RAG and AID biology

B淋巴细胞通过其细胞表面称为抗体的特殊受体识别和摧毁病毒和细菌。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)。第一种机制通过结合相关的DNA片段来组装抗体基因的5个末端。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，以促进消除入侵的病原体。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。自体免疫等复杂疾病长期以来一直与RAG和AID依赖的活性有关，这两种酶复合体是混杂的，因为它们还可以破坏非免疫球蛋白基因，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。这些异常中的主要是染色体易位，它导致人类B细胞淋巴瘤(例如Burkitt淋巴瘤和多发性骨髓瘤)的形成。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，我们从几个方面加深了对艾滋病生物学和B细胞转化的理解： I)我们已经解决了RPA招募的性质与AID介导的DNA断裂有关。如上所述，AID通过在细胞周期的G1期诱导免疫球蛋白基因和癌基因的DNA断裂来促进染色体易位。RPA是一种单链DNA结合蛋白，与S期受损的DNA结合，促进同源修复相关因子的组装，如RAD51。值得注意的是，RPA沉积也标志着AID介导的损害部位。由于细胞周期阶段的差异，科学家们认为RPA可能在免疫球蛋白基因重组中发挥作用，而不是它的同源修复作用。在一月份出版的《细胞报告》杂志上发表的一篇手稿中，我们证明了RPA与切除的单链DNA不对称地结合，以响应AID、RAG或其他核酸酶造成的损伤。少量的RPA沉积在G1的AID靶点。然而，如果Rpa主要在同源重组中发挥作用，则S-G2/M细胞中的募集是广泛的，并与RAD51的聚集如预期的那样相关。因此，大多数RPA的募集都是抗体基因，代表着在细胞周期的S-G2/M期，基于同源的途径挽救了未修复的断裂。 Ii)AID在活化的B淋巴细胞中表达。这一过程是由全球范围内的信使核糖核酸合成增加启动的。然而，在免疫反应过程中驱动转录组扩增的机制尚不清楚。通过对全基因组范围内的ssDNA进行监测，我们最近在《细胞》杂志上表明，NAVE细胞的基因组即将迅速激活。在G0中，来自循环淋巴细胞中表达基因的90%的启动子加载了聚合酶，但没有融化，因此它们只支持基础转录。此外，我们还发现，从流产延伸到生产性延伸的转变是动力学上的限制，导致聚合酶积累在更接近转录起始点的位置。静止的淋巴细胞也限制TFIIH复合体的表达，包括参与启动子熔化和开放复合体延伸的XPB和XPD解旋酶。到目前为止，已经证明了在真核生物中控制全球基因表达的两个限速步骤：预起始复合体组装和聚合酶暂停。我们的出版物将启动子融化确定为第三个关键调控步骤，并提出这一机制确保了淋巴细胞对入侵病原体的快速反应。 3)AID介导的染色体易位使Myc蛋白失控，参与生理或病理生长、增殖、细胞凋亡、代谢和细胞分化。目前还没有统一的原则来统一Myc的行动，部分原因是Myc的目标清单不完整。为了在Myc受时间和生理调节的系统中观察Myc靶标的表达和功能，我们与美国国立卫生研究院的David Levens实验室合作，创建了一个帮助可视化Myc在体内表达的小鼠模型。在《细胞》杂志上发表的一篇文章中，我们使用这些小鼠分析了激活的B细胞和ES细胞中Myc、RNA聚合酶II和染色质修饰的转录和全基因组分布。从这一定量分析中发现了一个非常简单的规则：MYC不是基因活性的开关指示器，而是一个非线性的表达放大器，普遍作用于活跃的基因，除了在Myc之前强烈诱导的即刻早期基因。Myc作用的这一规律解释了文献中观察到的绝大多数Myc生物学，并为这种蛋白质如何转化B淋巴细胞提供了理论基础。

B lymphocytes recognize and destroy 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: V(D)J recombination, somatic hypermutation, and class switch recombination (CSR). The first mechanism assembles the 5 end of 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 facilitate the elimination of the invading pathogen. 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. Complex diseases such as autoimmunity have long been associated with RAG and AID-dependent activity and both enzyme complexes are promiscuous, in that they can also damage non-immunoglobulin genes, including oncogenes (tumor-inducing genes). This off-targeting activity can lead to DNA mutations and oncogene deregulation, resulting in malignant transformation. Predominant among these irregularities are chromosomal translocations, which drive the formation of B cell lymphomas (e.g. Burkitt lymphomas and multiple myeloma) in humans. 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 and B cell transformation in several ways: i) we have resolved the nature of RPA recruitment to AID-mediated DNA breaks. As mentioned above, AID promotes chromosomal translocations by inducing DNA breaks at immunoglobulin genes and oncogenes in the G1 phase of the cell cycle. RPA is a ssDNA-binding protein that associates with damaged DNA in the S phase and facilitates the assembly of factors involved in homologous repair such as Rad51. Notably, RPA deposition also marks sites of AID-mediated damage. Because of the discrepancy in cell cycle stages, scientists have suggested that RPA might have a role in immunoglobulin gene recombination outside its homologous repair one. In a manuscript published in the January issue of Cell Reports we have demonstrate that RPA associates asymmetrically with resected ssDNA in response to lesions created by AID, RAG, or other nucleases. Small amounts of RPA are deposited at AID targets in G1. However, recruitment in S-G2/M is extensive and associated with Rad51 accumulation as expected if RPA functions mainly in homologous recombination. Thus, most RPA recruitment are antibody genes represents salvage of un-repaired breaks by homology-based pathways during the S-G2/M phases of the cell cycle. ii) AID is expressed in activated B lymphocytes. This process is initiated by a global increase in mRNA synthesis. However, the mechanisms driving transcriptome amplification during the immune response are unknown. By monitoring ssDNA genome-wide, we have recently shown in the journal Cell that the genome of nave cells is poised for rapid activation. In G0, 90% of promoters from genes to be expressed in cycling lymphocytes are loaded with polymerases but unmelted and thus they support only basal transcription. Furthermore, we have found that the transition from abortive to productive elongation is kinetically limiting causing polymerases to accumulate nearer transcription start sites. Resting lymphocytes also limit expression of the TFIIH complex, including XPB and XPD helicases involved in promoter melting and open complex extension. To date, two rate-limiting steps have been shown to control global gene expression in eukaryotes: preinitiation complex assembly and polymerase pausing. Our publication identify promoter melting as a third key regulatory step and propose that this mechanism ensures a prompt lymphocyte response to invading pathogens. iii) The Myc protein, which is deregulated by chromosomal translocations mediated by AID, has been implicated in physiological or pathological growth, proliferation, apoptosis, metabolism, and cell differentiation. No principle yet unifies Myc action due partly to an incomplete inventory of Myc's targets. To observe Myc target expression and function in a system where Myc is temporally and physiologically regulated, we collaborated with David Levens laboratory from NCI and created a mouse model that helps visualize Myc expression in vivo. In a publication in Cell we used these mice to analyze the transcriptomes and the genome-wide distributions of Myc, RNA polymerase II, and chromatin modifications in activated B cells and ES cells. A remarkably simple rule emerged from this quantitative analysis: Myc is not an on-off specifier of gene activity, but is a nonlinear amplifier of expression, acting universally at active genes, except for immediate early genes that are strongly induced before Myc. This rule of Myc action thereore explains the vast majority of Myc biology observed in literature and provides a rationale as to how this protein transforms B lymphocytes.