Fig. 4.

Chromatin-mediated suppression of the MCM2-7 complex promotes genome integrity. a U2OS cells synchronized by double thymidine block were transfected with Control and SET8 siRNA. Cells were released into BrdU-containing medium from G1/S boundary and were mock or sucrose treated at 12 h post G1/S release. Cells were then fixed at 15 h post G1/S release and were immunostained with DAPI and γH2A.X antibody (Bar, 10 µm). b Scatter plot showing the quantification of BrdU intensity (a). Mean ± SD is indicated by red lines. n > 230, ****p < 0.0001 (ANOVA). c U2OS cells were synchronized, siRNA transfected, treated with sucrose, and fixed as in a. Samples were immunostained with an RPA2 antibody after pre-extraction. Scatter plot showing the quantification of RPA2 intensity. Mean ± SD is indicated by red lines. n > 150, ****p < 0.0001 (ANOVA). d U2OS cells were transfected with siMCM7 and synchronized with double thymidine block. SET8 was depleted 6 h before G1/S release. Cells were fixed at 15 h post release and processed for immunofluorescence staining with DAPI and γH2A.X antibody (Bar, 30 µm). e Scatter plot showing the quantification of γH2A.X intensity from cells in d. Mean ± SD is indicated by red lines. n > 50, ****p < 0.0001 (ANOVA). f U2OS cells were synchronized and transfected as in a. DDKi inhibitors (PHA-767491 and XL413) were added at 11 h post release from G1/S boundary. Cells were then collected at 15 h post release and processed for FACS staining with PI and γH2A.X antibody. Bars representing the percentage of γH2A.X-positive cells in the indicated samples (average ± SD of three independent experiments). ****p < 0.0001 (unpaired t test). n > 20,000 in each experiment. g Illustrative plot details the dynamics of chromatin compaction/decompaction over different phases of cell cycle. Loss of SET8 and H4K20me leads to a notable reduction in chromatin compaction status in cells exiting mitosis. h Illustrative plot details the dynamics of loading of replication licensing factors. In late mitosis, the licensing starts with loading of ORC complex that promotes loading of MCM2-7 complex throughout G1 phase. Loss of SET8 and H4K20me favors excessive loading of ORC and MCM2-7 complexes in daughter cells