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. 2018 Sep 12;9(1):3704.
doi: 10.1038/s41467-018-06066-8.

Histone H4K20 Methylation Mediated Chromatin Compaction Threshold Ensures Genome Integrity by Limiting DNA Replication Licensing

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Free PMC article

Histone H4K20 Methylation Mediated Chromatin Compaction Threshold Ensures Genome Integrity by Limiting DNA Replication Licensing

Muhammad Shoaib et al. Nat Commun. .
Free PMC article

Abstract

The decompaction and re-establishment of chromatin organization immediately after mitosis is essential for genome regulation. Mechanisms underlying chromatin structure control in daughter cells are not fully understood. Here we show that a chromatin compaction threshold in cells exiting mitosis ensures genome integrity by limiting replication licensing in G1 phase. Upon mitotic exit, chromatin relaxation is controlled by SET8-dependent methylation of histone H4 on lysine 20. In the absence of either SET8 or H4K20 residue, substantial genome-wide chromatin decompaction occurs allowing excessive loading of the origin recognition complex (ORC) in the daughter cells. ORC overloading stimulates aberrant recruitment of the MCM2-7 complex that promotes single-stranded DNA formation and DNA damage. Restoring chromatin compaction restrains excess replication licensing and loss of genome integrity. Our findings identify a cell cycle-specific mechanism whereby fine-tuned chromatin relaxation suppresses excessive detrimental replication licensing and maintains genome integrity at the cellular transition from mitosis to G1 phase.

Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
SET8 and H4K20 methylation regulate ground-state chromatin compaction in cells exiting mitosis. a U2OS cells were synchronized with double thymidine block. Control and SET8 siRNAs transfected 6 h before G1/S release. Cells were then blocked in mitosis with nocodazole for 4 h (T0) and released into G1 phase for 5 h (T5). MNase digestion was performed on mitotic arrested and G1 phase cells. Thymidine containing methyl-14C was added throughout the experiment. b Cells from a were fixed and stained with phospho-Histone H3S10 antibody and propidium iodide (PI) followed by flow cytometric analysis. c Immunoblots of total cell lysates prepared from the samples in a probed with the indicated antibodies. Asterisk (*) represents a non-specific band. d Graph showing MNase digestion profile. Levels of methyl-14C in the supernatant indicates the degree of chromatin decompaction over time when incubated with MNase. e U2OS cells stably expressing H2B-GFP alone (U2OSH2B-GFP) or with mCherry-tagged histone H2B (U2OSH2B-2FPs) were synchronized with single thymidine block. Cells were treated with either control or SET8 siRNA during the block. FRET measurements were taken before (T0) and 24 h after release (Bar, 10 µm). f Quantification of the FLIM-FRET chromatin compaction assay. Inside box plots, the thick line represents median, the boxes correspond to the mean FRET values upper or lower of the median, with whiskers extending to the highest and lowest value. n > 30 nuclei, ***p < 0.001, **p < 0.01 (ANOVA), ns not significant. g U2OS cells were synchronized and siRNA transfected as in a. Cells were fixed at 15 h post release for transmission electron microscope (TEM) visualization. h Quantification of the average pixel intensity ± SD (n > 15) of nuclei in g. **p < 0.01 (unpaired t test). i U2OS cells stably expressing H2B-GFP alone (U2OSH2B-GFP) or with mCherry-H2B (U2OSH2B-2FPs) were transduced with FLAG-tagged Histone H4WT or H4K20A mutant. Mock transduced cells were taken as control. FRET measurements were taken for all the samples (Bar, 10 µm). j Quantification of the mean FRET levels in Mock, H4K20WT-, and H4K20A-expressing cells. Box plots represents mean FRET values as defined earlier (f). n > 30 nuclei. ns not significant, ***p < 0.001 (ANOVA)
Fig. 2
Fig. 2
Maintenance of ground-state chromatin compaction ensures genome integrity a Design of the experiment. U2OS cells synchronized by double thymidine block were transfected with Control and SET8 siRNA. Cells were released into thymidine-free medium and harvested at the indicated time points. b Cells from a were fixed and stained with γH2A.X antibody and PI followed by flow cytometric analysis. c Bars represent percentage of γH2A.X-positive cells harvested at 15 h post G1/S release. Average ± SD of five independent experiments. ****p < 0.0001 (unpaired t test). n > 20,000 in each experiment. d U2OS cells synchronized as in a were harvested at 15 h post G1/S release and immunoblotted with the indicated antibodies. e U2OS cells synchronized and siRNA transfected as in a were fixed at 15 h post G1/S release and stained with DAPI and γH2A.X antibody. (Bar, 10 µm). f U2OS cells synchronized and siRNA transfected as in a were prepared for single-cell electrophoresis in neutral electrophoresis buffer at 15 h post G1/S release. g Relative comet-tail moments from experiments in f plotted as means ± S.E.M. *p < 0.05 (unpaired t test). n > 55. h U2OS cells expressing Dox-inducible FLAG-HA-tagged histone H4-wild type (H4K20WT) or histone H4 lysine 20 to alanine or arginine (H4K20A/R) mutant were synchronized with double thymidine block. Cells were fixed at 15 h post G1/S release and stained with γH2A.X antibody and PI followed by flow cytometric analysis. i Bars represent percentage of γH2A.X-positive cells from h. Average ± SD of three independent experiments. *p < 0.05, **p < 0.01 (ANOVA). n > 20,000 in each experiment. j U2OS cells synchronized and siRNA transfected as in a were mock and sucrose treated (125 mM) at 12 h post G1/S release and were fixed at 15 h post G1/S release. Bars represent 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. k U2OS cells were transfected with mCherry-tagged human RNF2 (RING1b) and were double thymidine synchronized, siRNA transfected and fixed as in (2e) and stained with DAPI and γH2A.X antibody. (Bar, 10 µm)
Fig. 3
Fig. 3
Chromatin compaction threshold restricts excessive loading of replication licensing factors. a U2OS cells were synchronized with double thymidine block, transfected with either siControl or siSET8 6 h before G1/S release, and fixed at 15 h post release. Cells were pre-extracted in CSK buffer containing 0.5% triton and immunostained with the indicated antibodies. Cells were counterstained with DAPI (Bar, 10 µm). b Scatter plot showing the quantification of ORC1 intensity from cells in a where mean ± SD is indicated by red lines. n > 150, ****p< 0.0001 (unpaired t test). n > 280. c Scatter plot showing the quantification of MCM2 intensity from cells in a where mean ± SD is indicated by red lines. n > 150, ****p < 0.0001 (unpaired t test). n > 280. d Chromatin fraction was prepared from synchronized U2OS cells depleted as in a, harvested at 15 h, and immunoblotted with the indicated antibodies. e Chromatin was prepared from cells synchronized and transfected with siRNAs as in a, treated with sucrose at 12 h post G1/S release, and harvested at 15 h. Samples were blotted with the indicated antibodies. f U2OS cells expressing DOX-inducible FLAG-HA-tagged histone H4WT or H4K20A mutant were synchronized as in a and fixed at 15 h post release. Cells were pre-extracted and immunostained with the indicated antibody as well as DAPI for DNA (Bar, 10 µm). g Scatter plot showing the quantification of ORC1 intensity from HA-positive cells in f where mean ± SD is indicated by red lines. n > 250, **p < 0.01 (unpaired t test). h U2OS cells expressing DOX-inducible FLAG-HA-tagged histone H4WT or H4K20A mutant were synchronized as in a and fixed at 15 h post release. Cells were pre-extracted and immunostained with the indicated antibody as well as DAPI for DNA (Bar, 10 µm). i Scatter plot showing the quantification of MCM2 intensity from HA-positive cells in h where mean ± SD is indicated by red lines. n > 180, ****p < 0.0001 (unpaired t test)
Fig. 4
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

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