Abstract
Mammalian telomeres protect chromosome ends from aberrant DNA repair1. TRF2, a component of the telomere-specific shelterin protein complex, facilitates end protection through sequestration of the terminal telomere repeat sequence within a lariat T-loop structure2,3. Deleting TRF2 (also known as TERF2) in somatic cells abolishes T-loop formation, which coincides with telomere deprotection, chromosome end-to-end fusions and inviability3,4,5,6,7,8,9. Here we establish that, by contrast, TRF2 is largely dispensable for telomere protection in mouse pluripotent embryonic stem (ES) and epiblast stem cells. ES cell telomeres devoid of TRF2 instead activate an attenuated telomeric DNA damage response that lacks accompanying telomere fusions, and propagate for multiple generations. The induction of telomere dysfunction in ES cells, consistent with somatic deletion of Trf2 (also known as Terf2), occurs only following the removal of the entire shelterin complex. Consistent with TRF2 being largely dispensable for telomere protection specifically during early embryonic development, cells exiting pluripotency rapidly switch to TRF2-dependent end protection. In addition, Trf2-null embryos arrest before implantation, with evidence of strong DNA damage response signalling and apoptosis specifically in the non-pluripotent compartment. Finally, we show that ES cells form T-loops independently of TRF2, which reveals why TRF2 is dispensable for end protection during pluripotency. Collectively, these data establish that telomere protection is solved by distinct mechanisms in pluripotent and somatic tissues.
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Data availability
The datasets generated during and/or analysed during the current study are included alongside the Article or are available from S.J.B and A.J.C on reasonable request. Data were searched against a recent copy of the UniProt M. musculus reference proteome UP000000589 (https://www.uniprot.org/proteomes/UP000000589). For gel source data, see Supplementary Fig. 1. All data are archived at the Francis Crick Institute or CMRI. Source data are provided with this paper.
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Acknowledgements
We thank members of the Boulton and Cesare laboratories for suggestions and discussions, and for critical reading of the manuscript; S. Page and the Australian Cancer Research Foundation Telomere Analysis Centre at the Children’s Medical Research Institute (CMRI) for microscopy infrastructure; the Crick BRF and GEMs for support with animal experiments; and T. de Lange for advice on G-overhang analysis. 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 Wellcome Trust Senior Investigator and Collaborative Grants. The laboratory of A.J.C. is supported by grants from the Australian NHMRC (1162886 and 1185870) and philanthropy from the Neil and Norma Hill Foundation.
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Authors and Affiliations
Contributions
P.R., P.M. and S.J.B. conceived the study; P.R. generated all cell lines and performed the large majority of experiments in this Article, including all those not listed below; V.B., A.M. and P.R. performed EmbryoScope and embryo IF–FISH experiments; G.R.K. adapted stem cell culture for T-loop visualization; D.V.L. performed all AiryScan imaging and analysis; R.B. provided protocols and reagents for directed differentiation of ES cells; and P.M., S.H. and A.P.S. performed PICh mass spectrometry analysis. P.R., D.V.L., V.B., G.R.K., A.M., S.H., P.M., A.J.C. and S.J.B. analysed data; P.R., A.J.C. and S.J.B. wrote the manuscript with editorial input from D.V.L., P.M., J.B. and K.K.N.
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Peer review information Nature thanks Ylli Doksani and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.
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Extended data figures and tables
Extended Data Fig. 1 TRF2-null ES cells do not undergo telomere fusions and show only a mild growth defect.
