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: 8499380
• 负责人: Patrick C. Swanson
• 金额: $ 26.67万
• 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/8499380
• 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（由申请人提供）：B淋巴细胞和T淋巴细胞构成我们适应性免疫系统的基础，这是基于结构多样化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。这一过程分为两个阶段：“切割阶段”，RAG1和RAG2蛋白结合并切割特定重组信号序列上的一对受体基因编码片段；“连接阶段”，由RAG1介导的切割产生的四个DNA末端通过非同源末端连接（non-homologous end- joining， NHEJ）修复途径重组、加工并重新连接。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积极协调的，但这些过程所涉及的机制尚不完全清楚。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分作为全长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通过泛素修饰途径连接和调节的。为了支持这种可能性，我们发现E3泛素连接酶的一种组分，称为VprBP，是小鼠B细胞经过前B细胞阶段发育所必需的。这种发育障碍部分被Bcl2的强制表达所挽救，但在这些条件下，大多数新兴成熟B细胞表达Ig¿轻链。这些数据使我们得出了该提议的具体假设：VprBP对于大的Igh和Igk位点的高效和高保真重排是必要的，而不是更紧凑的Igl位点。为了验证这一假设，我们将在缺乏或存在强制Bcl2表达的vprbp缺陷B细胞中，建立在Igh、Igk和Igl位点的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，我们将利用病毒abelson转化的前b细胞系，确定V(D)J重组的哪些步骤会因VprBP功能的丧失而被特异性破坏。更多地了解诸如VprBP等与RAG蛋白相互作用的因子如何引导DNA断裂的引入、感知和修复，将提高我们对导致V(D)J重组受损和异常的机制的理解，这分别是某些形式的免疫缺陷和淋巴细胞恶性肿瘤的基础。