Phosphorylation of TRF2 promotes its interaction with TIN2 and regulates DNA damage response at telomeres

Abstract Protein phosphatase magnesium-dependent 1 delta (PPM1D) terminates the cell cycle checkpoint by dephosphorylating the tumour suppressor protein p53. By targeting additional substrates at chromatin, PPM1D contributes to the control of DNA damage response and DNA repair. Using proximity biotinylation followed by proteomic analysis, we identified a novel interaction between PPM1D and the shelterin complex that protects telomeric DNA. In addition, confocal microscopy revealed that endogenous PPM1D localises at telomeres. Further, we found that ATR phosphorylated TRF2 at S410 after induction of DNA double strand breaks at telomeres and this modification increased after inhibition or loss of PPM1D. TRF2 phosphorylation stimulated its interaction with TIN2 both in vitro and at telomeres. Conversely, induced expression of PPM1D impaired localisation of TIN2 and TPP1 at telomeres. Finally, recruitment of the DNA repair factor 53BP1 to the telomeric breaks was strongly reduced after inhibition of PPM1D and was rescued by the expression of TRF2-S410A mutant. Our results suggest that TRF2 phosphorylation promotes the association of TIN2 within the shelterin complex and regulates DNA repair at telomeres.


INTRODUCTION
Genome instability is one of the hallmarks of cancer cells ( 1 ).DNA damage response dri v en by Ataxia telangiectasia mutated (ATM) and Ataxia telangiectasia and Rad3related protein (ATR) kinases represents a surveillance mechanism that protects genome integrity by orchestrating a temporal cell cycle arrest and DNA repair (2)(3)(4).DNA double strand breaks (DSBs) are repaired either by nonhomologous end joining (NHEJ) or by homologous recombination (HR).Protein phosphatase magnesium-dependent 1 delta (PPM1D, also known as WIP1) promotes recovery from the G2 checkpoint by counteracting activities of the tumour suppressor p53 and KRAB-interacting protein 1 (KAP1) ( 5 , 6 ).In addition, PPM1D terminates DNA damage response by directly targeting ATM, histone H2AX, BRCA1 and other proteins at the chromatin flanking the DNA lesions (7)(8)(9)(10).Amplification of the PPM1D locus or gain-of-function mutations in the last exon of PPM1D have been reported to promote tumorigenesis by inhibiting p53 pathway and are commonly found in various solid tumours and haematological malignancies (11)(12)(13)(14).
Although essential for pre v enting global genome instability, DNA repair at the ends of chromosomes needs to be acti v ely suppr essed to pr e v ent the fusion of telomeric DNA ( 15 ).Integrity of the telomeres is protected by the shelterin complex comprising of telomeric repeat-binding factor 1 (TRF1), telomeric repeat-binding factor 2 (TRF2), TRF2-interacting telomeric protein 1 (TERF2IP; further r eferr ed to as RAP1), TRF1-interacting nuclear protein 2 (TIN2; also known as TINF2), protection of telomeres protein 1 (POT1), and Adrenocortical dysplasia protein homolog (ACD, her eafter r eferr ed to as TPP1) ( 16 ).TRF1 and TRF2 form homodimers through the TRFH domains, and they bind the TTAGGG repeats in the doublestranded telomeric DNA through their C-terminal Myb domains ( 17 ).In addition, the N-terminal basic domain of TRF2 can bind branched DNA structures and the double stranded DNA also wraps around the TRFH domain of TRF2 (18)(19)(20).The heterodimer comprising of TPP1 and POT1 associates with the single-stranded DNA through two oligonucleotide / oligosaccharide-binding (OB) folds of POT1 ( 21 , 22 ).In addition, TPP1 also promotes the recruitment of the telomerase ( 23 ).TIN2 bridges the TRF1 and TRF2 homodimers with TPP1 and pre v ents acti vation of Nucleic Acids Research, 2023, Vol. 51, No. 3 1155 ATR by stabilizing TPP1-POT1 at telomeric ssDNA (24)(25)(26).Similarly, TIN2 promotes TRF2 binding to telomeres thus protecting telomeric DNA from uncapping and from activation of ATM (26)(27)(28)(29).Structural studies have revealed that TIN2 interacts with the TRFH domains of TRF1 and TRF2, and with a short motif between the residues 392-408 of TRF2 (hereafter referred to as a TIN2-binding motif, TBM) ( 30 , 31 ).Due to its unique DNA-binding ability, TRF2 promotes the folding of the telomeric DNA into a lasso-like structur e r eferr ed to as a t-loop that pre v ents activation of ATM ( 15 , 32 , 33 ).In addition, the basic domain of TRF2 has been reported to pre v ent unwinding of the t-loops wher eas r ecruitment of the Regulator of telomere elongation helicase 1 (RTEL1) by TRF2 promotes telomere unwinding during the replication ( 20 , 34 , 35 ).Loss of TRF2 leads to exposure of the DNA end, causing activation of ATM followed by ubiquitination-dependent recruitment of 53BP1 (forming nuclear patches termed Telomere dysfunction-Induced Foci (TIFs)) and subsequent fusion of telomeres by NHEJ (36)(37)(38).In contrast to TRF2, TRF1 is r equir ed for r eplication of the telomeric DNA and its loss leads to telomeric fragility ( 39 ).Single-molecule imaging re v ealed the ability of TIN2 and TRF2 to compact the telomeric DNA in vitro ; howe v er, the importance of DNA de-compaction for DNA repair at telomeres still remains unclear (40)(41)(42)(43).
Here, we aimed to identify new substrates of PPM1D a t chroma tin.Using proximity biotinyla tion assay and immunoprecipitation, we identified the shelterin complex as a major interacting partner of PPM1D in human cells.Confocal microscopy confirmed a close association between PPM1D and shelterin at telomeres in various cell types.Since PPM1D directly interacted with TRF2 in vitro , we evaluated the ability of PPM1D to dephosphorylate TRF2 in cells.We found tha t ATR phosphoryla ted TRF2 a t S410 upon CRISPR Cas9-mediated induction of DNA breaks at telomeres.Inhibition or loss of PPM1D significantly increased the le v el of TRF2-S410 phosphorylation.In addition, PPM1D dephosphorylated TRF2 in vitro .Importantly, increased phosphorylation of TRF2-S410 in cells treated with PPM1D inhibitor promoted the association of TIN2 with the damaged telomeres and pre v ented recruitment of the DNA repair factor 53BP1.Inversely, the expression of a non-phosphorylatable mutant TRF2-S410A r escued the r ecruitment of 53BP1 to DSBs at telomeres in cells treated with PPM1D inhibitor.Furthermore, ov ere xpression of PPM1D impeded with assembly of the shelterin at telomeres and promoted telomeric fusions.We conclude that ATR and PPM1D control the binding of TIN2 at telomeres by inversely regulating the phosphorylation of TRF2 at S410.

Cells
Human hTERT-immortalized RPE1 cells (here referred to as RPE), HEK293, human br east adenocar cinoma MCF7 or human osteosarcoma U2OS cells were grown in DMEM supplemented with 6% FBS (Gibco), Penicillin and Streptomycin.U2OS-PPM1D-KO cells with a knock-out of PPM1D wer e described pr eviously ( 44 ).HeLa cells with doxy cy cline-inducib le knock-down of TRF2 were described previously ( 45 ).HeLa-shTRF2 cells were transfected by pEGFP-TRF2 or pEGFP-TRF2-S410A and selected with geneticin followed by single cell clone expansion.RPE1 cells transfected with pCW57-GFP-P2A-PPM1D-A380 plasmid were selected by geneticin for 3 weeks followed by single clone expansion and expression of the catalytic domain of PPM1D was induced by doxy cy cline.All cells were regularly tested for mycoplasma infection using MycoAlert kit (Lonza).Plasmid DNA transfection was performed using polyethylenimine in ratio 1:6.Stable cell lines were gener ated by tr ansfection of HEK293 cells with plasmid pBIOID2-HA or pBIOID2-PPM1D-D314A followed by 3 weeks selection with geneticin and expansion of single cell clones.Silencer Select siRNAs were transfected using RNAiMAX (both Thermo Scientific) at final concentration 5 nM and cells were analyzed after 2 days.Alternati v ely, two subsequent rounds of siRNA transfection were performed and cells were analyzed after 4 days.Expression of Cas9 was induced in iCut-RPE1 cells by overnight treatment with doxy cy cline and Shield-1 (1 M, Aobious) and telomeric DNA damage was generated by transfection of the synthetic sgRNA TT AGGGTT AGGGTT AGGGTT (Sigma) as described previously ( 46 , 47 ).sgRNA was transfected by Lipofectamine RNAiMAX (Thermofisher) at final concentration 5 nM.

