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: 8345107
• 负责人: Patrick C. Swanson
• 金额: $ 28.74万
• 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/8345107
• 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. PUBLIC HEALTH RELEVANCE: V(D)J recombination is a DNA rearrangement process that is responsible for generating a diverse population of B and T cells that together provide highly specific immunity to pathogenic microorganisms. V(D)J recombination is both essential and potentially dangerous to the host: defects in V(D)J recombination account for about 30% of human cases of severe combined immunodeficiency, yet certain forms of leukemia and lymphoma frequently bear molecular signatures implicating an aberrant V(D)J recombination event in their genesis. The research proposed here will investigate how VprBP, a component of an ubiquitin ligase complex that we have found associates with V(D)J recombinase, regulates V(D)J recombination during B cell development, which will improve our understanding of how this process contributes to B cell immune diversification and disease.

描述（由申请人提供）：B 和 T 淋巴细胞构成了我们的适应性免疫系统的基础，该系统基于结构多样的表面抗原受体对外来分子的特异性识别。这些受体的结构多样性源于体细胞 DNA 重排过程，称为 V(D)J 重组，该过程在淋巴细胞发育过程中组装抗原受体基因。该过程分两个阶段进行：“切割阶段”，在此期间，RAG1 和 RAG2 蛋白在特定的重组信号序列处结合并切割一对受体基因编码片段；以及“连接阶段”，在此期间，由 RAG 介导的切割产生的四个 DNA 末端通过非同源末端连接 (NHEJ) 修复途径进行重组、加工和重新连接。新出现的证据表明，DNA 断裂的引入、感知和修复是通过 RAG 蛋白与其他因子的相互作用来积极协调的，但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成分作为与全长 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是由泛素修饰途径连接和调节的。为了支持这种可能性，我们证明了 E3 泛素连接酶成分之一（称为 VprBP）是小鼠 B 细胞发育经过前 B 细胞阶段所必需的。这种发育障碍可以通过强制 Bcl2 表达得到部分挽救，但在这种情况下，大多数新兴的成熟 B 细胞都表达 Ig¿ 轻链。这些数据引导我们得出该提议的具体假设：VprBP 对于大 Igh 和 Igk 基因座的高效和高保真重排是必需的，但对于更紧凑的 Igl 基因座则不是必需的。为了检验这一假设，我们将在缺乏或存在强制 Bcl2 表达的 VprBP 缺陷 B 细胞中确定 Igh、Igk 和 Igl 位点处 V(D)J 重组中间体和产物的频率和保真度，以及这些位点的可及性。此外，我们将使用病毒 Abelson 转化的前 B 细胞系来确定 V(D)J 重组的哪些步骤因 VprBP 功能的丧失而被特异性破坏。更多地了解与 RAG 蛋白相互作用的因子（例如 VprBP）如何指导 DNA 断裂的引入、感知和修复，将提高我们对导致某些形式的免疫缺陷和淋巴恶性肿瘤的受损和异常 V(D)J 重组的机制的理解。 公共卫生相关性：V(D)J 重组是一种 DNA 重排过程，负责产生多样化的 B 细胞和 T 细胞群体，这些细胞共同提供针对病原微生物的高度特异性免疫力。 V(D)J 重组对宿主来说既重要又具有潜在危险：V(D)J 重组缺陷约占人类严重联合免疫缺陷病例的 30%，但某些形式的白血病和淋巴瘤经常带有暗示其发生过程中存在异常 V(D)J 重组事件的分子特征。这里提出的研究将调查 VprBP（我们发现与 V(D)J 重组酶相关的泛素连接酶复合物的一个组成部分）如何在 B 细胞发育过程中调节 V(D)J 重组，这将提高我们对这一过程如何促进 B 细胞免疫多样化和疾病的理解。