RIF1 is necessary to maintain the global epigenetic state in human cells.. RIF1 is necessary to maintain the global epigenetic state in human cells.

DNA is replicated in a defined temporal order termed the replication timing (RT) program, which is correlated with genome compartmentalization with early replicating chromatin located mainly in the A compartment and late replicating chromatin in the B compartment (Dixon et al 2012, Moindrot et al 2012, Ryba et al 2010, Yaffe et al 2010). Similarly, active histone modifications and transcriptional permissiveness are associated with early replication while a repressive chromatin state is associated with late replication (Hiratani et al 2009, Riveria-Mulia et al 2015, Lubelsky et al 2014). However, the mechanistic interplay between RT, chromatin state, and genome compartmentalization is largely unknown. Here we report RT is upstream of epigenome formation and compartmentalisation in human embryonic stem cells (hESCs) and the cancer cell line HCT116. Knockout (KO) of the conserved RT control factor RIF1, rather than causing discrete RT switches, leads to dramatically increased cell to cell heterogeneity of RT genome wide, despite RIF1’s enrichment in late replicating chromatin. RIF1 KO hESCs have a nearly random RT program, unlike any other cell type to date, yet cells self-renew without signs of differentiation. Those regions that do retain RT consist of large H3K9me3 domains, which are prevalent in HCT116 but rare in hESCs, revealing two independent mechanisms of RT regulation that are used to different extents in different cell types. RIF1 KO cells also showed genome wide downregulation of H3K27ac peaks and enrichment of H3K9me3 at large domains that remained late replicating, while H3K27me3 and H3K4me3 were also re-distributed genome wide but in a cell type specific manner. Histone modification changes correlated with gene expression changes and global reorganisation of genome compartments. Some RT/compartment shifts were associated with specific TAD boundary shifts, but overall we observed a genome wide strengthening of TAD structures. Our findings support a model in which RT controlled by RIF1 plays a key role in establishing epigenetic state and genome architecture in human cells. Overall design: Examination of E/L repli-seq, high resolution repli-seq, ChIP-seq of 4 different histone modifications, Hi-C, RNA-seq, and single cell RNA-seq of two cell lines both WT and RIF1 KO with two replicates each. Single cell repli-seq of WT and RIF1 KO for one other cell line.

DNA以特定的时间顺序进行复制，这一过程被称为复制时序（replication timing, RT）程序，其与基因组区室化密切相关：早期复制的染色质主要定位于A区室，而晚期复制的染色质则定位于B区室（Dixon et al 2012, Moindrot et al 2012, Ryba et al 2010, Yaffe et al 2010）。类似地，活性组蛋白修饰与转录许可性状态均与早期复制相关，而染色质抑制性状态则与晚期复制相关（Hiratani et al 2009, Riveria-Mulia et al 2015, Lubelsky et al 2014）。然而，复制时序、染色质状态与基因组区室化之间的机制互作目前仍知之甚少。本研究发现，在人类胚胎干细胞（human embryonic stem cells, hESCs）与癌细胞系HCT116中，复制时序先于表观基因组形成与区室化发生。保守的复制时序调控因子RIF1的敲除（knockout, KO）并不会引发离散的复制时序切换，反而会显著增加全基因组范围内细胞间的复制时序异质性，尽管RIF1富集于晚期复制的染色质区域。与目前已报道的所有细胞类型不同，RIF1敲除的人类胚胎干细胞的复制时序程序近乎随机，但细胞仍可自我更新，且未出现分化迹象。那些仍保留复制时序的区域多为大型H3K9me3结构域——这类结构域在HCT116细胞中较为普遍，但在人类胚胎干细胞中却极为罕见，这揭示了两种独立的复制时序调控机制，且不同细胞类型对这两种机制的使用程度存在差异。RIF1敲除细胞还表现出全基因组范围内H3K27ac峰的下调，以及在仍保持晚期复制的大型结构域中H3K9me3的富集；同时H3K27me3与H3K4me3也在全基因组范围内发生了重分布，但呈现细胞类型特异性的模式。组蛋白修饰的变化与基因表达变化及基因组区室的整体重构相关。部分复制时序/区室的改变与特定TAD（Topologically Associating Domain）边界偏移相关，但总体而言，我们观察到全基因组范围内TAD结构的增强。本研究的发现支持如下模型：由RIF1调控的复制时序在建立人类细胞的表观遗传状态与基因组架构过程中发挥关键作用。整体实验设计：对两种细胞系（野生型与RIF1敲除型，各设两个生物学重复）进行E/L复制测序（repli-seq）、高分辨率复制测序（high resolution repli-seq）、4种不同组蛋白修饰的染色质免疫共沉淀测序（ChIP-seq）、Hi-C、RNA测序（RNA-seq）以及单细胞RNA测序。此外，还对另一种细胞系的野生型与RIF1敲除型样本进行了单细胞复制测序。