Immunofluor escence microscop y
Cells grown on coverslips were washed in PBS, fixed by 4% PFA for 15 min and permeabilized with 0.2% Triton-X100 for 5 min.Where indicated, cells were pre-extracted prior fixation in 25 mM HEPES pH 7.4, 50 mM NaCl, 1 mM EDTA, MgCl 2 , 300 mM Sucrose, 0.5% Triton X-100 for 5 min.After washing in PBS, coverslips were blocked with 1% BSA in PBS for 30 min, incubated with primary antibodies for 2 h at room temperature and subsequently with Alexa Fluor secondary antibodies (Thermo Scientific) for 1 h.After incubation with DAPI for 2 min, coverslips were washed with water and mounted with Vectashield.For proximity ligation assay (PLA), coverslips were stained with the indicated primary antibodies followed by incubation with PLA probes (Merck, Duolink In Situ PLA Probe Anti-Rabbit PLUS and MINUS, DUO92002, DUO92004) for 1 h at 37 • C, ligation for 30 min at 37 • C, and polymerase reaction for 2 h at 37 • C according to the manufactur er's protocol (Mer ck, Duolink In Situ Detection Reagents Red, DUO92008).For immunofluorescence-FISH, coverslips were fixed, permeabilized, and blocked as described above.After dehydration with 70%, 95% and 100% ethanol for 3 min each, the coverslips were incubated for 10 min at 80 • C face down on a slide with 20 l of hybridization solution (10 mM Tris-HCl pH 7.2, 60% formamide, 0.4 M TelC-Cy5 PNA probe (Panagene), and 0.5% blocking reagent (Roche, 10% stock in 100 mM maleic acid pH 7.5 and 150 mM NaCl).Hybridization was perf ormed f or 2 h a t room tempera ture in a humidified chamber in dark.The coverslips were then washed twice for 10 min in wash buffer 1 (10 mM Tris-HCl pH 7.2, 70% formamide) and twice for 5 min in PBS.Incubation with primary antibodies was performed overnight at 4 • C, followed with PBS wash and incubation with secondary antibodies for 1 h at room temperature.The coverslips were then stained with DAPI, rinsed in water and mounted using Vectashield.For the high content microscop y, images wer e acquir ed us-ing Olympus ScanR equipped with 60 ×/ 1.42 OIL objecti v e and analyzed using ScanR analysis software.Confocal imaging was performed using Leica DMi8 equipped with HC PL APO 63 ×/ 1.40 OIL CS2 objecti v e. Images wer e acquir ed as Z-stacks of fiv e planes with v o xel size 44 × 44 × 129.7 nm and 3D-deconvolved using Huygens Professional (Scientific Volume Imaging) based on the theor etical point spr ead function.Metaphase spr eads wer e imaged using Leica DM6000 equipped with a HCX PL APO 63 ×/ 1.40 OIL objecti v e and a sCMOS Leica DFC 900 camera.

Metaphase FISH
Cells were synchronised in late G2 phase by treatment with 9 M R0-3306 (MedChemExpress) for 16 h.After washing with PBS, cells wer e r eleased into media supplemented with 0.1 ug / ml colcemid (Sigma) and incubated for 3 h.Subsequently, cells were trypsinised, pelleted at 300 g for 5 min and resuspended in 5 ml of warm 75 mM KCl.After incubation for 30 min at 37 • C, cell suspension was mixed with 1.25 ml of fixati v e solution (methanol:acetic acid, 3:1) while vortexing.After centrifugation, cells were 3 × washed with fixati v e solution.Finally, cells wer e r esuspended in 200-800 l of fixati v e solution to achie v e concentration 4 × 10 6 cells / ml, and dropped onto frozen slides from distance of 30 cm.Slides were air dried overnight, washed 3 × 5 min in PBS and hybridisation was performed as described above.After washing in wash buffer 1 and three times 10 min in wash buffer 2 (100 mM Tris-HCl pH 7.2, 150 mM NaCl, 0.08% Tween 20), slides were stained with DAPI, PBS washed, dehydrated with 70%-95%-100% ethanol series, and mounted in Vectashield.

Sample pr epar ation f or imaging of telomeric loops
For super-resolution imaging of telomeric loops, we used modified protocol from Doksani et al.P ar ental U2OS and PPM1D KO cells were trypsinized, washed with PBS and resuspended in 5 volumes of ice-cold nuclei extraction (NE) buffer (10 mM HEPES-KOH pH 7.9, 10 mM KCl, 1.5 MgCl 2 , 0.5 mM DTT, 0.5 mM PMSF) supplemented with cOmplete protease inhibitor cocktail (Roche).After 5 min of incubation, cells were pelleted at 500 g for 5 min at 4 • C and resuspended in 2 volumes on NE buffer.Nuclei were released from cells using Dounce homogeniser and collected with centrifugation at 800 g for 5 min at 4 • C. Nuclei were resuspended in nuclei wash (NW) buffer (10 mM Tris-HCl pH 7.4, 15 mM NaCl, 60 mM KCl, 5 mM EDTA) in concentration 1-2 × 10 7 nuclei / ml, and incubated with 100 g / ml of Trioxalen (Sigma) on ice while stirring in the dark for 5 min.Crosslinking was performed by exposing 2 ml of nuclei suspension at a 6-well plate to 365 nm light for 30 min on ice.Cross-linked nuclei were centrifuged at 800 g for 5 min a t 4 • C , washed with ice-cold NW buf fer, and resuspended a t 1 × 10 7 nuclei / ml.The nuclei suspension was diluted 10 × in spreading buffer (10 mM Tris-HCl pH 7.4, 10 mM EDTA, 0.05% SDS, 1 M NaCl) pre-warmed at 37 • C, and 100 l of the suspension was immediately dispersed on 13 mm round 1.5H coverslips using cytospin at 600 rpm for 2 min.Coverslips were dried at room temperature for 1 h and fixed in methanol:acetic acid (3:1) for 1h.Coverslips were dehydrated with 70%-95%-100% ethanol series and hybridized with TelC-Cy5 PNA probe overnight at 4 • C in a humidified chamber pr otected fr om light.After washing with wash buffer 1 and wash buffer 2, coverslips were washed in water, air-dried and mounted with Vectashield.

Structured illumination imaging
Thr ee dimensional-structur ed illumination microscop y (3D-SIM) was performed using DeltaVision OMX ™ V4 equipped with Blaze Module (GE Healthcare) and a PLAN APO N 60 ×/ 1.42 OIL objecti v e.A 568 nm OPSL laser was used for excitation and a pco.edge 5.5 sCMOS camera for signal detection.Raw images wer e acquir ed in a z-stack with 125 nm step, 8 z slices, 15 images per slice, pixel size 80 nm.The image reconstruction was performed using SoftWorX software (GE Healthcare).Blinded analysis of telomeres in maximal projection images was done as previousl y described ( 33 ).Onl y telomere without ga ps in telomere staining > 500 nm wer e scor ed.Branched and overlapping telomeres (30-60% of molecules) were excluded from analysis.

Pr oximity biotin ylation assa y and mass spectr ometry
HEK293 stably transfected with empty pBIOID2 or pBIOID2-PPM1D-D314A were grown in media supplemented with 50 M biotin for 5 h, then cells were collected, washed in cold PBS and lysed under dena tura ting conditions in lysis buffer (50 mM Tris pH 7.4, 1.0% SDS, 1 mM dithiothreitol, supplemented with cOmplete protease inhibitor).Protein lysates were diluted with four volumes of PBS and sonicated 3 × for 30 s. Cell lysates were cleared by centrifuga tion a t 15 000 g for 10 min and biotinylated proteins were pulled down by incubation with Dynabeads M-280 Streptavidin for 90 min.After washing twice in lysis buffer and twice with PBS, on-bead trypsin digestion was performed and peptides were analyzed by mass spectrometry using Orbitrap Fusion instrument (Q-OT-qIT, Thermo Scientific).All data were analyzed and quantified using MaxQuant (version 1.6.2.1) and P erseus softw ares ( 50 , 51 ).Thr ee biological r eplicates wer e analyzed and median peptide intensities were compared.Statistical significance was calculated using Student's t-test and hits with FDR < 0.05 were considered significant.

In vitro phosphatase assay
Expression and purification of human His-PPM1D was described previously ( 52 ).EGFP-TRF2 was purified from tr ansiently tr ansfected U2OS cells using GFP trap and a high salt IP buffer (50 mM Tris pH 8.0, 1 M NaCl, 1% Tween20, 0.1% NP-40, 10% glycerol, 2 mM EDTA, 3 mM EGTA, 10 mM MgCl 2 ) supplemented with PhosSTOP and protease inhibitors.Beads were washed with a phosphatase buf fer and incuba ted with mock or with 150 ng of the purified His-PPM1D for 20 min at 37 • C. Reaction was stopped by addition of 4 × concentrated Laemli buffer.

