Function of the AID C terminus in Ig class switching

AID C 末端在 Ig 类别转换中的功能

• 批准号: 8292343
• 负责人: Janet M. Stavnezer
• 金额: $ 20.76万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-08-21 至 2014-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8292343
• 关键词: Adaptor Signaling Protein; Amino Acids; Amino Acids Activation; Antibodies; B-Cell Activation; B-Cell Lymphomas; B-Lymphocytes; Binding; Biological Assay; C-terminal; Cell Cycle; Cells; Chromosomal translocation; Chromosome Deletion; Complex; DNA; DNA Binding; DNA biosynthesis; Data; Deaminase; Enzyme Activation; Enzymes; Estrogen Receptors; G1 Phase; Generations; Genes; Genetic Recombination; Grant; Immune response; Immunoglobulin Class Switching; Immunoglobulin Somatic Hypermutation; Immunoglobulin Switch Recombination; Lead; Length; Mus; Mutation; Nonhomologous DNA End Joining; Process; Protein Binding; Proteins; Recruitment Activity; Relative (related person); Role; S Phase; Testing; activation-induced cytidine deaminase; c-myc Genes; cancer type; cell type; chromatin immunoprecipitation; in vivo; novel; prevent; receptor binding; repaired; replication factor A

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.

描述（由申请人提供）：这是基于我们的初步结果的探索性赠款的应用，表明激活诱导的胞苷脱氨酶（AID）的C末端对于在抗体类别开关重组（CSR）期间重组步骤（CSR）非常重要。几年来，c的C末端10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）中似乎没有任何作用，但这一过程也取决于AID。同样，AID C末端对于防止IGH和C-MYC基因座之间的染色体易位很重要。我们已经获得了新的结​​果，表明在诱导经历CSR的脾脏B细胞中，AID与IG开关的结合与参与引入DNA断裂的其他酶合作，特别是UNG和MSH2-MSH6，并且这种结合取决于AID C终端。在AID - / - 小鼠脾脏B细胞中使用逆转录病毒过表达，我们检测到辅助与S的结合？和s ??在染色质免疫沉淀（CHIP）测定中，而c末端删除的辅助（？aid）不使用S区域DNA进行芯片。同样，在S处的芯片也检测到UNG和MSH2-MSH6。在表达辅助的辅助 - / - 细胞中，未在表达辅助的细胞中检测到它们，这表明这些修复蛋白的结合取决于辅助c末端，辅助和这些蛋白的结合可能是合作的，即共同依赖性。与这些蛋白质与s合作结合的假设一致？ DNA，在UNG - / - AID - / - B细胞或MSH2 - / - AID - / - B细胞中，无法在s处检测到已转导的全lengt AID？通过芯片。这些结果表明，为了获得AID，UNG和MSH2-MSH6的结合，可以稳定地结合以通过芯片在S区域检测到，它们必须彼此合作结合DNA，并且这种合作结合取决于AID C末端。 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非同源最终连接（NHEJ）。为了检验这些假设，我们提出了3个具体目的：1）研究AID与UNG和MSH2-MSH6之间相互作用的机制。 2）确定援助募集UNG和MSH2-MSH6是否对于在G1阶段在S区域创建DSB以及在G1阶段的维修中是否很重要。在正常的脾脏B细胞中，依赖辅助的s？ DSB仅限于G1阶段。但是，在其他细胞类型中，UNG和MSH2-MSH6在S期间由DNA复制复合物募集到DNA。 3）确定辅助C末端对于募集NHEJ涉及的酶至S区域是否重要，从而将CSR引导到NHEJ。 公共卫生相关性：该项目研究了启动抗体类转换的酶（AID）的功能，这是生成有效的抗体免疫反应所必需的。这种酶会引发DNA中断裂的形成，当一切顺利时，这会导致有效的免疫反应。但是，援助会导致附带损害，从而导致染色体缺失和易位，以及最常见的癌症类型B细胞淋巴瘤。我们将调查如何调节AID来引入DNA断裂，从而导致适当的抗体类切换。