Role of VprBP in B cell development and V(D)J recombination

VprBP 在 B 细胞发育和 V(D)J 重组中的作用

• 批准号: 8688270
• 负责人: Patrick C. Swanson
• 金额: $ 27.64万
• 依托单位: CREIGHTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-07-01 至 2016-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8688270
• 关键词: Accounting; Address; Affect; Animals; Antigen Receptors; B-Cell Development; B-Lymphocytes; Benign; Biological Assay; Cell Line; Cell Lineage; Cell physiology; Chromosomal Rearrangement; Cleaved cell; Code; Complex; Complication; Cullin Proteins; DNA; DNA Repair; DNA Repair Pathway; DNA Sequence Analysis; DNA Sequence Rearrangement; DNA biosynthesis; Data; Defect; Development; Disease; Distal; Epigenetic Process; Event; Exhibits; Family; Foundations; Frequencies; Functional RNA; G22P1 gene; Gene Rearrangement; Genes; Genetic Recombination; Genetic Transcription; Germ Lines; Health; Histones; Human; Immune; Immune system; Immunity; Immunodeficiency and Cancer; Immunologic Deficiency Syndromes; Knowledge; Lead; Length; Light; Lymphocyte; Malignant Neoplasms; Malignant lymphoid neoplasm; Mature B-Lymphocyte; Measures; Mediating; Modification; Molecular Profiling; Mus; Mutation; Nonhomologous DNA End Joining; Pathway interactions; Peptide Signal Sequences; Phase; Phosphotransferases; Population Heterogeneity; Process; Protein Binding; Proteins; Receptor Gene; Recurrence; Relative (related person); Research; Resolution; Reverse Transcriptase Polymerase Chain Reaction; Role; Services; Severe Combined Immunodeficiency; Southern Blotting; Staging; Sterility; Surface Antigens; Syndrome; T-Cell Receptor; T-Lymphocyte; Testing; Ubiquitin; Ursidae Family; V(D)J Recombination; VDJ Recombinases; Viral; Work; XRCC5 gene; base; chromatin immunoprecipitation; histone modification; improved; leukemia/lymphoma; microorganism; prevent; receptor; repaired; ubiquitin ligase; ubiquitin-protein ligase

DESCRIPTION (provided by applicant): B and T lymphocytes form the foundation of our adaptive immune system, which is based on specific recognition of foreign molecules by structurally diverse surface antigen receptors. Structural diversity in these receptors originates from a somatic DNA rearrangement process, called V(D)J recombination, that assembles the antigen receptor genes during lymphocyte development. This process proceeds in two phases: a "cleavage phase" during which the RAG1 and RAG2 proteins bind and cleave a pair of receptor gene coding segments at specific recombination signal sequences, and a "joining phase" during which the four DNA ends resulting from RAG-mediated cleavage are reorganized, processed and rejoined via the non-homologous end- joining (NHEJ) repair pathway. Emerging evidence suggests that the introduction, sensing, and repair of DNA breaks is actively coordinated through RAG protein interactions with other factors, but the mechanisms involved in these processes are not fully understood. We have identified two NHEJ repair factors, Ku70 and Ku80, and components of an E3 ubiquitin ligase complex as interaction partners with full-length RAG1. These findings lead us to speculate that the cleavage and joining phases of V(D)J recombination are connected and regulated by a ubiquitin modification pathway. In support of this possibility, we show that one of the E3 ubiquitin ligase components, called VprBP, is required for B cell development past the pro-B cell stage in mice. This developmental block is partially rescued by enforced Bcl2 expression, but under these conditions most emerging mature B cells express the Ig¿ light chain. These data lead us to the specific hypothesis of this proposal: that VprBP is necessary for efficient and high-fidelity rearrangement of the large Igh and Igk loci, but not the more compact Igl locus. To test this hypothesis, we will establish the frequency and fidelity V(D)J recombination intermediates and products at the Igh, Igk, and Igl loci, as well as the accessibility of these loci, in VprBP-deficient B cells in the absence or presence of enforced Bcl2 expression. In addition, we will determine which step(s) of V(D)J recombination are specifically disrupted by loss of VprBP function using viral Abelson-transformed pre-B cell lines. Greater knowledge of how factors interacting with the RAG proteins, such as VprBP, guide the introduction, sensing, and repair of DNA breaks will improve our understanding of the mechanisms contributing to impaired and aberrant V(D)J recombination that underlie certain forms of immunodeficiency and lymphoid malignancy, respectively.

描述（由应用提供）：B和T淋巴细胞构成了我们的适应性免疫系统的基础，该系统基于通过结构上多元化的表面抗原受体对外国分子的特定识别。这些受体的结构多样性源自躯体DNA重排过程，称为V（d）J重组，在淋巴细胞发育过程中聚集了抗原受体基因。该过程分为两个阶段：一个“切割阶段”，在此过程中，RAG1和RAG2蛋白在特定的重组信号序列上结合并清除了一对受体基因编码段，以及一个“连接阶段”，在此期间“连接阶段”在此过程中，由RAG介导的裂解引起的四个DNA结束是通过重新加工的，经过处理的，并通过不结合的结束（导致）的最终连接（er）。新兴的证据表明，通过与其他因素的破布蛋白相互作用，引入，感测和修复DNA断裂是积极协调的，但是这些过程中涉及的机制尚未完全了解。我们已经确定了两个NHEJ修复因子KU70和KU80，以及E3泛素连接酶复合物的组件是与全长RAG1的相互作用伙伴。这些发现使我们推测，V（d）J重组的裂解和连接阶段通过泛素修饰途径连接并调节。为了支持这种可能性，我们表明E3泛素连接酶成分（称为VPRBP）是B细胞发育经过小鼠的pro-B细胞阶段所必需的。该发育块部分由强制BCL2表达响应，但是在这些条件下，大多数出现的成熟B细胞表达了IG链。这些数据使我们提出了该提案的具体假设：VPRBP对于大型IGH和IGK基因座的有效和高保真重排是必要的，但不是更紧凑的IGL基因座。为了检验这一假设，我们将在IGH，IGK和IGL基因座以及这些基因座的可访问性中建立频率和保真度V（d）J重组中间体和产物，在不存在或没有强制性BCL2表达的情况下，在VPRBP缺乏的B细胞中。此外，我们将使用病毒abelson转换的前B细胞系的VPRBP函数丧失，确定V（d）J重组的哪个步骤被特异性破坏。更多了解因素如何与抹布蛋白相互作用的知识，例如VPRBP，指导DNA断裂的引入，感测和修复将提高我们对有助于受损和异常的V（d）J重组的机制的理解，这些机制分别是某些形式的免疫缺陷和淋巴性恶性肿瘤的形式。