Peptide pull down
Biotin-Ahx-ISRLVLEEDpSQSTEPSAGLNamide (TRF2-pS410) and Biotin-Ahx-ISRLVLEEDSQSTEPSAGLN-amide (TRF2-CTRL) peptides were synthesized (Genscript), dissolved in ammonia water and then diluted to 1 mg / ml in TBST (150 mM NaCl, 3 mM KCl, 25 mM Tris pH 8.0, 10% glycerol, 1 mM DTT, 0.1% Tween20).Peptide pull down was performed as described ( 53 ).Dynabeads M-280 Streptavidin (Thermo Fisher Scientific) were incubated with peptides (20 g) in TBST for 60 min and then beads were washed 3 times with TBST.Coupled beads were incubated with Hela nuclear extr act (6 mg / ml, Ipr aCell) for 90 min at 4 • C and then were washed 3 times with TBST and once with PBS.Proteins bound to the beads were digested by trypsin and peptides were analyzed by mass spectrometry.Three independent experiments were compared in one MS measurement.

Fluor escence anisotrop y assay
Purification of human TIN2 was described previously ( 54 ).TRF2-pS410 and TRF2-CTRL peptides fluorescently labelled at N-terminus with carboxyfluorescein (FAM; ex 494 nm, em 518 nm) were synthesized by GenScript.Peptides (3 nM) in a 1.5 ml quartz-glass cuvette with a magnetic stirr er wer e titrated with TIN2 to a final concentration of 500 nM in 50 mM NaCl in 50 mM phosphate buffer, pH 7.0 a t 25 • C .Fluor escence anisotrop y change upon addition of TIN2 was measured at ex 490 nm, em 520 nm with excitation and emission slits 9 nm.Fluorescence anisotropy was measur ed thr ee times, and averaged with a relati v e standar d de viation al ways lower than 3%.The value of the dissociation constant was determined by non-linear least square fits according to the equation: r = r MAX c / ( K D + c ) using Orig-inPro 2022 (OriginLab Corporation) ( 20 ).

Cell proliferation assay
Cell survival assay was performed as described ( 10 ).Briefly, cells were seeded to 96-well pla tes a t 100-130 cells / well, and treated as indicated.Se v en days after tr eatment, r esazurin was added in fresh media at final concentration 30 g / ml.Fluorescence at e xcitation wav elength 560 nm and emission 590 nm was measured using Envision plate reader (PerkinElmer, Waltham, MA, USA) after 2 h incubation.

Statistical analysis
Sta tistic was calcula ted using PRISM 5 (GraphPad Software).Only two-tailed test were used.Student's t-test were performed under the assumption of normality.As a nonparametric test, we used Mann-Whitney statistics.All experiments wer e r eproduced with similar r esults at least two times.

PPM1D interacts with components of the shelterin complex
Protein phosphatase magnesium-dependent 1 delta (PPM1D) is a chromatin-bound protein with poor solubility making analysis of its interacting partners a major challenge ( 8 ).To identify proteins forming a complex with PPM1D, we established a stable HEK293 cell line expressing a phosphatase-dead PPM1D-D314A fused with a proximity-dependent biotin identification (BioID2) tag and control cells expressing empty BioID2 ( 55 , 56 ).After incubating with biotin, cells were extracted under dena tura ting conditions and biotinylated proteins were isolated using streptavidin beads and subsequently identified by mass spectrometry (Figure 1 A, Supplementary Table S1).This analysis revealed that three components of the shelterin complex (namely TRF2, TRF1 and RAP1) and telomere-associated exonuclease DCLRE1B (also known as Apollo) were significantly enriched in complex with PPM1D-D314A-BioID2 fusion protein.To confirm the results from the proximity biotin labelling assay, we performed immunoprecipitation from HEK293 cells expressing EGFP-PPM1D or empty EGFP.We found that EGFP-PPM1D specifically interacted with TRF2 and RAP1 (Figure 1 B).In addition, EGFP-TRF2 and EGFP-TRF1 pulled down endogenous PPM1D from MCF7 cells indica ting tha t PPM1D interacts with shelterin in various cell types (Figure 1 C).To map the interaction between PPM1D and the shelterin, we performed immunoprecipitation with the full length EGFP-PPM1D, a mutant containing the catalytic domain of PPM1D (PPM1D-A380) or a mutant comprising of the unstructured C-terminal region of PPM1D (PPM1D-CT) (Figure 1 D, E).Due to the presence of two NLS sequences located at the C-terminal region and within the B-loop, respecti v ely, the catalytic domain of PPM1D as well as the C-terminal fragment of PPM1D localized in the nucleus (Figure 1 F) ( 57 ).Howe v er, onl y the catal ytic domain of PPM1D but not the C-terminal tail co-immunoprecipitated with TRF2 (Figure 1 E).Moreover, isolated EGFP-TRF2 (but not EGFP alone) was able to pull down purified His-PPM1D in vitro, suggesting that the interaction between TRF2 and PPM1D is direct (Supplementary Figure S1A).
Finally, we tested the interaction between PPM1D and shelterin in cells using a proximity ligation assay ( 43 ).We observed distinct nuclear foci in MCF7 cells when probing for PPM1D and RAP1 (Figure 1 G).Similarly, two distinct sets of antibodies directed against PPM1D and TRF2 showed a strong nuclear PLA signal in MCF7 and U2OS cells (Figure 1 H, Supplementary Figure S1B).Importantly, the specificity of the observed PLA signal was confirmed by a strong reduction caused by treating cells with GSK2830371 (further r eferr ed to as PPM1Di) that pr omotes a pr oteasomal degradation of PPM1D (Figure 1 G, Supplementary Figure S1C) ( 44 , 49 ).Similarly, depletion of TRF2 by RNA interference suppressed the PLA signal thus supporting our conclusion that PPM1D and TRF2 interact in the cell nuclei (Figure 1 H).
Taken together, we conclude that PPM1D interacts through its catalytic domain with se v eral components of the shelterin complex in various cell types regardless of the type of telomere maintenance, including telomerase proficient MCF7 cells and alternati v e telomere lengthening (ALT)dependent U2OS cells.

PPM1D is present at telomeres
Apart from functions at telomeres, TRF2 and TRF1 were reported to localize also to other chromatin compartments (58)(59)(60)(61).Ther efor e, we wonder ed wher e the interaction between PPM1D and the shelterin complex occurred at subcellular le v el.To this end, we transfected U2OS cells with a plasmid expressing an enzymatically inactive dCas9-mCherry reporter together with a telomere-specific sgRNA and we visualized telomeres by confocal microscopy ( 62 ).In parallel, we probed cells with validated antibodies against PPM1D and TRF2 (Supplementary Figure S1D, E) ( 8 ).As expected, signal from the dCas9-mCherry telomeric reporter overlapped with the staining for TRF2 (Figure 2 A).As expected, we observed a dotted nuclear pattern reflecting the localization of PPM1D to the chromatin (Figure 2 A) ( 8 ).In addition, we noticed that a fraction of spots recognized by PPM1D antibody localized at telomer es (Figur e 2 A).To investigate possible contribution of the stochastic cluster over lap, we r andomized PPM1D signal distribution using Interaction Factor package in ImageJ and compared r andom over lap with non-r andom values ( 63 ).We confirmed that the e xperimentally observ ed ov erlap between PPM1D and the telomeric staining in U2OS cells was statistically significant (Figure 2 B).In addition, we observed that PPM1D was present at a pproximatel y 60% of telomeres probably reflecting a dynamic interaction between PPM1D and the shelterin complex (Figure 2 B).Finally, we used an identical experimental approach to determine PPM1D distribution in MCF7 cells (Figure 2 C).We noted that TRF2 foci in MCF7 cells were smaller than in U2OS cells probably reflecting shorter telomeres in MCF7 cells compared to the ALT-positi v e U2OS cells.Ne v ertheless, we found that a fraction of endogenous PPM1D localized at telomeres recognized by TRF2 staining in MCF7 cells (Figure 2 D).Interestingly, the fraction of telomeres positi v e for PPM1D was comparable in MCF7 and U2OS cells (Figure 2 D).In summary, we conclude that PPM1D can associate with telomeres in various cell types.
To identify the regions in PPM1D that are necessary for its localization at telomeres, we transfected cells with plasmids expressing EGFP-tagged PPM1D or its deletion mutants.We found that the wild-type EGFP-PPM1D, a deletion mutant lacking the Proline-rich r egion (r eferr ed to as Pro) and a PPM1D-A380 mutant comprising of the catalytic domain between amino acids 1-380 all were enriched in TRF2 foci (Figure 2 E, Supplementary Figure S1F, G).In contrast, the unstructured C-terminal fragment  of PPM1D failed to accumulate in TRF2-positi v e foci although it showed a strong nuclear staining (Figure 2 E).Finally, a deletion mutant lacking amino acids 246-251 of the B loop (r eferr ed to as B) localized to the nucleus but it failed to co-localize with TRF2 (Figure 2 E, Supplementary Figure S1F, G).Thus, the microscopic analysis re v ealed that the B loop in the catalytic domain of PPM1D mediates its localiza tion a t telomeres, which is in good agreement with the data from immunoprecipitation (Figure 1 E).In addition, the observed difference between intensities of the wildtype EGFP-PPM1D and the EGFP-PPM1D-A380 mutant suggests that the C-terminal tail of PPM1D may be involved in negati v e regula tion of PPM1D localiza tion a t telomeres.