a, b, Growth curves of Trf2f/f ES cells and ear fibroblasts after treatment with OHT or EtOH. Cells were counted and re-seeded every 2 or 3 days (mean ± s.d., n = 3 biologically independent experiments). c, Bright-field images of the indicated cells 7 days after treatment with EtOH or OHT. d, Western blot of whole-cell extracts from Trf2f/f ES cells at the indicated times after treatment with EtOH or OHT. e, Flow cytometry determination of DNA content for the indicated cells 96 h after OHT or EtOH treatment (≥10,000 cells per condition, n = 3 independent experiments). f, Western blot of whole-cell extracts from indicated cells 96 h after treatment with EtOH or OHT. IR, 2 h post-treatment with 20 Gy ionizing radiation. g, Gating strategy for flow cytometry determination of DNA content. Example shown is Trf2f/f ear fibroblast EtOH sample from Fig. 1d. Similar strategies to gate live cells and singlets were applied for all flow cytometry experiments. h, Western blot of whole-cell extracts from three independently generated Trf2f/f ES cell clones 96 h after treatment with EtOH or OHT. i, Mitotic chromosome spreads from Trf2f/f ES cells 10 days after treatment with OHT or EtOH. The DNA is stained with DAPI (red) and the telomeres by FISH (green), with the mean percentage of telomere fusions from j indicated. Scale bar, 5 μm, j, Quantification of the experiment shown in i. Mean ± s.d., ≥60 spreads per condition across 3 independent experiments, unpaired two-tailed t-test; ns, not significant. k. Western blot of whole-cell extracts from Trf2f/f ES cells 10 days after treatment with EtOH or OHT.
Extended Data Fig. 2 The telomeric DDR is attenuated in ES cells lacking TRF2.
a, In-gel hybridization assay for single-stranded telomeric G-overhang DNA after Trf2 deletion. Left, TelC signals under the native condition. Right, same gel re-hybridized after in situ denaturation of the DNA. Overhang signals (bottom) were normalized to the total telomeric signals (right) and compared to Trf2f/f ear fibroblasts treated with EtOH, which was given an arbitrary value of 1 in each of three independent experiments. The mean of these normalized values is shown for each condition (n = 3 biologically independent experiments, representative example shown). b–d, Quantification of telomere fragility (b), heterogeneity (c) and loss (d) from mitotic chromosome spreads shown in Fig. 1b. Mean ± s.d., ≥70 spreads per condition across 3 independent experiments, unpaired two-tailed t-tests. e, Representative examples of the phenotypes quantified in b–d with relevant abnormalities marked with arrows. The DNA is stained with DAPI (red) and the telomeres by FISH (green). f, IF–FISH analysis to detect endogenous TRF2 in Trf2f/f ES cells 96 h after treatment with OHT or EtOH. TRF2 was detected by immunofluorescence (green), telomeric DNA with FISH (red) and the DNA with DAPI (blue). g, IF–FISH analysis to detect exogenous MYC–TRF2 in Trf2f/f ES cells stably expressing MYC–TRF2 or a vector-only control 96 h after treatment with OHT. MYC–TRF2 was detected by immunofluorescence (green), telomeric DNA with FISH (red) and the DNA with DAPI (blue). h, IF–FISH analysis to detect TRF1 in Trf1f/f ES cells 48 h after treatment with OHT or EtOH. TRF1 was detected by immunofluorescence (green), telomeric DNA with FISH (red) and the DNA with DAPI (blue). i, Cartoon depicting the SILAC–PICh workflow used in j, k. j, k, Results of SILAC–PICh mass spectrometry of Trf2f/f ES cells (j) and Trf2f/f ear fibroblasts (k) 96 h after OHT or EtOH treatment. Data are shown as the fold change of heavy/light ratio for each protein in duplicate experiments, for which opposite labelling with heavy/light isotopes was used. Shelterin subunits and select DNA repair factors are indicated in the plot. NA indicates protein was not detected in that experiment. l, Venn diagram showing overlap of the proteins identified by SILAC–PICh in wild-type Trf2f/f ES cells and Trf2f/f ear fibroblasts (EtOH conditions). m, IF–FISH analysis to detect γH2AX TIF in Trf2f/f ES cells and Trf2f/f ear fibroblasts 96 h after treatment with OHT or EtOH. γH2AX was detected by immunofluorescence (red), telomeric DNA with FISH (white) and the DNA with DAPI (blue). n, Quantification of the experiment shown in m. Mean ± s.d., ≥200 cells per condition across 3 independent experiments, one-way ANOVA. o, Quantification of the experiment shown in m. Each dot represents percentage of cells with >3 TIF in each of 3 independent experiments analysing around 100 cells per condition per experiment, bars represent mean ± s.d. of these values, one-way ANOVA. p, Quantification from experiment shown in Fig. 1g. Each dot represents percentage of cells with >3 TIF in each of 4 independent experiments analysing around 100 cells per condition per experiment, bars represent mean ± s.d. of these values, one-way ANOVA. q, Quantification of IF–FISH to detect 53BP1 TIF in the indicated cells, 96 h after treatment with EtOH or OHT. 53BP1 was detected by immunofluorescence (green), telomeric DNA with FISH (red) and DNA with DAPI (blue). c1, c2 and c3 indicate three independently derived clones. Mean ± s.d., ≥200 cells per condition across 3 independent experiments, one-way ANOVA. ns, not significant, ****P ≤ 0.0001. Scale bars, 5 μm (e–h, m).
