Article | Published:

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

Nature Structural & Molecular Biology volume 24, pages 3039 (2017) | Download Citation

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.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from $8.99

All prices are NET prices.

References

  1. 1.

    Shelterin: the protein complex that shapes and safeguards human telomeres. Genes Dev. 19, 2100–2110 (2005).

  2. 2.

    , & Long G tails at both ends of human chromosomes suggest a C strand degradation mechanism for telomere shortening. Cell 88, 657–666 (1997).

  3. 3.

    et al. Mammalian telomeres end in a large duplex loop. Cell 97, 503–514 (1999).

  4. 4.

    T-loops and the origin of telomeres. Nat. Rev. Mol. Cell Biol. 5, 323–329 (2004).

  5. 5.

    , , & Super-resolution fluorescence imaging of telomeres reveals TRF2-dependent T-loop formation. Cell 155, 345–356 (2013).

  6. 6.

    A theory of marginotomy. The incomplete copying of template margin in enzymic synthesis of polynucleotides and biological significance of the phenomenon. J. Theor. Biol. 41, 181–190 (1973).

  7. 7.

    Origin of concatemeric T7 DNA. Nat. New Biol. 239, 197–201 (1972).

  8. 8.

    , & Telomeric 3′ overhangs derive from resection by Exo1 and Apollo and fill-in by POT1b-associated CST. Cell 150, 39–52 (2012).

  9. 9.

    et al. Specific association of human telomerase activity with immortal cells and cancer. Science 266, 2011–2015 (1994).

  10. 10.

    , , , & Telomerase activity in human germline and embryonic tissues and cells. Dev. Genet. 18, 173–179 (1996).

  11. 11.

    et al. Protein composition of catalytically active human telomerase from immortal cells. Science 315, 1850–1853 (2007).

  12. 12.

    , , , & Evidence for an alternative mechanism for maintaining telomere length in human tumors and tumor-derived cell lines. Nat. Med. 3, 1271–1274 (1997).

  13. 13.

    , , , & Alternative lengthening of telomeres is characterized by high rates of telomeric exchange. Cancer Res. 64, 2324–2327 (2004).

  14. 14.

    et al. Telomerase-negative immortalized human cells contain a novel type of promyelocytic leukemia (PML) body. Cancer Res. 59, 4175–4179 (1999).

  15. 15.

    , , , & Telomere elongation in immortal human cells without detectable telomerase activity. EMBO J. 14, 4240–4248 (1995).

  16. 16.

    & Mammalian 5′ C-rich telomeric overhangs are a mark of recombination-dependent telomere maintenance. Mol. Cell 42, 224–236 (2011).

  17. 17.

    et al. Extra-chromosomal telomere repeat DNA in telomerase-negative immortalized cell lines. Biochem. Biophys. Res. Commun. 247, 765–772 (1998).

  18. 18.

    & Telomeric DNA in ALT cells is characterized by free telomeric circles and heterogeneous T-loops. Mol. Cell. Biol. 24, 9948–9957 (2004).

  19. 19.

    et al. DNA C-circles are specific and quantifiable markers of alternative-lengthening-of-telomeres activity. Nat. Biotechnol. 27, 1181–1185 (2009).

  20. 20.

    & How stem cells age and why this makes us grow old. Nat. Rev. Mol. Cell Biol. 8, 703–713 (2007).

  21. 21.

    Telomere dynamics and aging. Prog. Mol. Biol. Transl. Sci. 125, 89–111 (2014).

  22. 22.

    et al. Telomerase-mediated telomere elongation from human blastocysts to embryonic stem cells. J. Cell Sci. 127, 752–762 (2014).

  23. 23.

    et al. Telomere shortening and loss of self-renewal in dyskeratosis congenita induced pluripotent stem cells. Nature 474, 399–402 (2011).

  24. 24.

    et al. C. elegans telomeres contain G-strand and C-strand overhangs that are bound by distinct proteins. Cell 132, 745–757 (2008).

  25. 25.

