RAG and AID biology

B淋巴细胞是免疫系统的细胞，通过其细胞表面称为抗体的特殊受体识别并清除病毒和细菌。这些受体对病原体的亲和力和特异性在很大程度上取决于组装和精制这些蛋白质的三个遗传过程：V（D）J重组、体细胞超突变和类别转换重组（CSR）。第一种机制通过结合相关的DNA片段组装抗体基因。重组由RAG 1和RAG 2酶催化。另一方面，体细胞超突变引入随机点突变以增加抗体对所讨论的病原体的结合亲和力。最后，CSR引入了进一步的变化，以规定如何消除病原体。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。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。由RAG和AID（染色体易位）诱导的典型染色体不规则性是人类B细胞淋巴瘤形成的原因。伯基特淋巴瘤和多发性骨髓瘤是主要的例子。因此，阐明RAG和AID活性如何在正常条件下调节和在肿瘤发生期间解除调节是关键。本财政年度，我们通过以下几个重要方式进一步加深了对艾滋病生物学的理解： （一） 人类伯基特淋巴瘤（BL）分为两种主要的临床变异：地方性形式，影响感染疟疾的非洲儿童;和散发形式，分布在世界其他地区。然而，虽然零星易位斩首的Myc癌基因调控元件，控制其表达，大多数地方性事件发生数百个酶远离Myc。这些重排的起源以及它们如何在如此远的距离解除癌基因的调控尚不清楚。为了解决这个问题，我们已经概括了地方性BL样易位在小鼠浆细胞瘤。在发表在《美国国家科学院院刊》上的研究中，我们表明Myc的长距离失调与免疫球蛋白调节结构域与易位位点的物理相互作用成正比。因此，我们的研究揭示了重塑的程度，这些监管机构和地方性伯基特淋巴瘤的B细胞的转化提供了一个基本原理。 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。因此，我们研究的作用，全球核结构和DNA损伤的频率在染色体易位的发生，通过测量这些参数同时在培养的B淋巴细胞。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。我们在《自然》杂志上发表的研究结果表明，易位不是简单的随机事件，而是核组织决定了哪些基因对易位，DNA断裂形成决定了染色体重排的频率。

B lymphocytes are cells of the immune system that recognize and get rid of viruses and bacteria though special receptors on their cell surface called antibodies. The affinity and specificity of these receptors for pathogens depends to a great extent on three genetic processes that assemble and refine these proteins: V(D)J recombination, somatic hypermutation, and class switch recombination (CSR). The first mechanism assembles the antibody gene by combining related DNA segments. The recombination is catalyzed by the RAG1 and RAG2 enzymes. Somatic hypermutation on the other hand introduces random point mutations to increase the binding affinity of the antibody for the pathogen in question. Lastly, CSR introduces further changes to dictate how pathogens are eliminated. Both somatic hypermutation and switch recombination are carried out by a B cell specific enzyme: Activation-Induced Cytidine Deaminase (AID). 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, 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. A typical chromosomal irregularity induced by RAGs and AID (chromosomal translocations) are responsible for the formation of B cell lymphomas in humans. Burkitt lymphomas and multiple myeloma are prime examples. Thus, unraveling how RAG and AID activities are regulated under normal conditions and deregulated during tumorigenesis is key. This fiscal year we have furthered our understanding of AID biology in several important ways: i) Human Burkitt lymphomas (BL) are divided into two main clinical variants: the endemic form, affecting African children infected with malaria; and the sporadic form, distributed across the rest of the world. However, while sporadic translocations decapitate the Myc oncogene regulatory elements that control its expression, most endemic events occur hundreds of kilobases away from Myc. The origin of these rearrangements and how they deregulate oncogenes at such distances has been unclear. To solve this problem we have recapitulated endemic BL-like translocations in mouse plasmacytomas. In the study published in the Proceedings of the National Academy of Sciences we show that long-range deregulation of Myc is directly proportional to the physical interaction of immunoglobulin regulatory domains with translocated sites. Our studies thus uncover the extent of remodeling by these regulators and provide a rationale to the transformation of B cells in endemic Burkitt lymphomas. 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. We therefore 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 published in Nature 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.