Abstract

Myotonic dystrophy type 1 (DM1) is caused by expansion of a CTG repeat in the DMPK gene, where expansion size and somatic mosaicism correlates with disease severity and age of onset. While it is known that the mismatch repair protein MSH2 contributes to the unstable nature of the repeat, its role on other disease-related features, such as CpG methylation upstream of the repeat, is unknown. In this study, we investigated the effect of an MSH2 knock-down (MSH2KD) on both CTG repeat dynamics and CpG methylation pattern in human embryonic stem cells (hESC) carrying the DM1 mutation. Repeat size in MSH2 wild-type (MSH2WT) and MSH2KD DM1 hESC was determined by PacBio sequencing and CpG methylation by bisulfite massive parallel sequencing. We found stabilization of the CTG repeat concurrent with a gradual loss of methylation upstream of the repeat in MSH2KD cells, while the repeat continued to expand and upstream methylation remained unchanged in MSH2WT control lines. Repeat instability was re-established and biased towards expansions upon MSH2 transgenic re-expression in MSH2KD lines while upstream methylation was not consistently re-established. We hypothesize that the hypermethylation at the mutant DM1 locus is promoted by the MMR machinery and sustained by a constant DNA repair response, establishing a potential mechanistic link between CTG repeat instability and upstream CpG methylation. Our work represents a first step towards understanding how epigenetic alterations and repair pathways connect and contribute to the DM1 pathology.

中文翻译：

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MSH2 敲低显示 1 型强直性营养不良人类胚胎干细胞中 DMPK 基因座的 CTG 重复稳定性和伴随的上游去甲基化

摘要

1 型强直性营养不良 (DM1) 是由DMPK基因中CTG 重复序列的扩增引起的，其中扩增大小和体细胞嵌合与疾病严重程度和发病年龄相关。虽然已知错配修复蛋白 MSH2 导致重复序列的不稳定性质，但其对其他疾病相关特征（例如重复序列上游的 CpG 甲基化）的作用尚不清楚。在这项研究中，我们研究了MSH2敲低 (MSH2KD) 对携带 DM1 突变的人类胚胎干细胞 (hESC) 中 CTG 重复动力学和 CpG 甲基化模式的影响。MSH2 野生型 (MSH2WT) 和MSH2KD 中的重复大小DM1 hESC 通过 PacBio 测序确定，CpG 甲基化通过亚硫酸氢盐大规模平行测序确定。我们发现 CTG 重复序列的稳定性与 MSH2KD 细胞重复序列上游甲基化的逐渐丧失同时发生，而重复序列继续扩大且上游甲基化在 MSH2WT 控制系中保持不变。重新建立重复不稳定性并倾向于在MSH2 上扩展MSH2KD 系中的转基因重新表达，而上游甲基化并未始终如一地重新建立。我们假设突变 DM1 基因座的高甲基化由 MMR 机制促进并由持续的 DNA 修复反应维持，在 CTG 重复不稳定性和上游 CpG 甲基化之间建立潜在的机制联系。我们的工作代表了了解表观遗传改变和修复途径如何连接并促成 DM1 病理的第一步。