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

描述(申请人提供)：B和T淋巴细胞构成了我们适应性免疫系统的基础，这是基于结构上不同的表面抗原受体对外来分子的特定识别。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。这一过程分两个阶段进行：“切割阶段”，RAG1和RAG2蛋白在特定的重组信号序列上结合和切割一对受体基因编码片段；“连接阶段”，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蛋白与其他因素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泛素连接酶复合体的成分作为与全长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泛素连接酶组分之一，称为VprBP，是小鼠B细胞发育通过前B细胞阶段所必需的。这种发育障碍可以通过强制表达bcl2来部分挽救，但在这种情况下，大多数新出现的成熟B细胞都表达Ig轻链。这些数据使我们得出了这一假设的具体假设：VprBP是大的IgH和Igk基因座高效和高保真重排所必需的，但不是更紧密的IGL基因座所必需的。为了验证这一假设，我们将建立在缺乏或存在强制表达bcl2的VprBP缺陷的B细胞中，在IgH、Igk和IGL基因座上的V(D)J重组中间体和产物的频率和保真度，以及这些基因座的可及性。此外，我们将使用病毒Abelson转化的Pre-B细胞系来确定V(D)J重组的哪一步(S)因VprBP功能的丧失而特异性中断。更多地了解与RAG蛋白相互作用的因素，如VprBP，如何指导DNA断裂的引入、传感和修复，将提高我们对V(D)J重组受损和异常重组的机制的理解，V(D)J重组分别是某些形式的免疫缺陷和淋巴系统恶性肿瘤的基础。 公共卫生相关性：V(D)J重组是一种DNA重排过程，负责产生不同的B和T细胞群体，这些细胞共同提供对病原微生物的高度特异性免疫。V(D)J重组对宿主既是必要的，也是潜在的危险：V(D)J重组缺陷约占人类严重联合免疫缺陷病例的30%，然而某些形式的白血病和淋巴瘤经常带有分子特征，在其发生过程中涉及异常的V(D)J重组事件。VprBP是我们发现的与V(D)J重组酶相关的泛素连接酶复合体的一种成分，它将研究VprBP如何在B细胞发育过程中调节V(D)J重组，这将加深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。

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.