Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2017 Jan;24(1):30-39.
doi: 10.1038/nsmb.3335. Epub 2016 Dec 5.

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

Teresa Rivera  1 , Candy Haggblom  1 , Sandro Cosconati  2 , Jan Karlseder  1
Affiliations

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

Teresa Rivera et al. Nat Struct Mol Biol. 2017 Jan.

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.

PubMed Disclaimer

Conflict of interest statement

Statement: I declare that the authors have no competing financial interest as defined by Nature Publishing Group, or other interests that might be perceived to influence the results and discussion reported in this paper.

Figures

Figure 1
Figure 1. hESCs contain cytosine-rich telomeric overhang and extrachromosomal telomeric repeats
(a) Schematic representation of 2D gel electrophoresis, which allows the separation of DNA according to the molecular weight (1D) and conformation (2D). DNA overhangs and extrachromosomal ss DNA are visualized under native conditions. Linear or circular ds telomeric DNA are detected on denatured gels. (b) Native and denatured 2D gels of genomic DNA from HUES6 cells, probed for DNA of G-rich (top panels) or C-rich (bottom panels) telomeric sequence. 5′-3′ (RecJf) or 3′-5′ (Exo1) exonuclease treatment prior to separation is indicated. Red arrows point at the arcs resulting from circular dsDNA (T-circles). (c) C-circle assay in U20S, IMR90, HUES6, H1 and H9 cells. 100 and 50 ng of restriction-digested genomic DNA were used in each assay. The dot blot was hybridized with 32P end-labelled (CCCTAA)5 oligonucleotide probe. (d) Quantification of C-circles in U2OS, IMR90, HUES6, H1 and H9 cells. Levels are calculated relative to those of U20S cells. Data represent means ± s.e.m of four experiments. ****P <0.0001, **P <0.01, *P <0.05 (two-tailed Student’s t-test).
Figure 2
Figure 2. Telomerase-dependent telomere elongation in hESCs
(a) Representative images of CO-FISH metaphases from U2OS, HUES6, H1 and H9 cells. White arrows indicate t-SCEs. Green, (CCCTAA)n probe; Red, (TTAGGG)n probe. DNA was stained with DAPI (blue). Scale bar, 10 μm. Quantification of t-SCEs from CO-FISH analysis of the indicated cells is shown on the right. Data represent means ± s.e.m of three experiments (≥30 metaphases/experiment). ****P < 0.0001, ***P <0.001, **P <0.01 (two-tailed Student’s t-test). (b) TRAP assay of vector control and dominant negative (DN) TERT transduced H9 cells. Quantification is shown on the right. Data represent relative telomerase activity compared to control cells, n=3, (mean ± s.d.). **P <0.01 (two-tailed Student’s t-test). (c) Telomere restriction fragment length assay of H9 cells following control or TERT-DN transduction. Uncropped blot image is shown in Supplementary Data Set 1.
Figure 3
Figure 3. Telomere elongation stimulates the formation of extrachromosomal telomeric repeats in hESCs
(a) Telomere restriction fragment length assay of HUES6 cells and HUES6 cells stably expressing vector control or hTR on passage 3 after selection. (b) 1D gels of restriction digested genomic DNA from parental HUES6 and HUES6 cells 34 and 42 passages post control or hTR transduction. The gels were probed for G- and C-rich telomeric DNA under native and denatured conditions. The overhang signal in native gels was normalized against the total amount of telomeric DNA in denatured gels. Data represent means ± s.d of three independent experiments. (c) T-circle assay of 1 μg of digested genomic DNA from HUES6, vector control or hTR cells. The presence or absence of ϕ29 DNA polymerase is indicated. Quantification of T-circle products (arrow) is calculated relative to untreated cells and normalized against the signal from the reaction lacking ϕ29 polymerase. Data represent means ± s.e.m of three independent experiments. *P <0.05 (two-tailed Student’s t-test). (d) C-circle assay for 100 ng of digested genomic DNA from HUES6, vector control or hTR cells. Quantification of C-circle levels (bottom) from three independent experiments. Data represent means ± s.d. **P <0.01 (two-tailed Student’s t-test). (e) Representative images of CO-FISH metaphases from HUES6, control or hTR cells. Scale bar, 10 μm. The quantification of t-SCE events showing means ± s.e.m from three independent experiments is shown on the right. Ns, not significant (two-tailed Student’s t-test).
Figure 4
Figure 4. Reprogramming of human differentiated cells induces the accumulation of cytosine-rich telomeric overhang and extrachromosomal telomeric repeats
