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

基本信息

项目摘要

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(由申请方提供):生发中心B细胞中的免疫球蛋白(IG)基因经历高速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。这些过程由活化诱导的脱氨酶蛋白(AID)启动,其产生G:U错配,所述G:U错配通过复制、碱基切除修复和错配修复(MMR)进行处理,以产生抗体可变区和开关区的体细胞超突变所需的突变。MMR导致约50%的这些突变,其中大部分是在A:T碱基中。虽然典型的MMR通常保持基因组的完整性和稳定性,但在IG基因处,它似乎已经转移到MMR的易错非典型途径,其介导广泛的突变和重组。本研究通过检测新的具有功能分离突变的转基因小鼠来研究B细胞如何调节和募集易出错的MMR到免疫球蛋白基因:1)MSH 6 DNA结合域中的Msh 6 E433 A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有缺陷; 2)在遗传性非息肉病性癌症(HNPCC)患者中鉴定了MSH 6的PWWP结构域中的Msh 6S 144 I突变,并且该突变在结合H3 K36 me 3组蛋白修饰方面存在缺陷,并且可能在将MSH 6募集到染色质方面存在缺陷;和3)Msh 2G 674 D和Msh 6 T1217 D小鼠携带模拟其它HNPCC突变的ATP酶突变体,并且在经历募集下游因子所需的构象变化方面有缺陷。我们将对突变小鼠的IG可变区和转换区进行深度测序,以确定MMR中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求这些表型的机制。为了在原代B细胞中实现这一点,我们将使用在其内源基因座中表达TAP标记的MSH 6和EXO 1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,我们发现染色质重塑蛋白在CH 12 F3细胞的shRNA筛选中抑制CSR,并且是MMR相互作用组的一部分。我们相信,解剖出的作用,每个域的MSH 6和MSH 2和确定的差异,在MMR复合物和染色质重塑因子负责V区突变和类转换重组将揭示如何错误倾向MMR主要局限于IG基因,并最终如何在这些过程中的调节变化导致癌症在许多组织。

项目成果

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MATTHEW D SCHARFF其他文献

MATTHEW D SCHARFF的其他文献

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{{ truncateString('MATTHEW D SCHARFF', 18)}}的其他基金

Hybridoma (Monoclonal Antibody) Core
杂交瘤(单克隆抗体)核心
  • 批准号:
    7706296
  • 财政年份:
    2008
  • 资助金额:
    $ 37.58万
  • 项目类别:
IMMUNO-ONCOLOGY
免疫肿瘤学
  • 批准号:
    7506791
  • 财政年份:
    2007
  • 资助金额:
    $ 37.58万
  • 项目类别:
PROGRAM LEADERS
项目负责人
  • 批准号:
    7506775
  • 财政年份:
    2007
  • 资助金额:
    $ 37.58万
  • 项目类别:
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:
    8403693
  • 财政年份:
    2003
  • 资助金额:
    $ 37.58万
  • 项目类别:
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:
    7758277
  • 财政年份:
    2003
  • 资助金额:
    $ 37.58万
  • 项目类别:
Mismatch Repair in V region mutation and isotype switch
V区突变和同种型转换中的错配修复
  • 批准号:
    7076248
  • 财政年份:
    2003
  • 资助金额:
    $ 37.58万
  • 项目类别:
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
  • 财政年份:
    2003
  • 资助金额:
    $ 37.58万
  • 项目类别:
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:
    7579404
  • 财政年份:
    2003
  • 资助金额:
    $ 37.58万
  • 项目类别:
Mismatch Repair in V region mutation and isotype switch
V区突变和同种型转换的错配修复
  • 批准号:
    6925507
  • 财政年份:
    2003
  • 资助金额:
    $ 37.58万
  • 项目类别:
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:
    8900937
  • 财政年份:
    2003
  • 资助金额:
    $ 37.58万
  • 项目类别:

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