Mitotic bookmarking by H3K27 acetylation is critical for rapid transcriptional, but not architectural, resetting of stem cell-related genes and enhancers upon G1 entry [PRO-seq]. Mitotic bookmarking by H3K27 acetylation is critical for rapid transcriptional, but not architectural, resetting of stem cell-related genes and enhancers upon G1 entry [PRO-seq]

The identity of dividing cells is challenged during mitosis, as transcription is halted and chromatin architecture drastically altered. How cell type-specific gene expression and genomic organization are faithfully reset upon G1 entry in daughter cells remains elusive. To address this issue, we characterized at a genome-wide scale the dynamic transcriptional and architectural resetting of mouse pluripotent stem cells (PSCs) upon mitotic exit. This revealed distinct patterns of transcriptional reactivation with rapid induction of stem cell genes and their enhancers, a more gradual recovery of metabolic and cell cycle genes, and a weak and transient activation of lineage-specific genes only during G1. Topological reorganization at different hierarchical levels also occurred in an asynchronous manner and showed an overall weak coordination with transcriptional reactivation. Chromatin interactions around active promoters and enhancers, and particularly super enhancers, reformed at a faster rate than CTCF/Cohesin-bound structural loops. Interestingly, although regions with mitotic retention of the active histone mark H3K27ac associated both with faster transcriptional and architectural resetting, depletion of this mark specifically during mitosis perturbed transcriptional reactivation of H3K27ac-bookmarked genes without affecting chromatin topology. Our study provides an integrative map of the topological and transcriptional changes that lead to the resetting of pluripotent stem cell identity during mitotic exit, and reveals distinct patterns and features that balance the dual requirements for self-renewal and differentiation. Overall design: PRO-seq of mouse iPSCs after p300 inhibition and recovery, both in asychronous and M-to-G1 cell cycle populations. DMSO treatment was used as a control, and three replicates per condition/time point were analyzed, except for mitotic time points which had only two replicates.

细胞分裂过程中的身份特性在有丝分裂（mitosis）阶段会受到扰动，此时转录活动被暂停，染色质（chromatin）结构也发生显著重塑。子代细胞进入G1期时，细胞类型特异性基因表达与基因组组织如何被精准重置，这一问题至今仍未被阐明。为解决这一问题，我们在全基因组范围内解析了小鼠多能干细胞（pluripotent stem cells, PSCs）在有丝分裂退出过程中的动态转录与结构重置过程。该研究揭示了独特的转录重激活模式：干细胞基因及其增强子（enhancer）被快速诱导，代谢基因与细胞周期基因的恢复过程更为平缓，而仅在G1期才会出现谱系特异性（lineage-specific）基因的弱且短暂的激活。不同层级的染色质拓扑结构重组以异步方式发生，且整体上与转录重激活的协同性较弱。活跃启动子（promoter）与增强子周围，尤其是超级增强子（super enhancers）周围的染色质相互作用，其重建速率快于被CTCF/黏连蛋白（Cohesin）结合的结构环。值得注意的是，尽管在有丝分裂中保留活跃组蛋白修饰（histone mark）H3K27ac的区域既与更快的转录与结构重置相关，但在有丝分裂期间特异性清除该修饰，会扰动被H3K27ac印记的基因的转录重激活，却不会影响染色质拓扑结构。本研究绘制了一幅整合性图谱，展示了有丝分裂退出过程中驱动多能干细胞身份重置的拓扑结构与转录变化，并揭示了兼顾自我更新（self-renewal）与分化（differentiation）双重需求的独特模式与特征。实验设计总览：对经过p300抑制与恢复处理的小鼠诱导多能干细胞（induced pluripotent stem cells, iPSCs）进行PRO测序（PRO-seq），样本涵盖非同步化细胞群体以及M期到G1期的细胞周期群体。以二甲基亚砜（dimethyl sulfoxide, DMSO）处理作为对照，除有丝分裂时间点仅设置2次生物学重复外，每个处理条件/时间点均设置3次生物学重复并进行分析。