Nuclear Architecture Organized by Rif1 Underpins the Replication-Timing Program

Abstract

Summary DNA replication is temporally and spatially organized in all eukaryotes, yet the molecular control and biological function of the replication-timing program are poorly understood. A role for three-dimensional chromatin organization has been proposed. Rif1 is required for normal genome-wide regulation of replication timing, but its molecular function is poorly understood. Here we show that in mouse embryonic stem cells Rif1 coats late replicating domains and, together with Lamin B1 identifies the majority of the late replicating genome. Rif1 is an essential determinant of replication timing of non-Lamin B1-bound late domains. We further demonstrate that Rif1 defines and restricts the interactions between replication-timing domains during G1, thereby revealing a novel function of Rif1 as organizer of nuclear architecture. Loss of Rif1 affects both number and replication-timing specificity of the interactions between replication-timing domains. In addition, during S-phase Rif1 ensures temporal coordination of replication of interacting domains. In summary our study identifies Rif1 as the first molecular link between nuclear architecture organization and replication-timing establishment in mammals.

Telomere-binding factors in the regulation of DNA replication

“…Genome-wide analyses of Rif1 in mouse embryonic stem (ES) cells showed that Rif1 binds to large domains which coincide with the late-replicating domain and lamin B1-assoicated domain (Foti et al, 2016). This is consistent with the result from fission yeast, supporting the notion that chromatin-bound Rif1 suppresses origin firing.…”

“…In the absence of Rif1, chromatin loops are lost and chromatin is decondensed and becomes more dynamic (Masai et al, unpublished data). The 4C-Seq assays indicated that chromatin interactions between different timing domains increase in the absence of Rif1 (Foti et al, 2016), reflecting a more mobile nature of the chromosomes. Red and blue lines indicate, respectively, mid-S/ late-and early-replicating chromosome segments.…”

“…Chromosome conformation capture sequencing (4C-Seq) analyses of Rif1 knockout ES cells provided direct evidence for Rif1's role in chromatin organization (Foti et al, 2016). It was shown that Rif1 knockout results in increased interaction among replication timing domains, suggesting that Rif1 restricts the interactions between different replication timing domains.…”

Reversal of DDK-Mediated MCM Phosphorylation by Rif1-PP1 Regulates Replication Initiation and Replisome Stability Independently of ATR/Chk1

“…Our data are consistent with the idea that loss of the phosphatase causes premature Mcm4 hyper-phosphorylation, thus causing promiscuous activation of replication origins and disruption of the timing program (Figure 7D, top). This function of Rif1 can occur concomitantly with its proposed role in organizing 3D chromatin structure, which was recently shown to affect replication timing and 3D nuclear organization in mouse cells (Foti et al., 2016). This altered 3D chromatin structure may cause improper regulation of MCM phosphorylation.…”

DNA replication origins—where do we begin?

“…Depletion of Rif1 results in a consolidation of early-and late-replicating domains to be instead replicated in mid-S phase (or randomly throughout S phase) (Cornacchia et al 2012). Rif1 localizes to late-replicating domains, many of which are concordant with LADs (Foti et al 2016). Late-replicating regions of the genome marked only by Rif1 are most susceptible to advancing their time of replication in the absence of Rif1 function.…”