TIRR regulates 53BP1 by masking its histone methyl-lysine binding function

Abstract

P53-binding protein 1 (53BP1) is a multi-functional double-strand break repair protein that is essential for class switch recombination in B lymphocytes and for sensitizing BRCA1-deficient tumours to poly-ADP-ribose polymerase-1 (PARP) inhibitors. Central to all 53BP1 activities is its recruitment to double-strand breaks via the interaction of the tandem Tudor domain with dimethylated lysine 20 of histone H4 (H4K20me2). Here we identify an uncharacterized protein, Tudor interacting repair regulator (TIRR), that directly binds the tandem Tudor domain and masks its H4K20me2 binding motif. Upon DNA damage, the protein kinase ataxia-telangiectasia mutated (ATM) phosphorylates 53BP1 and recruits RAP1-interacting factor 1 (RIF1) to dissociate the 53BP1–TIRR complex. However, overexpression of TIRR impedes 53BP1 function by blocking its localization to double-strand breaks. Depletion of TIRR destabilizes 53BP1 in the nuclear-soluble fraction and alters the double-strand break-induced protein complex centring 53BP1. These findings identify TIRR as a new factor that influences double-strand break repair using a unique mechanism of masking the histone methyl-lysine binding function of 53BP1.

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Figure 1: TIRR is a novel partner of 53BP1.
Figure 2: TIRR associates with the tandem Tudor domain of 53BP1.
Figure 3: TIRR overexpression compromises 53BP1 functions.
Figure 4: Disruption of 53BP1–TIRR complex by DNA damage-dependent ATM phosphorylation.
Figure 5: TIRR regulates 53BP1 functions.

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Acknowledgements

We are grateful to L. Xu for RIF1 antibody and S. Boulton for RIF1−/− MEFs. D.C. is supported by R01 AI101897-01 (National Institute of Allergy and Infectious Diseases) and R01CA142698-07 (National Cancer Institute), a Leukemia and Lymphoma Society Scholar Grant, the Claudia Adams Barr Program for Innovative Cancer Research, a Department of Defense Ovarian Cancer Award, a Breast SPORE Pilot Award, and a Robert and Deborah First Fund Award. G.M. is supported by National Institutes of Health grants R01 CA132878 and R01 GM116829, and a Mayo Clinic Brain Cancer SPORE Program Pilot Award (P50 CA108961). J.W.H. is supported by AG011085. J.C. is supported by National Institutes of Health/National Institute of Allergy and Infectious Disease grants (1RO1AI072194 and 1RO1AI124186) and a National Cancer Institute Cancer Center Support grant (P30CA008748). W.T.Y. is supported by a training grant from the National Cancer Institute (4T32CA009149-40).

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P.D., G.M. and D.C. designed the study. P.D. performed most of the experiments with assistance from M.E.B., K.M., S.C., Y.Z.H., X.F.F. and N.P. A.D. did the statistical analysis. G.C. and M.V.B. conducted NMR studies under G.M.’s supervision. A.K. conducted microscopy studies with the LacO fusion system under E.S.’s guidance. C.M. did quantitative MS under J.W.H.’s guidance. W.T.Y. did CSR assays under J.C.’s guidance. P.D., G.M. and D.C. wrote the paper.

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Correspondence to Dipanjan Chowdhury.

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The authors declare no competing financial interests.

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Reviewer Information Nature thanks D. Durocher, J. Jonkers and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Extended data figures and tables

Extended Data Figure 1 TIRR prevents the interaction of 53BP1 Tudor domain with a peptide derived from H4K20me2.

a, Soluble nuclear proteins were prepared from HeLa and HeLa cells in which TIRR has been knock out using CRISPR/cas9 system (HeLaΔTIRR) undamaged or irradiated at 10 Gy for 90 min. Endogenous 53BP1 was next immunoprecipitated and immunoblotted using the indicated antibody. b, Coomassie staining of Flag-tagged recombinant proteins purified from bacteria; ‘OD-Tudor’ encompasses the oligomerization, the Tudor and UDR domains, ‘OD’ the oligomerization domain and ‘Tudor-UDR’ the Tudor and UDR domains. c, Recombinant TIRR-His protein was incubated with the indicated Flag fusion protein in presence of 200 or 400 mM NaCl. Pull-down proteins were subjected to Flag (top) and His (bottom) immunoblotting. M, molecular size markers. d, Coomassie staining of Flag–OD–Tudor and Flag–OD–Tudor/TIRR–His complex purified from bacteria co-expressing both proteins. e, Pull-down of the indicated proteins with a biotinylated peptide derived from histone H4K20me2. Bound proteins were analysed by immunoblotting. f, Interaction of 53BP1 Tudor with TIRR probed using ITC. Integrated heat measurements from raw titration data and curve fitting with a standard one-site model are shown. Kd and stoichiometry (n) are indicated.

