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. 2014 May 8;54(3):512-25.
doi: 10.1016/j.molcel.2014.03.020. Epub 2014 Apr 3.

Dephosphorylation Enables the Recruitment of 53BP1 to Double-Strand DNA Breaks

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Free PMC article

Dephosphorylation Enables the Recruitment of 53BP1 to Double-Strand DNA Breaks

Dong-Hyun Lee et al. Mol Cell. .
Free PMC article

Abstract

Excluding 53BP1 from chromatin is required to attenuate the DNA damage response during mitosis, yet the functional relevance and regulation of this exclusion are unclear. Here we show that 53BP1 is phosphorylated during mitosis on two residues, T1609 and S1618, located in its well-conserved ubiquitination-dependent recruitment (UDR) motif. Phosphorylating these sites blocks the interaction of the UDR motif with mononuclesomes containing ubiquitinated histone H2A and impedes binding of 53BP1 to mitotic chromatin. Ectopic recruitment of 53BP1-T1609A/S1618A to mitotic DNA lesions was associated with significant mitotic defects that could be reversed by inhibiting nonhomologous end-joining. We also reveal that protein phosphatase complex PP4C/R3β dephosphorylates T1609 and S1618 to allow the recruitment of 53BP1 to chromatin in G1 phase. Our results identify key sites of 53BP1 phosphorylation during mitosis, identify the counteracting phosphatase complex that restores the potential for DDR during interphase, and establish the physiological importance of this regulation.

