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）：这是一项探索性资助申请，旨在建立我们的初步结果，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）期间的重组步骤很重要。几年来已经知道AID的C末端10个氨基酸对于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，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是重要的。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与参与将DNA断裂引入S区的其他酶（特别是UNG和Msh 2-Msh 6）协同结合到IG开关（S）区，并且这种结合依赖于AID C末端。利用逆转录病毒在AID的AID-/-小鼠脾B细胞中的过表达，我们检测了AID与S？而S？在染色质免疫沉淀（ChIP）试验中，而C末端缺失的AID（？AID）不与S区DNA进行ChIP。同样，UNG和Msh 2-Msh 6也被ChIP在S？在Aid-/-细胞表达中的作用AID，但在表达AID的细胞中未检测到它们，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，即共依赖的。与这些蛋白质结合协同S？DNA，在ung-/-aid-/- B细胞或msh 2-/-aid-/- B细胞中，转导的完全脱辅基AID不能在S？通过Chip这些结果表明，为了使AID和UNG以及Msh 2-Msh 6在S区充分稳定地结合以被ChIP检测到，它们必须彼此协同结合DNA，并且这种协同结合依赖于AID C末端。我们提出验证以下假设：（1）AID C末端通过中间蛋白将UNG和Msh 2-Msh 6募集到S区，以及（2）AID的C末端对于在细胞周期的G1期将UNG和Msh 2-Msh 6募集到S区是重要的，和（3）AID的C末端对于随后形成DNA断裂的步骤是重要的，所述DNA断裂将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个具体的目的：1）研究AID与UNG和Msh 2-Msh 6之间的相互作用机制。2)确定AID募集UNG和Msh 2-Msh 6是否对G1期S区DSB的产生以及G1期DSB的修复重要。在正常脾B细胞，艾滋病依赖性S？DSB仅限于G1阶段。然而，在其他细胞类型中，UNG和Msh 2-Msh 6在S期期间被DNA复制复合物募集到DNA。3)确定AID C末端是否对将参与NHEJ的酶募集到S区并由此将CSR导向NHEJ重要。 公共卫生关系：本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应答所必需的。这种酶启动DNA断裂的形成，当一切顺利时，会导致有效的免疫反应。然而，艾滋病可能造成附带损害，导致染色体缺失和易位，并导致B细胞淋巴瘤，这是最常见的癌症类型。我们将研究AID是如何被调节以引入DNA断裂，从而导致适当的抗体类别转换的。