Abstract
The protection of telomere ends by the shelterin complex prevents DNA damage signalling and promiscuous repair at chromosome ends. Evidence suggests that the 3′ single-stranded telomere end can assemble into a lasso-like t-loop configuration1,2, which has been proposed to safeguard chromosome ends from being recognized as DNA double-strand breaks2. Mechanisms must also exist to transiently disassemble t-loops to allow accurate telomere replication and to permit telomerase access to the 3′ end to solve the end-replication problem. However, the regulation and physiological importance of t-loops in the protection of telomere ends remains unknown. Here we identify a CDK phosphorylation site in the shelterin subunit at Ser365 of TRF2, whose dephosphorylation in S phase by the PP6R3 phosphatase provides a narrow window during which the RTEL1 helicase can transiently access and unwind t-loops to facilitate telomere replication. Re-phosphorylation of TRF2 at Ser365 outside of S phase is required to release RTEL1 from telomeres, which not only protects t-loops from promiscuous unwinding and inappropriate activation of ATM, but also counteracts replication conflicts at DNA secondary structures that arise within telomeres and across the genome. Hence, a phospho-switch in TRF2 coordinates the assembly and disassembly of t-loops during the cell cycle, which protects telomeres from replication stress and an unscheduled DNA damage response.
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Data availability
The mass spectrometry proteomics dataset is publicly available through ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD014843. Source Data for Figs. 1–4 and Extended Data Figs. 1–8 are available with the online version of the paper. All other data are available from the corresponding author upon reasonable request.
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Acknowledgements
We thank members of the Boulton and Cesare laboratories for suggestions, discussions and critical reading of the manuscript. We thank N. O’Reilly and D. Joshi for peptide synthesis, the Australian Cancer Research Foundation Telomere Analysis Centre at the Children’s Medical Research Institute (Sydney) for imaging support and A. Colomba for providing reagents. G.S. is supported by an EMBO advanced fellowship (ALTF 1656-2014). P.K. and P.R. are supported by the Crick Institute core funding. The work in the Chowdhury laboratory is supported by the National Institutes of Health (NIH) R01 CA208244. The work in the Boulton laboratory is supported by the Francis Crick Institute, which receives its core funding from Cancer Research UK (FC0010048), the UK Medical Research Council (FC0010048), and the Wellcome Trust (FC0010048); a European Research Council (ERC) Advanced Investigator Grant (TelMetab); and a Wellcome Trust Senior Investigator Grant. The Cesare laboratory is supported by National Health and Medical Research Council of Australia (1106241) and the Cancer Institute NSW (11/FRL/5-02).
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S.J.B. and G.S. conceived the study; G.S., P.K., P.R., D.V.L., A.J.C. and S.J.B. designed experiments; G.S., P.K., P.R., D.V.L., P.M., V.B., X.-F.Z. and H.R.F. conducted experiments; G.S., P.K., P.R., D.V.L., P.M., X.-F.Z., H.R.F., A.P.S., D.C., A.J.C. and S.J.B. analysed data; S.J.B., G.S. and A.J.C. wrote the manuscript with editorial help from P.K., P.R., P.M. and D.V.L.
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Extended data figures and tables
Extended Data Fig. 1 TRF2 is phosphorylated at Ser365.
a, Annotated spectrum for the TRF2 phosphorylated peptide. The data were acquired on the LTQ Orbitrap Velos and processed in MaxQuant v.1.3.0.5 with the database search performed against the canonical sequences Homo sapiens from UniProt. For the spectrum shown, the posterior error probability value was 0.018258 and the localization score for the site was 1 DLVLPTQALPAS(1)PALK. b, HEK 293 cells were released from a double-thymidine block (left) or a thymidine plus nocodazole block (right). Cells were subjected to SDS–PAGE analysis and progression through the cell cycle was monitored by immunoblotting with cell cycle markers as indicated. Asterisks indicate time points after synchronization. c, PCR analysis of genomic DNA isolated from Trf2F/− MEFs stably expressing empty vector, wild-type or mutant TRF2, 96 h after infection with control or Cre-expressing adenovirus. d, Western blotting analysis of the cells described in c to monitor loss of endogenous TRF2 after Cre expression and to determine complementation efficiency with ectopic wild-type and mutant TRF2. The asterisk indicates endogenous TRF2. In a–d, the experiments were independently repeated at least twice with similar results.
Extended Data Fig. 2 Mutations of TRF2 at the Ser365 or Ser367 phospho-site do not affect interaction with shelterin components.
a, Whole-cell extracts from HEK 293 cells stably expressing empty vector, wild-type or mutant Myc-tagged TRF2 as indicated were immunoprecipitated with anti-Myc antibody or normal mouse IgG. Protein complexes were analysed with antibodies against RAP1, TRF1 and Myc. b, Trf2F/F MEFs expressing wild-type or phospho-dead (Ser367Ala mutant) TRF2 were transfected with either control siRNA (non-targeting control, NTC) or siRNA against RAP1 (siRAP1) and treated with 4-OHT for 96 h. Whole-cell extracts were analysed 72 h later as indicated. c, Quantification (left) and representative images (right) of chromosome fusions in the Trf2F/F MEFs depicted in b performed 96 h after 4-OHT treatment (n = 30 metaphases analysed). Data are mean ± s.e.m. P values were determined by one-way ANOVA. In a–d, the experiments were independently repeated at least twice with similar results.
