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，它们在其细胞表面上被称为抗体的特殊受体识别和处置病原体，例如病毒和细菌。免疫系统如何通过抗体分子识别和消除病原体在很大程度上取决于靶向B细胞抗体基因的三种遗传过程：V（d）J重组，体细胞超伪装和类转换重组（CSR）。第一个机制从变量（V），多样性（D）和连接（J）基因段组装了重（H）和光（L）链抗体基因。这种重组是由RAG1和RAG2复合物催化的，在个体发育过程中受到严格调节。另一方面，体细胞超突变在免疫反应期间在活化的成熟B细胞中的N末端部分引入随机点突变。在增加病原体的抗体的结合亲和力期间，突变与细胞选择相结合。最后，CSR改变了抗体基因的C末端部分，以多元化病原体的消除方式多样化。 B细胞特异性酶：激活诱导的胞苷脱氨酶（AID）进行了体细胞超突变和开关重组。该蛋白质修饰了DNA的化学性质，将胞苷转化为另一个称为尿嘧啶的碱，这是一种称为胞苷脱氨酸的过程。由于尿嘧啶是诱变的，因此辅助活性吸引了大量的修复酶进入免疫球蛋白基因座。这些酶可以忠实地修复DNA病变，也可以将其转化为单链断裂，分别涉及超誉和企业社会责任。 缺乏这些酶的人类和动物强调了破布和帮助对免疫反应的重要性，这些酶非常容易感染并表现出肠道绒毛的肠道依赖性增生。相反，诸如自身免疫之类的复杂疾病长期以来一直与抹布和依赖辅助活性有关。此外，抹布和援助本质上都是混杂的，因为它们还可以靶向非免疫球蛋白基因，包括肿瘤基因（肿瘤诱导基因）。这种不靶向活性会导致DNA突变和癌基因放松管制，从而导致恶性转化。此外，抹布和辅助介导的DNA断裂还可以重组或使致癌物与免疫球蛋白基因座密切接近，这是一种称为易位的染色体不规则性。染色体易位负责人类B细胞淋巴瘤的形成。汉堡和多发性骨髓瘤是主要的例子。这些论点强调了揭示在正常条件下如何调节抹布和援助活动并在肿瘤发生过程中进行管制的重要重要性。这个财政年度，我们在两项单独的研究中进一步了解了抹布和援助活动： i）迄今为止，染色体畸变的研究主要仅限于在肿瘤和肿瘤细胞系中发现的事件。尽管我们对基因组重排在癌症中的重要性有了很多了解，但不可能发展对控制其起源的细胞和分子需求的理解。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层测序TC-Seq对这些事件进行分类的技术。我们的结果和分析揭示了转录和物理接近在重组发生中的重要性，并确定了成熟B细胞中辅助介导的易位的热点。这些发现发表在2011年9月号的Cell。 ii）淋巴细胞染色体易位的起源已归因于选择随机重排，靶向DNA损伤（抹布和AID活动）或转运伙伴之间频繁的核相互作用。但是，这些过程的个人贡献尚未直接或大规模测量。在第二组实验中，我们通过在培养的B淋巴细胞中同时测量这些参数，研究了全球核结构和DNA损伤在染色体易位的发生中的作用。在没有复发性DNA损伤的情况下，IGH或C-MYC和所有其他基因之间的易位与它们的接触频率直接相关。相比之下，与复发位置指导的DNA损伤相关的易位与DNA双链断裂形成的速率成正比，这是通过在损伤部位的复制蛋白A（RPA）的积累来衡量的。我们的发现表明，易位不仅是随机事件，而且核组织确定了哪种基因对转移器，而DNA断裂形成控制了复发性染色体重排的速率。目前正在审查描述这些结果的手稿。