RAG and AID biology

B淋巴细胞是一种免疫系统细胞，通过其细胞表面称为抗体的特殊受体识别和处置病原体，如病毒和细菌。免疫系统如何通过抗体分子识别和清除病原体，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、体细胞超突变和类切换重组(CSR)。第一种机制从可变(V)、多样性(D)和连接(J)基因片段组装重链(H)和轻链(L)抗体基因。这种由RAG1和RAG2复合体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过程中在激活的成熟B细胞中引入抗体基因N端部分的随机点突变。在细胞选择过程中的突变增加了抗体对病原体的结合亲和力。最后，CSR改变抗体基因的C末端部分，使病原体被消除的方式多样化。体细胞的超突变和开关重组都是通过一种B细胞特异性酶：激活诱导胞苷脱氨酶(AID)来实现的。这种蛋白质改变了DNA的化学性质，将胞苷转化为另一种称为尿嘧啶的碱基，这一过程被称为胞苷脱氨作用。由于尿嘧啶具有诱变性，艾滋病活性会吸引过多的修复酶到免疫球蛋白基因座上。这些酶既可以忠实地修复DNA损伤，也可以将它们转化为单链或双链断裂，分别是高突变和CSR的中间环节。 RAGS和AID在免疫反应中的重要性在缺乏这些酶的人和动物中得到强调，这些人和动物非常容易受到感染，并表现出肠道菌群依赖性的肠道绒毛增生。相反，像自身免疫这样的复杂疾病长期以来一直与RAG和AID依赖的活动有关。此外，RAG和AID本质上都是混杂的，因为它们也可以针对非免疫球蛋白基因，包括癌基因(肿瘤诱导基因)。这种脱靶活动可能导致DNA突变和癌基因失控，导致恶性转化。此外，RAG和AID介导的DNA断裂也可以重组癌基因或使其接近免疫球蛋白基因位点，这是一种称为易位的染色体异常。染色体易位是人类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。这些论点强调了了解RAG和AID活性在正常情况下是如何调节的，以及在肿瘤发生过程中如何解除调节的重要性。本财年，我们通过两项独立的研究加深了我们对RAG和AID活动的理解： I)到目前为止，对染色体异常的研究主要局限于在肿瘤和肿瘤细胞系中发现的事件。虽然我们已经了解了许多关于基因组重排在癌症中的重要性，但还不可能理解支配它们发生的细胞和分子要求。为了检测短期培养中原代细胞的基因组重排(在非选择性条件下)，我们开发了一种通过深度测序来对这些事件进行分类的技术，TC-seq。我们的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AID介导的成熟B细胞易位的热点。这些发现发表在2011年9月的《细胞》杂志上。 Ii)淋巴细胞染色体易位的起源被归因于随机重排的选择、靶向DNA损伤(RAG和AID活性)或易位伙伴之间频繁的核相互作用。然而，还没有直接或大规模地衡量这些进程的个别贡献。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，研究了全球核结构和DNA损伤频率在染色体易位发生中的作用。在没有反复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，与反复发生的定点DNA损伤相关的易位与DNA双链断裂形成的速度成正比，这是通过在损伤部位积累复制蛋白A(RPA)来衡量的。我们的发现表明，易位不是简单的随机事件，而核组织决定了哪些基因对易位，DNA断裂形成控制着反复发生的染色体重排的速度。描述这些结果的手稿目前正在审查中。

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.