AID biology

B淋巴细胞是免疫系统细胞，通过其细胞表面上称为抗体的特殊受体识别和处理病原体，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靶向B细胞抗体基因的两个遗传过程：体细胞超突变和类别转换重组。第一种机制在抗体基因的N末端部分引入随机点突变。在免疫应答期间与细胞选择偶联的突变增加了抗体对病原体的结合亲和力。第二种机制改变（通过基因重组）抗体基因的C末端部分，这反过来又决定了免疫系统消除病原体的策略。体细胞超突变和开关重组都是由一种新发现的酶进行的：激活诱导的胞苷脱氨酶或AID。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，这一过程称为胞苷脱氨基。由于尿嘧啶具有致突变性，AID活性会吸引过多的修复酶到免疫球蛋白位点，试图将所有尿嘧啶还原为胞苷。然而，虽然自发的胞苷脱氨基被忠实地修复，但以不完全理解的方式，艾滋病介导的脱氨基导致形成突变和DNA断裂，这分别是体细胞超突变和开关重组的基础。 AID在免疫应答中的重要性在AID缺陷的人和动物中突出，其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，长期以来一直与艾滋病依赖性超突变有关。此外，AID不仅被募集到抗体基因，而且越来越多的证据表明许多其他基因，包括癌基因（肿瘤诱导基因）可以是AID的生理靶点。 如上所述，因为AID活性导致DNA突变，所以癌基因可以通过AID靶向而变得失调，导致易感个体中表达AID的细胞的恶性转化。此外，艾滋病介导的DNA断裂也可以重组或将癌基因带到免疫球蛋白位点附近，这是一种称为易位的染色体不规则性。染色体易位代表了另一种机制，其中细胞癌基因在人类B细胞淋巴瘤中变得失调，Burkits和多发性骨髓瘤是AIDS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需要了解调节AID表达和活性的分子途径。本财政年度，我们加深了对援助条例的理解，并在两份单独的手稿中发表： （一） 如上所述，AID诱导Igh基因处的DNA损伤，其不仅对于抗体基因恒定结构域的重组至关重要，而且还涉及cMyc癌基因和Igh基因座之间的染色体易位。这些染色体病变通常导致人类（伯基特淋巴瘤）和小鼠（浆细胞瘤）的B细胞肿瘤发展。然而，由于cMyc易位也存在于来自健康个体的淋巴细胞中，因此仍然不清楚AID活性是否直接影响B细胞转化的动力学。使用肿瘤小鼠模型，该实验室的博士后研究员Makiko Takizawa已经表明，AID表达水平通过确定肿瘤诱导期间出现的携带cMyc易位的细胞的数量来直接确定B细胞肿瘤发展的发生率。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）第二阶段 由于AID的肿瘤诱导活性，B淋巴细胞已经发展出多种机制来调节免疫应答期间的AID。一些研究表明，AID在转录水平上受到严格调控，通过翻译后修饰，与特定辅因子的相互作用，以及通过运输和区室化。 与F合作。来自洛克菲勒大学的Nina Papavasiliou最近发现，AID也以一种不太传统的方式进行调节：通过microRNA，miR-155进行转录下调。MicroRNA是一类非编码的1830 nt RNA，其通过靶向其同源信使RNA进行降解或翻译抑制而作为基因表达的转录后调节因子。使用表达荧光形式的AID（AID-GFP）的BAC转基因小鼠，我们表明AID是miR-155负调控的直接靶标。在AIDmRNA中的miR-155靶标被消除的小鼠中，在免疫应答期间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。重要的是，Michel Nunssenzweig及其同事在同一期Immunity上发表的一篇论文中还表明，与野生型小鼠相比，miR-155缺陷小鼠的B细胞携带的染色体易位明显更多，表明miR-155保护基因组免受AID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错误定位的程度。我们的目标是确定AID是如何被招募并靶向非免疫球蛋白基因的，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.