PPM1D counteracts ATR-dependent phosphorylation of TRF2 at S410
As PPM1D localizes at telomeres, we wondered if it could regula te the phosphoryla tion of the shelterin components either in context of the cell cycle progression or following DNA damage at telomeres.Since PPM1D has been implicated in termination of the global DNA damage response, we have focused on the phosphorylations triggered by exposure of cells to genotoxic stress.Unfortunately, commercial antibodies raised against se v eral phosphopeptides in TRF2 and TRF1 did not show sufficient le v el of sensitivity and specificity pre v enting us from testing the effect of PPM1D activity (data not shown).Ther efor e, we generated an affinity purified rabbit polyclonal antibody against the phosphorylated S410 of TRF2 that is conserved across species, matches a consensus motif for A TM / A TR and PPM1D and has previously been detected in cells exposed to ionizing radiation or to treatment with cytarabine (Supplementary Figure S1H) (64)(65)(66).First, we tested the reactivity of pTRF2-S410 antibody using the wild-type EGFP-TRF2 or the EGFP-TRF2-S410A mutant immunopurified from HEK293 cells.Importantly, we observed a strong reduction of the signal in the alanine mutant, confirming that the pTRF2-S410 antibody predominantly recognizes the phosphorylated form of TRF2 in immunoblotting (Figure 3 A).In addition, we found that the basal le v el of pTRF2-S410 signal that was increased by treatment of the cells with hydroxyurea and / or PPM1D inhibitor which is consistent with the DNA damage-induced phosphorylation of TRF2 that is counteracted by PPM1D (Figure 3 B).In agreement with this possibility, we found that purified His-PPM1D dephosphorylated the purified TRF2 at S410 in vitro (Figure 3 C).Next, we used control HeLa cells or cells with doxy cy cline-induced knock-down of TRF2 and exposed them to ionizing radiation (60 Gy) ( 45 ).In non-treated cells, the phosphorylation of endogenous TRF2 was below the detection limit in the nuclear extracts.On the other hand, the e xtensi v e DNA damage induced the signal of pTRF2-S410 antibody and importantly, the specificity was confirmed by depletion of the TRF2 (Figure 3 D).As expected, treatment of cells with PPM1Di decreased the le v el of PPM1D protein and induced ␥ H2AX staining ( 8 , 44 , 49 ).In addition, we found that inhibition of PPM1D further increased the pTRF2-S410 signal suggesting that PPM1D might dephosphorylate pTRF2-S410 (Figure 3 D).To validate specificity of the pTRF2-S410 antibody in immunoflu-or escence microscop y, we depleted endogenous TRF2 in U2OS cells by RNAi and treated them or not with PPM1D inhibitor (Figure 3 E).As expected, we observed a strong induction of the pTRF2-S410 signal at telomeres upon treatment of control cells with PPM1D inhibitor.Importantly, the signal was lost upon depletion of TRF2, thus confirming specificity of the antibody (Figure 3 E).Further, we observed an increase in pTRF2-S410 phosphorylation in U2OS-PPM1D-KO cells and the signal was significantly reduced by expression of the FLAG-PPM1D confirming that the observed phenotype was indeed caused by the loss of PPM1D (Figure 3 F).We conclude that PPM1D counteracts the TRF2-S410 phosphorylation at telomeres.
As the basal le v el of pTRF2-S410 signal in non-treated cells was relati v ely low, we searched for conditions that would stimulate the phosphorylation of TRF2.Upon exposure to ionizing radiation, DSBs are randomly generated across the genome making interpretation of e v ents observ ed at telomeres difficult.To induce DSBs specifically at telomeres, we transfected cells with a plasmid expressing Cas9 and a sgRNA targeting the telomeric repeats ( 67 ).Consistent with previous reports, we observed formation of the telomeric dysfunction-induced foci (TIFs) defined by recruitment of 53BP1 and by phosphorylation of H2AX at S139 (called ␥ H2AX) (Figure 3 G, H, Supplementary Figure S1I) ( 68 ).As expected, DSBs induction eventually resulted in telomere clustering that we observed as r educed telomer e count and increased area of the foci (Supplementary Figure S2A-C) ( 69 ).In addition, we noted an increased ␥ H2AX signal in cells lacking PPM1D, which is in agreement with the previously described role of PPM1D in dephosphoryla ting H2AX a t chroma tin (Figure 3 H, Supplementary Figure S1I) ( 8 ).Importantl y, telomeric DN A damage also strongly induced the TRF2-S410 phosphoryla tion a t telomeres and the signal was further increased in U2OS-PPM1D-KO cells (Figure 3 I, Supplementary Figure S2D).Of note, pTRF2-S410 signal was significantly enriched at telomeres in U2OS-PPM1D-KO cells without any induction of telomeric damage suggesting that PPM1D may constantly dephosphorylate TRF2 at telomer es (Figur e 3 I, Supplementary Figure S2D).
Finally, we induced DSBs at telomeres in cells treated with small molecule inhibitors of the major protein kinases responding to DNA damage and assayed the impact on protein phosphoryla tion a t telomeres.Similarly to DSBs induced by TRF1-FokI, we observed that inhibition of ATM reduced the le v el of ␥ H2AX at telomer e br eaks induced by Cas9 (Figure 3 J) ( 70 ).In contrast, pTRF2-S410 phosphorylation was insensiti v e to the inhibition of ATM but was reduced upon treatment with ATR inhibitor (Figure 3 K, Supplementary Figure S2E).Similarly, we observed that RNAimediated depletion of ATR (but not ATM) supressed the le v el of pTRF2-S410 phosphorylation (Supplementary Figure S2F-H).We conclude that following induction of DSBs at telomeres, TRF2 phosphorylation at S410 is inversely regulated by ATR and PPM1D.

