GSE160563
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.