Functional characterization of DNA methylation in the oligodendrocyte lineage [RNASeq_Dnmt1cKO_OPC]

Oligodendrocytes derive from progenitors (OPC) through the interplay of epigenetic and transcriptional events. By integrating high-resolution methylomics, RNA-sequencing and multiple transgenic lines, this study defines the role of DNMT1 in developmental myelination. We detected hypermethylation of genes related to cell cycle and neurogenesis during differentiation of OPC, and yet, genetic ablation of Dnmt1 resulted in inefficient OPC expansion and severe hypomyelination associated with ataxia and tremors in mice. This phenotype was not caused by lineage switch or massive apoptosis, but was characterized by a profound defect of differentiation, associated with massive changes in exon-skipping and intron-retention splicing events, and by the activation of an endoplasmic reticulum stress response. Therefore, loss of Dnmt1 in OPC is not sufficient to induce a lineage switch, but acts as an important determinant of the coordination between RNA splicing and protein synthesis, necessary for myelin formation. mRNA profiles of P5 mouse Olig1+/+;Dnmt1flox/flox;Pdgfra-GFP and Olig1cre/+;Dnmt1flox/flox;Pdgfra-GFP sorted OPC were generated by RNA-sequencing, in triplicate, using Illumina HiSeq 2500.少突胶质细胞（Oligodendrocytes）由少突胶质祖细胞（OPC）通过表观遗传与转录事件的协同互作分化而来。本研究整合高分辨率甲基化组学、RNA测序（RNA-sequencing）及多种转基因品系，明确了DNMT1在发育性髓鞘形成过程中的功能。我们在少突胶质祖细胞分化阶段检测到与细胞周期及神经发生相关基因的高甲基化修饰；然而，敲除Dnmt1会导致少突胶质祖细胞增殖效率低下，并使小鼠出现与共济失调、震颤相关的严重髓鞘发育不全。该表型并非由谱系转换或大规模细胞凋亡引发，其核心特征为分化过程存在严重缺陷，伴随外显子跳跃与内含子保留等大量可变剪接事件的异常改变，同时激活内质网应激应答通路。因此，在少突胶质祖细胞中敲除Dnmt1并不会引发谱系转换，而是作为关键调控因子协调RNA剪接与蛋白质合成的动态平衡，而这一平衡是髓鞘形成所必需的。本研究通过Illumina HiSeq 2500平台进行三次生物学重复的RNA测序，获取了P5小鼠Olig1+/+;Dnmt1flox/flox;Pdgfra-GFP与Olig1cre/+;Dnmt1flox/flox;Pdgfra-GFP分选的少突胶质祖细胞的mRNA表达谱。