Extended Data Fig. 3 The DDR and NHEJ are functional in ES cells.
a, Immunofluorescence to detect γH2AX (green) and 53BP1 (red) in untreated Trf2f/f ES cells and Trf2f/f ear fibroblasts or 1 h after treatment with 0.5 Gy ionizing radiation. DNA stained with DAPI (blue). b, Quantification from experiment shown in a. Each dot represents percentage of cells with >4 foci in each of 2 independent experiments analysing ≥100 cells per condition per experiment, bars represent mean ± s.d. of these values, one-way ANOVA. c, Flow cytometry determination of DNA content for the indicated cells at the indicated times after ionizing radiation treatment, 96 h after treatment with EtOH or OHT. n = 3 independent experiments, ≥10,000 cells per condition in each experiment. d, DNA from Trf2f/f ES cells and Trf2f/f MEFs was collected, subjected to pulsed-field gel electrophoresis and stained with EtBr at the indicated times after irradiation with 20 Gy. The band of DNA resulting from double-strand breaks (DSBs) is indicated. e, Quantification of the experiment in d. The DNA band resulting from DNA double-strand breaks was quantified and normalized to the unbroken DNA. The level of DNA resulting from double-strand breaks was subsequently compared to the sample collected 10 min after ionizing radiation from that condition, which was normalized as 100% double-strand breaks remaining. Mean ± s.d. n = 3 biologically independent experiments for MEF samples, n = 4 independent experiments for ES cell samples. f, Survival of non-targeting control, Ku70 knockout or Lig4-knockout Trf1f/f Trf2f/f ES cells after exposure to the indicated doses of ionizing radiation as measured using CellTiter-Glo, 4 days after ionizing radiation treatment. n = 3 (NTC and Ku70 KO) or 2 (Lig4 KO) biologically independent experiments, mean ± s.e.m. Individual clonal knockout lines are denoted c1 and c2. g, h, Western blot of whole-cell extracts from the cells used in f. Asterisk indicates a non-specific band. i, Ninety-six h after treatment with EtOH or OHT, Trf2f/f ES cells were grown asynchronously or treated for 16 h with mimosine to induce G1 arrest. DNA content from these cells was determined using flow cytometry at 0 h, 4 h and 9 h after release from mimosine. j, Mitotic chromosome spreads from asynchronous cells and cells released from mimosine block for 9 h as in i. The DNA was stained with DAPI (red) and the telomeric DNA with FISH (green). The mean percentage of fused chromosomes from k is indicated. k, Quantification from experiment shown in i, j. Mean ± s.d., ≥70 spreads per condition across 3 independent experiments, unpaired two-tailed t-test. l, Mitotic chromosome spreads from Trf2f/f ES cells 96 h after treatment with OHT and 24 h after mock treatment or treatment with 2 Gy ionizing radiation. Samples are stained as in j and the mean percentage of telomere fusions from m is indicated. m, Quantification of experiment shown in l. Mean ± s.d., ≥50 spreads per condition across 2 independent experiments, unpaired two-tailed t-test. ns, not significant, ****P ≤ 0.0001. Scale bars, 5 μm;.
Extended Data Fig. 4 TPP1 and TRF1 function are conserved in ES cell and somatic tissues.