    , , , & Control of telomere length by a trimming mechanism that involves generation of t-circles. EMBO J. 28, 799–809 (2009).

  26. 26.

    , , , & Normal mammalian cells negatively regulate telomere length by telomere trimming. Hum. Mol. Genet. 20, 4684–4692 (2011).

  27. 27.

    & Unusual telomeric DNAs in human telomerase-negative immortalized cells. Mol. Cell. Biol. 29, 703–713 (2009).

  28. 28.

    , & Homologous recombination generates T-loop-sized deletions at human telomeres. Cell 119, 355–368 (2004).

  29. 29.

    et al. Rapid induction of alternative lengthening of telomeres by depletion of the histone chaperone ASF1. Nat. Struct. Mol. Biol. 21, 167–174 (2014).

  30. 30.

    et al. Telomeres acquire embryonic stem cell characteristics in induced pluripotent stem cells. Cell Stem Cell 4, 141–154 (2009).

  31. 31.

    et al. Telomere elongation in induced pluripotent stem cells from dyskeratosis congenita patients. Nature 464, 292–296 (2010).

  32. 32.

    et al. Molecular insights into the heterogeneity of telomere reprogramming in induced pluripotent stem cells. Cell Res. 22, 757–768 (2012).

  33. 33.

    et al. Rapid and highly efficient generation of induced pluripotent stem cells from human umbilical vein endothelial cells. PLoS One 6, e19743 (2011).

  34. 34.

    et al. The metabolome of induced pluripotent stem cells reveals metabolic changes occurring in somatic cell reprogramming. Cell Res. 22, 168–177 (2012).

  35. 35.

    et al. SMARCAL1 maintains telomere integrity during DNA replication. Proc. Natl. Acad. Sci. USA 112, 14864–14869 (2015).

  36. 36.

    et al. Stabilization of quadruplex DNA perturbs telomere replication leading to the activation of an ATR-dependent ATM signaling pathway. Nucleic Acids Res. 37, 5353–5364 (2009).

  37. 37.

    et al. Mammalian DNA2 helicase/nuclease cleaves G-quadruplex DNA and is required for telomere integrity. EMBO J. 32, 1425–1439 (2013).

  38. 38.

    et al. DNA2 drives processing and restart of reversed replication forks in human cells. J. Cell Biol. 208, 545–562 (2015).

  39. 39.

    et al. Mammalian telomeres resemble fragile sites and require TRF1 for efficient replication. Cell 138, 90–103 (2009).

  40. 40.

    & 5′ C-rich telomeric overhangs are an outcome of rapid telomere truncation events. DNA Repair (Amst.) 12, 238–245 (2013).

  41. 41.

    , , , & Xrcc3 and Nbs1 are required for the production of extrachromosomal telomeric circles in human alternative lengthening of telomere cells. Cancer Res. 67, 1513–1519 (2007).

  42. 42.

    & Alternative lengthening of telomeres: models, mechanisms and implications. Nat. Rev. Genet. 11, 319–330 (2010).

  43. 43.

    et al. Telomere maintenance in telomerase-deficient mouse embryonic stem cells: characterization of an amplified telomeric DNA. Mol. Cell. Biol. 20, 4115–4127 (2000).

  44. 44.

    et al. An increase in telomere sister chromatid exchange in murine embryonic stem cells possessing critically shortened telomeres. Proc. Natl. Acad. Sci. USA 102, 10256–10260 (2005).

  45. 45.

    et al. Zscan4 regulates telomere elongation and genomic stability in ES cells. Nature 464, 858–863 (2010).

  46. 46.

    et al. Genetic and molecular identification of three human TPP1 functions in telomerase action: recruitment, activation, and homeostasis set point regulation. Genes Dev. 28, 1885–1899 (2014).

  47. 47.

    et al. Defective telomere elongation and hematopoiesis from telomerase-mutant aplastic anemia iPSCs. J. Clin. Invest. 123, 1952–1963 (2013).

