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A balance between elongation and trimming regulates telomere stability in stem cells

Abstract

Telomere length maintenance ensures self-renewal of human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs); however, the mechanisms governing telomere length homeostasis in these cell types are unclear. Here, we report that telomere length is determined by the balance between telomere elongation, which is mediated by telomerase, and telomere trimming, which is controlled by XRCC3 and Nbs1, homologous recombination proteins that generate single-stranded C-rich telomeric DNA and double-stranded telomeric circular DNA (T-circles), respectively. We found that reprogramming of differentiated cells induces T-circle and single-stranded C-rich telomeric DNA accumulation, indicating the activation of telomere trimming pathways that compensate telomerase-dependent telomere elongation in hiPSCs. Excessive telomere elongation compromises telomere stability and promotes the formation of partially single-stranded telomeric DNA circles (C-circles) in hESCs, suggesting heightened sensitivity of stem cells to replication stress at overly long telomeres. Thus, tight control of telomere length homeostasis is essential to maintain telomere stability in hESCs.

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Figure 1: hESCs contain C-rich telomeric overhang and extrachromosomal telomeric repeats.
Figure 2: Telomerase-dependent telomere elongation in hESCs.
Figure 3: Telomere elongation stimulates the formation of extrachromosomal telomeric repeats in hESCs.
Figure 4: Reprogramming of human differentiated cells induces the accumulation of C-rich telomeric overhang and extrachromosomal telomeric repeats.
Figure 5: DNA replication stress causes the accumulation of C-circles in hESCs.
Figure 6: XRCC3 and Nbs1 contribute to the formation of 5′ C-rich telomeric DNA and T-circles and regulate telomere length in hESCs.
Figure 7: Proposed model for telomere length regulation in hESCs.

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Acknowledgements

We are grateful to K. Collins (University of California, Berkeley) for generously sharing the pBABE-hTR plasmid and R. Alvarez Rodriguez (the Salk Institute) and members of J.C.I. Belmonte's laboratory (the Salk Institute) for sharing reagents. Packaging plasmids pCMV-gag-pol-PA and pCMV-VSVg were provided by G. Pao (the Salk Institute); pBABE-U3-hTR was provided by K. Collins (University of California, Berkeley); third-generation lentiviral vector was provided by R.A. Rodriguez; packaging plasmids pMDL, Rev and VSVg were provided by O. Singer (the Salk Institute). We thank the members of the Salk Institute's Stem Cell Core for expert advice and members of J.K.'s laboratory for comments. T.R. was supported by the Glenn Center for Research on Aging and CIRM training grant TG2-01158. J.K. is supported by the Salk Institute Cancer Center Core Grant (P30CA014195), the NIH (R01GM087476, R01CA174942), the Donald and Darlene Shiley Chair, the Highland Street Foundation, the Fritz B. Burns Foundation, the Emerald Foundation and the Glenn Center for Aging Research.

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Authors

Contributions

T.R. designed and performed the experiments and wrote the manuscript. C.H. carried out experiments. S.C. provided RHPS4. J.K. designed experiments, supervised the work and wrote the manuscript.

Corresponding author

Correspondence to Jan Karlseder.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Analysis of extrachromosomal T-circles, 5’ C-rich telomeric DNA and APBs in hESCs.

(a) Native and denatured 2D gels of genomic DNA from hES cell lines (H1 and H9), hybridized with radioactively labeled (CCCTAA)5 and (TTAGGG)4 oligonucleotides. Red arrows indicate the arc resulting from T-circles.

(b) Immunofluorescence for PML (red) and TRF2 (green) in hESCs (H1, H9 and HUES6). U2OS was included as a positive control for APB. DNA was stained with DAPI (blue). Scale bar, 10 μm.

Supplementary Figure 2 Self-renewal and differentiation potential of hESCs with elongated telomeres.

(a) Relative gene expression analysis of the indicated genes following the transduction of vector control or hTR. Mean values represent fold change compared to parental HUES6, n=3. Error bars represent s.e.m. ***P <0.001 (two-tailed Student’s t-test).