TRF2 phosphorylation at S410 increases its binding to TIN2
Se v eral recent studies have identified regions within individual shelterin components tha t media te pr otein-pr otein interactions and are critically needed for folding of the shelterin complex ( 16 , 25 , 30 , 31 , 71 ).For instance, the TRF1 TRFH (residues 58-268) and TRF2 TRFH (residues 84-287) domains interact with a TRFH-binding motif (TBM) of TIN2 (residues 256-276) ( 30 ).In addition, TRFH domain of TIN2 interacts with a recently described TBM2 region of TRF2 (residues 392-408) ( 31 ).As the published crystal structure of TIN2 TRFH -TRF2 TBM2 interaction interface lacks the structural information for S410, we used Alphafold2 Colab to predict the position of residues 392-420 of TRF2 ( 30 ).The structural alignment of Alphafold2 model showed a perfect overlap with the crystal structure (with RMSD 0.252 Å ) (Supplementary Figure S3A).In this model, S410 of TRF2 is positioned opposite the positi v ely charged residues of TIN2 TRFH suggesting that phosphorylation of S410 might strengthen the TRF2-TIN2 interaction by formation of salt bridges between the phosphate and basic residues in the AA50-56 region of TIN2 (Supplementary Figure S3A).To experimentally test the impact of TRF2 TBM2 phosphorylation on TRF2-TIN2 interaction, we designed se v eral independent assays.First, we performed a pull down from the nuclear extracts using biotinyla ted phosphoryla ted or non-phosphoryla ted peptides of TRF2 as baits.Mass spectrometry analysis re v ealed tha t the phosphoryla ted but not the non-phosphoryla ted TRF2 peptide specifically pulled down TIN2, TPP1 and POT1 (Figure 4 A, Supplementary Table S2).Second, we quantified the binding affinities of the purified TIN2 with short, fluorescently labelled TRF2 oligopeptides containing phosphorylated or non-phosphorylated S410 (Figure 4 B).Analysis of the binding isotherms showed that the binding affinity for unmodified TRF2-S410 oligopeptide was K D = 240 ± 80 nM that corresponded well to the pr eviously r eported affinity for TRF2-TIN2 binding ( 71 ).When S410 was phosphorylated, we observed a significant increase of the binding affinity with the corresponding K D = 180 ± 30 nM.To confirm the data from the in vitro assays, we tested the interaction between TRF2 and TIN2 in cells by immunoprecipitation (Figure 4 C).Consistent with the pr evious r eports, we observed that the wild-type EGFP-TRF2 interacted with TIN2 ( 25 ).In addition, the non-phosphorylatable EGFP-TRF2-S410A mutant pulled down a reduced but still detectable level of TIN2, suggesting tha t modifica tion of S410 is not absolutely r equir ed for the basal interaction between TRF2-TIN2 (Figure 4 C).This finding is in agreement with the previous report where interaction was observed with a TRF2 TBM2 fragment (residues 392-408) lacking the S410 site ( 72 ).Interestingly, howe v er, we observed an increased interaction between the phosphorylation mimicking EGFP-TRF2-S410E m utant, w hich is consistent with increased binding affinity between TRF2 and TIN2 upon phosphorylation (Figure 4 C).
To test if the TRF2 interaction with TIN2 is regulated by PPM1D, we performed the PLA assay in parental U2OS cells, U2OS-PPM1D-KO and U2OS cells treated with PPM1D inhibitor.We observed that loss or inhibition of PPM1D significantly increased the interaction between TRF2 and TIN2 (Figure 4 D, E, Supplementary Figure S3B).Consistent with this, we found that TIN2 was enriched at telomeres in U2OS cells treated with PPM1D inhibitor compared to the non-treated cells (Figure 4 F).Similarly, intensity of the TIN2 signal at telomeres was increased in U2OS-PPM1D-KO cells compared to the parental U2OS cells and the le v el was rescued by expression of the wild-type EGFP-PPM1D (Figure 4 G).Importantly, total protein levels of TRF2 and TIN2 remained unchanged in U2OS-PPM1D-KO cells thus excluding the possibility that the observed enrichment of TIN2 at telomeres is a consequence of alter ed protein expr ession (Figur e 4 H).In contrast to TIN2, we did not observe any increase in TRF2 accumulation at telomeres in cells lacking PPM1D suggesting that the incr eased r ecruitment of TIN2 depends on phosphorylation status of TRF2 rather than a change of its total le v els at the telomer e (Figur e 4 I).As TIN2 mediates the recruitment of and bound proteins were analyzed by immunoblotting.( B ) HEK293 stably expressing EGFP-TRF2 were treated with DMSO, HU (2 mM), PPM1Di (1 M) or combination of both for 18 h.Cell extracts were incubated with GFP trap and bound proteins were analyzed by immunoblotting.( C ) In vitro phosphatase assay.EGFP-TRF2 was isolated from cells by GFP Trap in a buffer containing 1 M NaCl.Beads were washed with a phosphatase buffer and incubated with mock or with the purified His-PPM1D for 20 min a t 37 • C .Le v el of TRF2-S410 phosphorylation was analyzed by immunoblotting.( D ) HeLa cells stably transfected with inducible TRF2 shRNA were treated with mock or with doxy cy cline (2 g / ml) for 7 days and were exposed or not to IR (60 Gy).Where indicated, cells were incubated with PPM1Di for the last 12 h.Nuclear extracts were separated on 4-20% SDS-PAGE gel and analyzed by immunoblotting.( E ) U2OS cells after two consecutive transfections of control or TRF2 siRNA were treated or not with PPM1D inhibitor (2 M, 4 h), fixed and co-stained for TIN2 (telomeric marker) and pTRF2-S410.Plotted is the mean pTRF2-S410 intensity in TIN2-positi v e foci, each dot r epr esents a single cell (n = 500).Bars indicate mean ± SD, statistical significance was evaluated using Mann-Whitney test (**** P < 0.0001).Representati v e experiment is shown from two independent repeats.The scale bar in r epr esentati v e images corresponds to 10 m. ( F ) P ar ental U2OS, U2OS-PPM1D-KO and U2OS-PPM1D-KO cells stab ly e xpr essing FLAG-PPM1D wer e tr eated or not with PPM1D inhibitor for 24 h.Cells were pre-extracted, fixed and stained for TRF2 and pTRF2-S410.Plotted is the mean pTRF2-S410 intensity in TRF2-positi v e foci, each dot r epr esents a single cell ( n = 300).Bar indicates mean ± SD, statistical significance was evaluated using Mann-Whitney test (**** P < 0.0001).Representati v e e xperiment is shown from two independent repeats.( G ) U2OS cells were transfected with plasmids coding for Cas9-EGFP with or without the telomeric repeat-targeting sgRNA.After 24 h, cells wer e fix ed, hybridized with telomeric FISH probe, and stained for 53BP1 (TIF marker).The scale bar r epr esent 10 m).Bar indicates mean ± SD, n = 300.( H ) P ar ental U2OS or U2OS-PPM1D-KO cells were transfected with plasmids coding for Cas9-EGFP with or without telomeric repeat-targeting sgRNA.After 24 h, cells were fixed and stained for ␥ H2AX.Mean nuclear intensity is plotted ± SD, n ≥ 208.Statistical significance was evaluated using Mann-Whitney test (**** P < 0.0001).Representati v e e xperiment is shown from thr ee independent r epeats.( I ) U2OS cells wer e transfected as in H and were stained for TRF2 and pTRF2-S410.Plotted is the mean pTRF2-S410 intensity in TRF2-positi v e foci.Bars indicate mean ± SD, n ≥ 150.Statistical significance was evaluated using Mann-Whitney test (**** P < 0.0001).Representati v e e xperiment is shown from two independent repeats.( J ) U2OS cells were transfected with plasmids coding for Cas9-EGFP with or without the telomeric repeat-targeting sgRNA and were treated with indicated inhibitors for 20 h.After fixation, the intensity of ␥ H2AX signal in TRF2 foci was determined by ScanR microscopy.Bars indicate median ± SD, n ≥ 161.Statistical significance was evaluated using Mann-Whitney test (**** P < 0.0001).Representati v e e xperiment is shown from two independent repeats.( K ) U2OS cells wer e tr eated as in (J) and wer e probed with TRF2 and pTRF2-S410 antibodies.Plotted is the mean pTRF2-S410 intensity in TRF2 foci.Bars indicate median ± SD, n ≥ 249.Statistical significance was evaluated using Mann-Whitney test (**** P < 0.0001).Representati v e e xperiment is shown from two independent repeats.TPP1-POT1 to TRF1 / 2 we also evaluated the amount of TPP1 at telomeres.We observed that inhibition or loss of PPM1D increased the le v el of TPP1 at telomeres confirming that the activity of PPM1D may regulate assembly of the shelterin complex at telomeres (Figure 4 J, K).Similarly to U2OS cells, we observed that inhibition of PPM1D increased the phosphorylation of TRF2-S410 as well as the le v els of TIN2 and TPP1 at telomeres in MCF7 cells, suggesting that the impact of PPM1D activity on recruitment of shelterin components to telomeres is not restricted to cells with alternati v e lengthening of telomeres (Supplementary Figure S3C, D and E).
TRF2 and TIN2 jointly protect telomeric ends by promoting formation of t-loop and ther efor e we asked if manipulation with the strength of TRF2:TIN2 binding by removing PPM1D activity could affect t-loop formation.To this end, we pr epar ed chromatin spr eads from the par ental U2OS and U2OS-PPM1D-KO cells and determined fractions of the linear or looped chromosome ends by 3D-SIM microscopy as previously described ( 33 ).Consistent with published literature, we observed t-loops in about 25% of chromosomes ( 33 , 73 ).Howe v er, we did not find any significant differences between parental U2OS and U2OS-PPM1D-KO cells (Figure 4 L, Supplementary Figure S3F) suggesting that PPM1D activity does not interfere with formation of the t-loop.On the other hand, we cannot exclude tha t dif ferences in organisa tion of the chromosome ends caused by loss of PPM1D are below the sensitivity of the assay because we were unable to conclusively categorize about a half of the imaged telomeres.

