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消除病原体，在很大程度上取决于针对B细胞抗体基因的两个遗传过程：体细胞高突变和类开关重组。第一种机制在抗体基因的N端部分引入随机点突变。在免疫反应过程中，与细胞选择有关的突变增加了抗体与病原体的结合亲和力。第二种机制(通过基因重组)改变抗体基因的C末端部分，这反过来决定了免疫系统用来消除有问题的病原体的策略。体细胞的超突变和开关重组都是由一种新发现的酶：激活诱导胞苷脱氨酶或AID进行的。AID酶改变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。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，试图将所有尿嘧啶还原为胞苷。然而，虽然自发的胞苷脱氨得到了忠实的修复，但AID介导的脱氨导致了形成突变和DNA断裂，这分别是体细胞超突变和开关重组的基础。 AID在免疫反应中的重要性在AID缺乏的人和动物中得到强调，这些人和动物对感染高度敏感，并表现出肠道菌群依赖性的肠道绒毛增生。相反，复杂的疾病，如自身免疫，长期以来一直与依赖艾滋病的超突变有关。此外，AID不仅被招募到抗体基因中，而且越来越多的证据表明，包括癌基因(肿瘤诱导基因)在内的许多其他基因也可以成为AID的生理靶点。如上所述，由于AID活性导致DNA突变，癌基因可以通过AID靶向被解除调控，导致表达AID的细胞在易感个体中恶性转化。此外，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，这是一种称为易位的染色体异常。染色体易位是人类B细胞淋巴瘤细胞癌基因失控的另一种机制，Burkits和多发性骨髓瘤是AID诱导的易位导致肿瘤发展的主要例子。这些重要的临床考虑强调了了解调控AID表达和活性的分子途径的必要性。本财年，我们通过两个不同的手稿，进一步加深了我们对艾滋病监管的理解： 1)如上所述，AID导致IgH基因DNA损伤，这些DNA损伤不仅对抗体基因恒定域的重组至关重要，而且还参与了cMyc癌基因和IgH基因之间的染色体易位。这些染色体损伤通常会导致人类(Burkitts淋巴瘤)和小鼠(浆细胞瘤)发生B细胞肿瘤。然而，由于cMyc易位也存在于健康个体的淋巴细胞中，目前尚不清楚AID活性是否直接影响B细胞转化的动力学。该实验室的博士后Makiko Takizawa使用肿瘤小鼠模型表明，通过确定在肿瘤诱导过程中出现的cMyc转位承载细胞的数量，AID的表达水平直接决定了B细胞肿瘤的发生。这些研究预测，在肿瘤易感个体中，cMyc易位细胞的实际数量具有临床意义。 Ii)由于AID的肿瘤诱导活性，B淋巴细胞在免疫应答过程中发展了多种调节AID的机制。一些研究表明，AID在转录水平、翻译后修饰、与特定辅因子的相互作用以及贩运和区隔等方面受到严格调控。在与洛克菲勒大学的F.Nina Papavasiliou的合作中，我们最近表明AID也以一种不那么传统的方式受到调控：通过microRNA，miR-155转录下调。MicroRNAs是一类非编码的1830nT RNAs，通过靶向同源信使RNAs的降解或翻译抑制，发挥基因表达的转录后调节作用。使用表达AID荧光版本的BAC转基因小鼠(AID-GFP)，我们发现AID是miR-155负调控的直接靶点。在去除AID mRNA中miR-155靶点的小鼠中，免疫反应过程中AID蛋白的表达增强，导致抗体分子成熟缺陷，这显然是由于AID过度活性所致。重要的是，在同一期《免疫》杂志上发表的一篇论文中，Michel Nunssenzweig和他的同事还表明，miR-155缺陷小鼠的B细胞比野生型B细胞携带明显更多的染色体易位，这表明miR-155保护基因组免受艾滋病诱变活性的影响。 目前，我们正在使用深度测序技术来确定小鼠和人类B细胞基因组中AID错误靶向的程度。我们的目标是查明AID是如何招募到非免疫球蛋白基因并针对非免疫球蛋白基因的，比如cMyc癌基因。

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 two genetic processes targeting B cell antibody genes: somatic hypermutation and class switch recombination. The first mechanism introduces random point mutations at the N terminal portion of the antibody gene. Mutations coupled to cell selection during the immune response increase the binding affinity of the antibody for the pathogen. The second mechanism changes (via gene recombination) the C terminal portion of the antibody gene, which in turns dictates the strategy used by the immune system to eliminate the pathogen in question. Both somatic hypermutation and switch recombination are carried out by a newly discovered enzyme: Activation-Induced cytidine Deaminase or AID. The AID enzyme 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, which attempt to revert all uracils into cytidines. However, while spontaneous cytidine deamination is faithfully repaired, in a manner not fully understood AID-mediated deamination leads to the formation mutations and DNA breaks, which are at the basis of somatic hypermutation and switch recombination respectively. The importance of AID in the immune response is highlighted in AID deficient humans and animals, 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 AID-dependent hypermutation. Moreover, AID is not only recruited to antibody genes, but increasing evidence indicate that many other genes, including oncogenes (tumor-inducing genes) can be physiological targets of AID. Because, as stated above, AID activity leads to DNA mutations, oncogenes can become