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CDK phosphorylation of TRF2 controls t-loop characteristics throughout the cell cycle

Abstract

The security of telomere ends by the shelterin complex avoids DNA damage signalling and promiscuous repair work at chromosome ends. Proof recommends that the 3 ′ single-stranded telomere end can assemble into a lasso-like t-loop setup 1,2, which has actually been proposed to secure chromosome ends from being acknowledged as DNA double-strand breaks 2 Systems should likewise exist to transiently dismantle t-loops to permit accurate telomere duplication and to allow telomerase access to the 3 ′ end to fix the end-replication problem. However, the guideline and physiological significance of t-loops in the security of telomere ends stays unknown. Here we identify a CDK phosphorylation website in the shelterin subunit at Ser365 of TRF2, whose dephosphorylation in S stage by the PP6R3 phosphatase offers a narrow window during which the RTEL1 helicase can transiently gain access to and loosen up t-loops to assist in telomere duplication. Re-phosphorylation of TRF2 at Ser365 outside of S stage is required to launch RTEL1 from telomeres, which not only safeguards t-loops from promiscuous unwinding and improper activation of ATM, but likewise counteracts replication conflicts at DNA secondary structures that develop within telomeres and throughout the genome. A phospho-switch in TRF2 coordinates the assembly and disassembly of t-loops throughout the cell cycle, which protects telomeres from duplication stress and an unscheduled DNA damage response.

Information schedule

The mass spectrometry proteomics dataset is publicly offered through ProteomeXchange Consortium by means of the PRIDE partner repository with the dataset identifier PXD014843 Source Data for Figs. 1– 4 and Extended Data Figs. 1– 8 are readily available with the online version of the paper. All other data are offered from the corresponding author upon reasonable request.

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Acknowledgements

We thank members of the Boulton and Cesare laboratories for recommendations, discussions and important reading of the manuscript. We thank N. O’Reilly and D. Joshi for peptide synthesis, the Australian Cancer Research Study Structure Telomere Analysis Centre at the Children’s Medical Research Institute (Sydney) for imaging support and A. Colomba for offering 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 financing. The operate 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 gets its core funding from Cancer Research study UK (FC0010048), the UK Medical Research Study Council (FC0010048), and the Wellcome Trust (FC0010048); a European Research Council (ERC) Advanced Private Investigator Grant (TelMetab); and a Wellcome Trust Elder Private Investigator Grant. The Cesare lab is supported by National Health and Medical Research Council of Australia (1106241) and the Cancer Institute NSW (11/ FRL/5-02).

Author details

Author notes

  1. These authors contributed equally: Grzegorz Sarek, Panagiotis Kotsantis

Associations

  1. The Francis Crick Institute, London, UK

    • Grzegorz Sarek
    • , Panagiotis Kotsantis
    • , Phil Ruis
    • , Pol Margalef
    • , Valerie Borel
    • , Helen R. Flynn
    • , Ambrosius P. Snijders
    • & Simon J. Boulton
  2. Genome Integrity Unit, Kid’s Medical Research study Institute, University of Sydney, Westmead, New South Wales, Australia

    • David Van Ly
    • & Anthony J. Cesare
  3. School of Medicine, The University of Notre Dame Australia, Sydney, New South Wales, Australia

    • David Van Ly
  4. Dana-Farber Cancer Institute, Harvard Institute of Medication, Boston, MA, USA

    • Xiao-Feng Zheng
    • & Dipanjan Chowdhury

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Contributions

S.J.B. and G.S. developed 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. performed 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 information; S.J.B., G.S. and A.J.C. wrote the manuscript with editorial assistance from P.K., P.R., P.M. and D.V.L.

Corresponding author

Correspondence to.
Simon J. Boulton.

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Completing interests

The authors declare no completing interests.

Peer evaluation details Nature thanks Eric Gilson, Joachim Lingner and the other, confidential, reviewer( s) for their contribution to the peer evaluation of this work. The information were gotten 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. Data are mean ± s.e.m. P worths were identified by one-way ANOVA.
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Extended Data Fig. 3 Inhibition of MEK– ERK signalling pathway does not impact TRF2 phosphorylation at Ser365 or Ser367

a, Amount screen for TRF2-biotinylated peptides. Slot-blot assay in which biotin-tagged TRF2 peptides were nurtured with streptavidin-coated beads to make sure that the appropriate quantities were utilized in the peptide pull-down assay. b, HEK 293 cells (left) or Rtel1 F/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 individually duplicated at least twice with comparable results.