(b) TRAP assay of untreated, vector control and hTR transduced HUES6 cells. Quantification of telomerase activity relative to untreated cells is shown on the right, n=3. Data represent means ± s.d. **P <0.01 (two-tailed Student’s t-test).

(c) Expression of pluripotent markers in HUES6 with elongated telomeres. Immunofluorescence analysis of Sox2 (red), SSEA-4 (green), top panels; Oct4 (red), TRA-1-60 (green), bottom panels of parental, vector control and hTR expressing HUES6 cells. DNA is stained with DAPI. Scale bar, 20 μm.

(d,e) Embryoid body (EB)-mediated differentiation assay in HUES6, vector and hTR cells.

(d) Gene expression analysis of differentiation markers for the mesodermal (ACTA2, MSX1), endodermal (GATA6, AFP, ALB, FOXA2) and ectodermal (PAX6, beta-III tubulin, FGF5) germ layers in the indicated cells. Data represent means ± s.e.m., n=3.

(e) Immunofluorescence analysis of the three embryonic germ layers, AFP (endoderm), SMA (mesoderm) and TUJ1 (ectoderm) 14 days following differentiation of HUES6, vector and hTR cells. DNA was stained with DAPI (blue). Scale bar, 40 μm.

Supplementary Figure 3 IMR90 iPSC lines demonstrate pluripotency in vitro.

(a) Karyotype analysis demonstrating that IMR90-iPS cell lines maintain normal chromosomal integrity.

(b) Immunofluorescence analysis of pluripotent markers in IMR90-iPSCs. Cells were stained for Sox2 (red) and TRA-1-60 (green) (left panels) or Oct4 (red) and SSEA-4 (green) (right panels) and DAPI (blue). Scale bar, 20 μm.

(c) Gene expression analysis of pluripotent markers (Nanog, Oct4, Sox2) in IMR90-iPSCs and EB derived IMR90-iPSCs. Data represent relative expression to hESC (HUES6) of three independent experiments (mean ± s.d).

(d) IMR90-iPSCs were used in embryoid body (EB)-mediated differentiation assays and stained by immunofluorescence for AFP (endoderm), SMA (mesoderm), TUJ1 (ectoderm) and Cdx2 (trophectodermal) markers of the embryonic germ layers. DNA was stained with DAPI. Scale bar, 40 μm.

(e) Gene expression analysis of differentiation markers for the mesodermal (ACTA2, MSX1), endodermal (GATA6, AFP, ALB, FOXA2) and ectodermal (PAX6, beta-III tubulin, FGF5) germ layers in EB from IMR90-iPSCs. Data represent relative expression to EB derived from hESCs (HUES6), n=3 (mean ± s.e.m.).

Supplementary Figure 4 Extrachromosomal T-circle formation and telomere elongation is induced after cell reprogramming.

(a) T-circle assay from IMR90, IMR90-iPSCs and HUES6 cells. The presence or absence of ϕ29 DNA polymerase is indicated. Black arrow indicates T-circle products.

(b) Absence of APBs in IMR90-iPSCs. Immunofluorescence for PML (red) and TRF2 (green) in IMR90-iPSCs. DNA was stained with DAPI. Scale bar, 10 μm.

(c) Gene expression analysis of TERT and pluripotent markers (Nanog, Sox2) in IMR90-iPSCs, HUVEC-iPSCs and Keratinocytes-iPSCs. Data represent mean ± s.e.m. relative expression to hESC (HUES6), n=3.

(d) TRF analysis of IMR90-iPSCs, HUVEC-iPSCs, Keratinocytes-iPSCs and HUES6 cells.

Supplementary Figure 5 Telomere elongation in IMR90 cells induces accumulation of ECTRs.

(a) TRAP assay of untreated, vector control and hTERT transduced IMR90 cells.

(b) TRF analysis of vector control and hTERT transduced IMR90 cells at different population doublings (PD) following transduction.

