SIRT1 regulates DNA damage signaling through the PP4 phosphatase complex

Abstract

The Sirtuin family of NAD+-dependent enzymes plays an important role in maintaining genome stability upon stress. Several mammalian Sirtuins have been linked directly or indirectly to the regulation of DNA damage during replication through Homologous recombination (HR). The role of one of them, SIRT1, is intriguing as it seems to have a general regulatory role in the DNA damage response (DDR) that has not yet been addressed. SIRT1-deficient cells show impaired DDR reflected in a decrease in repair capacity, increased genome instability and decreased levels of γH2AX. Here we unveil a close functional antagonism between SIRT1 and the PP4 phosphatase multiprotein complex in the regulation of the DDR. Upon DNA damage, SIRT1 interacts specifically with the catalytical subunit PP4c and promotes its inhibition by deacetylating the WH1 domain of the regulatory subunits PP4R3α/β. This in turn regulates γH2AX and RPA2 phosphorylation, two key events in the signaling of DNA damage and repair by HR. We propose a mechanism whereby during stress, SIRT1 signaling ensures a global control of DNA damage signaling through PP4.

Graphical Abstract

Figure 1.

Identification of a novel protein complex containing SIRT1 and the PP4 phosphatase complex. (A) Schematic representation of the SIRT1 purification from HeLaS3 nuclear extracts (NE) including the buffers used (BC or BD) and a range of KCl gradient concentrations (from 40 mM to 1 M) depending on each chromatographic step (see Methods and Supplementary information). (B, C) Silver stained SDS-gel (B) and western-blot (C) of the anion-exchange MonoQ column, the final chromatography step of the purification. Fractions 23–25 were pooled and analyzed by mass spectrometry. The analysis showed that PP4 complex components PP4c, PP4R2, PP4R3α and PP4R3β co-fractionate with SIRT1 (Supplementary Table S1).

Figure 2.

SIRT1-PP4 complex formation is boosted by oxidative stress conditions. (A) SIRT1 and the PP4 complex interaction with SIRT1-FLAG IP experiments in HeLa cells non-stress (NT) or under 5mM H₂O₂ (2h), 2mM hydroxyurea (4h, HU) and 10 and 20 μM camptothecin (2 h, CPT). (B) Endogenous IP using α-SIRT1 antibodies between the PP4 complex (PP4c, PP4R2, PP4R3α/β) and SIRT1 from whole cell extracts of U2OS cells previously treated or not (C) with 2 mM H₂O₂ for 1 h. As a negative control the same cell extracts without SIRT1 antibody (no Ab) was used. Inputs (I) and elutions (E) are shown. (C) FRET analysis of SIRT1-YFP (acceptor) and PP4c-CFP (donor) in HeLa cells treated during 1 h with 2 mM H₂O₂. Cells were imaged with confocal fluorescence microscopy and fluorescence of CFP-PP4c was measured before and after photobleaching in control and oxidative stress conditions. Left, schematic representation of the process. Right, quantification of detected FRET efficiency is shown from n = 3 experiments. Two-tailed t-test analysis (**P< 0.01). (D) SIRT1 and the PP4 complex cofractionate in fractions around 600 kDa (fractions 13–16). Glycerol gradient analysis (12.5–35%) of nuclear extract from HeLa cells expressing FLAG-SIRT1 and treated with H₂O₂ before harvest (see Methods and Supplementary information). (E) HA and FLAG co-immunoprecipitations of HA-PP4c and FLAG-SIRT1, SIRT1 catalytically inactive point mutant H363Y (SIRT1m) or SIRT6 in whole-cell extracts from HeLa cells expressing the indicated constructs treated or not with 2 mM H₂O₂ for 1 h before harvest. (F) Time course experiment from 0 to 24 h of SIRT1/PP4 complex assembly upon oxidative stress. PP4c (FLAG) immunoprecipitation of whole-cell extracts from HeLa cells expressing HA-tagged SIRT1 and PP4R2, FLAG-tagged PP4C and Myc-tagged PP4R3α/β treated with H₂O₂ 2 mM for 1h and harvested at the indicated times.

Figure 3.

