Replication timing maintains the global epigenetic state in human cells

Abstract

5 6DNA is replicated in a defined temporal order termed the replication timing (RT) program. RT is 2 7 spatially segregated in the nucleus with early/late replication corresponding to Hi-C A/B 2 8 chromatin compartments, respectively. Early replication is also associated with active histone 2 9 modifications and transcriptional permissiveness. However, the mechanistic interplay between 3 0 RT, chromatin state, and genome compartmentalization is largely unknown. Here we report that 3 1RT is central to epigenome maintenance and compartmentalization in both human embryonic 3 2 stem cells (hESCs) and cancer cell line HCT116. Knockout (KO) of the conserved RT control 3 3 factor RIF1, rather than causing discrete RT switches as previously suspected, lead to 3 4 dramatically increased cell to cell heterogeneity of RT genome wide, despite RIF1's enrichment 3 5 in late replicating chromatin. RIF1 KO hESCs have a nearly random RT program, unlike all prior 3 6 RIF1 KO cells, including HCT116, which show localized alterations. Regions that retain RT, 3 7 which are prevalent in HCT116 but rare in hESCs, consist of large H3K9me3 domains revealing 3 8 two independent mechanisms of RT regulation that are used to different extents in different cell 3 9 types. RIF1 KO results in a striking genome wide downregulation of H3K27ac peaks and 4 0 enrichment of H3K9me3 at large domains that remain late replicating, while H3K27me3 and 4 1H3K4me3 are re-distributed genome wide in a cell type specific manner. These histone 4 2 modification changes coincided with global reorganization of genome compartments, 4 3 transcription changes and a genome wide strengthening of TAD structures. Inducible 4 4 degradation of RIF1 revealed that disruption of RT is upstream of genome compartmentalization 4 5 changes. Our findings demonstrate that disruption of RT leads to widespread epigenetic mis-4 6 regulation, supporting previously speculative models in which the timing of chromatin assembly 4 7 at the replication fork plays a key role in maintaining the global epigenetic state, which in turn 4 8 5 8 chromatin correspond to A-and B-compartments respectively as defined by high throughput 5 9chromatin conformation capture (Hi-C) (2). Despite these close correlations, the mechanistic link 6 0 between RT and the accurate maintenance of chromatin through cell cycles remains elusive. 1Prior work has shown that histones and their modifications are both recycled from parental 6 2 chromatin and added and modified de novo after passage of the replication fork with different 6 3 chromatin states showing differing dynamics of reassembly (3, 4). It has long been 6 4 hypothesized that RT influences chromatin maintenance. Indeed, microinjection of plasmids into 6 5 mammalian nuclei revealed that plasmids replicated in early S phase were decorated with 6 6 acetylated histones, while those replicated later in S phase were devoid of acetylated histones 6 7 (5). However, there is still no direct evidence implicating RT in epigenetic state maintenance, 6 8 largely due to t...

The RIF1-long splice variant promotes G1 phase 53BP1 nuclear bodies to protect against replication stress

“…Debatisse and Rosselli interpreted this suppression as due to earlier replication of fragile site DNA in lymphoblastoid cells in the absence of RIF1. The lack of any effect in our HCT116-based line may reflect cell line-specificity of RIF1 effects on replication timing ( Gnan et al, 2020 ; Klein et al, 2019 ).…”

An alignment free approach confirms semantic properties of species proteomes

“…Another concerted but only recently explained process is the conservation of replication timing order, which has been shown to orchestrate the global epigenetic state of individual cells. The principle behind this process is the continuation of previously established functional interpretation of the information stored in the genes (Klein et al, 2021), which follows our rationale. Beside random protein samples, we showed that even highly homologous groups of proteins categorized under different protein families' express more affinity towards proteome context than their family counterparts when being modeled semantically.…”

Chromatin accessibility of primary human cancers ties regional mutational processes with tissues of origin

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