Nuclear organisation and replication timing are coupled through RIF1–PP1 interaction

Three-dimensional genome organisation and replication timing are known to be correlated, however, it remains unknown whether nuclear architecture overall plays an instructive role in the replication-timing programme and, if so, how. Here we demonstrate that RIF1 is a molecular hub that co-regulates both processes. Both nuclear organisation and replication timing depend upon the interaction between RIF1 and PP1. However, whereas nuclear architecture requires the full complement of RIF1 and its interaction with PP1, replication timing is not sensitive to RIF1 dosage. The role of RIF1 in replication timing also extends beyond its interaction with PP1. Availing of this separation-of-function approach, we have therefore identified in RIF1 dual function the molecular bases of the co-dependency of the replication-timing programme and nuclear architecture.


Statistics
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Data
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Life sciences study design
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Sample size
The sample size was chosen on the basis of the published literature. Data from 3 different individuals per genotype provides enough information to evaluate the spread of the data and the inter-sample variability.
Data exclusions In one FACS for Fig. 1e, one wild type and one Rif1 DPP1 were excluded because not enough cells had been collected.

Replication
We have included the replicates in the experiments shown.
Randomization Each experiemental group included all the genotypes.

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April 2020 Flow Cytometry Plots Confirm that: The axis labels state the marker and fluorochrome used (e.g. CD4-FITC).
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Methodology Sample preparation
For quantification of the expression of the different RIF1-HA alleles described in the paper by intra-cellular FACS, the samples were prepared as follows: After four days of OHT treatment, cells were collected and counted. 3?106 cells were fixed in 400 μl of DPBS/2% Paraformaldehyde (Sigma P-6148) for 10 minutes at room temperature shaking. Paraformaldehyde was then diluted to 0.2% and next cells were washed in cold DPBS. After 2 minutes permeabilisation in 200 μl PBS-Triton X-100 0.1%, cells were incubated 5 minutes in saponin solution (COMPONENT E from kit C10424, Thermo Fisher Scientific) at room temperature and anti-HA antibody (Covance monoclonal HA.11 clone 16B12 #MMS-101R, RRID:AB_291262) was added at 1:500. After 1 hour at room temperature rotating, cells were washed twice in DPBS/2% FBS, resuspended in 200 μl of saponin solution with goat anti-mouse Alexa Fluor 647 1:1000 (Thermo Fisher Scientific A-21235, RRID:AB_2535804) and incubated for 1 hour rotating in the dark. After washing twice samples were resuspended in 400 μl of saponin solution with DAPI 2.5 g/ ml (Thermo Fisher Scientific D1306) and analyzed on an LSR II FACS (BD). Data were processed using R version 3.5.1. The confidence intervals (CI) of the median shown in Fig. 1B were calculated by bootstrap.
For the FACS analysis of chromatin association of RIF1-HA encoded by the different alleles described in this paper, the samples were processed as above, except, fixation was preceded by 3 minutes incubation in CSK buffer (25 mM HEPES pH 7.4, 50 mM NaCl, 1 mM EDTA, 3 mM MgCl2, 300 mM sucrose, 0.5% Triton X-100 and complete protease inhibitor cocktail tablet). Pre-extracted cells were subsequently fixed in 3%PFA/sucrose for 30 minutes at room temperature shaking. The samples for the analysis of cell cycle distribution were prepared as follows:After four days of OHT treatment, cells were pulsed for 30 minutes with 10 μM EdU (Invitrogen A10044). Cells were then washed with cold DPBS (Thermo Fisher Scientific 14190094), collected, counted and fixed in 75 %. EtOH Samples were kept at -20 oC for at least overnight. 7.5?105 cells were then processed for click-chemistry detection of EdU. After washing in cold DPBS, cells were permeabilised in DPBS/1% FBS/0.01 % Triton X-100 (Sigma 93426-250ML) for 10 minutes on ice. After washing twice, cells were incubated in 900 μl of DPBS with 10 mM Na-Ascorbate (Sigma A7631-25G), 1 μM Alexa Fluor 647 Azide (Thermo Fisher Scientific A10277) and CuSO4 0.1 M (Sigma C1297) for 30 minutes at room temperature in the dark, rotating. Cells were washed in DPBS/1% FBS/0.5% Tween 20 (Sigma P9416-100ML) for 10 minutes and then twice in cold DPBS/1% FBS. After 1 hour incubation in 300 μl of DPBS/1%FBS /DAPI 2.5 g/ml (Thermo Fisher Scientific D1306), the samples were analyzed using an LSR II FACS (BD). The data acquired were analysed using Flowjo software and plotted in R 3.5.1. To calculate the percentages of cells in early, mid and late S-phase in Supp. Fig. 2C, we have defined the S-phase substages based on the intensities of the PI/EdU signals in the wild type, drawn the gates and applied them to all the samples. Cell population abundance When sorting has been performed, the single fraction were re-run individually to check for purity.

Gating strategy
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