Increased PPM1D activity impairs assembly of the shelterin complex
As the interaction of TRF2 and TIN2 responded to the inhibition of PPM1D, we asked if increased activity of PPM1D might interfere with function of the shelterin complex at telomeres.Indeed, we found that ov ere xpression of the wild-type PPM1D decreased the amount of TIN2 at telomer es (Figur e 5 A).In addition, we observed that expression of the A380 fragment of PPM1D (that showed the strongest targeting to the telomeres in Figure 2 E) efficiently stripped TIN2 from the telomeres (Figure 5 A).Of note, expression of the A380 fragment of PPM1D also reduced the intensity of TRF2 staining at telomeres suggesting that assembly of the shelterin may be impaired after dephosphorylation by PPM1D (Figure 5 B).
To study consequences of the increased PPM1D expression, we de v eloped a doxy cy cline-inducib le RPE1-PPM1D-A380 cells (Supplementary Figure S4A), and followed formation of TIFs upon treatment with doxy cy cline for 10 days (Figure 5 C).Although the fraction of cells with > 3 TIFs was slightly higher in cells treated with doxy cy cline compared to control cells, the difference was not statistically significant (Figure 5 C).As PPM1D can target ␥ H2AX and ATM, we hypothesised that failure to form TIFs could be caused by overall suppression of DDR by PPM1D activity ( 8 , 74 ).Ther efor e, we tr eated RPE1-PPM1D-A380 cells for 10 days to allow formation of potential telomeric damage and then treated cells with PPM1D inhibitor just before fixation to allow activation of DDR.Indeed, transient PPM1D inhibition increased activity of ATM as documented by increased le v el of KAP1-S824 phosphorylation (Figure 5 C).Consistently, upon transient inhibition of PPM1D, we observed a significant increase of TIF formation in cells expressing PPM1D-A380 suggesting that these cells experienced telomeric damage (Figure 5 C).Next, we analyzed telomeric damage in RPE1-PPM1D-A380 cells by telomeric FISH in metaphase spreads (Figure 5 D).We noted that the fraction of telomeric fusions was doubled in RPE1-PPM1D-A380 cells treated with doxy cy cline compared to control cells Figure 5 D) confirming that increased PPM1D activity in cells promotes damage of the telomeric DNA.
Finally, we asked if the phosphorylation of TRF2 is required for cell proliferation.To this end, we used HeLa cells identified by mass spectrometry ( n = 3).Plotted are -log P values of proteins enriched or reduced in condition with TRF2-pS410 peptide.The line delineates the statistical significance (FDR < 0.1).( B ) Fluorescently-labelled TRF2-pS410 and TRF2-CTRL peptides were titrated with purified TIN2 to a final concentration of 500 nM.Fluorescence anisotropy change was measured and dissociation constant values for unmodified and modified oligopeptides were calculated as described in Methods.( C ) HEK293 cells stably expressing EGFP-TRF2 were treated with DMSO or with PPM1D inhibitor for 4 h.EGFP-TRF2 was immunoprecipitated from cell extracts with GFP Trap.Proteins were separated by SDS-PAGE and binding of TIN2 was determined by immunoblotting.Numbers at the bottom indicate the TIN2 signal relati v e to the total immunoprecipitated TRF2 and normalized to the wild-type TRF2.Representati v e result from three experiments is shown.( D ) TRF2:TIN2 interaction was determined in parental U2OS and U2OS-PPM1D-KO cells by PLA.Mean PLA foci count is plotted ± SD, n = 500.Statistical significance was evaluated using Mann-Whitney test (**** P < 0.0001).Representati v e experiment is shown from two independent repeats.( E ) TRF2:TIN2 interaction was determined in U2OS cells treated with DMSO or PPM1Di by PLA.Mean PLA foci count is plotted ± SD, n = 500.Statistical significance was evaluated using Mann-Whitney test (**** P < 0.0001).Representati v e e xperiment is shown from two independent repeats.( F ) U2OS cells were treated or not with PPM1Di for 24 h, pre-extracted, fixed and stained with TRF2 (m-Santa Cruz) and TIN2 (Rb-Novus) antibodies.Mean TIN2 intensity in TRF2 foci is plotted ± SD, n = 300.Statistical significance was evaluated using Mann-Whitney test (**** P < 0.0001).Representati v e e xperiment is shown from two independent repeats.The scale bar r epr esents 10 m. ( G ) P ar ental U2OS, U2OS-PPM1D-K O and U2OS-PPM1D-K O stab ly e xpr essing FLAG-PPM1D cells wer e tr eated or not with PPM1Di for 24 h.Cells wer e pr e-extracted, fixed and stained for TIN2 and TRF2.Mean TIN2 intensity in TRF2 foci ± SD is plotted, n = 300.Statistical significance was evaluated using Mann-Whitney test (**** P < 0.0001).Representati v e e xperiment is shown from two independent repeats.( H ) Le v els of TRF2 and TIN2 were analyzed in whole cell extracts from the parental U2OS and U2OS-PPM1D-KO cells by immunoblotting.Importin staining was used as a loading control.( I ) Cells from G were analysed for TRF2 intensity in TRF2 foci.Plotted is mean ± SD, n = 300.( J ) U2OS cells wer e tr eated or not with PPM1Di for 24 h, pr e-extracted, fixed and stained with TRF2 and TPP1 antibodies.Mean TPP1 intensity in TRF2 foci normalized to the mean nuclear TPP1 intensity ± SD is plotted, n > 300.Statistical significance was evaluated using Mann-Whitney test (**** P < 0.0001).The scale bars in r epr esentati v e images corresponds to 10 m and 1 m respecti v ely.( K ) Parental U2OS, U2OS-PPM1D-KO cells and U2OS-PPM1D-KO stab ly e xpr essing FLAG-PPM1D cells wer e pr e-extracted, fixed and stained for TPP1 and TRF2.Mean TPP1 intensity in TRF2 foci normalized to the mean nuclear TPP1 intensity ± SD is plotted, n > 300.Statistical significance was evaluated using Mann-Whitney test (**** P < 0.0001, *** P < 0.001).( L ) Chromosome spreads from parental U2OS and U2OS-PPM1D-KO cells were hybridized with TAACCC FISH-probe and imaged by 3D-SIM.Plotted is a fraction of telomeres that formed t-loops.More than 203 telomeres were quantified per condition in each experiment ( n = 3).Significance was determined by unpaired t -test.with inducible knock down of endogenous TRF2 and stably reconstituted them with the wild-type or S410A mutant TRF2 (Figure 5 E).After 12 days of doxy cy cline treatment, we compared relati v e proliferation and found that two independent clones expressing S410A TRF2 proliferated significantly worse than the cells expressing the wild-type TRF2 (Figure 5 F) suggesting that impaired phosphorylation of TRF2 leads to suppression of cell proliferation.

