Mismatch repair in V region mutation and isotype switching

描述(由申请人提供)：生发中心B细胞中的免疫球蛋白(Ig)基因经历了高比率的体细胞突变和重组，以实现亲和力成熟和同型转换，从而产生保护性抗体。这些过程是由激活诱导的脱氨酶蛋白(AID)启动的，它产生G：U错配，通过复制、碱基切除修复和错配修复(MMR)来产生抗体可变区和开关区的体细胞超突变所需的突变。这些突变中约50%是由MMR引起的，其中大部分位于A：T碱基。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，它似乎已经转移到MMR的一条容易出错的非规范途径，这介导了广泛的突变和重组。在这里，我们建议通过检测新的具有功能分离突变的转基因小鼠来研究B细胞如何调节和招募容易出错的MMR到免疫球蛋白基因：1)MSH6 DNA结合域的Msh6E433A突变位于识别和结合错配碱基的位置，并且通过MMR的非规范途径在修复氧化损伤方面存在缺陷；2)MSH6的PWWP域的Msh6S144I突变在遗传性非息肉病癌症(HNPCC)患者中被发现，并且在结合H3K36me3组蛋白修饰和招募MSH6与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，并且在吸收下游因子所需的构象变化方面存在缺陷。我们将使用对突变小鼠的Ig可变区和切换区的深度测序来确定MMR中的这些突变对体细胞超突变和类切换重组的影响。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蛋白的化学计量比和相互作用来寻找导致这些表型的机制。为了在原代B细胞中实现这一点，我们将对表达TAP标记的MSH6和EXO1内源基因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，我们将通过检测染色质重塑蛋白的作用来分析染色质重塑的作用。我们在CH12F3细胞的shRNA屏幕上发现了染色质重塑蛋白抑制CSR的作用，这些蛋白是MMR互动组的一部分。我们相信，剖析MSH6和MSH2每个结构域的作用，并确定导致V区突变和类开关重组的MMR复合体和染色质重塑因子的差异，将揭示容易出错的MMR如何在很大程度上限于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。

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.