Figure 1: Rif1 Coordinates the RT program of hESCs.

(A) Parental (WT) and RIF1 knockout (KO) high resolution Repli-seq (as described in Zhao, 2020) of a 20Mb segment of chromosome 1 in the human ESC line WA09 (H9) (top) and in human colorectal carcinoma, HCT116 cells (bottom). (from Klein et al, 2021). Nascent DNA that was replicated in each of 16 temporal windows of S phase was sequenced and the density of reads displayed as a heat map (Y-axis rows). Most cells in a WT population replicate any given genomic region within a defined temporal window of S phase. In RIF1 KO cells, either the entire genome (H9) or large segments of the genome (HCT116) are replicated stochastically. HCT116 differs from H9 in having late replicating regions that are unaffected by RIF1 KO. (B) Model of Rif1 dependent RT control. During early S-phase in WT cells Rif1 shields late replicating chromatin domains from replication initiation factors. This directs the replication initiation factors to early replicating chromatin domains. During late S-phase, Rif1 is removed from late replicating domains, allowing their access to initiation factors. KO of Rif1 allows late replicating domains to compete with early domains for limiting replication initiation factors, resulting in an increase in the heterogeneity of replication timing.