  48. 48.

    et al. Poly(A)-specific ribonuclease (PARN) mediates 3′-end maturation of the telomerase RNA component. Nat. Genet. 47, 1482–1488 (2015).

  49. 49.

    et al. Impaired telomere maintenance and decreased canonical WNT signaling but normal ribosome biogenesis in induced pluripotent stem cells from X-linked dyskeratosis congenita patients. PLoS One 10, e0127414 (2015).

  50. 50.

    & The DNA damage machinery and homologous recombination pathway act consecutively to protect human telomeres. Cell 127, 709–720 (2006).

  51. 51.

    et al. Disruption of telomere maintenance by depletion of the MRE11/RAD50/NBS1 complex in cells that use alternative lengthening of telomeres. J. Biol. Chem. 282, 29314–29322 (2007).

  52. 52.

    et al. Chromosome instability and defective recombinational repair in knockout mutants of the five Rad51 paralogs. Mol. Cell. Biol. 21, 2858–2866 (2001).

  53. 53.

    et al. Nbn heterozygosity renders mice susceptible to tumor formation and ionizing radiation-induced tumorigenesis. Cancer Res. 63, 7263–7269 (2003).

  54. 54.

    & How telomeres are replicated. Nat. Rev. Mol. Cell Biol. 8, 825–838 (2007).

  55. 55.

    et al. Increased telomere fragility and fusions resulting from TRF1 deficiency lead to degenerative pathologies and increased cancer in mice. Genes Dev. 23, 2060–2075 (2009).

  56. 56.

    et al. SMARCAL1 catalyzes fork regression and Holliday junction migration to maintain genome stability during DNA replication. Genes Dev. 26, 151–162 (2012).

  57. 57.

    et al. A short G1 phase imposes constitutive replication stress and fork remodelling in mouse embryonic stem cells. Nat. Commun. 7, 10660 (2016).

  58. 58.

    , , & TeloTool: a new tool for telomere length measurement from terminal restriction fragment analysis with improved probe intensity correction. Nucleic Acids Res. 42, e21 (2014).

  59. 59.

    et al. Derivation of human embryonic stem cells in defined conditions. Nat. Biotechnol. 24, 185–187 (2006).

  60. 60.

    & Telomerase RNA level limits telomere maintenance in X-linked dyskeratosis congenita. Genes Dev. 20, 2848–2858 (2006).

  61. 61.

    , , & Reduced histone biosynthesis and chromatin changes arising from a damage signal at telomeres. Nat. Struct. Mol. Biol. 17, 1218–1225 (2010).

  62. 62.

    , , & Phosphorylation and regulation of Akt/PKB by the rictor-mTOR complex. Science 307, 1098–1101 (2005).

  63. 63.

    et al. A high proliferation rate is required for cell reprogramming and maintenance of human embryonic stem cell identity. Curr. Biol. 21, 45–52 (2011).

  64. 64.

    et al. Spontaneous occurrence of telomeric DNA damage response in the absence of chromosome fusions. Nat. Struct. Mol. Biol. 16, 1244–1251 (2009).

  65. 65.

    , & Senescence induced by altered telomere state, not telomere loss. Science 295, 2446–2449 (2002).

  66. 66.

    , , , & Ku suppresses formation of telomeric circles and alternative telomere lengthening in Arabidopsis. Mol. Cell 27, 163–169 (2007).

Download references

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.

Author information

Affiliations

  1. Molecular and Cellular Biology Department, The Salk Institute for Biological Studies, La Jolla, California, USA.

    • Teresa Rivera
    • , Candy Haggblom
    •  & Jan Karlseder
  2. DiSTABiF, Second University of Naples, Caserta, Italy.

    • Sandro Cosconati

Authors

  1. Search for Teresa Rivera in:

  2. Search for Candy Haggblom in:

  3. Search for Sandro Cosconati in:

  4. Search for Jan Karlseder in:

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.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Jan Karlseder.

Integrated supplementary information

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–7 and Supplementary Table 1

  2. 2.

    Supplementary Data Set 1

    Uncropped gels and blots

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/nsmb.3335