The temporal order of DNA replication [replication timing (RT)] is correlated with chromatin modifications and three-dimensional genome architecture; however, causal links have not been established, largely because of an inability to manipulate the global RT program. We show that loss of RIF1 causes near-complete elimination of the RT program by increasing heterogeneity between individual cells. RT changes are coupled with widespread alterations in chromatin modifications and genome compartmentalization. Conditional depletion of RIF1 causes replication-dependent disruption of histone modifications and alterations in genome architecture. These effects were magnified with successive cycles of altered RT. These results support models in which the timing of chromatin replication and thus assembly plays a key role in maintaining the global epigenetic state.

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...

Human cells lacking RIF1 are highly sensitive to replication inhibitors, but the reasons for this sensitivity have been enigmatic. Here we show that RIF1 must be present both during replication stress and in the ensuing recovery period to promote cell survival. Of two isoforms produced by alternative splicing, we find that RIF1-Long alone can protect cells against replication inhibition, but RIF1-Short is incapable of mediating protection. Consistent with this isoform-specific role, RIF1-Long is required to promote the formation of the 53BP1 nuclear bodies that protect unrepaired damage sites in the G1 phase following replication stress. Overall, our observations show that RIF1 is needed at several cell cycle stages after replication insult, with the RIF1-Long isoform playing a specific role during the ensuing G1 phase in damage site protection.

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