(a) Restriction-digested genomic DNA from human IMR90, and IMR90-iPS cell lines was electrophoresed in two dimensions and probed for DNA of G-rich (left panels) or C-rich (right panels) telomeric sequence under native and denatured conditions. Red arrows indicate T-circles. (b) C-circle assay in IMR90, H9, HUES6 and IMR90-iPS cell lines. The dot blot was hybridized with 32P end-labeled (CCCTAA)5 oligonucleotide probe. (c) Representative images of CO-FISH metaphases from IMR90 and IMR90-iPS cells. U2OS cells were included as a positive control. White arrowheads indicate t-SCEs. Scale bar, 10 μm. Quantification of t-SCE from CO-FISH analysis is shown on the right. Data represent means ± s.d of three independent experiments (≥25 metaphases/experiment). Ns, not significant (two-tailed Student’s t-test).
Figure 5
Figure 5. DNA replication stress causes the accumulation of C-circles in hESCs
(a) HUES6+hTR cells were treated with 0.2 mM, 3 mM HU, 5 μM Aphidicolin (Aph) or 0.5 μM RHPS4 for 24 h before analyzed for C-circle formation. 100 ng of digested genomic DNA was used in each condition. The quantification (bottom) is from five independent experiments. C-circle levels are calculated relative to the levels in control treated cells. Data represent means ± s.e.m. *P <0.05 (two-tailed Mann-Whitney test). Uncropped blot image is shown in Supplementary Data Set 1. (b) C-circle assay for 100 ng of digested genomic DNA from untreated HUES6+hTR or transfected with the indicated siRNAs. The quantification (bottom) is calculated relative to the levels in siControl treated cells. Data represent means ± s.e.m of three independent experiments. ***P <0.001 (two-tailed Student’s t-test). (c) Quantification of metaphase chromosomes with MTS in hESCs with overextended telomeres relative to control cells. Data represent means ± s.e.m of six independent experiments (≥30 metaphases/experiment). *P <0.05 (two-tailed Student’s t-test). Representative images of chromosomes with MTS (white arrowheads) are shown on the right. DAPI (blue), telomere FISH (green). Scale bar, 5 μm. (d) Quantification of γ-H2AX associated telomeres in meta-TIF assays in parental, vector control or hTR overexpressing HUES6 cells. The mean and s.d of three independent experiments are shown (≥25 metaphases/experiment). **P <0.01 (two-tailed Student’s t-test). Representative image from meta-TIF assay is shown on the right. DAPI (blue), telomere FISH (green) and γ-H2AX (red) IF. Scale bar, 10 μm.
Figure 6
Figure 6. XRCC3 and Nbs1 contribute to the formation of 5′ C-rich telomeric DNA and T-circles and regulate telomere length in hESCs
(a) HUES6 cells were transfected with siControl or siXRCC3 and analyzed for G-rich and C-rich telomeric DNA in 1D gels under native and denatured conditions. Quantification of the overhangs signal is shown on the right. The amount of G-rich and C-rich ss telomeric DNA in native gels was normalized against the total amount of telomeric DNA in denatured gels. Values are calculated relative to untreated cells from five independent experiments (means ± s.e.m). *P <0.05; ns, not significant (two-tailed Student’s t-test). (b) T-circle assay from HUES6 cells infected with shScramble or shRNAs against Nbs1. Samples were analyzed 5 days post-transduction. Relative T-circle levels normalized against the signal without ϕ29 DNA polymerase from three independent experiments are shown on the right (means ± s.e.m). *P <0.05, **P <0.01 (two-tailed Student’s t-test). (c) Telomere length was analyzed by TRF analysis 7 days following the indicated knockdowns of XRCC3 and Nbs1. Mean telomere length is shown in each condition calculated with TeloTool software. Quantification of telomere length fold change relative to control samples is shown on the right. Data represent means ± s.e.m between four independent experiments. ****P < 0.0001, *P <0.05 (two-tailed Student’s t-test).
Figure 7
Figure 7
Proposed model for telomere length regulation in hESCs. (a) Telomere elongation occurs through the telomerase dependent pathway, and is counteracted by trimming mechanisms mediated by XRCC3 and Nbs1 (b). The activity of XRCC3 and Nbs1 is required to promote the resolution of T-loops giving rise to ss 5′ C-rich telomeric DNA and T-circles. Both HR factors are essential to compensate for excessive telomere elongation. (c) Long telomeres are prone to replicative stress resulting in the formation of C-circles. We speculate that C-circles are generated by resolution of replication intermediates after replication fork stalling.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. de Lange T. Shelterin: the protein complex that shapes and safeguards human telomeres. Genes Dev. 2005;19:2100–10. - PubMed
    1. Makarov VL, Hirose Y, Langmore JP. Long G tails at both ends of human chromosomes suggest a C strand degradation mechanism for telomere shortening. Cell. 1997;88:657–66. - PubMed
    1. Griffith JD, et al. Mammalian telomeres end in a large duplex loop. Cell. 1999;97:503–14. - PubMed
    1. de Lange T. T-loops and the origin of telomeres. Nat Rev Mol Cell Biol. 2004;5:323–9. - PubMed
    1. Doksani Y, Wu JY, de Lange T, Zhuang X. Super-resolution fluorescence imaging of telomeres reveals TRF2-dependent T-loop formation. Cell. 2013;155:345–56. - PMC - PubMed

Publication types

LinkOut - more resources

-