Disabling leading and lagging strand histone transmission results in parental histones loss and reduced cell plasticity and viability [SCAR-seq]. Disabling leading and lagging strand histone transmission results in parental histones loss and reduced cell plasticity and viability [SCAR-seq]

In the process of DNA replication, the first steps in restoring the chromatin landscape involve parental histone recycling and new histone deposition. Disrupting histone recycling to either the leading or lagging strand induces asymmetric histone inheritance, impacting epigenome maintenance and cellular identity. However, the order and kinetics of these effects remain elusive. Here, we employ inducible mutants to dissect the early and late consequences of impaired histone recycling. Simultaneous disruption of both leading (POLE4) and lagging strand (MCM2-2A) recycling pathways impairs transmission of parental histones to newly synthesized DNA, with release of some parental histones to the soluble pool. Subsequently, H3K27me3 accumulates aberrantly during chromatin restoration in a manner preceding gene expression changes. Loss of histone inheritance and the ensuing chromatin restoration defects alter gene expression in embryonic stem cells, challenges differentiation programs and cell viability. Our findings demonstrate the importance of efficient transmission of histone-based information during DNA replication for maintaining chromatin landscapes, differentiation potential, and cellular viability. Overall design: SCAR-seq (Sister Chromatids After Replication) measuring histone partition in nascent chromatin of H4K20me2, H4K20me0 in POLE4-dTAG (1 clone) and POLE4-dTAG/MCM2-2A (2 clones) mouse embryonic stem cells (DMSO and dTAG treatment, 2 replicates for histone partition scores, includes stranded input controls).

DNA复制过程中，恢复染色质景观的首个步骤涉及亲本组蛋白回收与新组蛋白沉积。将组蛋白回收干扰至前导链或滞后链会诱导不对称组蛋白遗传，进而影响表观基因组维持与细胞身份。然而，此类效应的发生顺序与动力学特征仍尚不明确。本研究利用诱导型突变体，解析组蛋白回收受损后的早期与晚期效应。同时干扰前导链（POLE4）与滞后链（MCM2-2A）的组蛋白回收通路，会损害亲本组蛋白向新合成DNA的传递，并使部分亲本组蛋白释放至可溶性池。随后，在染色质恢复过程中，H3K27me3会发生异常积累，且该过程先于基因表达变化出现。组蛋白遗传丧失及随之而来的染色质恢复缺陷，会改变胚胎干细胞的基因表达模式，干扰分化程序并影响细胞存活能力。本研究结果证实，DNA复制过程中高效传递基于组蛋白的遗传信息，对维持染色质景观、分化潜能与细胞存活能力至关重要。整体实验设计：采用SCAR-seq（Sister Chromatids After Replication，复制后姐妹染色单体测序）技术，检测POLE4-dTAG（1株细胞克隆）与POLE4-dTAG/MCM2-2A（2株细胞克隆）小鼠胚胎干细胞新生染色质中H4K20me2、H4K20me0的组蛋白分配情况；实验设置二甲基亚砜（DMSO）与dTAG处理组，组蛋白分配评分设置2次生物学重复，并包含链特异性输入对照。