(c) T-circle assay from IMR90 cells transduced as in (a). The presence or absence of ϕ29 DNA polymerase is indicated. Black arrow indicates T-circle products.

(d) C-circle assay from untreated, vector control and hTERT transduced IMR90 cells at PD40 and PD135. Quantification of C-circle levels (bottom) is shown relative to untreated IMR90 cells. Data represent means ± s.e.m of three experiments. *P <0.05; ns, not significant (two-tailed Student’s t-test).

Supplementary Figure 6 Replication stress induced in HUES6+hTR cells.

(a) HUES6+hTR cells were treated with 0.2 mM or 3 mM hydroxyurea (HU), 5 μM aphidicolin (Aph) and 0.5 μM RHPS4 for 24 hours, and cell cycle profiles were analyzed by flow cytometry. Untreated and DMSO treated cells were used as a control. The percentages of cells in G1, S and G2 phases are shown in the upper left corner in each plot. Cells were incubated with 10 μM BrdU for 30 minutes prior to the analysis. BrdU incorporation was analyzed by flow cytometry and represented in each condition. A representative experiment quantifying ≥ 10,000 cells per condition is shown.

(b) Relative gene expression of the indicated genes in HUES6+hTR cells 4 days after transfection of the indicated siRNAs. Values of gene expression are shown as fold change relative to the value of the sample control. Data represent mean ± s.e.m., n=3.

(c) Relative gene expression of the indicated genes in HUES6+vector or HUES6+hTR cells 10 days post-transduction with shScramble or shTERT. Values of gene expression are shown as fold change relative to the value of the sample control. Data represent mean ± s.e.m., n=3.

(d) Quantification of metaphase chromosomes with MTS in cells depicted in (c). Data represent means ± s.e.m of three independent experiments; ns, not significant (two-tailed Student’s t-test).

(e) TRF1 and TRF2 ChIP of parental, vector or hTR transduced HUES6 cells. Quantification of the ChIP assay relative to untreated cells is shown on the right.

Supplementary Figure 7 Contribution of XRCC3 and Nbs1 to ECTR formation.

(a) Expression of XRCC3 as fold change relative to untreated cells (HUES6) at 72 h following siControl or siXRCC3 transfection. Data represent mean ± s.d. of three independent experiments **P <0.01 (two-tailed Student’s t-test).

(b) T-circle assay from untreated, siControl and siXRCC3 HUES6 cells depicted in (a). The presence or absence of ϕ29 DNA polymerase is indicated. The quantification of T-circle levels relative to untreated cells is shown on the right. Data represent means ± s.e.m. of three independent experiments; ns, not significant (two-tailed Student’s t-test).

(c) Expression of Nbs1 as fold change relative to untreated cells (HUES6) 5 days post-transduction with shScramble or shRNAs (sh1 and sh2) against Nbs1. Data represent mean ± s.d. of three independent experiments **P <0.01, *P <0.05 (two-tailed Student’s t-test).

(d) C-circle assay from untreated, siControl or siXRCC3 HUES6 cells. Quantification of C-circle levels (bottom) is shown relative to untreated HUES6. Data represent means ± s.d of four experiments; ns, not significant (two-tailed Student’s t-test).

(e) C-circle analysis of HUES6 cells following the knockdown of Nbs1 using two different shRNAs. Quantification represents mean values relative to untreated HUES6 cells from three experiments. Error bars represent s.d., ns, not significant (two-tailed Student’s t-test).

(f) Expression of Nbs1, XRCC3 and Oct4 in XRCC3 and Nbs1 knockdown cells. Quantification represents relative gene expression to control depleted shScramble cells normalized against GAPDH expression from two experiments (mean ± s.d.).

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Rivera, T., Haggblom, C., Cosconati, S. et al. A balance between elongation and trimming regulates telomere stability in stem cells. Nat Struct Mol Biol 24, 30–39 (2017). https://doi.org/10.1038/nsmb.3335

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