Loss of PPM1D supresses recruitment of DNA repair proteins to the DSBs at telomeres
Finally, we investigated the consequence of altered PPM1D activity for DNA repair at telomeres.We induced DSBs at telomeres by Cas9 and compared recruitment of various DNA repair factors in control cells and in PPM1D-KO cells.We found no difference in recruitment of NBS1 suggesting that recognition of the DNA break by MRN complex was unaffected by the loss of PPM1D (Figure 6 A, Supplementary Figure S4B).In contrast, we observed that recruitment of 53BP1 protein to telomeric DSBs was significantly reduced in U2OS-PPM1D-KO cells (Figure 6 B, C).Similarly, formation of the 53BP1 foci upon Cas9-mediated DNA damage at telomeres was impaired in MCF7 and RPE1 cells treated with PPM1D inhibitor (Supplementary Figure S4C, D).Importantly, recruitment of 53BP1 to damaged telomeres was rescued in U2OS-PPM1D-KO cells by expression of the wild type EGFP-PPM1D (but not with the phosphatase dead D314A mutant) confirming that the phenotype was indeed caused by a loss of PPM1D activity (Figure 6 B, C).We also noted that the le v el of protein ubiquitination detected by FK2 antibody was reduced at damaged telomeres in U2OS-PPM1D-KO cells (Figure 6 D, Supplementary Figure S4E).Histone H2A ubiquitination is r equir ed for r ecruitment of 53BP1 and BRCA1 to DNA damage foci, and thus the lack of ubiquitina tion a t telomeres may explain the decreased le v el of 53BP1 in cells treated with PPM1D inhibitor ( 75 ).As the mouse TRF2 has previously been shown to recruit a deubiquitinating enzyme BRCC3 through a so-called iDDR region ( 36 ), we tested if the observed defect of 53BP1 binding upon inhibition of PPM1D could be rescued by depletion of BRCC3.Howe v er, we did not observe any difference in 53BP1 recruitment to the telomeric DSBs suggesting that the phosphorylation of TRF2 at S410 suppresses 53BP1 recruitment through a distinct molecular mechanism than the iDDR region (Supplementary Figure S4F).
Besides impaired formation of 53BP1 foci, we also observed strongly reduced recruitment of RAD51 to the telomeric breaks suggesting that the repair through homologous recombination is also impaired (Figure 6 E, F).To investigate if the effect of PPM1D inhibition on TRF2 phosphorylation and reduced recruitment of 53BP1 are functionally linked, we co-expressed Cas9 together with the telomeric sgRNA and various forms of TRF2 in cells treated or not with PPM1D inhibitor.Wher eas expr ession of the wild-type EGFP-TRF2 did not fully rescue recruitment of 53BP1 to damaged telomeres, expression of the EGFP-TRF2-S410A mutant significantly increased the le v el of 53BP1 at damaged telomer es (Figur e 6 G).This result suggests that PPM1D promotes recruitment of 53BP1 to DNA breaks at telomeres by dephosphorylating TRF2.
To evaluate the functional outcome of PPM1D inhibition at damaged telomeres, we determined the relati v e proliferation of RPE1-iCut cells upon induction of a mild telomeric DNA damage achie v ed by titrating down of the amount of telomeric sgRNA ( 46 ) (Figure 6 H, Supplementary Figure S4G).We found that PPM1D inhibition significantly suppressed proliferation of the RPE1-iCut cells that experienced telomeric DNA damage (Figure 6 H).We conclude that PPM1D activity is needed for the cell response to telomeric DNA damage although the precise molecular defect in DNA repair remains to be addressed by future research.
In the summary, we show that TRF2 is phosphoryla ted a t S410 upon DNA damage at telomeres by ATR which promotes its interaction with TIN2 and limits recruitment of 53BP1 to the breaks.Phosphorylation of TRF2 is re v ersed by the activity of PPM1D phospha tase tha t promotes recruitment of 53BP1 to telomeres (Figure 6 I).Physiological le v els of TRF2 phosphorylation ar e r equir ed for cell survival as increased TRF2 phosphorylation does not allow efficient repair, while impaired TRF2 phosphorylation su- is the mean NBS1 signal in TRF2 foci ± SD, n ≥ 171.Statistical significance was evaluated using Mann-Whitney test.Representati v e e xperiment is shown from two independent r epeats.( B ) P ar ental, U2OS-PPM1D-KO cells and U2OS-PPM1D-KO cells stab ly e xpressing FLAG-PPM1D variants were transfected with plasmids coding for Cas9-EGFP with or without the telomere-targeting sgRNA.After 24 h, cells wer e fix ed and stained for 53BP1, the scale bar r epr esents 10 m. ( C ) Quantification of (B).Plotted is the mean of 53BP1 foci count ± SD, n ≥ 221.Statistical significance was evaluated using Mann-Whitney test (**** P < 0.0001).Representati v e e xperiment is shown.( D ) Cells wer e tr eated as in A and wer e stained for TRF2 and conjugated ubiquitin using Fk2 antibody.Plotted is the mean FK2 signal in TRF2 foci ± SD, n ≥ 205.Statistical significance was evaluated using Mann-Whitney test (**** P < 0.0001).Representati v e e xperiment is shown from two independent r epeats.( E ) P ar ental and U2OS-PPM1D-KO cells wer e transfected as in (A), fixed, and stained for RAD51 and TRF2.Plotted is mean RAD51 intensity in TRF2 foci ± SD, n ≥ 161.Statistical significance was evaluated using Mann-Whitney test (**** P < 0.0001).Representati v e e xperiment is shown from two independent r epeats.( F ) Repr esentati v e images for (E), the scale bar r epr esents 10 m. ( G ) P ar ental and U2OS-PPM1D-KO cells were co-transfected with plasmids coding for GFP or GFP-TRF2 variants, and FLAG-Cas9 with or without the telomere-targeting sgRNA, and treated or not with PPM1Di for 24 h.Cells were fixed and stained for 53BP1 and FLAG.Only FLAG and GFP double positive cells were analyzed.Means of three independent experiments are plotted ± SD.Statistical significance was evaluated using unpaired t -test.Representati v e images are shown, the scale bar represents 10 m. ( H ) RPE1-iCut cells were treated overnight with doxycycline and Shield-1 and telomeric DNA damage was induced by transfection of indicated amounts of telomeric sgRNA.Cells were incubated with DMSO or PPM1D inhibitor for 7 days.Relati v e proliferation was determined by resazurin assay and was normalized to non-treated cells ( n = 3).( I ) Model of pTRF2-S410 function at telomere.Under basal conditions, non-phosphorylated TRF2 interacts with TIN2 through its TRFH domain and with telomeric DNA through its Myb domain.Induction of DSBs at telomeres leads to recruitment of DNA repair factors including 53BP1.Upon activation of ATR, TRF2 is phosphorylated at S410, which promotes tight binding of TIN2 and protects the broken telomere from recruitment of 53BP1.Loss of PPM1D activity leads to hyper-phosphorylation of TRF2 and pre v ents recruitment of 53BP1 to the telomeric DSBs, possibly decreasing the risk of the telomere fusion.
presses shelterin complex assembly and may lead to telomeric fusions.

DISCUSSION
Se v eral components of the shelterin complex were reported to undergo phosphorylation at various conditions, howe v er only some of these e v ents were thoroughly characterized ( 76 ).Most importantly, CDK-dependent phosphorylation of TRF2 at Ser365 pre v ents recruitment of the helicase RTEL1 to telomeres ( 35 ).During S phase, TRF2-Ser365 is dephosphorylated by PP6 phosphatase that promotes recruitment of RTEL1, unwinding the t-loops and telomere r eplication ( 34 , 35 ).Following exposur e of cells to ionizing radiation, TRF2 was reported to be transiently phosphoryla ted a t Thr230 allowing its association with DNA lesions outside the telomeres and promoting DNA repair (77)(78)(79).Howe v er, the role of TRF2 modification in DNA repair of the telomeric lesions has remained unclear.
Her e, we r eport a new phosphoryla tion of TRF2 a t S410 that is strongly induced by Cas9-mediated DSBs at telomeres.Using specific small-molecule inhibitors and RNA interference, we identify ATR as the major kinase responsible for TRF2-S410 modification at damaged telomeres.Further, we show that the le v el of TRF2-S410 phosphorylation is tightly regulated by PPM1D phosphatase that associates with TRF2 and localizes at the telomeres.Loss of PPM1D or inhibition of its enzymatic activity strongly induced TRF2-S410 phosphoryla tion a t telomeres and promoted recruitment of TIN2 and TPP1 to the telomeres.Since the S410 is located close to the TBM2 region responsible for the interaction with TIN2, we tested the impact of TRF2-S410 phosphorylation on this interaction.An unbiased pr oteomic appr oach re v ealed that the phosphorylated peptide spanning residues 403-417 of TRF2 (but not the non-phosphorylated counterpart), pulled down the TIN2-TPP1-POT1 trimer from the nuclear extract.Subsequently, a fluor escence anisotrop y assa y perf ormed with synthetic peptides and with purified TIN2 confirmed that TRF2 phosphoryla tion a t S410 increases the affinity between TRF2 and TIN2.When expressed in cells, the nonphosphorylatable TRF2-S410A mutant was able to interact with TIN2, which suggests that phosphorylation is not critically needed for mediating the interaction.On the other hand, the PLA assay re v ealed a stronger interaction between TRF2 and TIN2 upon inhibition of PPM1D that increases the le v el of TRF2 phosphorylation at S410.As TRF2 and TIN2 protect the ends of telomeres by promoting t-loop formation, we tested if the activity of PPM1D affects the ar chitectur e of the telomeric ends through r egulating the shelterin comple x assemb ly.To address this, we imaged the telomeres in psoralen-crosslinked chromatin spreads using Structured Illumination Microscopy and determined the fractions of linear and closed telomeres.Consistent with the published literature, we observed t-loops in about 25% of telomeres in parental cells ( 33 ).Ne v ertheless, fraction of the t-loops was comparable in U2OS-PPM1D-KO cells suggesting that PPM1D activity may not affect the t-loop forma tion.As approxima tely half of the imaged telomeres is excluded from the analysis due to inconclusi v e shape, we also cannot rule out the possibility that the assay is not sen-siti v e enough to detect mild differences in the t-loop forma tion.Alterna ti v ely, the acti vity of PPM1D may impact a higher-order organization of the telomeres mediated in cis and trans by TRF2 and TIN2 ( 40 ).
The main finding of this study is that PPM1D is needed for DNA damage response at telomeric DSBs (Figure 6 I).When PPM1D activity was present, cells recruited DNA repair factors to the DSBs loca ted a t telomeres.Conversely, loss or inhibition of PPM1D impaired recruitment of the DNA repair factors 53BP1 and RAD51 to the broken telomeres.As the non-phosphorylatable TRF2-S410A mutant rescued the recruitment of 53BP1 significantly better than the wild-type TRF2, we concluded that phosphorylation of TRF2 inhibits DNA damage response at telomeres.The dimerization domain and the iDDR region (corresponding to residues 449-473 of human TRF2) within the hinge domain of TRF2 were previously shown to supress the DNA damage response at telomeres by pre v enting activation of ATM and by inhibiting the RNF168-dependent ubiquitination, respecti v ely ( 36 ).We found that the formation of 53BP1 foci at telomeric DSBs was not rescued by depletion of the BRCC3 or UBR5 in U2OS-PPM1D-KO cells suggesting that PPM1D affects DDR independently of the iDDR region in TRF2.We hypothesize that DSBinduced phosphorylation of TRF2 may allow cells to reestablish the telomere organization by promoting TRF2 association with TIN2-TPP1-POT1.An increased assembly of the shelterin may then interfere with the recruitment of 53BP1 to the break, thus limiting the risk of telomeric fusions.In contrast, dephosphorylation of TRF2 and weakening its interaction with TIN2-TPP1-POT1 could make the telomer e mor e accessible to the recruitment of the DNA repair proteins.
We also noted that ov ere xpression of PPM1D decreased the le v els of TRF2 at telomeres which is in line with the disassembly of the shelterin after dephosphorylation of its components.Howe v er, we did not observe the formation of the TIFs upon ov ere xpression of PPM1D, possibly due to the ability of PPM1D to efficiently suppress the activity of ATM ( 7 , 80 ).We propose that PPM1D activity needs to be tightly balanced at telomeres to allow the recruitment of DNA repair proteins to DSBs while pre v enting disassembly of the shelterin from the telomeres.Of note, high levels of acti v e PPM1D ar e commonly pr esent in cancer cells due to amplification of the chromosomal locus 17q23 or due to gain-of-function mutations in the last exon of PPM1D ( 11 , 12 , 65 , 81 , 82 ).It is tempting to speculate that besides the established role of the over expr essed PPM1D in overriding the cell cycle checkpoint, the increased activity of PPM1D could promote genome instability in cancer cells by interfering with the telomere functions.

