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Comparative Study
. 2008 Oct;9(10):1019-26.
doi: 10.1038/embor.2008.162. Epub 2008 Aug 29.

PP4 Is a Gamma H2AX Phosphatase Required for Recovery From the DNA Damage Checkpoint

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Free PMC article
Comparative Study

PP4 Is a Gamma H2AX Phosphatase Required for Recovery From the DNA Damage Checkpoint

Shinichiro Nakada et al. EMBO Rep. .
Free PMC article

Erratum in

  • EMBO Rep. 2008 Dec;9(12):1251

Abstract

Phosphorylation of histone H2AX on Ser 139 (gammaH2AX) is one of the earliest events in the response to DNA double-strand breaks; however, the subsequent removal of gammaH2AX from chromatin is less understood, despite being a process tightly coordinated with DNA repair. Previous studies in yeast have identified the Pph3 phosphatase (the PP4C orthologue) as important for the dephosphorylation of gammaH2AX. By contrast, work in human cells attributed this activity to PP2A. Here, we report that PP4 contributes to the dephosphorylation of gammaH2AX, both at the sites of DNA damage and in undamaged chromatin in human cells, independently of a role in DNA repair. Furthermore, depletion of PP4C results in a prolonged checkpoint arrest, most likely owing to the persistence of mediator of DNA damage checkpoint 1 (MDC1) at the sites of DNA lesions. Taken together, these results indicate that PP4 is an evolutionarily conserved gammaH2AX phosphatase.

Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Figure 1
Figure 1
PP4C and PP2ACα dephosphorylate H2AX in vitro. (AD) Purified histone substrates isolated from cycling (N) or irradiation-treated U2OS cells (I) were incubated with varying amounts of immunopurified wild-type (WT) Flag-PP4C (A,C,D), Flag-PP2ACα (B), Flag-PP1C (A,B), phosphatase-dead Flag-PP4C-H56Q and PP4C-R86A mutants (C) and Flag-PP4R2 (D). Phosphatase reactions were followed by immunoblotting and probed with the indicated antibodies. H3, histone H3.
Figure 2
Figure 2
PP4 reduces γH2AX independently of a role in DNA repair in vivo. (A,B) U2OS cells were transfected with siRNAs against PP4C, PP2ACα/β (A), PP4R2 (B) or a non-targeting control sequence. Cells were irradiated with 10 Gy and collected at the indicated times after IR for immunoblotting with the indicated antibodies. Signal intensity was analysed using Image-J (http://rsb.info.nih.gov/ij/). Signal intensity of γH2AX is normalized against that of histone H3. (C) Cells were irradiated with 50 Gy and collected at the indicated times after IR for neutral comet assays. A representative image of the nuclei is presented. (D) Quantification of the comet tail moments of the experiment in (C). Tail moments for each condition were calculated on a minimum of 75 cells for each data point, normalized against the average comet tail moment of the mock-irradiated control, and shown as a box-and-whisker plot. Outliers are indicated as dots. The ordinate is a square-root scale. Data were statistically analysed using the Wilcoxon test. P-values are adjusted for several comparisons with the Bonferroni method. *P<0.05; **P<0.01; ***P<0.0001; NS, no significance. (E) U2OS cells transfected with siRNAs against either PP4C or a control sequence were irradiated with 10 Gy. At 1 h post-IR, the ATM (KU55933) and DNA-PK (KU57788) inhibitors were added to the tissue culture medium (t=0). Samples were then collected at the indicated time points and processed for immunoblotting with the indicated antibodies. Signal intensity was analysed using Image-J. Signal intensity of γH2AX is normalized against that of histone H3. ATM, ataxia telangiectasia mutated; DNA-PK, DNA-dependent protein kinase; H3, histone H3; IR, irradiation; siRNA, small interfering RNA.
Figure 3
Figure 3
PP4 dephosphorylates γH2AX at the sites of DNA damage. (A,B) U2OS cells transfected with siRNAs against PP4C, PP2ACα/β or a control sequence were irradiated with 1.5 Gy and processed for γH2AX (A,B) and MDC1 (B) immunofluorescence and DAPI counterstaining at the indicated time points. Images were taken with a × 40 objective (A) or with a × 63 objective (B). Scale bar, 23 μm (A) and 16 μm (B). (CF) Quantification of cells with >5 γH2AX (C), >10 γH2AX (D,F) and >5 MDC1 (E) foci. At least 300 cells were analysed per experiment. Cells were treated with KU55933 and KU57788 12 min post-IR (F). Error bars represent standard error of mean of independent duplicated experiments (C) or standard deviation of three independent experiments (DF). A × 63 oil immersion objective (C,E) or a × 40 water immersion objective (D,F) was used. (G) U2OS cells transfected with siRNAs against PP4C or a control sequence were collected at the indicated time points immediately before (no IR), or 1 or 6 h after irradiation (10 Gy), then fractionated as described in Méndez & Stillman (2000) into whole-cell lysates (WCLs), nuclear soluble (S3) or chromatin (P3) fractions, and analysed by immunoblotting using the indicated antibodies. DAPI, 4,6-diamidino-2-phenylindole; H3, histone H3; IR, irradiation; MDC1, mediator of DNA damage checkpoint 1; siRNA, small interfering RNA.
Figure 4
Figure 4
PP4 promotes recovery from the G2/M checkpoint arrest. (A) Schematic diagram of the checkpoint recovery experiment. S-phase cells were first arrested by incubation with aphidicolin, then mock-treated (No IR) or irradiated with an X-ray dose of 3 Gy. At 1 h post-treatment, nocodazole was added to the media and cells were fixed at various time points for processing by flow cytometry. (B) U2OS cells transfected with siRNAs against PP4C or a non-targeting control sequence were collected 1 and 8 h after mock (No IR) or 3 Gy irradiation and processed for flow cytometry with an antibody against phospho-histone H3 and propidium iodide (PI). The boxes represent the mitotic cells. (C) U2OS cells transfected with siRNAs against PP4C or a non-targeting control sequence were mock-treated (No IR) or irradiated (3Gy), and processed as described in (A). The percentage of mitotic cells (as determined by phospho-histone H3 staining) was calculated for each indicated time point. The experiment was repeated three times and the error bars represent the standard deviation. (D) Percentage of mitotic cells (as determined by phospho-histone H3 staining) calculated for each time point for the mock-irradiated (No IR, left) or irradiated (3 Gy, right) samples. The onset of mitosis represented by the intersecting lines was arbitrarily fixed at 0.4%. (E) Model of action of PP4 and dephosphorylation of γH2AX. IR, irradiation; siRNA, small interfering RNA.

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