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提纯这些蛋白的三个遗传过程：V(D)J重组、体细胞超突变和类开关重组(CSR)。第一种机制通过组合相关的DNA片段来组装抗体基因。重组是由RAG1和RAG2酶催化的。另一方面，体细胞超突变引入随机点突变，以增加抗体对所述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。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。由RAGS和AID(染色体易位)引起的典型染色体异常是人类B细胞淋巴瘤形成的原因。伯基特淋巴瘤和多发性骨髓瘤就是最好的例子。因此，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是如何解除调节的是关键。本财年，我们从几个重要方面加深了对艾滋病生物学的理解： I)人类Burkitt淋巴瘤分为两个主要临床变种：地方性淋巴瘤，影响感染疟疾的非洲儿童；散发性淋巴瘤，分布于世界其他地区。然而，虽然零星易位导致控制其表达的Myc癌基因调控元件断头，但大多数地方性疾病发生在距离Myc数百个碱基的地方。这些重排的起源以及它们如何在如此远的距离解除对癌基因的调控一直不清楚。为了解决这个问题，我们重述了小鼠浆细胞瘤中的地方性BL样易位。在发表在《美国国家科学院院刊》上的这项研究中，我们发现Myc的长期放松调控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，我们的研究揭示了这些调节因子重塑的程度，并为地方性Burkitt淋巴瘤中B细胞的转化提供了理论基础。 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。因此，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。

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.