Extended Data Fig. 3 Inhibition of MEK–ERK signalling pathway does not affect TRF2 phosphorylation at Ser365 or Ser367.
a, Quantity screen for TRF2-biotinylated peptides. Slot-blot assay in which biotin-tagged TRF2 peptides were incubated with streptavidin-coated beads to ensure that the correct amounts were used in the peptide pull-down assay. b, HEK 293 cells (left) or Rtel1F/F MEFs (right) were pre-treated with vehicle control (DMSO) or with 25 μM of MEK1/2 kinase inhibitor (U0126) for 48 h. Whole-cell extracts were subjected to SDS–PAGE analysis followed by immunoblotting with antibodies as indicated. In a and b, the experiments were independently repeated at least twice with similar results.
Extended Data Fig. 4 Identification of TRF2- and RTEL1-interacting phosphatases and protein phosphatase regulatory subunits.
a, Intensity-based absolute quantification (iBAQ) scatter plots comparing protein abundance in cells synchronized during S phase versus asynchronous control cells. Immunoprecipitates from asynchronous or S-phase-synchronized HEK 293 cells stably expressing Flag–haemagglutinin (HA)-tagged RTEL1 (top), N-terminal FLAP (Flag–GFP)-tagged RTEL1 (middle) or Myc–TRF2 (bottom) were separated by SDS–PAGE and stained with Coomassie blue to visualize proteins. Immunoprecipitations with haemagglutinin (top), GFP (middle) and Myc (bottom) antibodies were performed. The proteins along the entire length of the gel were extracted and analysed by liquid chromatography–tandem mass spectrometry (LC/MS–MS). b, HEK 293 cells stably expressing wild-type Myc–TRF2 were transfected with either non-target control or siRNA against protein phosphatase regulatory subunits, as specified. Three days later, protein levels were analysed with the indicated antibodies. c, FLAP-tagged RTEL1 HEK 293 cells expressing Myc-tagged wild-type TRF2 were transfected with either control siRNA or siRNA against PP4R2 or PP6R3. Whole-cell extracts were immunoprecipitated with anti-Flag antibody and immunocomplexes were analysed for Myc (TRF2) and Flag (RTEL1). Inputs (5%) are shown on the right. d, HEK 293 cells expressing wild-type Myc–TRF2 (left) or Flag–HA-tagged RTEL1 (right) were subjected to immunoprecipitation with normal rabbit IgG or antibodies against PP4R2 and PP6R3. Immune complexes were analysed by western blotting with the indicated antibodies. In b–d, the experiments were independently repeated at least twice with similar results.
Extended Data Fig. 5 PP6R3 controls phosphorylation of TRF2 at Ser365 or Ser367.
HEK 293 cells expressing wild-type Myc–TRF2 were transfected with a non-targeting control siRNA or siRNAs against protein phosphatase regulatory subunits (a) or catalytic subunits (b). Cells were collected, and whole-cell extracts were immunoprecipitated with anti-RTEL1 antibody. Immunocomplexes were resolved by SDS–PAGE and analysed by western blotting as indicated. c, HEK 293 cells (c) and Trf2F/− MEFs (d) expressing Myc-tagged wild-type TRF2 were transfected with control siRNA or siRNA targeting PP4R2 or PP6R3 (Pp4r2 or Pp6r3 for MEFs). Whole-cell extracts were immunoprecipitated with anti-TRF2 antibody, and immunocomplexes were resolved by SDS–PAGE and analysed for human phospho-TRF2 (pS365 TRF2; left panel in c) or mouse phospho-TRF2 (pS367 TRF2; left panel in d). e, Top, frequency of telomere loss and telomere fragility per metaphase in Rtel1F/F MEFs transfected with control siRNA or with Pp4r2 or Pp6r3 siRNA (n = 58 (NTC), n = 57 (Pp4r2), and n = 55 (Pp6r3) of analysed metaphases). Efficiency of siRNA knockdown was determined by western blotting with PP6R3 and PP4R2 antibodies as indicated. Data are mean ± s.e.m. P values determined by one-way ANOVA. Bottom, representative images of the telomere FISH experiments. The arrowheads show loss of telomere signal. Red, telomere PNA FISH; blue, DAPI. f, Phi29-dependent telomere circles (top) detected in cells as indicated in e. The extent of [32P] incorporation was quantified (bottom) from the autoradiographs, and the level of [32P] incorporation by cells transfected with control siRNA was arbitrarily assigned a value of 100%. Data are mean ± s.d. and from two independent experiments. P values determined by one-way ANOVA. In a–f, the experiments were independently repeated at least twice with similar results.
