Role of Non-Core RAG1 in DSB Repair, Cell Survival, and Cell Cycle Progression

非核心 RAG1 在 DSB 修复、细胞存活和细胞周期进展中的作用

• 批准号: 8655783
• 负责人: Julie Horowitz
• 金额: $ 4.27万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-05-01 至 2016-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8655783
• 关键词: Allergens; Antigen Receptors; Autoimmunity; B-Cell Development; B-Lymphocytes; BCL-2 Protein; CDK2 gene; Cell Cycle; Cell Cycle Checkpoint; Cell Cycle Progression; Cell Cycle Regulation; Cell Survival; Cells; Chromatin; Chromatin Structure; Cleaved cell; Code; DNA; DNA Double Strand Break; DNA Repair; Data; Defect; Detection; Development; Double Strand Break Repair; Enzymes; Excision; Exhibits; Failure; Flow Cytometry; G1 Phase; Gene Rearrangement; Genes; Genetic Recombination; Genetic Transcription; Genomic Instability; Health; Histones; Human; Immune system; Immunoglobulins; Immunologic Deficiency Syndromes; Lymphocyte; Lymphocyte antigen; Lymphoid; Lymphoma; Malignant Neoplasms; Measures; Mediating; Molecular; Mus; Nonhomologous DNA End Joining; Nucleotides; Oncogenic; Organism; Phenotype; Phosphorylation; Phosphotransferases; Physiological; Prevention; Process; Process Measure; Proteins; Rag1 Mouse; Receptor Gene; Repression; Retroviridae; Role; S Phase; Signal Pathway; Signal Transduction; Signaling Molecule; Southern Blotting; Structure of thyroid parafollicular cell; T-Cell Receptor; T-Cell Receptor Genes; Tertiary Protein Structure; Testing; Transcript; Transcription Factor AP-1; Transcriptional Activation; Up-Regulation; artemis; ataxia telangiectasia mutated protein; base; conserved helix-loop-helix ubiquitous kinase; density; endonuclease; human H2AX protein; immunoglobulin B; interest; knock-down; leukemia/lymphoma; mutant; novel; pathogen; prevent; public health relevance; repaired; response; small hairpin RNA; tumor; ubiquitin-protein ligase

DESCRIPTION (provided by applicant): The RAG1/RAG2 endonuclease catalyzes assembly of antigen receptor genes in developing G1 phase lymphocytes through the induction of DNA double-strand breaks (DSBs), which are repaired by non- homologous end-joining (NHEJ) proteins. DSB repair defects and inability to coordinate DSB repair with cell survival and cell cycle progression result in genomic instability that can cause leukemias and lymphomas. The RAG proteins each consist of "core" endonuclease regions, and "non-core" regions required for normal assembly of lymphocyte antigen receptors and normal early lymphocyte development. RAG1 non-core regions include protein domains that ubiquitylate histones and interact with the additional ubuitylating enzymes to prevent aberrant deletion and/or insertion of nucleotides during repair of RAG-mediated DSBs. These phenotypes may result from direct defects in DSB repair in G1 phase cells and/or an inability to coordinate repair with cell survival and cell cycle progression. I have found that mice expressing truncated "core" Rag1 proteins (Rag1C/C mice) exhibit impaired late B cell development and that this defect is tied to decreased transcription of antigen receptor genes and failure to induce survival signals following RAG-mediated DSBs. I have observed that Rag1C/C pre-B cells show loss of coding ends following Rag-generated DSBs, suggesting that non-core regions of Rag1 may have a role in preventing aberrant resection of these DNA ends. Together, these data form the basis of my hypothesis that, in addition to serving with RAG2 as the V(D)J endonuclease, RAG1 functions as a chromatin-modifying enzyme to promote normal DSB repair and as a signaling molecule to coordinate DSB repair with cell survival and cell cycle progression. To test this, I will confirm that non-cor Rag1 regions protect RAG-generated DNA coding ends from aberrant resection in G1 cells by analyzing recombination of chromosomally-integrated substrates in wild-type and Rag1C/C pre-B cells. Further, I will test the ability of non-core Rag1 regions to facilitate DNA repair via promoting H2AX phosphorylation and H2AX, H2A, and H2B ubiquitylation following RAG DSBs. To test the ability of non-core Rag1 to coordinate RAG DSB repair with cell survival and cell cycle regulation, I will first determine how non-core Rag1 regions promote NF¿B-dependent transcriptional activation of the pro-survival Pim2 kinase in response to RAG DSBs. I will use retroviruses to investigate if Rag activates NF¿B via ubiquitylation of NEMO. I will then investigate if non-core Rag1 regions promote NF¿B-dependent activation of the CDK2-AP1 cell cycle checkpoint protein to prevent unrepaired RAG DSBs from entering the cell cycle by knocking down and over-expressing CDK2-AP1. Successful completion of these studies should demonstrate novel functions of Rag1 as a histone-modifying factor and signaling molecule that promotes cell survival and cell cycle regulation, which will further our understanding of how developing lymphocytes integrate DNA repair with cell survival and cell cycle progression to yield a protective adaptive immune system and prevent formation of translocations and tumors.