Extended Data Fig. 4 Identification of TRF2- and RTEL1-interacting phosphatases and protein phosphatase regulatory subunits.

a, Intensity-based outright quantification (iBAQ) scatter plots comparing protein abundance in cells integrated throughout S phase versus asynchronous control cells. Immunoprecipitates from asynchronous or S-phase-synchronized HEK 293 cells stably revealing 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 carried out. The proteins along the whole length of the gel were extracted and evaluated 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. 3 days later on, protein levels were analysed with the suggested 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 revealed on the. 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 separately repeated at least twice with comparable 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 fixed by SDS– PAGE and analysed by western blotting as suggested. c, HEK 293 cells ( c) and Trf2 F/ − MEFs ( d) revealing 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 Rtel1 F/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). Effectiveness of siRNA knockdown was identified by western blotting with PP6R3 and PP4R2 antibodies as indicated. Data are mean ± s.e.m. P values figured out by one-way ANOVA. Bottom, representative pictures of the telomere FISH experiments. The arrowheads show loss of telomere signal. Red, telomere PNA FISH; blue, DAPI. f, Phi29- reliant telomere circles (top) spotted in cells as suggested in e The level of [32P] incorporation was measured (bottom) from the autoradiographs, and the level of [32P] incorporation by cells transfected with control siRNA was arbitrarily appointed a worth of 100%. Information are mean ± s.d. and from 2 independent experiments. P worths figured out by one-way ANOVA. In a f, the experiments were individually repeated at least two times with similar outcomes.
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Extended Data Fig. 6 Replication problems in Trf2 F/ − MEFs in the absence of TRF2 phosphorylation at Ser365 or Ser367

a, Quantification of worldwide duplication fork characteristics (left) and rates of duplication fork development (right) of the IdU/CldU double pulse-labelling experiment in Trf2 F/ − MEFs matched with empty vector, wild-type or mutant TRF2, carried out 96 h after infection with control- or Cre-expressing adenovirus ( n represents variety of evaluated forks). Information 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 reproduce), mitotic disaster ( d; 500 nuclei per reproduce), and 53 BP1 foci frequency ( e; 150 nuclei per duplicate) in Trf2 F/ − MEFs complemented as in a Information are mean ± s.e.m. of 3 ( c, d) or 2 ( e) independent experiments. f, DNA damage in Trf2 F/ − MEFs matched as in a was approximated by counting the frequency of cells with five or more 53 BP1 foci. For each independent experiment ( n=2), a minimum of 150 nuclei of each condition were evaluated. All P worths were determined by one-way ANOVA.
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Extended Data Fig. 7 Suppression of constitutive binding of RTEL1 to the TRF2( S367 A) phospho-dead mutant saves replication problems 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 Trf2 F/F; Rtel1 F/F mouse ear fibroblasts stably revealing Myc– TRF2( S367 A), together with wild-type V5– RTEL1 (WT) or C4C4 mutant V5– RTEL1( R1237 H) (R/H), carried out 96 h after infection with Cre-expressing adenovirus. Information are mean ± s.e.m. of triplicate experiments. b, Metrology of replication fork characteristics (top) and fork asymmetry (bottom) from cells as in a Staining with anti-ssDNA antibody (right) was used to exclude damaged DNA tracks ( n represents number of evaluated forks). Box plots are as in Fig. 3e. Data are mean ± s.e.m. c e, Metrology of the frequency of micronuclei ( c; 500 nuclei per reproduce), mitotic catastrophe ( d; 500 nuclei per duplicate), and 53 BP1 foci ( e; 150 nuclei per duplicate) in Trf2 F/F; Rtel1 F/F mouse ear fibroblasts matched as suggested in a Information are mean ± s.e.m. of 3 ( c, d) or more ( e) independent experiments. f, DNA damage in Trf2 F/F; Rtel1 F/F mouse ear fibroblasts complemented as in a was approximated by counting the frequency of cells with five or more 53 BP1 foci. For each independent experiment ( n=2), a minimum of 150 nuclei of each condition were evaluated. All P worths were figured out by one-way ANOVA.
Source data.

Extended Data Fig. 8 TRF2( S367 A) mutation induces TIFs and hinders development of t-loops. d, Quantification (left) and representative images (right) of RPA staining at TIFs in Trf2 F/F; Rtel1 F/F mouse ear fibroblasts complemented as in c All P values were figured out by one-way ANOVA.
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Supplemental details

Supplementary Info

This file contains Supplementary Figure 1 (source data for main and Extended Data Figures, each panel consists of immunoblot analysis, with a dashed box suggesting the cropped area), and Supplementary Tables 1-2, which list antibodies and oligonucleotides utilized in the paper.

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Sarek, G., Kotsantis, P., Ruis, P. et al. CDK phosphorylation of TRF2 controls t-loop characteristics throughout the cell cycle.
Nature575, 523–527(2019) doi: 10.1038/ s41586-019-1744 -8

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