Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Mammalian Rap1 controls telomere function and gene expression through binding to telomeric and extratelomeric sites

Nature Cell Biology volume 12, pages 768–780 (2010)Cite this article

Subjects

Abstract

Rap1 is a component of the shelterin complex at mammalian telomeres, but its in vivo role in telomere biology has remained largely unknown to date. Here we show that Rap1 deficiency is dispensable for telomere capping but leads to increased telomere recombination and fragility. We generated cells and mice deleted for Rap1; mice with Rap1 deletion in stratified epithelia were viable but had shorter telomeres and developed skin hyperpigmentation in adulthood. By performing chromatin immunoprecipitation coupled with ultrahigh-throughput sequencing, we found that Rap1 binds to both telomeres and to extratelomeric sites through the (TTAGGG)2 consensus motif. Extratelomeric Rap1-binding sites were enriched at subtelomeric regions, in agreement with preferential deregulation of subtelomeric genes in Rap1-deficient cells. More than 70% of extratelomeric Rap1-binding sites were in the vicinity of genes, and 31% of the genes deregulated in Rap1-null cells contained Rap1-binding sites, suggesting a role for Rap1 in transcriptional control. These findings place a telomere protein at the interface between telomere function and transcriptional regulation.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Generation of Rap1Δ/Δ cells.
Figure 2: Rap1 deletion does not affect the binding of other shelterins to telomeres.
Figure 3: Rap1-deleted MEFs show increased fragility (MTSs) and recombination but no fusions.
Figure 4: Deletion of Rap1 in stratified epithelia leads to telomere shortening and skin hyperpigmentation in adulthood.
Figure 5: Role for RAP1 in subtelomeric silencing.
Figure 6: Differentially expressed genes on abrogation of Rap1.
Figure 7: Genome-wide mapping of RAP1-binding sites.
Figure 8: De novo identification of a consensus RAP1-binding site.

Similar content being viewed by others

Accession codes

Accessions

Gene Expression Omnibus

References

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

    Article  CAS  Google Scholar 

  2. Shore, D. & Nasmyth, K. Purification and cloning of a DNA binding protein from yeast that binds to both silencer and activator elements. Cell 51, 721–732 (1987).

    Article  CAS  Google Scholar 

  3. Li, B., Oestreich, S. & de Lange, T. Identification of human Rap1: implications for telomere evolution. Cell 101, 471–483 (2000).

    Article  CAS  Google Scholar 

  4. Kyrion, G., Liu, K., Liu, C. & Lustig, A. J. Rap1 and telomere structure regulate telomere position effects in Saccharomyces cerevisiae. Genes Dev. 7, 1146–1159 (1993).

    Article  CAS  Google Scholar 

  5. Marcand, S., Gilson, E. & Shore, D. A protein-counting mechanism for telomere length regulation in yeast. Science 275, 986–990 (1997).

    Article  CAS  Google Scholar 

  6. Hecht, A., Laroche, T., Strahl-Bolsinger, S., Gasser, S. M. & Grunstein, M. Histone H3 and H4 N-termini interact with SIR3 and SIR4 proteins: a molecular model for the formation of heterochromatin in yeast. Cell 80, 583–592 (1995).

    Article  CAS  Google Scholar 

  7. Tanny, J. C., Dowd, G. J., Huang, J., Hilz, H. & Moazed, D. An enzymatic activity in the yeast Sir2 protein that is essential for gene silencing. Cell 99, 735–745 (1999).

    Article  CAS  Google Scholar 

  8. Carmen, A. A., Milne, L. & Grunstein, M. Acetylation of the yeast histone H4 N terminus regulates its binding to heterochromatin protein SIR3. J. Biol. Chem. 277, 4778–4781 (2002).

    Article  CAS  Google Scholar 

  9. Imai, S., Armstrong, C. M., Kaeberlein, M. & Guarente, L. Transcriptional silencing and longevity protein Sir2 is an NAD-dependent histone deacetylase. Nature 403, 795–800 (2000).