SIRT1 inhibits PP4 phosphatase activity. (A) High throughput microscopy (HTM) immunofluorescence analysis of γH2AX in Wt and Sirt1^−/− cells treated or not with 7.5 Gy IR for 1 h and analyzed at the indicated times after irradiation. Data are representative of three independent experiments. Two-tailed t-test (****P< 0.001). At least 300 nuclei were analyzed and the mean with SEM is shown for independent cultures. (B) In vitro γH2AX phosphatase activity of a titration of PP4 complexes (1×, 2×, 3×) purified from HeLa cells treated or not with 2 mM H₂O₂ for 1 h and incubated –/+ SIRT1 in presence or absence of NAD⁺. (C) In vitro phosphatase activity as in (B) of PP4 complex purified from Wt and Sirt1^−/− MEFs under oxidative stress 2 mM H₂O₂ for 1 h. (D) Assay as in (B) and (C) Purified PP4 complexes in the presence of Ex-527 (1 μM, 10 μM) and 1 mM nicotinamide (NAM). (E) Western-blot of γH2AX, H2AX and the indicated proteins of extracts from HEK293T overexpressing the PP4 complex in presence or absence of FLAG-SIRT1 WT or catalytically-inactive mutant H363Y. (F) Phosphatase assay of whole-cell extracts from Wt and Sirt1^−/− cells previously treated with Okadaic Acid (OA) (50 nM) or DMSO (control) for 24 h followed by 1 h incubation of 2 mM H₂O₂ before harvest. Quantification of the ratio of γH2AX/ H2AX in the phosphatase assay is shown.

Figure 4.

SIRT1 targets K64 in PP4R3α, β to regulate PP4 activity. (A) PP4 complex was purified from MEFs Wt and Sirt1^−/− under oxidative stress (2 mM H₂O₂ 1 h) overexpressing PP4 core complex (PP4c and PP4R2) and analyzed by MS to identify acetylated peptides in the PP4 complex upon loss of SIRT1. Left, Schematic representation of the experiment's pipeline. Right, Levels of the indicated proteins by western blot in the MEFs analyzed. (B) Summary of the acetylated peptides identified in the MS analysis of (A). Differential acetylation was identified in the endogenous PP4R3α and β subunits (see Methods, Supplementary information and Supplementary Table S2). (C) PP4R3α and β protein domains, the acetylation events identified in Sirt1^−/− but not in Wt cells, and their position in both subunits. (D) K64 and D67 are two conserved residues in WH1 domain of PP4R3α,β from Drosophila to humans. WH1 domain primary sequence of the homologs of both PP4R3α,β isoforms in Saccaromyces cerevisiae, Drosophila melanogaster, Xenopus laevis, zebrafish, mouse and humans PP4R3 homologs. (E) In vitro γH2AX phosphatase activity assay of human PP4 complex containing the indicated mutations expressed and purified from HeLa cells. A quantification of n = 3 experiments (Supplementary Figure S4D) is shown. Two-tailed t-test (*P< 0.05; **P< 0.01; ***P< 0.005). (F) Quantification of n = 4 experiments (measured by Western blot) as in 3E of the ratio γH2AX/H2AX in HEK293T cells overexpressing the PP4 complex containing PP4R3α/β WT or K64R −/+ SIRT1 treated with 2 mM H₂O₂ for 1 h. A representative image is shown in Supplementary Figure S3E. Two-tailed t-test assay was performed (*P< 0.005; **P< 0.01; ***P< 0.005). (G) MALDI-MS analysis of an in vitro K64ac deacetylation reaction by SIRT1. The chromatograms for the control reactions without neither SIRT1 nor NAD⁺ (Ctrl) and with SIRT1 but not NAD⁺ (SIRT1) are shown together with the complete reaction (SIRT1 + NAD⁺). The acetylated peptide was detected either alone or in form of a single sodium adduct. Only the complete reaction rendered deacetylation of both species found for the peptide (b and c) in contrast to the acetylated forms of the peptide (a) (see Materials and Methods). (H) Structure of human PP4R3α suggests that K64 and D67 are physically very close and have the potential to interact only if K64 is in its unacetylated form (right upper cartoon). Acetylation of K64 could break the interaction between K64 and D67, allowing D67 to turn to the substrate binding region and interact with the substrate through a lysine residue in the FXXP motif (main and right lower cartoon). (I) Pull-down experiments with HA resin of previously purified PP4 complexes containing PP4R3α WT/K64R/D67N and core histones. Both the PP4 complexes and the core histones were independently purified from HEK293cells previously treated with 2 mM H₂O₂. (J) γH2AX phosphatase activity of the PP4 complexes in (H).

Figure 5.

