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: 8525811
• 负责人: Julie Horowitz
• 金额: $ 4.22万
• 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/8525811
• 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 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（由申请方提供）：RAG 1/RAG 2核酸内切酶通过诱导DNA双链断裂（DSB）催化发育中G1期淋巴细胞中抗原受体基因的组装，DNA双链断裂（DSB）由非同源末端连接（NHEJ）蛋白修复。DSB修复缺陷和不能协调DSB修复与细胞存活和细胞周期进展导致基因组不稳定性，可引起白血病和淋巴瘤。RAG蛋白各自由淋巴细胞抗原受体的正常组装和正常早期淋巴细胞发育所需的“核心”核酸内切酶区域和“非核心”区域组成。RAG 1非核心区包括使组蛋白泛素化并与另外的泛素化酶相互作用以防止在RAG介导的DSB的修复期间核苷酸的异常缺失和/或插入的蛋白质结构域。这些表型可能是由于G1期细胞中DSB修复的直接缺陷和/或不能协调修复与细胞存活和细胞周期 进展我发现，表达截短的“核心”Rag 1蛋白的小鼠（Rag 1 C/C小鼠）表现出受损的晚期B细胞发育，并且这种缺陷与以下因素的转录降低有关： 抗原受体基因和在RAG介导的DSB后未能诱导存活信号。我观察到Rag 1C/C pre-B细胞在Rag-generated DSB后表现出编码末端的丢失，这表明Rag 1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，这些数据形成了我的假设的基础，除了作为V（D）J核酸内切酶的RAG 2，RAG 1的功能作为染色质修饰酶，以促进正常的DSB修复和作为信号分子，以协调DSB修复与细胞存活和细胞周期的进展。为了验证这一点，我将通过分析野生型和Rag 1C/C前B细胞中染色体整合底物的重组，证实非cor Rag 1区域保护了G1细胞中RAG产生的DNA编码末端不被异常切除。此外，我将测试非核心Rag 1区域通过促进H2 AX磷酸化和H2 AX，H2 A和H2 B泛素化在RAG DSB后促进DNA修复的能力。为了测试非核心Rag 1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Rag 1区域如何促进NF κ B依赖的促存活Pim 2激酶的转录激活以响应RAG DSB。我将使用逆转录病毒来研究Rag是否通过NEMO的泛素化激活NF B。然后，我将研究非核心Rag 1区域是否促进CDK 2-AP 1细胞周期检查点蛋白的NF κ B依赖性激活，以通过敲低和过表达CDK 2-AP 1来防止未修复的RAG DSB进入细胞周期。这些研究的成功完成将证明Rag 1作为组蛋白修饰因子和信号分子的新功能，促进细胞存活和细胞周期调节，这将进一步加深我们对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，以产生保护性适应性免疫系统并防止易位和肿瘤形成的理解。