    Article  CAS  Google Scholar 

  10. Conrad, M. N., Wright, J. H., Wolf, A. J. & Zakian, V. A. Rap1 protein interacts with yeast telomeres in vivo: overproduction alters telomere structure and decreases chromosome stability. Cell 63, 739–750 (1990).

    Article  CAS  Google Scholar 

  11. Buchman, A. R., Kimmerly, W. J., Rine, J. & Kornberg, R. D. Two DNA-binding factors recognize specific sequences at silencers, upstream activating sequences, autonomously replicating sequences, and telomeres in Saccharomyces cerevisiae. Mol. Cell. Biol. 8, 210–225 (1988).

    Article  CAS  Google Scholar 

  12. Capieaux, E., Vignais, M. L., Sentenac, A. & Goffeau, A. The yeast H+-ATPase gene is controlled by the promoter binding factor TUF. J. Biol. Chem. 264, 7437–7446 (1989).

    CAS  PubMed  Google Scholar 

  13. Sfeir, A., Kabir, S., van Overbeek, M., Celli, G. B. & de Lange, T. Loss of Rap1 induces telomere recombination in the absence of NHEJ or a DNA damage signal. Science 327, 1657–1661 (2010).

    Article  CAS  Google Scholar 

  14. Martinez, P. 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).

    Article  CAS  Google Scholar 

  15. Tejera, A. et al. TPP1 is required for TERT recruitment, telomere elongation during nuclear reprogramming, and normal skin development in mice. Dev. Cell 18, 775–789 (2010).

    Article  CAS  Google Scholar 

  16. Yang, X., Figueiredo, L. M., Espinal, A., Okubo, E. & Li, B. Rap1 is essential for silencing telomeric variant surface glycoprotein genes in Trypanosoma brucei. Cell 137, 99–109 (2009).

    Article  CAS  Google Scholar 

  17. Rodriguez, C. I. et al. High-efficiency deleter mice show that FLPe is an alternative to Cre-loxP. Nat. Genet. 25, 139–140 (2000).

    Article  CAS  Google Scholar 

  18. Tan, M., Wei, C. & Price, C. M. The telomeric protein Rap1 is conserved in vertebrates and is expressed from a bidirectional promoter positioned between the Rap1 and KARS genes. Gene 323, 1–10 (2003).

    Article  CAS  Google Scholar 

  19. d'Adda di Fagagna, F. et al. A DNA damage checkpoint response in telomere-initiated senescence. Nature 426, 194–198 (2003).

    Article  CAS  Google Scholar 

  20. Takai, H., Smogorzewska, A. & de Lange, T. DNA damage foci at dysfunctional telomeres. Curr. Biol. 13, 1549–1556 (2003).

    Article  CAS  Google Scholar 

  21. Silver, D. P. & Livingston, D. M. Self-excising retroviral vectors encoding the Cre recombinase overcome Cre-mediated cellular toxicity. Mol. Cell 8, 233–243 (2001).

    Article  CAS  Google Scholar 

  22. Blanco, R., Munoz, P., Flores, J. M., Klatt, P. & Blasco, M. A. Telomerase abrogation dramatically accelerates TRF2-induced epithelial carcinogenesis. Genes Dev. 21, 206–220 (2007).

    Article  CAS  Google Scholar 

  23. Munoz, P., Blanco, R., Flores, J. M. & Blasco, M. A. XPF nuclease-dependent telomere loss and increased DNA damage in mice overexpressing TRF2 result in premature aging and cancer. Nat. Genet. 37, 1063–1071 (2005).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  25. Park, J. I. et al. Telomerase modulates Wnt signalling by association with target gene chromatin. Nature 460, 66–72 (2009).

    Article  CAS  Google Scholar 

  26. Tarutani, M. et al. Tissue-specific knockout of the mouse Pig-a gene reveals important roles for GPI-anchored proteins in skin development. Proc. Natl Acad. Sci. USA 94, 7400–7405 (1997).

