Genome-wide maps of chromatin state in pluripotent and lineage-committed cells.

We applied sequential ChIP-bisulfite-sequencing for H3K4me1 and H3K27ac in mouse embryonic stem cells (ESCs). By obtaining over ~4*10^9 bases of sequence from chromatin immunoprecipitated DNA which was bisulfite treated, we identified enhancers that were differentially methylated between these two marks. We found a global gain of CpG methylation, primarily in H3K4me1-marked nucleosomes during mouse ESC differentiation. This gain occurred largely in enhancer regions that regulate genes critical for differentiation. We showed that higher levels of DNA methylation in H3K4me1 comparing to H3K27ac indicates cellular heterogeneity of enhancer states. We then used single-cell RNA-seq profiles to show that this heterogeneity correlates with gene expression during ESC differentiation. Furthermore, we showed that heterogeneity of enhancer methylation correlates with transcription start site methylation. Our results provide insights into enhancer-based functional variation in complex biological systems. Overall design: Examination of H3K4me1 and H3K27ac DNA that was sequentially bisulfite treated in embryonic stem cells and differentiated cells

本研究针对小鼠胚胎干细胞（embryonic stem cells, ESCs）中的H3K4me1与H3K27ac组蛋白修饰标记，采用了连续染色质免疫沉淀-亚硫酸氢盐测序（sequential ChIP-bisulfite-sequencing）技术。通过对经亚硫酸氢盐处理的染色质免疫沉淀DNA进行测序，获得了约4×10^9个碱基的测序序列，最终鉴定出两类标记间存在差异甲基化的增强子。
研究发现，在小鼠胚胎干细胞分化过程中，整体CpG甲基化水平呈现全局性升高，且该升高主要发生在带有H3K4me1标记的核小体中。该甲基化升高主要集中于调控分化关键基因的增强子区域内。
本研究证实，相较于H3K27ac标记，H3K4me1标记区域的DNA甲基化水平更高，这一现象反映了增强子状态的细胞异质性。随后，我们借助单细胞RNA测序（single-cell RNA-seq）谱数据分析，证实该异质性与小鼠胚胎干细胞分化过程中的基因表达水平相关。此外，本研究还发现增强子甲基化的异质性与转录起始位点（transcription start site）的甲基化水平存在关联。
本研究结果为解析复杂生物系统中基于增强子的功能变异提供了全新视角。
实验整体设计：对胚胎干细胞及分化细胞中带有H3K4me1与H3K27ac标记的DNA进行连续亚硫酸氢盐处理后开展测序分析。