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.
To protect us from pathogenic organisms, it is necessary for us to produce antibodies to every possible antigen and to rapidly change those antibodies as the pathogen changes. We do this by introducing many mutations into the genes that encode the antibody molecules as we respond to infection and this genetic instability is accomplished through hypermutation and the shifting of mismatch repair to make it highly error prone. We will use mice that have genetic defects in mismatch repair to examine how this repair process contributes to antibody diversity and sometimes also causes high mutation rates in proto-oncogenes that lead to B cell and other malignancies.
|Wang, Xiaohua; Duan, Zhi; Yu, Guojun et al. (2018) Human Immunodeficiency Virus Tat Protein Aids V Region Somatic Hypermutation in Human B Cells. MBio 9:|
|Lee, Kyeryoung; Tosti, Elena; Edelmann, Winfried (2016) Mouse models of DNA mismatch repair in cancer research. DNA Repair (Amst) 38:140-6|
|Rein, Katrin; Yanez, Diana A; Terré, Berta et al. (2015) EXO1 is critical for embryogenesis and the DNA damage response in mice with a hypomorphic Nbs1 allele. Nucleic Acids Res 43:7371-87|
|Wei, Lirong; Chahwan, Richard; Wang, Shanzhi et al. (2015) Overlapping hotspots in CDRs are critical sites for V region diversification. Proc Natl Acad Sci U S A 112:E728-37|
|van Oers, J M M; Edwards, Y; Chahwan, R et al. (2014) The MutS? complex is a modulator of p53-driven tumorigenesis through its functions in both DNA double-strand break repair and mismatch repair. Oncogene 33:3939-46|
|Wang, Xiaohua; Fan, Manxia; Kalis, Susan et al. (2014) A source of the single-stranded DNA substrate for activation-induced deaminase during somatic hypermutation. Nat Commun 5:4137|
|Schaetzlein, Sonja; Chahwan, Richard; Avdievich, Elena et al. (2013) Mammalian Exo1 encodes both structural and catalytic functions that play distinct roles in essential biological processes. Proc Natl Acad Sci U S A 110:E2470-9|