    Article  CAS  Google Scholar 

  27. Ramirez, A., Bravo, A., Jorcano, J. L. & Vidal, M. Sequences 5′ of the bovine keratin 5 gene direct tissue- and cell-type-specific expression of a lacZ gene in the adult and during development. Differentiation 58, 53–64 (1994).

    CAS  PubMed  Google Scholar 

  28. Benetti, R., Garcia-Cao, M. & Blasco, M. A. Telomere length regulates the epigenetic status of mammalian telomeres and subtelomeres. Nat. Genet. 39, 243–250 (2007).

    Article  CAS  Google Scholar 

  29. Azzalin, C. M., Reichenbach, P., Khoriauli, L., Giulotto, E. & Lingner, J. Telomeric repeat containing RNA and RNA surveillance factors at mammalian chromosome ends. Science 318, 798–801 (2007).

    Article  CAS  Google Scholar 

  30. Schoeftner, S. & Blasco, M. A. Developmentally regulated transcription of mammalian telomeres by DNA-dependent RNA polymerase II. Nat. Cell Biol. 10, 228–236 (2008).

    Article  CAS  Google Scholar 

  31. Estep, P. W. 3rd, Warner, J. B. & Bulyk, M. L. Short-term calorie restriction in male mice feminizes gene expression and alters key regulators of conserved aging regulatory pathways. PLoS ONE 4, e5242 (2009).

    Article  Google Scholar 

  32. Crowley, V. E., Yeo, G. S. & O'Rahilly, S. Obesity therapy: altering the energy intake-and-expenditure balance sheet. Nat. Rev. Drug Discov. 1, 276–286 (2002).

    Article  CAS  Google Scholar 

  33. Leone, T. C. et al. PGC-1α deficiency causes multi-system energy metabolic derangements: muscle dysfunction, abnormal weight control and hepatic steatosis. PLoS Biol. 3, e101 (2005).

    Article  Google Scholar 

  34. Pavesi, G., Mereghetti, P., Mauri, G. & Pesole, G. Weeder web: discovery of transcription factor binding sites in a set of sequences from co-regulated genes. Nucleic Acids Res. 32, W199–W200 (2004).

    Article  CAS  Google Scholar 

  35. Bae, N. S. & Baumann, P. A Rap1/TRF2 complex inhibits nonhomologous end-joining at human telomeric DNA ends. Mol. Cell 26, 323–334 (2007).

    Article  CAS  Google Scholar 

  36. Sarthy, J., Bae, N. S., Scrafford, J. & Baumann, P. Human Rap1 inhibits non-homologous end joining at telomeres. EMBO J. 28, 3390–3399 (2009).

    Article  CAS  Google Scholar 

  37. Armanios, M. Y. et al. Telomerase mutations in families with idiopathic pulmonary fibrosis. N. Engl. J. Med. 356, 1317–1326 (2007).

    Article  CAS  Google Scholar 

  38. Mitchell, J. R., Wood, E. & Collins, K. A telomerase component is defective in the human disease dyskeratosis congenita. Nature 402, 551–555 (1999).

    Article  CAS  Google Scholar 

  39. Tsakiri, K. D. et al. Adult-onset pulmonary fibrosis caused by mutations in telomerase. Proc. Natl Acad. Sci. USA 104, 7552–7557 (2007).

    Article  CAS  Google Scholar 

  40. Vulliamy, T. et al. The RNA component of telomerase is mutated in autosomal dominant dyskeratosis congenita. Nature 413, 432–435 (2001).

    Article  CAS  Google Scholar 

  41. Yamaguchi, H. et al. Mutations in TERT, the gene for telomerase reverse transcriptase, in aplastic anemia. N. Engl. J. Med. 352, 1413–1424 (2005).

