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)RAG1和RAG2酶在V、D和J片段的组装中起着独特的作用。RAG1包含与抗原受体基因重组信号序列相互作用的结构域，以及DNA切割所必需的关键氨基酸。RAG2的功能还不是很清楚。它与RAG1相互作用，增强DNA结合的特异性和亲和力，对催化是必不可少的。虽然RAG2本身没有可检测到的DNA结合活性，但它包含一个蛋白质结构域，形成一个与染色质(DNA包裹在其周围的蛋白质支架)特异的结合口袋。为了准确地确定抗原受体基因的RAG结合的特异性，我们利用了一种新开发的高通量测序技术。我们发现RAG2在体内以一种基于其与染色质支架结合的能力的模式在整个基因组(24,000个位点)上结合。这种异常的混杂回避了一个问题，即重组是如何主要针对抗原受体基因的。由于RAG1和RAG2酶是成对工作的，一个明显的机制是对RAG1DNA募集进行严格的调控。为了验证这一想法，我们目前正在用同样的测序技术研究RAG1在小鼠和人类T和B细胞发育中的结合。我们希望这些研究一方面有助于解释RAGS对抗原受体基因的偏好的本质，另一方面RAGS的混杂如何促进小鼠和人类白血病染色体易位的发展。一篇关于这些发现的手稿最近发表在《细胞》杂志上。 Ii)在第二组实验中，我们还定位了激活的B细胞中的AID结合。辅助活性只发生在单链DNA上，在转录过程中被RNA聚合酶取代和稳定。我们的测序分析显示，艾滋在B淋巴细胞中募集到了惊人的6000个基因。奇怪的是，在这些基因中，AID在聚合酶停滞的位置被招募得最多。我们认为，这一特征为AID提供了攻击DNA的机会之窗。我们结果的另一个突出特征是，在某些情况下，AID对癌基因的招募超过了在抗原受体基因座位上的测量。这是意想不到的，因为后者的突变负荷是前者的10-100倍。因此，仅凭AID结合密度不能完全解释AID的活性。那么艾滋病特异性的本质是什么呢？一些证据表明，单链DNA结合蛋白RPA通过进一步稳定单链DNA来帮助AID。当我们在小鼠基因组中定位RPA时，我们发现与AID相反，辅助蛋白只招募到抗原受体基因。因此，结论是RPA很可能是提供特异性的酶。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) The RAG1 and RAG2 enzymes play unique roles in the assembly of V, D, and J segments. RAG1 contains domains that interact with recombination signal sequences of antigen receptor genes, as well as key amino-acids that are essential for DNA cleavage. The functions of RAG2 are less well understood. It interacts with RAG1, enhances the specificity and affinity of DNA binding, and is essential for catalysis. While RAG2 has no detectable DNA binding activity by itself, it contains a protein domain that forms a binding pocket specific for chromatin (the protein scaffold around which DNA is packed). To determine exactly how specific RAG biding is for antigen receptor genes, we made use of a newlydeveloped high-throughput sequencing technique. We found that RAG2 binds in vivo across the entire genome (24,000 sites) in a pattern that would be expected based on its ability to bind to the chromatin scaffold. This extraordinary promiscuity begs the question of how recombination is targeted predominantly to antigen receptor genes. Since the RAG1 and RAG2 enzymes work in pairs, one obvious mechanism would be a tight regulation in RAG1 DNA recruitment. To test this idea, we are currently investigating RAG1 binding in developing T and B cells form mice and humans by the same sequencing technique. We hope these studies will help explain on the one hand the nature of RAGs preference for antigen receptor genes, and on the other how RAGs promiscuity promotes the development of chromosomal translocations in mouse and human leukemias. A manuscript relating these findings was recently published in the journal Cell. ii) In a second set of experiments, we have also mapped AID binding in activated B cells. AID activity only occurs at sites of single-stranded DNA, which is displaced and stabilized by RNA polymerases during transcription. Our sequencing analysis shows AID recruitment to an astonishing 6,000 genes in B-lymphocytes. Curiously, within these genes AID is maximally recruited at sites where the polymerase is stalled. We believe that this feature provides AID with a window of opportunity to attack DNA. Another prominent feature of our results is that AID recruitment to oncogenes in some instances exceeds that measured at antigen receptor gene loci. This is unexpected because the magnitude of the mutation load at the latter is between 10-100 fold higher than the former. Thus, AID activity cannot be fully explained on the basis of AID binding densities alone. What is then the nature of AID specificity? Several lines of evidence indicate that the single stranded DNA binding protein RPA helps AID by further stabilizing single stranded DNA. When we mapped RPA in the mouse genome we found that in contrast to AID, the helper protein is only recruited to antigen receptor genes. The conclusion therefore is that RPA is likely the enzyme that provides specificity. The manuscript describing these results is currently under review.