The mismatch-repair proteins MSH2 and MSH6 interact with the imprinting control regions through the ZFP57-KAP1 complex (RRBS)

Genomic imprinting is controlled by CpG-rich regions (ICRs) acquiring differential methylation in the female and male germline and maintaining it in a parental origin-specific manner in somatic cells. Despite their expected mutation rate due to spontaneous deamination of methylated cytosines, ICRs maintain their CpG-richness and show conservation of CpG-bearing transcription binding sites in mammals. In order to gain further insights into the mechanisms of maintenance of methyl CpGs, we sought to identify the proteins interacting with the methylated allele of the ICRs, by determining the interactors of ZFP57 that recognizes a specific methylated ICR motif in mouse ESCs. Several proteins including factors involved in mRNA processing/splicing, chromatin organization, transcription and DNA repair were identified through a tagged approach coupled to LC–MS/MS analysis. The demonstration of the components of the post-replicative mismatch-repair (MMR) complex as ZFP57 interactors prompted us to investigate the DNA binding profile of MSH2 and MSH6 by chromatin immunoprecipitation and sequencing. We found that these proteins were enriched at the methylated allele of the ICRs and their binding was mediated by the ZFP57/KAP1 complex, suggesting that the MMR complex is recruited to these loci to preserve their genetic integrity. In this study we perfomed Chromatin immunoprecipitation and sequencing (ChIP-seq) of Msh2 and Msh6 and methylation analysis (RRBS) in mESCs.

基因组印记（Genomic Imprinting）由CpG富集区域——印记控制区（Imprinting Control Regions, ICRs）调控，这些区域在雌性与雄性生殖系中获得差异甲基化，并在体细胞中以亲本起源特异性的方式维持该甲基化状态。尽管甲基化胞嘧啶会因自发脱氨而产生预期的突变率，但ICRs仍能维持其CpG富集特性，并在哺乳动物中保留带有CpG位点的转录结合位点的保守性。为进一步深入解析甲基化CpG位点的维持机制，我们尝试鉴定能够与ICRs的甲基化等位基因结合的蛋白质：通过结合标签蛋白技术与液相色谱-串联质谱（Liquid Chromatography-Tandem Mass Spectrometry, LC-MS/MS）分析，鉴定出可识别小鼠胚胎干细胞（mouse Embryonic Stem Cells, ESCs）中特定甲基化ICR基序的ZFP57的互作蛋白，其中包括参与mRNA加工/剪接、染色质组织、转录及DNA修复的多种因子。复制后错配修复（Mismatch Repair, MMR）复合体的组分被鉴定为ZFP57的互作蛋白，这一发现促使我们通过染色质免疫共沉淀测序（Chromatin Immunoprecipitation and Sequencing, ChIP-seq）探究MSH2与MSH6的DNA结合谱。我们发现，这些蛋白在ICRs的甲基化等位基因区域富集，且其结合依赖于ZFP57/KAP1复合体，这表明MMR复合体被招募至这些位点以维持其遗传完整性。本研究对小鼠胚胎干细胞（mouse Embryonic Stem Cells, mESCs）中的Msh2与Msh6实施了染色质免疫共沉淀测序（ChIP-seq），并开展了基于简化代表性亚硫酸氢盐测序（Reduced Representation Bisulfite Sequencing, RRBS）的甲基化分析。