a, Western blot of whole-cell extracts from indicated cells 96 h after EtOH or OHT treatment, and 72 h after siRNA transfection. b, TPP1 expression in the indicated cells analysed by RT–qPCR. Data were normalized to the ES cell Trf2f/f EtOH control siRNA (siCon) condition, which was arbitrarily assigned a value of 1. Mean ± s.d., n = 3 biologically independent experiments, one-way ANOVA. c, Quantification of 53BP1 TIF from cells shown in d. Mean ± s.d., ≥300 cells per condition across 3 independent experiments, one-way ANOVA, **P = 0.0013. d, 53BP1 TIF in the indicated cells. 53BP1 was detected by immunofluorescence (green), telomeres by FISH (red) and DNA with DAPI (blue). e, Mitotic chromosome spreads from indicated cells 96 h after OHT or EtOH treatment, and 72 h after siRNA transfection. Telomeres stained with FISH (green) and the DNA with DAPI (red). The mean percentage of fused telomeres from f is shown. f, Quantification of telomere fusions from e. Mean ± s.d., 90 spreads per condition across 4 independent experiments, one-way ANOVA. g, Flow cytometry determination of DNA content for the indicated cells 96 h after OHT or EtOH treatment. n = 3, ≥10,000 cells per condition. h, Telomeric RPA-pSer33 foci in Trf1f/f ES cells 72 h after treatment with EtOH or OHT. RPA-pSer33 was detected by immunofluorescence (green), telomeres by FISH (red) and DNA with DAPI (blue). i, Quantification of the experiment in h. Mean ± s.d., ≥300 cells per condition across 3 independent experiments, unpaired two-tailed t-test. j, 53BP1 TIF in Trf1f/f ES cells 72 h after treatment with OHT or EtOH. Samples are stained as in d. k, Quantification of j. Mean ± s.d., ≥300 cells per condition across 3 independent experiments, unpaired two-tailed t-test. Scale bars, 5 μm; ns, not significant, ****P ≤ 0.0001.
Extended Data Fig. 5 ES cells require shelterin for chromosome end protection.
a, Equal numbers of the indicated cells were seeded and treated with EtOH or OHT. The cells were counted at 72 h and the number of cells in each condition normalized to the ES cell Trf1+/+ Trf2f/f EtOH condition, which was given an arbitrary value of 1. Mean ± s.d., n = 3 biologically independent experiments. b, Flow cytometry determination of DNA content for the indicated cells 48 or 72 h after OHT or EtOH treatment. n = 3, ≥10,000 cells per condition. c, 53BP1 TIF in the indicated cells 72 h after treatment with OHT or EtOH. 53BP1 was detected by immunofluorescence (green), the telomeres with FISH (red) and DNA with DAPI (blue). Scale bar, 5 μm. d, Quantification of the experiment shown in c. Mean ± s.d., roughly 300 cells per condition across 3 independent experiments, one-way ANOVA, ****P ≤ 0.0001. e, Western blot of whole-cell extracts from Trf2f/f ES cells at indicated times after treatment with 2 Gy or 20 Gy ionizing radiation.
Extended Data Fig. 6 Shelterin-free ES cells activate a full DDR equivalent to that in shelterin-free somatic cells.
a, Western blotting of whole-cell extracts from Trf1f/f Trf2f/f ES cells at indicated times after EtOH or OHT treatment. b, c, Quantification of telomere fusions (b) or γH2AX TIF (c) in Trf1f/f Trf2f/f ES cells at the indicated times after OHT or EtOH treatment. Mean ± s.d., ≥70 mitotic spreads per condition for b or ≥175 cells per condition for c, across 3 independent experiments (n = 2 for 12 h condition), one-way ANOVA. d, 53BP1 TIF in the indicated cells, 48 h after EtOH or OHT treatment or infection with AdGFP or AdCre. 53BP1 was detected by immunofluorescence (green), telomeres by FISH (red) and DNA with DAPI (blue). e, Quantification of the experiment shown in d. Mean ± s.d., ≥200 cells per condition across 2 independent experiments, one-way ANOVA. f, Quantification of γH2AX TIF in the indicated cells 48 after treatment with EtOH or OHT or infection with AdGFP or AdCre. Mean ± s.d., ≥180 cells per condition across 3 independent experiments, one-way ANOVA. g, Western blotting of whole-cell extracts from Trf1f/f Trf2f/f ES cells and Trf1f/f Trf2f/f MEFs at the indicated times after EtOH or OHT treatment or infection with AdGFP or AdCre. h, Quantification of telomere fragility from mitotic chromosome spreads shown in Fig. 2f, 72 h after EtOH or OHT treatment. Mean ± s.d., ≥70 spreads per condition examined over 3 experiments, one-way ANOVA. i, γH2AX TIF in the indicated cells 36 h after EtOH or OHT treatment. γH2AX was detected by immunofluorescence (green) telomeric DNA with FISH (red) and the DNA with DAPI (blue). j, Quantification of the experiment shown in i. Mean ± s.d., ≥250 cells per condition across 3 independent experiments, one-way ANOVA. Scale bars, 5 μm; ns, not significant; ****P ≤ 0.0001.
