Function of the AID C terminus in Ig class switching

描述(由申请人提供)：这是一项探索性拨款的申请，以我们的初步结果为基础，该结果表明，激活诱导的胞苷脱氨酶(AID)的C末端对于抗体类别转换重组(CSR)中的重组步骤是重要的。已知AID的C端10个氨基酸对CSR非常重要，尽管它们在抗体基因的体细胞超突变(SHM)过程中似乎没有任何作用，这一过程也依赖于AID。此外，AID C末端对于防止IgH和c-myc基因座之间的染色体易位也很重要。我们的新结果表明，在被诱导为CSR的脾B细胞中，AID与参与将DNA断裂引入S区域的其他酶协同结合到Ig开关(S)区域，特别是UNG和Msh2-MSH6，并且这种结合依赖于AID C末端。利用逆转录病毒在艾滋病小鼠脾B细胞中的过度表达，检测艾滋病病毒与S的结合。S呢？？在染色质免疫沉淀(CHIP)检测中，C端缺失的AID(？AID)不与S区域DNA发生芯片反应。同样，在S的芯片中也检测到了UNG和Msh2-MSH6。但在表达AID的细胞中未检测到，提示这些修复蛋白的结合依赖于AID的C末端，AID与这些蛋白的结合可能是协同的，即相互依赖的。与这些蛋白质与S协同结合的假设一致吗？S未检测到转导全长AID的Un-/-Aid-/-B细胞或MsH2-/-Aid-/-B细胞中的DNA？用芯片。这些结果表明，Aid、UNG和Msh2-MSH6要足够稳定地结合到S区域的芯片上，它们必须相互协同结合DNA，这种协同结合依赖于AID C末端。我们建议检验假设(1)AID的C末端通过中间蛋白将UNG和Msh2-MSH6招募到S区域，(2)在细胞周期的G1期，AID的C末端对于将UNG和Msh2-MSH6招募到S区域是重要的，(3)AID的C末端对于DNA断裂的形成是重要的，DNA断裂将CSR导向非同源末端连接(NHEJ)。为了验证这些假说，我们提出了三个具体目标：1)研究AID与UNG和Msh2-MSH6的相互作用机制。2)研究AID对UNG和Msh2-MSH6的募集是否对S区G1期双链断裂的产生和G1期双链断裂的修复起重要作用。在正常脾B细胞中，艾滋病依赖的S？双链断裂被限制在G1相。然而，在其他类型的细胞中，UNG和Msh2-MSH6在S期被DNA复制复合体招募到DNA中。3)确定AID C末端是否对将NHEJ相关酶招募到S区域，从而将CSR导向NHEJ起重要作用。 公共卫生相关性：该项目调查启动抗体类别转换的酶(AID)的功能，这是产生有效的抗体免疫反应所必需的。这种酶启动DNA断裂的形成，当一切顺利时，会产生有效的免疫反应。然而，艾滋病会造成附带损害，导致染色体缺失和易位，并导致最常见的癌症--B细胞淋巴瘤。我们将研究AID如何被调控以引入DNA断裂，从而导致适当的抗体类别转换。

DESCRIPTION (provided by applicant): This is an application for an exploratory grant to build on our preliminary results suggesting that the C terminus of activation-induced cytidine deaminase (AID) is important for the recombination step during antibody class switch recombination (CSR). It has been known for several years that the C terminal 10 amino acids of AID are very important for CSR, although they do not appear to have any role during somatic hypermutation (SHM) of antibody genes, a process also dependent upon AID. Also, the AID C terminus is important for preventing chromosomal translocations between the IgH and c-myc loci. We have obtained novel results indicating that in splenic B cells induced to undergo CSR, AID binds to Ig switch (S) regions cooperatively with other enzymes involved in introducing DNA breaks into S regions, specifically UNG and Msh2-Msh6, and that this binding is dependent upon the AID C terminus. Using retroviral over-expression in aid-/- mouse splenic B cells of AID, we detect AID binding to S? and S?? in chromatin immunoprecipitation (ChIP) assays, whereas C terminal deleted AID (?AID) does not ChIP with S region DNA. Likewise, both UNG and Msh2-Msh6 are also detected by ChIP at S? in aid-/- cells expressing ?AID, but they are not detected in cells expressing AID, suggesting that the binding of these repair proteins depends on the AID C terminus, and the binding of AID and these proteins might be cooperative, i.e. co-dependent. Consistent with the hypothesis that these proteins bind cooperatively to S? DNA, in ung-/-aid-/- B cells or in msh2-/-aid-/- B cells, transduced full lengt AID cannot be detected at S? by ChIP. These results suggest that in order for AID and UNG and Msh2-Msh6 to bind sufficiently stably to be detected by ChIP at S regions, they must bind DNA cooperatively with each other, and this cooperative binding depends on the AID C terminus. We propose to test the hypotheses (1) that the AID C terminus recruits UNG and Msh2-Msh6 to S regions via an intermediary protein, and (2) that the C terminus of AID is important for recruiting UNG and Msh2-Msh6 to S regions during the G1 phase of the cell cycle, and (3) that the C terminus of AID is important for steps subsequent to formation of DNA breaks that direct CSR towards non-homologous end-joining (NHEJ). In order to test these hypotheses we propose 3 Specific Aims: 1) To investigate the mechanism of interaction between AID and UNG and Msh2-Msh6. 2) To determine if the recruitment of UNG and Msh2-Msh6 by AID is important for creating DSBs in S regions during G1 phase and also for their repair during G1 phase. In normal splenic B cells, AID-dependent S? DSBs are restricted to the G1 phase. However, in other cell types, UNG and Msh2- Msh6 are recruited by the DNA replication complex to DNA during S phase. 3) To determine if the AID C terminus is important for recruiting enzymes involved in NHEJ to S regions and thereby directing CSR toward NHEJ. PUBLIC HEALTH RELEVANCE: This project investigates the function of the enzyme (AID) that initiates antibody class switching, which is required for generation of an effective antibody immune response. This enzyme initiates formation of breaks in DNA which when everything goes well leads to an effective immune response. However, AID can cause collateral damage, which leads to chromosomal deletions and translocations and to B cell lymphomas, the most common type of cancer. We will investigate how AID is regulated to introduce DNA breaks that lead to proper antibody class switching.