Extended Data Figure 2 Ectopic TIRR restores RAD51 foci formation in BRCA1-mutant MEFs.

a, Brca1-mutant MEFs stably transduced with an empty vector (EV) or TIRR-FH were transfected with the indicated siRNA. RAD51 was stained 6 h after 5 Gy irradiation (mean ± s.d., n = 3). b, Quantification of CSR at 96 h or 72 h after stimulation from splenic B cells transduced with pMIG or pMIG–TIRR, and stimulated with LPS and IL-4 for 72 h. c, AID, Sμ germline (Sμ GLT) and Sγ1 germline (Sγ1 GLT, black) transcripts in splenic B cells stimulated for 48 h (mean ± s.d., n = 3 mice). d, Immunoblotting of AID and TIRR from lysates prepared from splenic B cells stimulated for 72 h.

Extended Data Figure 3 TIRR expression level remains unchanged after irradiation.

a, TIRR-FH-expressing U2OS cells were irradiated at 10 Gy and collected next at the indicated time. Whole-cell extracts were analysed by immunoblotting using the indicated antibody. b, U2OS whole-cell extracts were prepared 90 min after a 10 Gy irradiation and analysed by immunoblotting using the indicated antibody. c, RPE-1 cells stably expressing TIRR-FH wild-type (WT) or double mutated (K10R, K151R) were irradiated at 10 Gy for 90 min followed by TIRR-FH immunoprecipitation. Pull-down proteins were subjected to immunoblotting with the indicated antibodies. d, U2OS cells were first transfected with the indicated siRNA. The day after, cells were transfected with a vector encoding Ubiquitin fused to an N-terminal His tag. Forty-eight hours later, cells were irradiated at 10 Gy for 90 min when indicated followed by His pull-down. Pull-down proteins were subjected to immunoblotting with the indicated antibodies. e, Laser stripes examined by immunofluorescence 15 min after irradiation in RPE-1 cells expressing TIRR-FH (left) or RPE-1 cells (right).

Extended Data Figure 4 RIF1 depletion affects 53BP1 foci formation in various cellular models.

a, RPE-1 cells deprived of endogenous ATM using CRISPR/cas9 system (RPE-1 ΔATM) were stably transfected with GFP–TIRR. GFP–TIRR was next pull-down and retained proteins were analysed by immunoblotting. The star represents a non-specific band. b, Flag immunoprecipitation from extracts prepared from indicated siRNA-transfected TIRR-FH-expressing RPE-1 cells. c, Immunofluorescence of 53BP1 and RIF1 in siRNA-transfected U2OS cells irradiated at 10 Gy for 90 min. The graph represents the percentage of cells harbouring more than ten 53BP1 foci. d, Same as c in RPE-1 cells (mean ± s.d., n = 2). e, Kinetics of 53BP1 foci formation in RPE-1 ΔRIF1 cells. Results are expressed as number of foci per cell (top) and as percentage of cells harbouring more than ten 53BP1 foci (bottom) (mean ± s.d., n = 2). Deletion of RIF1 was checked by immunoblotting. f, Same as in e in MEF RIF1−/− cells.

Extended Data Figure 5 TIRR depletion affects mainly the stability of soluble nuclear 53BP1.

a, Immunoblotting of siRNA-transfected Hela cell extracts. TIRR expression has been rescued with an siRNA-resistant construct. b, Nuclear proteins were sequentially extracted by increasing amount of salt from siRNA-transfected U2OS cells. After salt extraction, the resulting pellet corresponds to the insoluble material (insoluble). The amount of 53BP1 present in each fraction was measured by immunoblotting and quantification using ImageJ. c, Relative TIRR and 53BP1 protein distribution in the RPE-1 nucleus as measured by mass spectrometry. d, U2OS-soluble nuclear extract (1 mg) was loaded onto a sucrose gradient of 5–30% and ultracentrifuged for 4 h at 40,000 rpm. Fractions were collected from the top of the gradient and immunoblotted. The amount of 53BP1 and TIRR was quantified using ImageJ. e, Quantification of TIRR mRNA expression in siRNA-transfected Brca1-mutant MEFs. TIRR level was normalized to 5S expression.