Extended Data Fig. 7 Shelterin-free ES cell telomeres are substrates for both ATM and ATR kinases and fuse primarily via ATM signalling and NHEJ.
a, IF–FISH analysis to detect telomeric ATM-pSer1987 foci in CSK-extracted Trf1f/f Trf2f/f ES cells 36 h after treatment with OHT or EtOH. ATM-pSer1987 was detected by immunofluorescence (green), telomeres by FISH (red) and DNA with DAPI (blue). b, Quantification from experiment shown in a. Mean ± s.d., n = 2 independent experiments, ≥100 cells per condition per experiment, unpaired two-tailed t-test. c, IF–FISH analysis to detect telomeric RPA-pSer33 foci in CSK-extracted Trf1f/f Trf2f/f ES cells 36 h after treatment with OHT or EtOH. RPA-pSer33 (green) was detected by immunofluorescence, telomeres by FISH (red) and DNA with DAPI (blue). d, Quantification from experiment shown in c. Mean ± s.d., n = 4 independent experiments, ≥100 cells per condition per experiment, unpaired two-tailed t-test. e, IF–FISH analysis to detect telomeric ATM-pSer1987 foci in CSK-extracted Trf1f/f Trf2f/f MEFs 48 h after infection with AdGFP or AdCre. ATM-pSer1987 was detected by immunofluorescence (green), telomeres by FISH (red) and DNA with DAPI (blue). f, Quantification from experiment shown in e. Mean ± s.d., n = 2 independent experiments, ≥150 cells per condition per experiment, unpaired two-tailed t-test. g, IF–FISH analysis to detect telomeric RPA-pSer33 foci in CSK-extracted Trf1f/f Trf2f/f MEFs 48 h after infection with AdGFP or AdCre. RPA-pSer33 was detected by immunofluorescence (green), telomeres by FISH (red) and DNA with DAPI (blue). h, Quantification from experiment shown in g. Mean ± s.d., n = 2 independent experiments, ≥150 cells per condition per experiment, unpaired two-tailed t-test. i, Mitotic chromosome spreads from the indicated cells 72 h after treatment with OHT or EtOH, mock-treated or treated with an ATR inhibitor (ATRi). The DNA is stained with DAPI (red) and the telomeres by FISH (green). The mean percentage of fused chromosome ends from Fig. 2k is noted. j, Western blotting of whole-cell lysates from the indicated cells, 72 h after infection with AdGFP or AdCre. Asterisk indicates a non-specific band. k, Quantification of telomere fusions in the indicated cells 72 h after infection with AdGFP or AdCre. Cells were either mock-treated or treated with an ATRi. n ≥ 48 spreads per condition analysed over 2 biologically independent experiments, one-way ANOVA. ns, not significant; ****P ≤ 0.0001. l, Western blotting of whole-cell lysates from Ku70-knockout or non-targeting control Trf1f/f Trf2f/f ES cells 72 h after treatment with EtOH or OHT. m, Mitotic chromosome spreads from the indicated cells 72 h after treatment with OHT or EtOH. Cells were stained as in i and the mean percentage of fused chromosome ends from Fig. 2l is shown. n, Western blotting of whole-cell lysates from Lig4-knockout or non-targeting control Trf1f/f Trf2f/f ES cells 72 h after treatment with EtOH or OHT. Asterisk indicates a non-specific band. o, Mitotic chromosome spreads from the indicated cells 72 h after treatment with OHT or EtOH. Cells were stained as in i. The mean percentage of chromosome ends fused from Fig. 2m is noted. Scale bars, 5 μm; ****P ≤ 0.0001. For b, d, f, g, each dot represents the value from each independent experiment, bars represent mean ± s.d. of these values.