Figures

Figure 1
Figure 1. 53BP1 is a bonafide substrate of PP4C/PP4R3β
A. Schematic representation of human 53BP1 and alignment of the region flanking T1609 and S1618 showing evolutionary conservation of this region. Sequence alignment was performed with ClustalW2 (http://www.ebi.ac.uk/Tools/msa/clustalw2). B. Interaction of 53BP1 with PP4C and PP4R3β. U2OS cells, stably expressing FH-Empty vector (FH-c) or FH-53BP1 were harvested after 8h synchronization in mitosis (M) using 100 ng/ml Nocodazole or left in an asynchronous state (Asyn). Whole cell extracts were immunoprecipitated with anti-FLAG agarose beads and analyzed by immunoblotting using indicated antibodies. C. Impact of PP4C and PP4R3β silencing on 53BP1 phosphorylation. HeLa cells were transfected with indicated siRNAs against PP4 subunits for 60h and synchronized in mitosis with 100 ng/ml Nocodazole for 8h. Cells were released by mitotic shake-off into media without drug and harvested after 5h (G1 phase). Whole-cell lysates were probed with 53BP1 phospho-1618 (p1618) and 53BP1 phospho-1609/1618 (p1609–1618) antibodies. Total 53BP1 and tubulin were used as loading controls. Phospho-Ser10-histone H3 (pS10-H3) was used to indicate mitotic (M) cells. Cells were probed in parallel with antibodies against PP4R1, PP4R2, PP4R3α, PP4R3β, and PP4C to determine knockdown efficiency and specificity. D. Kinetics of 53BP1 hyperphosphorylation in PP4C/PP4R3β silenced cells during transition from mitosis to G1 phase. Upper panel: Schematic to study kinetics of 53BP1 hyperphosphorylation. Lower panel: HeLa cells transfected with scrambled control, PP4C or PP4R3β siRNAs, synchronized in mitosis with 100 ng/ml Nocodazole, released by mitotic shake-off, and harvested at indicated time points for western blot analysis. Cell lysates were probed with indicated antibodies. E. Inhibition of kinases PLK-1 and p38-MAPK in PP4C/PP4R3β silenced cells does not affect hyperphosphorylation of 53BP1 at T1609 and S1618. Upper panel: Schematic to study PLK-1 and p38-MAPK inhibition in PP4C/PP4R3β silenced cells. Lower panel: HeLa cells were transfected with indicated siRNAs and treated with Nocodazole as in Fig 1D. Kinase inhibitors against PLK-1 (BI2536, 20nM) and p38-MAPK (SB202190, 10µM) were added to the cells1h following release from mitosis. Cells were harvested at various time-points after mitotic release and lysates were probed with indicated antibodies. F. Recombinant PP4C incubated with mitotic extracts shows a dose-dependent decrease in phosphorylation at T1609 and S1618 whereas the catalytically inactive PP4C D82A mutant protein fails to dephosphorylate even at high concentrations. Extract was also incubated with λ-protein phosphatase (PP) as a positive control.
Figure 2
Figure 2. PP4C/PP4R3β silencing abrogates 53BP1 foci in G1 cells
A, B: Schematic shows protocol for assessing 53BP1 activity in G1 cells irradiated immediately after mitotic shake-off. PP4C or PP4R3β was depleted by siRNA transfection of HeLa cells (A) or Fucci-RPE1 cells (B). Mitotic cells were collected after 8h Nocodazole treatment, irradiated immediately after release (10 Gy), fixed and co-stained with antibodies for 53BP1 (A, B) and γ-H2AX (A) at indicated times in G1 phase. CDT1/RFP is an internal Fucci cell marker that illuminates cells in G1 phase. Quantification shows percent 53BP1 positive cells in G1. Cells displaying ≥ 10 foci were counted as positive. The data are expressed as mean ± S.D; n = 3. More than 100 cells were quantified per condition.
Figure 3
Figure 3. Constitutive phosphorylation of 53BP1 at T1609 and S1618 impedes foci formation at DSBs
A. Phosphomimetic 53BP1 mutant is not recruited to DSBs. Upper panel: Schematic to study 53BP1 focal recruitment. Lower left panel: HeLa cells stably expressing full-length FH-53BP1 (WT, AA, or ED) were prepared for immunofluorescence after mitotic shake-off and 10Gy IR as described in Fig 2A,B. 53BP1 foci were visualized using anti-FLAG antibody. γ-H2AX was stained to mark sites of DNA damage. Lower right panel: Quantification shows percent 53BP1 positive cells in G1 for WT and phospho-mutants. Cells displaying ≥ 10 foci were counted as positive. The data is expressed as mean ± S.D; n = 3; > 100 cells quantified per mutant. Lower right panel inset: Immunoblot showing equal expression levels of different FH-53BP1 constructs (WT, AA, and ED). Irradiated cells released into G1 were analyzed in parallel for expression using anti-FLAG antibody. Tubulin was used as a loading control. B. The failure of 53BP1 to form foci in PP4R3β silenced cells is rescued by expressing the 53BP1 AA mutant. Left panel: HeLa cells expressing FH-53BP1 WT or indicated phosphomutants were transfected with control siRNA or siRNA against PP4R3β and prepared for immunofluorescence as described in Figure 2A,B. Anti-FLAG antibody was used to visualize 53BP1. γ-H2AX was stained to mark sites of DNA damage. Right panel: Quantification shows percent 53BP1 positive cells in G1 for WT and phospho-mutants. Cells displaying ≥ 10 foci were counted as positive. The data is expressed as mean ± S.D; n = 3; > 100 cells quantified per mutant. C. Kinetics of 53BP1 recruitment to DNA lesions. To quantify the kinetics of 53BP1 recruitment to DSBs, a multi-photon laser (MPL) system was used. U2OS cells stably expressing full-length GFP-53BP1 (WT, AA, or ED) were treated with Nocodazole for 6h and released into G1. Left panels: Representative freeze-frame images from live-cell movies for each sample. A total of 10 cells were monitored per condition for 53BP1 recruitment to DSBs. Right panel: Quantification of 53BP1 recruitment was done by comparison of average signal intensity at 8 min in individual cells represented by a single dot; mean ± S.D; n = 3.
Figure 4
Figure 4. Constitutive phosphorylation of 53BP1 at T1609 and S1618 alters 53BP1 recruitment to nuclear bodies and induces resistance to PARP inhibitor
