Mismatch repair in V region mutation and isotype switching

V区突变和同种型转换中的错配修复

• 批准号: 9132482
• 负责人: MATTHEW D SCHARFF
• 金额: $ 34.44万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE, INC
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-07-01 至 2018-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9132482
• 关键词: ATP phosphohydrolase; Affect; Affinity; Affinity Chromatography; Antibodies; Antibody Diversity; Antigens; B-Lymphocytes; Base Excision Repairs; Binding; Cancer Patient; Cells; Characteristics; Chromatin; Chromatin Remodeling Factor; Complementary DNA; Complex; Core Protein; DNA Binding Domain; DNA Double Strand Break; DNA Sequence; Data; EXO1 exonuclease; EXO1 gene; Event; Exonuclease; Frequencies; Genes; Genetic; Genetic Recombination; Genome Stability; HTATIP gene; Health; Hot Spot; ISWI; Immunoglobulin Class Switching; Immunoglobulin Genes; Immunoglobulin Somatic Hypermutation; Immunoglobulin Switch Recombination; Immunoglobulins; Immunoprecipitation; Infection; Inherited; Knock-in Mouse; Knockout Mice; Lead; MLH1 gene; MSH2 gene; MSH6 gene; Malignant Neoplasms; Mass Spectrum Analysis; Mediating; Mismatch Repair; Modeling; Molecular; Mus; Mutant Strains Mice; Mutate; Mutation; Organism; PMS2 gene; PWWP Domain; Pathway interactions; Pattern; Phenotype; Process; Proteins; Proteomics; Proto-Oncogenes; Recruitment Activity; Regulation; Repair Complex; Replication-Associated Process; Resolution; Role; SMARCA5 gene; Site; Somatic Mutation; Spleen; Structure; Structure of germinal center of lymph node; T-Lymphocyte; Tissues; Western Blotting; activation-induced cytidine deaminase; base; chromatin remodeling; deep sequencing; histone modification; knock-down; mutant; null mutation; oxidative damage; pathogen; pol Gene Products; protein protein interaction; repaired; research study; scaffold; small hairpin RNA; stoichiometry; transition mutation

DESCRIPTION (provided by applicant): The immunoglobulin (Ig) genes in germinal center B cells undergo a high rate of somatic mutation and recombination to achieve the affinity maturation and isotype switching that generates protective antibodies. These processes are initiated by activation induced deaminase protein (AID) which generates G:U mismatches that are processed by replication, base excision repair and mismatch repair (MMR) to produce the mutations required for somatic hypermutation of antibody variable and switch regions. MMR is responsible for ~50% of these mutations most of which are in A:T bases. While canonical MMR normally maintains the integrity and stability of the genome, at the Ig gene it appears to have shifted to an errorprone non-canonical pathway of MMR, which mediates extensive mutation and recombination. Here we propose to examine how B cells regulate and recruit error prone MMR to immunoglobulin genes by examining new genetically modified mice with separation-of-function mutations: 1) the Msh6E433A mutation in the DNA binding domain of MSH6 is at the site that recognizes and binds the mismatched base and is defective in repairing oxidative damage through the non-canonical pathway of MMR; 2) the Msh6S144I mutation in the PWWP domain of MSH6 was identified in hereditary non-polyposis cancer (HNPCC) patients and is defective in binding the H3K36me3 histone modification and presumably in recruiting MSH6 to chromatin; and 3) the Msh2G674D and Msh6T1217D mice carry ATPase mutants that model other HNPCC mutations and are defective in undergoing conformational changes required to recruit downstream factors. We will use deep sequencing of the Ig variable and switch regions of the mutant mice to determine the impact of these mutations in MMR on somatic hypermutation and class switch recombination. This will provide a high-resolution phenotype of each of the MMR mutations. We will seek the mechanisms responsible for these phenotypes by determining the stoichiometry and interactions of the core MMR proteins in wild type and mutant B cells. To accomplish this in primary B cells, we will carry out immunoprecipitation (IP), tandem affinity purification (TAP), western blot analysis and mass spectrometry using mice expressing TAP-tagged MSH6 and EXO1 in their endogenous loci. Finally we will analyze the role of chromatin remodeling by examining the role of chromatin remodeling proteins that we have found to inhibit CSR in an shRNA screen of CH12F3 cells and that are part of the MMR interactome. We believe that dissecting out the role of each of the domains of MSH6 and of MSH2 and identifying the differences in the MMR complexes and chromatin remodeling factors responsible for V region mutation and class switch recombination will reveal how error prone MMR is largely restricted to the Ig gene and ultimately how changes in the regulation of these processes leads to cancer in many tissues.

描述（申请人提供）：生发中心B细胞中的免疫球蛋白（Ig）基因经历高频率的体细胞突变和重组，以实现亲和力成熟和同种型转换，从而产生保护性抗体。这些过程由激活诱导脱氨酶蛋白 (AID) 启动，该蛋白产生 G:U 错配，通过复制、碱基切除修复和错配修复 (MMR) 处理，产生抗体可变区和开关区体细胞超突变所需的突变。这些突变中约 50% 是由 MMR 造成的，其中大部分发生在 A:T 碱基中。虽然规范的 MMR 通常维持基因组的完整性和稳定性，但在 Ig 基因上，它似乎已转变为容易出错的非规范 MMR 途径，介导广泛的突变和重组。在此，我们建议通过检查具有功能分离突变的新型转基因小鼠来研究 B 细胞如何调节和招募易出错的 MMR 到免疫球蛋白基因：1）MSH6 DNA 结合域中的 Msh6E433A 突变位于识别和结合错配碱基的位点，并且在通过 MMR 的非经典途径修复氧化损伤方面存在缺陷； 2) 在遗传性非息肉病癌症 (HNPCC) 患者中发现了 MSH6 PWWP 结构域中的 Msh6S144I 突变，该突变在结合 H3K36me3 组蛋白修饰方面存在缺陷，并且可能在将 MSH6 招募到染色质方面存在缺陷； 3) Msh2G674D 和 Msh6T1217D 小鼠携带模拟其他 HNPCC 突变的 ATPase 突变体，并且在招募下游因子所需的构象变化方面存在缺陷。我们将使用突变小鼠的 Ig 变量和转换区域的深度测序来确定 MMR 中的这些突变对体细胞超突变和类别转换重组的影响。这将提供每个 MMR 突变的高分辨率表型。我们将通过确定野生型和突变型 B 细胞中核心 MMR 蛋白的化学计量和相互作用来寻找导致这些表型的机制。为了在原代 B 细胞中实现这一目标，我们将使用在内源基因座中表达 TAP 标记的 MSH6 和 EXO1 的小鼠进行免疫沉淀 (IP)、串联亲和纯化 (TAP)、蛋白质印迹分析和质谱分析。最后，我们将通过检查染色质重塑蛋白的作用来分析染色质重塑的作用，我们发现这些蛋白在 CH12F3 细胞的 shRNA 筛选中可以抑制 CSR，并且是 MMR 相互作用组的一部分。我们相信，剖析 MSH6 和 MSH2 每个结构域的作用，并识别 MMR 复合物和负责 V 区突变和类别转换重组的染色质重塑因子的差异，将揭示容易出错的 MMR 在很大程度上局限于 Ig 基因，以及最终这些过程的调节变化如何导致许多组织中的癌症。