RAG and AID biology

RAG 和 AID 生物学

• 批准号: 8344717
• 负责人: rafael c casellas
• 金额: $ 327.4万
• 依托单位: NATIONAL INSTITUTE OF ARTHRITIS AND MUSCULOSKELETAL AND SKIN DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8344717
• 关键词: Animals; Antibodies; Antibody Affinity; Architecture; Autoimmunity; Automobile Driving; B-Cell Lymphomas; B-Lymphocytes; Bacteria; Binding; Bioinformatics; Biology; C-terminal; Cataloging; Catalogs; Cell surface; Cells; Chemicals; Chromosomal Rearrangement; Chromosomal translocation; Chromosome abnormality; Complex; Coupled; Cytidine; DNA; DNA Damage; DNA Double Strand Break; DNA Sequence Rearrangement; DNA lesion; Deamination; Development; Disease; Enzyme Activation; Enzymes; Event; Exhibits; Fc Receptor; Frequencies; Gene Mutation; Gene Targeting; Genes; Genetic Processes; Genetic Recombination; Genetic Transcription; Genomics; Goals; Growth; Human; Hyperplasia; Immune response; Immune system; Immunoglobulin Class Switching; Immunoglobulin Somatic Hypermutation; Immunoglobulin Switch Recombination; Immunoglobulins; Individual; Infection; J segment gene; Laboratories; Lead; Learning; Light; Lymphocyte; Malignant - descriptor; Malignant Neoplasms; Manuscripts; Mature B-Lymphocyte; Measures; Mediating; Molecular; Molecular Biology; Multiple Myeloma; Mutation; N-terminal; Nature; Nuclear; Oncogene Deregulation; Oncogenes; Peripheral; Point Mutation; Process; Proteins; Publishing; Random Allocation; Reaction; Recurrence; Role; Site; Techniques; Transgenic Organisms; Tumor Cell Line; Uracil; V(D)J Recombination; Virus; activation-induced cytidine deaminase; base; c-myc Genes; interest; intestinal villi; pathogen; receptor; repair enzyme; repaired; replication factor A; research study; tool; tumor; tumorigenesis

B lymphocytes are the immune system cells that recognize and dispose pathogens such as viruses and bacteria though special receptors on their cell surface known as antibodies. How the immune system recognizes and eliminates pathogens via antibody molecules depends to a great extent on three genetic processes targeting B cell antibody genes: V(D)J recombination, somatic hypermutation, and class switch recombination (CSR). The first mechanism assembles heavy (H) and light (L) chain antibody genes from variable (V), diversity (D), and joining (J) gene segments. This recombination, which is catalyzed by the RAG1 and RAG2 complex, is tightly regulated during ontogeny. Somatic hypermutation on the other hand introduces random point mutations at the N terminal portion of the antibody gene in activated, mature B cells during the immune response. Mutations coupled to cell selection during increase the binding affinity of the antibody for the pathogen. Lastly, CSR changes the C terminal portion of the antibody gene to diversify how pathogens are eliminated. Both somatic hypermutation and switch recombination are carried out by a B cell specific enzyme: Activation-Induced cytidine Deaminase (AID). This protein modifies the chemical nature of DNA, converting cytidines into another base called uracil, a process known as cytidine deamination. Because uracils are mutagenic, AID activity attracts a plethora of repair enzymes to the immunoglobulin loci. These enzymes can either faithfully repair the DNA lesions, or convert them into single or double strand breaks, which are intermediate to hypermutation and CSR respectively. The importance of RAGs and AID in the immune response is highlighted in humans and animals deficient for these enzymes, which are highly susceptible to infection and exhibit gut flora-dependent hyperplasia of intestinal villi. Conversely, complex diseases such as autoimmunity have long been associated with RAG and AID-dependent activity. Moreover, both RAGs and AID are promiscuous by nature, in that they can also target non-immunoglobulin genes, including oncogenes (tumor-inducing genes). This off-targeting activity can lead to DNA mutations and oncogene deregulation, resulting in malignant transformation. In addition, RAG and AID-mediated DNA breaks can also recombine or bring oncogenes into close proximity of the immunoglobulin loci, a chromosomal irregularity known as a translocation. Chromosomal translocations are responsible for the formation of B cell lymphomas in humans. Burkits and multiple myeloma are prime examples. These arguments underscore the important of unraveling how RAG and AID activity is regulated under normal conditions and deregulated during tumorigenesis. This fiscal year we have furthered our understanding of RAG and AID activities in two separate studies: i) To date, the study of chromosomal aberrations has been primarily limited to events identified in tumors and tumor cell lines. Although we have learned a great deal about the importance of genomic rearrangements in cancer, it has not been possible to develop an understanding of the cellular and molecular requirements that govern their genesis. To examine genomic rearrangements in primary cells in short term cultures (under non-selective conditions), we developed a technique to catalog these events by deep sequencing, TC-seq. Our results and analysis reveal the importance of transcription and physical proximity in recombinogenesis, and identifies hotspots for AID-mediated translocations in mature B cells. These findings are published in the 2011 September issue of Cell. ii) The origin of lymphocyte chromosomal translocations has been ascribed to selection of random rearrangements, targeted DNA damage (RAG and AID activity), or frequent nuclear interactions between translocation partners. However, the individual contributions of these processes have not been measured directly or at a large scale. In a second set of experiments we have examined the role of global nuclear architecture and frequency of DNA damage in the genesis of chromosomal translocations by measuring these parameters simultaneously in cultured B lymphocytes. In the absence of recurrent DNA damage, translocation between Igh or c-myc and all other genes is directly related to their contact frequency. In contrast, translocations associated with recurrent site-directed DNA damage are proportional to the rate of DNA double strand break formation, as measured by accumulation of replication protein A (RPA) at the site of damage. Our findings demonstrate that translocations are not simply random events but that nuclear organization determines which gene pairs translocate and that DNA break formation governs the rate of recurrent chromosomal rearrangements. The manuscript describing these results is currently under review.