A. Recruitment of 53BP1 to replication stress induced nuclear bodies in G1. U2OS cells, stably expressing full-length GFP-53BP1 (WT, AA or ED) were exposed to low dose of Aphidicolin (0.2 µM) for 12 h and stained with anti-Cyclin A, which illuminates S/G2 cells. Representative images and quantification of 53BP1 accumulation in nuclear bodies are shown; >100 Cyclin A negative cells were quantified; mean ± S.D; n = 3. B. Radiosensitivity of 53BP1 phosphomutants. Endogenous 53BP1 was silenced in HeLa cells with siRNA and replaced with siRNA-resistant 53BP1 constructs (WT, AA, or ED). At 72 h after siRNA transfection, cells were irradiated at the indicated doses and viability was evaluated by clonogenic survival. Immunoblots performed to confirm siRNA efficiency and expression of siRNA-resistant constructs are shown. Data are expressed as mean ± S.D. n =3. C. Phosphomimetic 53BP1 mutant rescues PARP inhibitor sensitivity in BRCA1-deficient cells. As in Figure 3B, endogenous 53BP1 was replaced with siRNA-resistant 53BP1 constructs (WT, AA, or ED) in the BRCA1-deficient ovarian cancer line UWB1.289, and sensitivity to clinical-grade PARP inhibitor ABT888 was assessed by CellTiter-Glo® colorimetric viability assay. Immunoblots performed to confirm siRNA efficiency and expression of siRNA-resistant constructs are shown. Data are expressed as mean ± S.D. n =3.
Figure 5
Figure 5. Constitutive phosphorylation of 53BP1 alters sub-nuclear localization and prevents its direct interaction with RNF168-ubiquitinated mononucleosomes
A. Sub-nuclear localization of 53BP1 is altered by hyperphosphorylation of T1609/S1618. U2OS cells, expressing full length FH-tagged 53BP1 (WT, AA or ED) were synchronized in mitosis using Nocodazole and biochemical fractionation was conducted at indicated time points after 10 Gy IR. Nuclear soluble (NS) and chromatin-enriched (Chr) fractions were probed using anti-FLAG antibody. KU70 and histone H3 (H3) was probed for loading and fractionation controls, respectively. B. Interaction of 53BP1 with H4K20Me2. U2OS cells expressing full-length FH-tagged 53BP1 (WT, AA, or ED) or empty vector (FH-c) were synchronized in mitosis, exposed to 10Gy IR immediately after release, and harvested at indicated time points. 53BP1 was immunopurified using anti-FLAG agarose beads. The immunoprecipitate was analyzed by immunoblotting using antibodies against H4K20Me2 and RPA2, which is a known interacting partner of 53BP1. Total 53BP1 in the input and pull-down fraction is also shown. C. Pull-down assays of RNF168-ubiquitinated H4KC20me2 nucleosomes with GST-fusion proteins. Left panel: Installation of a dimethyl-lysine analogue at the mutated cysteine of H4K20C and ubiquitination of mononucleosomes was confirmed by immunoblotting using specific antibodies. Right panel: Pull-down assays of non-ubiquitinated and RNF168-ubiquitinated H4KC20me2 nucleosomes with the indicated GST-fusion proteins. Input lanes represent 33% of the nucleosome amount used in the pull-down assays; n = 3. D. Phosphorylation of 53BP1 foci-forming region (FFR) by incubation with mitotic extract prevents its interaction with RNF168-ubiquitinated H4KC20me2 nucleosomes. GST-fusion proteins were pre-incubated with a HeLa mitotic extract in presence of 1mM ATP and a combination of PLK-1 inhibitor (BI2536, 100nM) and p38-MAPK inhibitor (SB202190, 10µM) (Kinase-i) when indicated. Following pre-incubation, GST proteins were further incubated with RNF168-ubiquitinated H4KC20me2 nucleosomes. Input represents 33% of the nucleosomes amount used in the pull-down assays; n = 3.
Figure 6
Figure 6. Loss of phosphorylation at T1609 and S1618 of 53BP1 allows its recruitment to DNA lesions in mitotic cells and promotes genomic instability
A. Premature recruitment of 53BP1-AA mutant to DNA lesions during mitosis. To examine the kinetics of 53BP1 foci formation, U2OS cells expressing RFP-H2B were transfected with different GFP-53BP1 constructs (WT, AA, ED), treated with 0.2 uM Aphidicolin for 12 hrs and released prior to live cell imaging. Left panels: representative time-lapse still images of cells expressing RFP-H2B and GFP tagged 53BP1-WT (top panels) or 53BP1-AA (middle panels) or 53BP1-ED (bottom panels) during G2, mitosis (early and late) and the following G1 phase. Time is shown in minutes (t=0, anaphase onset). Upper right panels: late mitotic cells (red box region) with premature 53BP1-AA foci formation are illustrated in overlaid images along with high magnification images (boxes 1 and 2). GFP-53BP1 and RFP-H2B are shown in green and red, respectively. Bottom right panels: Corresponding quantification of the timing of 53BP1 foci formation (p <0.0001, non-parametric t-test). Averages are shown in red bars. B, C. Premature mitotic 53BP1-AA foci formation is accompanied by increased mitotic defects. B. U2OS cells expressing RFP-H2B were transfected with different GFP-53BP1 constructs (WT, AA) and exposed to 0.5Gy IR during mitosis prior to live cell imaging. Overlaid images with GFP-53BP1 (green) and RFP-H2B (red, also shown at the bottom with RFP-H2B only). Cells containing premature mitotic 53BP1-AA foci progress through cell cycle with increased mitotic defects, such as lagging chromosomes (dashed boxes) and micronuclei (MN, boxes). C. Left panels: Corresponding mitotic defects that persist as MN in G1. Note that some MN contains 53BP1-AA (Box, 130 min, GFP in primary nuclei is overexposed to accentuate 53BP1 signal in MN). Upper right panel: Corresponding quantification for the timing of 53BP1 foci formation in indicated conditions (p <0.0001, non-parametric t-test). Lower right panel: percentage of cells that display lagging chromosomes and MN in indicated conditions. Only newly arising defects in cells exiting mitosis were scored (p <0.0001, non-parametric t-test). Errors bars indicate S.E.M. D. U2OS cells expressing different GFP-53BP1 constructs (WT or AA) were treated with Nocodazole for 6h. Resulting mitotic cells were irradiated at low-dose (0.5 Gy) and released into normal media. At 6h following release, cells were fixed and co-stained with antibodies for γ-H2AX and CREST (kinetochore marker) to visualize kinetochore-positive (CREST+) MN. Where indicated, transfected cells were pretreated with DNA-PK inhibitor (DNA-PKi) (NU7441, 10µM) 1h prior to irradiation. Quantification of CREST and CREST+ MN in cells expressing WT or AA GFP-53BP1 with or without DNA-PKi is shown. The data are expressed as mean ± S.D; n = 3 (> 100 cells were quantified). Refer to Supplementary Figure 5B for representative CREST staining.

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