Substained DNA demethylation and discordant chromatin accessibility dynamics define enhancer regulation during neural progenitor cell differentiation [6base]. Substained DNA demethylation and discordant chromatin accessibility dynamics define enhancer regulation during neural progenitor cell differentiation [6base]

Epigenetic mechanisms govern the transcriptional activity of lineage-specifying enhancers; but recent work challenges the dogma that both chromatin accessibility and DNA hypomethylation are prerequisites for transcription, highlighting a need to understand their coordinated dynamics. We established a highly-resolved timeline of DNA demethylation, chromatin accessibility, and transcription factor (TF) occupancy during early human cell differentiation. We show >30,000 lineage-specifying enhancers undergo rapid and transient accessibility changes associated with distinct periods of TF expression. By contrast, enhancer DNA methylation changes are prolonged, unidirectional and delayed relative to chromatin dynamics, creating discordant epigenetic states. Using 6-base sequencing to detect methyl-intermediate, 5hmC, revealed that, for a subset of enhancers, TET-mediated, active demethylation begins prior to, and is maintained independently of, TF binding. In fact, machine learning models trained on 5-hydroxymethylation can predict of future chromatin states. Complete demethylation persists long after TF binding and accessibility have dissipated, suggesting that long-lasting hypomethylation of certain enhancers is a historical record of previous activity. Overall design: Timecourse NPC differentiation with collection of genomic DNA at three timepoints over 8 days to be used for evoC 6-base-seq

表观遗传机制调控谱系特异性增强子的转录活性；但近期研究对“染色质开放度与DNA低甲基化均为转录发生的必要条件”这一传统教条提出了挑战，凸显出解析二者协同动态变化的研究需求。本研究构建了人类早期细胞分化过程中DNA去甲基化、染色质开放度以及转录因子（Transcription Factor, TF）结合占据情况的高分辨率时间图谱。研究发现，超过30000个谱系特异性增强子会发生快速且瞬时的染色质开放度变化，且该变化与转录因子表达的不同阶段密切相关。与之形成对比的是，增强子的DNA甲基化变化相较于染色质动态变化更为持久、单向且发生滞后，从而形成不一致的表观遗传状态。通过6碱基测序技术检测甲基化中间产物5-羟甲基胞嘧啶（5-hydroxymethylcytosine, 5hmC），本研究发现，对于部分增强子而言，TET介导的主动去甲基化过程先于转录因子结合启动，且其维持过程不依赖于转录因子结合。事实上，基于5-羟甲基化修饰训练得到的机器学习模型，能够预测未来的染色质状态。完全去甲基化状态会在转录因子结合与染色质开放度消散后仍持续较长时间，这表明部分增强子的长期低甲基化状态是其过往活性的历史记录。实验整体设计：开展历时8天的神经前体细胞（Neural Progenitor Cell, NPC）分化时间进程实验，在8天内的3个时间点收集基因组DNA，用于evoC 6-base-seq。