Function of the AID C terminus in Ig class switching

描述（由申请人提供）：这是一份探索性拨款申请，基于我们的初步结果，表明激活诱导胞苷脱氨酶（AID）的C端对抗体类别转换重组（CSR）中的重组步骤很重要。尽管在抗体基因的体细胞超突变（SHM）过程中似乎没有任何作用，但多年来人们已经知道AID的C端10氨基酸对CSR非常重要，这一过程也依赖于AID。此外，AID C末端对于防止染色体在IgH和C -myc位点之间易位也很重要。我们已经获得了新的结果，表明在诱导进行CSR的脾B细胞中，AID与其他参与将DNA断裂引入S区域的酶（特别是UNG和Msh2-Msh6）合作结合到Ig开关(S)区域，并且这种结合依赖于AID C端。利用逆转录病毒在aid-/-小鼠脾B细胞中的过表达，我们检测了aid与S？和S ? ?在染色质免疫沉淀（ChIP）实验中，C端缺失AID (？AID)不与S区DNA ChIP。同样地，芯片在S？在aid-/-细胞表达？AID，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 C端，AID与这些蛋白的结合可能是合作的，即共依赖的。与这些蛋白质与S？DNA，在ung-/- AID -/- B细胞或msh2-/- AID -/- B细胞中，在S？通过芯片。这些结果表明，为了使AID和UNG以及Msh2-Msh6在S区足够稳定地结合并被ChIP检测到，它们必须相互合作结合DNA，而这种合作结合依赖于AID C端。我们拟验证以下假设：(1)AID的C端通过中间蛋白将UNG和Msh2-Msh6招募到S区；(2)AID的C端在细胞周期G1期将UNG和Msh2-Msh6招募到S区很重要；(3)AID的C端在DNA断裂形成的后续步骤中很重要，这些步骤将CSR导向非同源末端连接（NHEJ）。为了验证这些假设，我们提出了3个具体目标：1)研究AID与UNG和Msh2-Msh6相互作用的机制。2)确定AID对UNG和Msh2-Msh6的募集对于G1期S区dsb的产生和G1期dsb的修复是否重要。在正常的脾B细胞中，艾滋病依赖性S？dsb仅限于G1期。然而，在其他细胞类型中，UNG和Msh2- Msh6在S期被DNA复制复合体招募到DNA。3)确定AID C末端是否对招募NHEJ中涉及的酶到S区，从而将CSR导向NHEJ很重要。

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.