Figure 1 .
Figure 1.PPM1D interacts with component of the shelterin complex.( A ) HEK293 cells stably expressing PPM1D-D314A-BioID2 or empty BioID2 were lysed 5h after treatment with biotin.Biotinylated proteins were pulled down by streptavidin beads and bound proteins were analyzed by MS ( n = 3).Volcano plot shows -log P values for proteins enriched or reduced in PPM1D-BioID2 sample.Line delineates the statistical significance (FDR < 0.05).( B ) HEK293 cells were lysed 24 h after transfection with plasmids expressing EGFP or EGFP-PPM1D and cell extracts supplemented with bensonase were incubated with GFP trap.Bound proteins were analyzed by immunoblotting.( C ) MCF7 cells were transfected with plasmids expressing EGFP, EGFP-TRF1, or EGFP-TRF2.Cell extracts supplemented with bensonase were incubated with GFP trap.Binding of PPM1D was probed by immunoblotting.( D ) Scheme of EGFP-tagged PPM1D constructs used in the study.Shown are the catalytic domain in yellow, the basic loop in magenta, the Proline-rich loop in cyan and the NLS in grey.Note that an additional NLS is located within the B loop. ( E ) HEK293 cells were transfected with plasmids expressing EGFP, the wild type EGFP-PPM1D, EGFP-PPM1D-A380 corresponding to the catalytic domain, or EGFP-PPM1D-CT corresponding to the unstructured C-terminal tail of PPM1D.Cell extracts were incubated with GFP trap and binding of TRF2 was evaluated by immunoblotting.( F ) U2OS were transfected with plasmids coding for EGFP-PPM1D variants.Cells were fixed and visualized by wide-field microscopy, the scale bar represents 10 m.Representati v e images are shown.( G ) MCF7 cells were fixed and probed for interaction of PPM1D with RAP1 by PLA assay.W here indica ted, cells were treated with PPM1D inhibitor for 24 h.Mean count on nuclear PLA foci is plotted ± SD, n = 300.Statistical significance was evaluated using Mann-Whitney test, (**** P < 0.0001).Representati v e e xperiment is shown from two independent repeats.The scale bar in r epr esentati v e images corresponds to 10 m. ( H ) MCF7 cells were transfected twice with control siRNA (siNC) or siRNA to TRF2.After 6 days, cells were fixed and probed for interaction of PPM1D with TRF2 by PLA assay using two different pairs of antibodies (rabbit rb-PPM1D / mouse m-TRF2, mouse m-PPM1D / rabbit rb-TRF2).W here indica ted, cells were treated with PPM1D inhibitor for 18 h prior fixation.Mean count of the nuclear PLA foci is plotted ± SD, n = 500.Statistical significance was evaluated using Mann-Whitney test (**** P < 0.0001).Representati v e e xperiment is shown from two independent r epeats.The scale bar in r epr esentati v e images corresponds to 10 m.

Figure 2 .
Figure 2. PPM1D is present at telomeres.( A ) U2OS cells were co-transfected with plasmids coding for mCherry-dCas9 and telomeric repeat-targeting sgRNA.After 24h, cells wer e fix ed and stained for PPM1D and TRF2.Images show a single confocal plane processed with deconvolution.The scale bars r epr esent 10 m or 2 m, respecti v ely.( B ) Quantification of A. Area of the overlapping PPM1D and TRF2 signal was determined using Interaction Factor packa ge in Ima geJ.Subsequently, PPM1D signal was randomized for each image.Means of 20 randomizations are plotted together with experimentally observed values (left).Shown is also a fraction of telomeres that contain PPM1D signal (right).Values for 46 cells form two independent experiments are plotted with means ± SD.Statistical significance was evaluated using paired t -test (**** P < 0.0001).( C ) MCF7 cells were stained for PPM1D and TRF2 and imaged by confocal microscop y.A r epr esentati v e single deconvolv ed planes ar e shown.The scale bar r epr esents 10 m or 2 m respecti v ely.( D ) PPM1D and TRF2 signals from (C) were analyzed as in (B).Values for 51 cells form two independent experiments are plotted with means ± SD.Statistical significance was evaluated using paired t -test (**** P < 0.0001).( E ) U2OS cells were transfected with plasmids coding for individual EGFP-PPM1D variants.Cells were fixed, stained for TRF2 and imaged by confocal microscop y.A r epr esentati v e single deconvolved planes are shown.The scale bar r epr esents 10 or 2 m, r especti v ely.

Figure 3 .
Figure 3. TRF2 is phosphorylated at S410 by ATR and dephosphorylated by PPM1D.( A ) HEK293 cells were transfected with the wild-type EGFP-TRF2 (WT) or EGFP-TRF2-S410A (SA) mutant and incubated with PPM1Di for 18 h prior harv esting.Cell e xtr acts were incubated with GFP tr ap

Figure 5 .
Figure 5. Increased PPM1D activity at telomere impairs shelterin function.( A ) U2OS cells were fixed 24 h after transfection with the wild type or A380 mutant of PPM1D, and were stained with TRF2 and TIN2 antibodies.Relati v e TIN2 intensity in TRF2 foci is plotted ± SD, n ≥ 286.Statistical significance was evaluated using Mann-Whitney test (**** P < 0.0001).( B ) Plotted is the mean intensity of TRF2 staining in nuclear foci ± SD in cells from K. Statistical significance was evaluated using Mann-Whitney test (* P < 0.05, **** P < 0.0001), n ≥ 286.The scale bar in r epr esentati v e images corresponds to 10 m. ( C ) Expression of the catalytic domain of PPM1D was induced or not in RPE1-PPM1D-A380 cells by addition of doxy cy cline f or 10 da ys and where indicated, PPM1D inhibitor was added to the media 1 h prior fixation.Cells were hybridized with TAACCC FISH-probe, stained for 53BP1, and formation of TIFs was quantified by ScanR microscopy.Plotted is a fraction of cells with more than three TIFs.Mean ± SD is shown, statistical significance was evaluated by unpaired t -test ( n = 4).Whole cell lysates were evaluated by immunoblotting, phosphorylation of KAP1 at S824 is a marker of ATM activity, TurboGFP is a marker of PPM1D-A380 expression, the asterisk indicates a non-specific band.Note that PPM1D (Santa Cruz) reco gnizes onl y the endo genous full length PPM1D.( D ) Quantification of chromosome fusions in RPE1-PPM1D-A380 cells treated or not with doxy cy cline for 10 days.More than 1246 chromosomes per condition was analyzed in each of the three independent experiments.Mean ± SD is shown, statistical significance was evaluated by paired t -test.The scale bars in the r epr esentati v e images corresponds to 10 or 2 m, respecti v ely.( E ) HeLa cells with tetracy cline-inducib le knock down of endogenous TRF2 were stably reconstituted with the wild type or S410A mutant GFP-TRF2 and single cell clones were cultured in the absence or presence of doxy cy cline for 5 days.Whole cell lysates were analyzed by immunoblotting.( F ) Cells from E were seeded into 96 wells at 100 cells / well, and cultured for additional 7 days.Relati v e cell proliferation was determined by resazurin assay.Statistical significance was determined by unpaired t -test, n = 3.

Figure 6 .
Figure 6.Loss of PPM1D affects recruitment of DNA repair factors to telomeric breaks.( A ) Parental and U2OS-PPM1D-KO cells were transfected with plasmids coding for Cas9-EGFP with or without the telomere-targeting sgRNA.After 24 h, cells wer e fix ed and stained for NBS1 and TRF2.Plotted