deregulated by AID-targeting, resulting in malignant transformation of AID expressing cells in susceptible individuals. In addition, 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 represent another mechanism whereby cellular oncogenes become deregulated in B cell lymphomas in humans, Burkits and multiple myeloma are prime examples of AID-induced translocations leading to tumor development. These important clinical considerations emphasize the need to understand the molecular pathways that regulate AID expression and activity. This fiscal year we have furthered our understanding of AID regulation as published in two separate manuscripts: i) As discussed above, AID induces DNA lesions at Igh genes which are not only critical for recombination of antibody gene constant domains, but are also involved in chromosomal translocations between the cMyc oncogene and the Igh loci. These chromosomal lesions often lead to B cell tumor development both in humans (Burkitts lymphomas) and in mice (plasmacytomas). However, as cMyc translocations are also present in lymphocytes from healthy individuals, it has remained unclear whether AID activity directly influences the dynamics of B cell transformation. Using a tumor mouse model, Makiko Takizawa, a postdoctoral fellow in the lab, has shown that AID expression levels directly define the incidence of B cell tumor development by determining the number of cMyc translocation-bearing cells emerging during tumor induction. These studies predict that in tumor susceptible individuals the actual number of cMyc-translocated cells has clinical significance. ii) Because of AID tumor-inducing activity, B lymphocytes have developed a variety of mechanisms to regulate AID during the immune response. Several studies indicate AID is tightly regulated at the transcriptional level, by post-translational modifications, by interaction with specific cofactors, and by trafficking and compartmentalization. In collaboration with F. Nina Papavasiliou from the Rockefeller University we have recently shown AID is also regulated in a less traditional way: by transcriptional downregulation via a microRNA, miR-155. MicroRNAs are a class of non-coding 1830 nt RNAs that function as post-transcriptional regulators of gene expression by targeting their cognate messenger RNAs for degradation or translational repression. Using BAC transgenic mice expressing a fluorescent version of AID (AID-GFP), we showed that the AID is a direct target for miR-155 negative regulation. In mice where the miR-155 target in the AID mRNA was ablated there was enhanced expression of AID protein during the immune response leading to defects in the maturation of antibody molecules, apparently because of AID overactivity. Importantly, in a paper published in the same issue of Immunity, Michel Nunssenzweig and colleagues also showed that miR-155 deficient mice, B cells carry significantly more chromosomal translocations compared to wild type counterparts, indicating that miR-155 protects the genome against AID mutagenic activity. Currently, we are using deep-sequencing techniques to determine the extent of AID mistargeting in both the mouse and human B cell genomes. Our goal is to pinpoint how AID is recruited to and target non-immunoglobulin genes, like cMyc oncogenes.