Extended Data Fig. 6 Replication defects in Trf2F/− MEFs in the absence of TRF2 phosphorylation at Ser365 or Ser367.
a, Quantification of global replication fork dynamics (left) and rates of replication fork progression (right) of the IdU/CldU double pulse-labelling experiment in Trf2F/− MEFs complemented with empty vector, wild-type or mutant TRF2, performed 96 h after infection with control- or Cre-expressing adenovirus (n denotes number of analysed forks). Data are mean ± s.e.m. of triplicate experiments. Box plots are as in Fig. 3e. b, Representative images of the experiment from a. c–e, Quantification of micronuclei (c; 500 nuclei per replicate), mitotic catastrophe (d; 500 nuclei per replicate), and 53BP1 foci frequency (e; 150 nuclei per replicate) in Trf2F/− MEFs complemented as in a. Data are mean ± s.e.m. of three (c, d) or two (e) independent experiments. f, DNA damage in Trf2F/− MEFs complemented as in a was estimated by counting the frequency of cells with five or more 53BP1 foci. For each independent experiment (n = 2), a minimum of 150 nuclei of each condition were analysed. All P values were determined by one-way ANOVA.
Extended Data Fig. 7 Suppression of constitutive binding of RTEL1 to the TRF2(S367A) phospho-dead mutant rescues replication defects in MEFs.
a, Quantification of rates of replication fork progression (left) and representative images (right) of the IdU/CldU double pulse-labelling experiment in double-knockout Trf2F/F;Rtel1F/F mouse ear fibroblasts stably expressing Myc–TRF2(S367A), together with wild-type V5–RTEL1 (WT) or C4C4 mutant V5–RTEL1(R1237H) (R/H), performed 96 h after infection with Cre-expressing adenovirus. Data are mean ± s.e.m. of triplicate experiments. b, Quantification of replication fork dynamics (top) and fork asymmetry (bottom) from cells as in a. Staining with anti-ssDNA antibody (right) was used to exclude broken DNA tracks (n denotes number of analysed forks). Box plots are as in Fig. 3e. Data are mean ± s.e.m. c–e, Quantification of the frequency of micronuclei (c; 500 nuclei per replicate), mitotic catastrophe (d; 500 nuclei per replicate), and 53BP1 foci (e; 150 nuclei per replicate) in Trf2F/F;Rtel1F/F mouse ear fibroblasts complemented as indicated in a. Data are mean ± s.e.m. of three (c, d) or two (e) independent experiments. f, DNA damage in Trf2F/F;Rtel1F/F mouse ear fibroblasts complemented as in a was estimated by counting the frequency of cells with five or more 53BP1 foci. For each independent experiment (n = 2), a minimum of 150 nuclei of each condition were analysed. All P values were determined by one-way ANOVA.
Extended Data Fig. 8 TRF2(S367A) mutation induces TIFs and impairs formation of t-loops.
a, Quantification (top) of the cross-linking efficiency test (bottom) in the Trf2F/F MEFs stably expressing wild-type or S367A mutant Myc–TRF2 120 h after treatment with 4-OHT (n = 3 independent biological replicates). Data are mean ± s.e.m. b, Left, quantification of TIFs per interphase in Trf2F/− MEFs complemented with empty vector, wild-type Myc–TRF2, phospho-dead mutant TRF2(S367A), or phospho-mimetic Myc–TRF2(S367D) and Myc–TRF2(S367E) mutants 96 h after infection with Cre-expressing adenovirus. Right, representative interphase TIF images. c, Quantification of TIFs per interphase in Trf2F/F;Rtel1F/F mouse ear fibroblasts stably expressing Myc–TRF2(S367A) together with wild-type V5–RTEL1 or mutant V5–RTEL1(R1237H) 96 h after infection with GFP- or Cre-expressing adenovirus. d, Quantification (left) and representative images (right) of RPA staining at TIFs in Trf2F/F;Rtel1F/F mouse ear fibroblasts complemented as in c. Analysis was carried out 96 h after infection with Cre-expressing adenovirus. Data in b–d are mean ± s.d. from three independent experiments (n = 100 cells in each treatment group analysed per independent experiment). e, Measurement of linear and t-loop molecules shown in Fig. 4c (n = 3 biological replicates scoring ≥1,192 molecules per replicate). T-loop measurements are a sum of the loop and tail portions of the molecule. Data are mean ± s.e.m. f, Measurement of the loop portion of t-loops from the experiments depicted in Fig. 4c (n = 3 biological replicates scoring ≥1,192 molecules per replicate). All P values were determined by one-way ANOVA.
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This file contains Supplementary Figure 1 (source data for main and Extended Data Figures, each panel contains immunoblot analysis, with a dashed box indicating the cropped region), and Supplementary Tables 1-2, which list antibodies and oligonucleotides used in the paper.
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Sarek, G., Kotsantis, P., Ruis, P. et al. CDK phosphorylation of TRF2 controls t-loop dynamics during the cell cycle. Nature 575, 523–527 (2019). https://doi.org/10.1038/s41586-019-1744-8
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DOI: https://doi.org/10.1038/s41586-019-1744-8
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