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）：这是一份探索性资助申请，以我们的初步结果为基础，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) 期间的重组步骤很重要。几年前人们就知道 AID 的 C 末端 10 个氨基酸对于 CSR 非常重要，尽管它们似乎在抗体基因的体细胞超突变 (SHM) 过程中没有任何作用，而该过程也依赖于 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。我们获得了新的结​​果，表明在诱导进行 CSR 的脾 B 细胞中，AID 与参与将 DNA 断裂引入 S 区域的其他酶（特别是 UNG 和 Msh2-Msh6）协同结合到 Ig 开关 (S) 区域，并且这种结合是取决于 AID C 末端。使用逆转录病毒在aid-/-小鼠脾B细胞中过度表达AID，我们检测到AID与S？和S??在染色质免疫沉淀 (ChIP) 测定中，而 C 末端删除的 AID (?AID) 不能与 S 区 DNA 进行 ChIP。同样，UNG 和 Msh2-Msh6 也在 S? 处被 ChIP 检测到。在表达?AID的aid-/-细胞中，但在表达AID的细胞中没有检测到它们，这表明这些修复蛋白的结合依赖于AID C末端，并且AID和这些蛋白的结合可能是协同的，即共-依赖。与这些蛋白质协同结合S？的假设一致。 DNA，在 ung-/-aid-/- B 细胞或 msh2-/-aid-/- B 细胞中，转导的全长 AID 在 S 处无法检测到？通过 ChIP。这些结果表明，为了使 AID 和 UNG 以及 Msh2-Msh6 足够稳定地结合以通过 ChIP 在 S 区检测到，它们必须彼此协同结合 DNA，并且这种协同结合取决于 AID C 末端。我们建议检验以下假设：(1) AID C 末端通过中间蛋白将 UNG 和 Msh2-Msh6 招募到 S 区域，以及 (2) AID 的 C 末端对于将 UNG 和 Msh2-Msh6 招募到 S 区域非常重要(3) AID 的 C 末端对于 DNA 断裂形成后的步骤非常重要，从而将 CSR 导向非同源末端连接（NHEJ）。为了检验这些假设，我们提出了 3 个具体目标：1）研究 AID 和 UNG 以及 Msh2-Msh6 之间相互作用的机制。 2) 确定AID招募UNG和Msh2-Msh6对于G1期S区DSB的创建以及G1期修复是否重要。在正常脾 B 细胞中，AID 依赖性 S？ DSB 仅限于 G1 相。然而，在其他细胞类型中，UNG 和 Msh2-Msh6 在 S 期被 DNA 复制复合体募集到 DNA。 3) 确定 AID C 末端对于将 NHEJ 涉及的酶募集到 S 区域并从而将 CSR 导向 NHEJ 是否很重要。 公共卫生相关性：该项目研究启动抗体类别转换的酶 (AID) 的功能，这是产生有效抗体免疫反应所必需的。这种酶会引发 DNA 断裂的形成，当一切顺利时，就会产生有效的免疫反应。然而，AID 会造成附带损害，从而导致染色体缺失和易位以及 B 细胞淋巴瘤（最常见的癌症类型）。我们将研究如何调节 AID 以引入 DNA 断裂，从而导致正确的抗体类别转换。