会降低暴露于辐射和其他破坏DNA的药物后患淋巴系统恶性肿瘤的风险。 公共卫生相关性：V(D)J重组酶由RAG1和RAG2蛋白组成，对于适应性免疫系统的发展是必不可少的；然而，V(D)J重组的错误会导致基因组的不稳定性，从而增加淋巴系恶性肿瘤的风险。初步的新数据表明，V(D)J重组酶在暴露于DNA损伤剂时从基因组中隔离，这可能是为了减少异常重组反应。这项研究的目的是确定这种效应的机制基础，以及这种调控过程是否导致V(D)J重组酶活性降低。

DESCRIPTION (provided by applicant): The goal of this project is to determine how cellular localization of the V(D)J recombinase is regulated during genotoxic stress, and if this correlates with reduced DNA cleavage activity. Central to lymphocyte development is the assembly of immunoglobulin and T cell receptor genes from component gene segments. In this process, DNA double strand breaks (DSBs) are generated in an intermediate step by the V(D)J recombinase, which consists of RAG1 and RAG2. This is a particularly risky step, as DSBs are the most genotoxic form of DNA damage. In accordance with this, many lymphomas and leukemias contain characteristic chromosomal translocations between an oncogene and an antigen receptor locus, with breakpoints in the latter locus often attributed to RAG-mediated cleavage. Lymphocytes undergoing genotoxic stress would be at a greater risk of forming such deleterious chromosomal translocations, as more DNA ends would be available to aberrantly join with RAG-produced breaks. Furthermore, DNA damage induces cell cycle arrest in G1. However, as the V(D)J recombinase is stabilized in G1, prolonging this cell cycle phase would be expected to increase the probability of aberrant recombination events. In our preliminary studies using fluorescently tagged RAG proteins, we determined that DNA damage induced by ionizing radiation (IR) prompted a re-localization of the V(D)J recombinase in the cell, which in effect sequestered the enzyme complex from the genome. This was a transient effect, as the pre-DNA damage co-localization properties of the V(D)J recombinase were re- established upon DNA repair. Moreover, the IR-induced redistribution of the V(D)J recombinase was blocked by addition of an inhibitor to DNA damage response (DDR) kinases. Given these results, we hypothesize that during genotoxic stress, the DDR system sequesters the V(D)J recombinase from the genome by disrupting interactions between RAG2 and Histone H3 trimethylated on Lysine 4 (H3K4me3). In this project, we will use fluorescence microscopy combined with assays to monitor cellular protein-protein and protein-DNA interactions. Three specific aims are proposed. First, we will ascertain if spatial regulation of the V(D)J recombinase is a ubiquitous process independent of cell type. Second, we will determine if the interaction between RAG2 and H3K4me3-bound chromatin is disrupted upon IR exposure, and if this occurs independently of RAG1. In addition, the specific DDR factor that mediates this effect will be identified. Third, we will determine if the sequence-specific association with and cleavage activity on RAG recognition sites in the antigen receptor loci are affected by IR-induced DNA damage. This project will provide an important foundation for future studies in understanding mechanisms that regulate the V(D)J recombinase during genotoxic stress. The ability to enhance such a regulatory process may lead to decreased risks of developing lymphoid malignancies following exposure to radiation and other DNA damaging drugs. PUBLIC HEALTH RELEVANCE: The V(D)J recombinase, consisting of the RAG1 and RAG2 proteins, is essential for development of the adaptive immune system; however, errors in V(D)J recombination can result in genomic instabilities leading to an increased risk of lymphoid malignancies. Preliminary new data indicate the V(D)J recombinase is sequestered from the genome upon exposure to DNA damaging agents in a possible attempt to reduce aberrant recombination reactions. The goal of this study is to determine the mechanistic basis for this effect, and if such a regulatory process leads to reduced V(D)J recombinase activity.