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.