Extended Data Figure 6 Expression of TIRR protein in various cancer cell lines and alteration frequency of Nudt16L1 (TIRR) gene in cancer.

a, Immunoblotting of 53BP1 and TIRR from extracts prepared from various human cancer cell lines: A, Ovarian cancer cell lines; B, Breast cancer cell lines; C, paediatric glioma cell lines; D, pancreatic cell lines; E, lung cell lines. HCT116, colon cancer cells; Jurkat, acute T cell leukaemia; LnCap, prostate adenocarninoma. Epithelial cells from retinal pigment RPE-1 and ovarian surface HiO80 are immortalized cell lines. b, Data extracted from cBioportal (http://www.cBioportal.org) for alterations in the Nudt16L1 (TIRR) gene across different cancer types. Twenty-nine out of 34 cancer types exhibit amplifications in the Nudt16L1 gene.

Extended Data Figure 7 TIRR depletion modifies the interaction of 53BP1 with its partners.

a, HeLa cells deprived of endogenous TIRR (HeLaΔTIRR) were stably transduced with FH-53BP1 together with GFP–TIRR or GFP. FH-53BP1 partners were purified from total nuclear extracts (nuclear-soluble and chromatin extracts) 90 min after a 10 Gy irradiation and analysed by mass spectrometry. b, HeLaΔTIRR or parental HeLa cells were stably transduced with FH-53BP1. FH-53BP1 partners were purified from total nuclear extracts from undamaged or irradiated cells and analysed by immunoblotting after normalization of the amount of 53BP1 pull-down (see results). The star represents a non-specific band.

Extended Data Figure 8 TIRR depletion hypersensitizes human BRCA1-mutant cells to PARPi in a 53BP1-dependent manner.

Human ovarian cancer cell lines Cov362 (a) and UWB1.289 (b) were transfected with the indicated siRNA, followed by treatment with olaparib. Percentage survival was calculated by normalizing the survival from treatment with olaparib versus untreated cells (mean ± s.d., n = 3). Efficiency of siRNAs was checked by immunoblot using the indicated antibodies in the Cov362 cell line. c, Top: siRNA-transfected Brca1-mutant MEFs mock-transfected or overexpressing RNF168 were treated with olaparib. Percentage survival was calculated as in a and b (mean ± s.d., n = 3). Bottom: stable expression of Flag–RNF168 and efficiency of 53BP1 siRNA were checked by immunoblotting. d, Immunofluorescence of 53BP1 in BRCA1-mutant MEFs (mean ± s.d., n = 2).

Extended Data Figure 9 TIRR depletion increases radiosensitivity.

a, Whole-cell extracts from RPE-1 cells (CTRL) and from a polyclonal population (poly) and three RPE-1 clones deprived of endogenous TIRR using CRISPR/cas9 system were analysed by immunoblotting using the indicated antibodies. The amount of 53BP1 was quantified and normalized to β-tubulin using ImageJ. b, Kinetics of γH2AX foci formation in indicated cells after a 2 Gy irradiation. The graph represents the mean number of γH2AX foci per cell (mean ± s.d., n = 3). c, Clonogenic survival of indicated cells after irradiations (0 to 5 Gy). TIRR expression has been restored in clone 13 by stable expression of GFP–TIRR. Survival was expressed as a percentage of colonies formed relative to the non-irradiated control (mean ± s.d., n = 3).

Extended Data Figure 10 Model depicting the regulation of 53BP1 activity by TIRR.

Supplementary information

Supplementary Figure 1

This file contains the uncropped scans for Immunoblot and silver stained gels with protein sizes indicated in kDa. (PDF 3654 kb)

Supplementary Table 1

This file contains the mass spectrometry analyses of FH-53BP1-FFR complex purified from U2OS cells. (XLSX 51 kb)

Supplementary Table 2

This file contains the mass spectrometry analyses of TIRR-FH complex purified from U2OS cells. (XLSX 82 kb)

Supplementary Table 3

This file contains the mass spectrometry analyses of FH-53BP1 complex purified from HeLaTIRR stably expressing GFP-TIRR or GFP. Cells were irradiated at 10 Gy for 90 min before purification of 53BP1-complex by tandem affinity. (XLSX 80 kb)

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Drané, P., Brault, ME., Cui, G. et al. TIRR regulates 53BP1 by masking its histone methyl-lysine binding function. Nature 543, 211–216 (2017). https://doi.org/10.1038/nature21358

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