Cell-type specific DNA methylome signatures reveal epigenetic mechanisms for neuronal diversity and neurodevelopmental disorder

We report the application of an affinity purification approach (INTACT) to isolate tagged nuclei for GABAergic (GABA), Glutamatergic (Glu), and Purkinje neurons in mice. We performed whole-genome bisulfite sequencing (WGBS) to explore the cell type-specific DNA methylation signatures for the three neuronal types, together with matched transcriptomics. We found a substantial number of differentially methylated regions (DMRs) between cell types, characterized cell-type specific DMRs, and estimated the effects of DNA methylation on gene expression. Finally, we revealed that DNA methylation altered in a cell-type specific manner in a mouse model of Rett syndrome, a neurodevelopmental disorder caused by Mecp2 loss of function. The presented data emphasize the important role of DNA methylation-mediated epigenetic regulation in neuronal diversity and disease. Overall design: WGBS and RNA-Seq for mouse GABAergic, Glutamatergic and Purkinje neurons, as well as for GABAergic and Glutamatergic neurons with Mecp2 KO. Only male mice were included in this project. For the nuclei isolated from one mouse, half of them were used for DNA extraction, and another half were used for RNA-Seq extraction. Therefore, each sample has DNA and matched RNA for WGBS and RNA-Seq, respectively.

本研究报道了利用亲和纯化技术（INTACT）分离小鼠γ-氨基丁酸能（GABA）、谷氨酸能（Glu）及浦肯野神经元的标记细胞核的实验方法。我们通过全基因组亚硫酸氢盐测序（WGBS），解析了这三类神经元的细胞类型特异性DNA甲基化特征，并同步开展了匹配性转录组学分析。研究鉴定出大量细胞类型间的差异甲基化区域（DMRs），对细胞类型特异性DMRs进行了系统表征，并评估了DNA甲基化对基因表达的调控作用。此外，我们还发现，在由Mecp2功能丧失引发的神经发育疾病——雷特综合征的小鼠模型中，DNA甲基化呈现细胞类型特异性的异常改变。本研究数据凸显了DNA甲基化介导的表观遗传调控在神经元多样性形成与疾病发生发展中的关键作用。

实验设计概述：本研究对小鼠γ-氨基丁酸能、谷氨酸能与浦肯野神经元，以及Mecp2敲除（KO）的γ-氨基丁酸能和谷氨酸能神经元开展了WGBS与RNA测序（RNA-Seq）。本项目仅纳入雄性小鼠。从单只小鼠体内分离得到的细胞核被平均分为两份：一半用于DNA提取以进行WGBS，另一半用于RNA提取以开展RNA-Seq。因此，每个样本均配套有用于WGBS的DNA样品与用于RNA-Seq的RNA样品。