B淋巴细胞是免疫系统细胞，通过其细胞表面上称为抗体的特殊受体识别和处理病原体，如病毒和细菌。免疫系统如何通过抗体分子识别和消灭病原体在很大程度上取决于针对B细胞抗体基因的三个遗传过程：V（D）J重组、体细胞超突变和类别转换重组（CSR）。第一种机制从可变（V）、多样性（D）和连接（J）基因区段组装重（H）和轻（L）链抗体基因。这种由RAG 1和RAG 2复合物催化的重组在个体发育过程中受到严格调控。另一方面，体细胞超突变在免疫应答期间在活化的成熟B细胞中的抗体基因的N末端部分引入随机点突变。与细胞选择偶联的突变增加了抗体对病原体的结合亲和力。最后，CSR改变了抗体基因的C末端部分，以使病原体的消除方式多样化。体细胞超突变和开关重组都是通过B细胞特异性酶：激活诱导的胞苷脱氨酶（AID）进行的。这种蛋白质改变DNA的化学性质，将胞苷转化为另一种称为尿嘧啶的碱基，这一过程称为胞苷脱氨基。由于尿嘧啶是致突变的，AID活性吸引了大量的修复酶到免疫球蛋白位点。这些酶可以忠实地修复DNA损伤，或者将它们转化为单链或双链断裂，这分别是超突变和CSR的中间产物。 RAG和AID在免疫应答中的重要性在缺乏这些酶的人类和动物中突出，这些酶高度易受感染并表现出肠道菌群依赖性肠绒毛增生。相反，复杂的疾病，如自身免疫长期以来一直与RAG和艾滋病依赖性活动。此外，RAG和AID本质上都是混杂的，因为它们也可以靶向非免疫球蛋白基因，包括致癌基因（肿瘤诱导基因）。这种脱靶活性可导致DNA突变和癌基因失调，导致恶性转化。此外，RAG和AIDS介导的DNA断裂也可以重组或将癌基因带到免疫球蛋白位点附近，这是一种称为易位的染色体不规则性。染色体易位是导致人类B细胞淋巴瘤形成的原因。Burkits和多发性骨髓瘤就是最好的例子。这些论点强调了阐明RAG和AID活性在正常条件下如何调节以及在肿瘤发生期间如何解除调节的重要性。本财政年度，我们在两项独立的研究中进一步了解了RAG和AID的活动： （一） 迄今为止，染色体畸变的研究主要限于肿瘤和肿瘤细胞系中鉴定的事件。虽然我们已经了解了很多关于基因组重排在癌症中的重要性，但还不可能了解控制其发生的细胞和分子要求。为了在短期培养物（在非选择性条件下）中检查原代细胞中的基因组重排，我们开发了一种通过深度测序（TC-seq）来对这些事件进行分类的技术。我们的研究结果和分析揭示了转录和物理邻近在重组发生中的重要性，并确定了成熟B细胞中AIDS介导的易位热点。这些发现发表在2011年9月号的Cell上。 ii）第二阶段 淋巴细胞染色体易位的起源归因于随机重排的选择、靶向DNA损伤（RAG和AID活性）或易位伴侣之间频繁的核相互作用。然而，这些过程的单独贡献还没有被直接或大规模地测量。在第二组实验中，我们通过同时测量培养的B淋巴细胞中的这些参数，研究了染色体易位发生中的全局核结构和DNA损伤频率的作用。在没有复发性DNA损伤的情况下，Igh或c-myc与所有其他基因之间的易位与它们的接触频率直接相关。相反，与复发性定点DNA损伤相关的易位与DNA双链断裂形成的速率成比例，如通过损伤位点处复制蛋白A（RPA）的积累所测量的。我们的研究结果表明，易位不是简单的随机事件，但核组织决定哪些基因对易位和DNA断裂的形成管理率的经常性染色体重排。描述这些结果的手稿目前正在审查中。