SIRT1 regulates RPA2 phosphorylation through PP4. (A) IF analysis of γH2Ax and RPA2 foci formation in Wt or Sirt1^−/− MEFs under or not oxidative stress (2 mM H₂O₂ for 1 h), or ionizing irradiation (IR 30 min 7.5 Gy). (B) Western-blot analysis of the levels of the total pRPA2 in Wt and Sirt1^−/− MEFs previously treated with 7.5 Gy IR. Quantification of n = 3 experiments and representative experiment are shown. The shifted phosphorylated form of RPA2 is detected in the longer exposure blots and is marked with *. Two-tail analysis (**P< 0.01). (C) Western blot of SIRT1, RPA2 (total and S33ph) in whole cell extracts (WCE), nuclear soluble fraction (NS) and chromatin insoluble fraction (Chrom) of Wt or Sirt1^−/− U2OS cells previously treated with 2 mM H₂O₂ for 1 h. The levels of fibrillarin and histone H3 were also analyzed as controls of NS and Chrom fractions, respectively. (D) IF Time-course experiment of RPA2-S33P performed as in 3A. A similar experiment is shown by western blot in Figure S4B. (E) Co-IP between FLAG-RPA2 and HA-SIRT1 using FLAG and HA resin under untreated condition or oxidative stress (2 mM H₂O₂ for 1 h) in HeLa cells. (F) Endogenous IP with α-SIRT1 antibodies of RPA2 and SIRT1 from whole cell extracts of U2OS cells previously treated with 2 mM H₂O₂ for 1 h. Sirt1^−/− extracts were used as a negative control. (G) Levels of the indicated proteins and RPA2 marks upon shRNA-driven downregulation or not of PP4c in Wt and Sirt1^−/− MEFs untreated or irradiated with IR (7.5 Gy). Lanes 5 and 10, SIRT1 expression was re-introduced in Sirt1^−/− (indicated with *). (H) Similar experiment as in 3D with RPA2-S33P levels.

Figure 6.

The interplay between SIRT1 and PP4 regulates DNA repair and genome stability. (A)Downregulation of SIRT1 (siSIRT1) and/or PP4C (siPP4c) expression in U2OS cells using siRNAs (siScr: scramble). (B) Quantification of Propidium iodine (PI)/EdU based cell cycle analysis (n = 3) of the cells used in (A). (C) Cell cycle analysis as in (B) of U2OS cells transfected with an empty (C), wild-type PP4R3α,β (WT) or mutant PP4R3α,β K64Q (Q) expression vectors. (D) Neutral comet assay of U2OS cells Wt or Sirt1^−/− expressing siRNA scramble or against PP4c treated with 2 mM of H₂O₂ for 1 h (upper panel) or 1 μM camptothecin for 90 min (lower panel). A quantification of n = 3 experiments are shown. The % DNA in tail and tail moment are quantified. A similar analysis in absence of stress is included in Supplementary Figure S5E. Two-tailed t-test assay was performed (**P< 0.01; ***P< 0.005; ****P< 0.001). (E) in vivo HR reporter system in U2OS cells upon siRNA-driven downregulation of PP4c and/or SIRT1. Quantification of n = 3 experiments(bottom) are shown. Two-tailed t-test analysis was performed (*P< 0.05; ***P< 0.005; ****P< 0.001). (F, G) HR assays as in (E) upon treatment with either DMSO (control) or 100 μM of SIRT1 inhibitor Ex-527 (F) or upon overexpression of both PP4R3α and β WT or K64R (G). (H) in vivo NHEJ reporter system in U2OS cells upon siRNA-driven downregulation of PP4c and/or SIRT1. Quantification of n = 3 experiments(bottom) are shown. Two-tailed t-test analysis was performed (***P< 0.005; ****P< 0.001). (I–K) Chromosome aberration analysis of Giemsa stained chromosomes in U2OS cells Wt or Sirt1^−/− expressing siRNA scramble (siScr) or against PP4 (siPP4), as in (D), treated with 100 μM H₂O₂ for 30 min. (I) Representative images of each condition. Arrows in zoomed images indicate chromosome aberrations (for Sirt1^−/−, denote fusions; and for siPP4c and Sirt1^−/−/siPP4c, denote breaks/deletions). (J) Quantification of n = 20 metaphases from each genotype. Two-tailed t-test analysis was performed (*P< 0.05; ****P< 0.001). (K) Table showing frequencies of chromosome aberration types. Frequencies were calculated as the proportion of the number of each chromosome aberration detected in n = 20 metaphases/condition compared to the total number of chromosomes detected in these n = 20 metaphases.

Figure 7.

SIRT1 and PP4 inversely correlate in cancer. (A) Proteomics and phosphoproteomics data from tumor samples was obtained from the National Cancer Institute's Clinical Proteomic Tumor Analysis Consortium (CPTAC) analysis of tissues from the Breast Invasive Carcinoma (TCGA) dataset. (B) Heatmap of SIRT1, SIRT6, PPP4C protein abundance and pRPA2 (p238) and pTRIM28 (pS473) levels in samples from the study described in (A). Each column corresponds to one tumor sample. (C) Correlations between the indicated proteins and pRPA2 in each tumor sample were analyzed. Pearson correlation coefficient (r) and P-value (P) for each analysis are shown. (D) List of HDR-associated genes used in the analysis of (E). (E) Samples of the breast cancer cohort containing mutations or deep deletions in the genes in (D) were selected and analyzed as in (C). (F) Proposed model of the antagonistic interplay between SIRT1 and PP4 in HR repair.