描述（由申请人提供）：RAG1/RAG2核酸内切酶通过诱导DNA双链断裂（DSB）来催化发育中的G1期淋巴细胞中抗原受体基因的组装，所述DNA双链断裂由非同源末端连接（NHEJ）蛋白修复。 DSB 修复缺陷以及无法协调 DSB 修复与细胞存活和细胞周期进展会导致基因组不稳定，从而导致白血病和淋巴瘤。 RAG蛋白各自由“核心”核酸内切酶区域和淋巴细胞抗原受体正常组装和正常早期淋巴细胞发育所需的“非核心”区域组成。 RAG1 非核心区域包括泛素化组蛋白并与其他泛素化酶相互作用的蛋白质结构域，以防止 RAG 介导的 DSB 修复过程中核苷酸的异常删除和/或插入。这些表型可能是由于 G1 期细胞 DSB 修复的直接缺陷和/或无法协调修复与细胞存活和细胞周期造成的 进展。我发现表达截短“核心”Rag1 蛋白的小鼠（Rag1C/C 小鼠）表现出晚期 B 细胞发育受损，并且这种缺陷与 抗原受体基因和 RAG 介导的 DSB 后未能诱导生存信号。我观察到 Rag1C/C 前 B 细胞在 Rag 生成的 DSB 后显示出编码末端的丢失，这表明 Rag1 的非核心区域可能在防止这些 DNA 末端的异常切除中发挥作用。总之，这些数据构成了我的假设的基础，即除了与 RAG2 一起充当 V(D)J 核酸内切酶之外，RAG1 还可以作为染色质修饰酶促进正常的 DSB 修复，并作为信号分子协调 DSB 修复与细胞存活和细胞周期进展。为了测试这一点，我将通过分析野生型和 Rag1C/C pre-B 细胞中染色体整合底物的重组来确认非 cor Rag1 区域可保护 G1 细胞中 RAG 生成的 DNA 编码末端免受异常切除。此外，我将测试非核心 Rag1 区域通过促进 RAG DSB 后的 H2AX 磷酸化和 H2AX、H2A 和 H2B 泛素化来促进 DNA 修复的能力。为了测试非核心 Rag1 协调 RAG DSB 修复与细胞存活和细胞周期调节的能力，我将首先确定非核心 Rag1 区域如何响应 RAG DSB 促进促生存 Pim2 激酶的 NF?B 依赖性转录激活。我将使用逆转录病毒来研究 Rag 是否通过 NEMO 泛素化激活 NF¿B。然后，我将研究非核心 Rag1 区域是否促进 CDK2-AP1 细胞周期检查点蛋白的 NF¿B 依赖性激活，以通过敲低和过度表达 CDK2-AP1 来防止未修复的 RAG DSB 进入细胞周期。这些研究的成功完成应该证明Rag1作为组蛋白修饰因子和信号分子的新功能，可促进细胞存活和细胞周期调节，这将进一步我们了解发育中的淋巴细胞如何将DNA修复与细胞存活和细胞周期进程整合起来，以产生保护性适应性免疫系统并防止易位和肿瘤的形成。