Fig 2: PIAS4 is required to suppress multiple RIF1/53BP1 Nuclear Bodies. (A) Nuclear bodies containing RIF1 and 53BP1 were detected using specific antibodies. (B) U2OS cells were co-immunostained with RIF1 and Cyclin B1 antibodies. (C) U2OS cells were incubated with EdU for 60 minutes and immunostained with RIF1 antibody. (D) U2OS cells transfected with either control siRNA or PIAS4 siRNA were immunostained with RIF1 and 53BP1 antibodies. (E) Percentage of EdU positive cells containing different numbers of RIF1 nuclear bodies either in presence of in the absence of PIAS4. (F) Percentage of Edu positive cells containing different numbers of 53BP1 nuclear bodies either in presence of in the absence of PIAS4. Data represent at least two independent experiments; values represent mean ± SD.

Fig 3: Effect of RIF1 on origin activation in unperturbed S phase Specimen images of DNA fibers sequentially labeled with CldU and IdU. siControl‐ and siRIF1‐treated Flp‐In T‐REx 293 cells (with integrated GFP) were incubated for 4 h in the presence of 200 μM HU, with pulse‐labeling using CldU (visualized red) then IdU (visualized green) in the final 40 min.Distribution of origin distance in siCtrl (open bars) and siRIF1 (black bars) cell lines, treated as described in (A) but with no HU treatment. For statistical analysis of IOD data, Mann–Whitney–Wilcoxon test was performed using R (version 3).Distribution of origin distance in siCtrl (open bars) and siRIF1 (black bars) cell lines, treated as described in (A) including HU treatment. The area corresponding to IOD values shorter than 40 kb is marked by pink background.

Fig 4: Effect of RIF1 and its PP1 interaction on phosphorylation status of MCM proteinsPhosphorylation status of MCM4 and MCM2 was analyzed as in Fig 2B, in a biological duplicate experiment.

Fig 5: RIF1 associates with methylated H3K4 in vitro and in vivo(A) Chromatin immunoprecipitation profiles of murine RIF11 at H3K4me1 and H3K4me3 peak sites in mESCs from ENCODE. Data are from Foti et al. (2016).(B and C) Chromatin immunoprecipitation profiles of murine RIF1 and BLISS signals over H3K4me3-positive areas in mESCs that lie outside areas defined as TSS by ENCODE. Data are from Yan et al. (2017).(D) HeLa nuclear cell extracts were incubated with biotinylated histones and analyzed by immunoblotting.(E and F) In vitro transcribed/translated RIF1 was incubated with biotinylated histones and analyzed by immunoblotting.(G) HEK-293 cells were transfected with plasmids expressing the indicated RIF1 proteins, left for 48 h, and cell lysates incubated with purified biotinylated histones and analyzed by immunoblotting. Data in (D)–(G) represent =2 independent experiments.(H) (Upper) Upon DSB formation in G1, pre-existing H4K20me2 and H2AK15Ub recruit 53BP1 to sites of DSBs. H3K4me catalyzed by SETD1A and its co-factor BOD1L (1 and 2) stabilize the recruitment of RIF1 (3), allowing downstream cascades (not shown). This suppresses BRCA1-dependent resection and promotes NHEJ (4). (Lower) In the absence of H3K4me (5), RIF1 recruitment is destabilized (6), leading to inappropriate BRCA1-dependent resection of DSBs (7) and mis-repair.See also Figure S7.