Dramatic Increase in Cell-subset Specific Methylation Accompanies Mammalian Brain Development [hairpin BS-Seq]

Purpose: This study aims to identify cell-subset specific methylated (CSM) loci in mammalian brain methylomes and to uncover epigenetic regulatory networks associated with brain development. Methods: To generate methylation data from DNA double strands simultaneously, genome-scale hairpin bisulfite sequencing technique was used for the assessment of the levels of asymmetric DNA methylation in the human frontal cortical gray and white matters Results and conclusion: CSM loci increase dramatically during early stages of brain development. They are overrepresented in cis-regulatory elements such as active enhancers and promoters, and are enriched for GWAS variants associated with neurological-disorder-related diseases/traits. Co-methylated CSM clustering analysis indicated that the CSMs hypo-methylated in neurons are enriched for EGR1 binding sites.Our results show the functional relevance of CSM loci identified and provide insights into epigenetic complexity of mammalian brains. Overall design: We first explored asymmetric DNA methylation in human fetal and adolescent brain tissues using genome-scale hairpin bisulfite sequencing technique. Based on the finding that brain methylomes are with exceeding high methylation fidelity, we developed an analytical procedure to identify CSM loci and applied it to human and mouse brain methylomes throughout the life span. We further explored the functional relevance of CSM loci via integrative “omics” analysis with ChIP-Seq data for histone modifications, transcription factor (TF) bindings and GWAS disease/trait associated variants.

研究目的：本研究旨在鉴定哺乳动物脑甲基化组中细胞亚群特异性甲基化（cell-subset specific methylated, CSM）位点，并揭示与脑发育相关的表观遗传调控网络。
研究方法：为同时获取DNA双链的甲基化数据，本研究采用基因组规模发夹亚硫酸氢盐测序技术，对人类额叶皮层灰质与白质中的不对称DNA甲基化水平进行评估。
结果与结论：脑发育早期阶段，细胞亚群特异性甲基化位点的数量会显著增加。这类位点在顺式调控元件（如活性增强子与启动子）中占比偏高，且富集有与神经疾病/性状相关的全基因组关联研究（Genome-Wide Association Study, GWAS）变异。共甲基化CSM聚类分析显示，神经元中低甲基化的CSM位点富集于早期生长应答因子1（Early Growth Response 1, EGR1）结合位点。本研究结果证实了所鉴定CSM位点的功能相关性，并为解析哺乳动物大脑的表观遗传复杂性提供了新的认知视角。
整体实验设计：本研究首先利用基因组规模发夹亚硫酸氢盐测序技术，对人类胎儿与青少年脑组织中的不对称DNA甲基化模式展开探究。基于脑甲基化组具有极高甲基化保真度这一发现，我们开发了一套用于鉴定CSM位点的分析流程，并将其应用于全生命周期内的人类与小鼠脑甲基化组数据。此外，我们还通过整合‘组学’分析，结合组蛋白修饰、转录因子（Transcription Factor, TF）结合的染色质免疫沉淀测序（Chromatin Immunoprecipitation Sequencing, ChIP-Seq）数据以及GWAS疾病/性状关联变异，进一步探究了CSM位点的功能相关性。