Extended Data Fig. 8 TRF2 is required for telomere protection and viability upon loss of pluripotency.
a, Bright-field images of Trf2f/f epiblast stem cells 96 h after EtOH or OHT treatment. b–f, Gene expression in the indicated cells analysed by RT–qPCR 96 h after EtOH or OHT treatment. Data were normalized to expression in the ES cell Trf2f/f EtOH sample, which was arbitrarily assigned a value of 1 for each gene in each experiment. Mean ± s.d., n = 2 biologically independent experiments. g, h, γH2AX TIF images (g) and quantification (h), 96 h hours after EtOH or OHT treatment. Mean ± s.d. ≥300 cells across 4 independent experiments, unpaired two-tailed t-test, **** P ≤ 0.0001. γH2AX was detected by immunofluorescence (green), telomeres by FISH (red) and DNA with DAPI (blue). i, 53BP1 TIF in Trf2f/f epiblast stem cells 96 h after OHT or EtOH treatment. 53BP1 was detected by immunofluorescence (green), telomeric DNA by FISH (red) and the DNA with DAPI. j–n, Gene expression in the indicated cells analysed by RT–qPCR from 2–5 days after initiating differentiation. Data were normalized as described for b–f. ‘Diff day x’ refers to cells on day x of the differentiation protocol. Mean ± s.d., n = 3 biologically independent experiments. o–r, Gene expression in the indicated cells analysed by RT–qPCR. Data were normalized as described in b–f. Mean ± s.d., n = 3 biologically independent experiments. s, Western blots of whole-cell extracts from Trf2f/f ES cells differentiated as in Fig. 3e. t, u, Nanog and Fgf5 gene expression in the indicated cells analysed by RT–qPCR 96 h after treatment with EtOH or OHT. Data were normalized as described for b–f. Mean ± s.d., n = 3. v, Western blot of whole-cell extracts from the indicated cells 96 h after treatment with EtOH or OHT. Asterisk indicates a non-specific band. Scale bar, 5 μm (h, i).
Extended Data Fig. 9 TRF2 is required for blastocyst development before implantation.
a, Quantification of Nanog-positive and Nanog-negative cells in each of the embryos assessed in Fig. 5e, f. Mean ± s.d., one-way ANOVA, ns, not significant. b, Maximal projection of z-stacks from Trf2−/− embryos displaying pan-nuclear γH2AX-positive cells, which are highlighted with arrows. γH2AX (green) and Nanog (magenta) were detected by immunofluorescence, telomeres by FISH (red) and DNA with DAPI (blue). c, Quantification of percentage of cells within an embryo showing pan-nuclear γH2AX staining from experiment shown in b. Mean ± s.d., one-way ANOVA.
Extended Data Fig. 10 TRF2 is not required for T-loop formation in ES cells.
a, IF–FISH analysis to detect telomeric ATM-pSer1987 foci in Trf2f/f ES cells 96 h after treatment with OHT or EtOH. ATM-pSer1987 was detected by immunofluorescence (green), telomeres by FISH (red) and DNA with DAPI (blue). b, Quantification from experiment shown in a. Mean ± s.d., ≥300 cells across 3 independent experiments, unpaired two-tailed t-test. c, Schematic of TRF1 and the TRF2 variants used in this study21. d, Western blots of whole-cell extracts from Trf2f/f ES cells expressing wild-type, mutant or hybrid Trf2 alleles 96 h after treatment with EtOH or OHT. e, IF–FISH detection of γH2AX TIF in the indicated cells 96 h after treatment with OHT or EtOH. γH2AX was detected by immunofluorescence (green), telomeric DNA by FISH (red) and DNA with DAPI (blue). f, Quantification of the experiment shown in e. Mean ± s.d., ≥300 cells across at least 3 independent experiments, two-tailed t-test. g, Quantification (top) of the cross-linking efficiency test (bottom) in Trf2f/f ES cells 96 h after treatment with EtOH or OHT. Mean ± s.e.m., n = 3 biologically independent experiments, unpaired two-tailed t-test. h, Measurement of linear and T-loop molecules shown in Fig. 5f. Mean ± s.e.m., n = 3 biological replicates scoring ≥2,804 molecules per replicate. T-loop measurements are a sum of the loop and tail portions of the molecule. Scale bar, 5 μm (a, e). ns, not significant, ****P ≤ 0.0001.
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Ruis, P., Van Ly, D., Borel, V. et al. TRF2-independent chromosome end protection during pluripotency. Nature 589, 103–109 (2021). https://doi.org/10.1038/s41586-020-2960-y
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DOI: https://doi.org/10.1038/s41586-020-2960-y
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