    Article  CAS  Google Scholar 

  42. Moretti, P., Freeman, K., Coodly, L. & Shore, D. Evidence that a complex of SIR proteins interacts with the silencer and telomere-binding protein Rap1. Genes Dev. 8, 2257–2269 (1994).

    Article  CAS  Google Scholar 

  43. Schoeftner, S. et al. Telomere shortening relaxes X chromosome inactivation and forces global transcriptome alterations. Proc. Natl Acad. Sci. USA 106, 19393–19398 (2009).

    Article  CAS  Google Scholar 

  44. Bradshaw, P. S., Stavropoulos, D. J. & Meyn, M. S. Human telomeric protein TRF2 associates with genomic double-strand breaks as an early response to DNA damage. Nat. Genet. 37, 193–197 (2005).

    Article  CAS  Google Scholar 

  45. Deng, Z., Atanasiu, C., Burg, J. S., Broccoli, D. & Lieberman, P. M. Telomere repeat binding factors TRF1, TRF2, and hRap1 modulate replication of Epstein–Barr virus OriP. J. Virol. 77, 11992–12001 (2003).

    Article  CAS  Google Scholar 

  46. Mignon-Ravix, C., Depetris, D., Delobel, B., Croquette, M. F. & Mattei, M. G. A human interstitial telomere associates in vivo with specific TRF2 and TIN2 proteins. Eur J. Hum. Genet. 10, 107–112 (2002).

    Article  CAS  Google Scholar 

  47. Smogorzewska, A. et al. Control of human telomere length by TRF1 and TRF2. Mol. Cell. Biol. 20, 1659–1668 (2000).

    Article  CAS  Google Scholar 

  48. Zhang, P. et al. Nontelomeric TRF2–REST interaction modulates neuronal gene silencing and fate of tumor and stem cells. Curr. Biol. 18, 1489–1494 (2008).

    Article  Google Scholar 

  49. Mendez, J. & Stillman, B. Chromatin association of human origin recognition complex, cdc6, and minichromosome maintenance proteins during the cell cycle: assembly of prereplication complexes in late mitosis. Mol. Cell. Biol. 20, 8602–8612 (2000).

    Article  CAS  Google Scholar 

  50. Brummelkamp, T. R., Bernards, R. & Agami, R. A system for stable expression of short interfering RNAs in mammalian cells. Science 296, 550–553 (2002).

    Article  CAS  Google Scholar 

  51. Brown, M. et al. A recombinant murine retrovirus for simian virus 40 large T cDNA transforms mouse fibroblasts to anchorage-independent growth. J. Virol. 60, 290–293 (1986).

    CAS  PubMed  PubMed Central  Google Scholar 

  52. Herrera, E. et al. Disease states associated with telomerase deficiency appear earlier in mice with short telomeres. EMBO J. 18, 2950–2960 (1999).

    Article  CAS  Google Scholar 

  53. Samper, E., Goytisolo, F. A., Slijepcevic, P., van Buul, P. P. & Blasco, M. A. Mammalian Ku86 protein prevents telomeric fusions independently of the length of TTAGGG repeats and the G-strand overhang. EMBO Rep. 1, 244–252 (2000).

    Article  CAS  Google Scholar 

  54. Blasco, M. A. et al. Telomere shortening and tumor formation by mouse cells lacking telomerase RNA. Cell 91, 25–34 (1997).

    Article  CAS  Google Scholar 

  55. Benetti, R. et al. Suv4-20h deficiency results in telomere elongation and derepression of telomere recombination. J. Cell Biol. 178, 925–936 (2007).

    Article  CAS  Google Scholar 

  56. Bock, C. et al. BiQ Analyzer: visualization and quality control for DNA methylation data from bisulfite sequencing. Bioinformatics 21, 4067–4068 (2005).

    Article  CAS  Google Scholar 

  57. Glez-Pena, D., Gomez-Lopez, G., Pisano, D. G. & Fdez-Riverola, F. WhichGenes: a web-based tool for gathering, building, storing and exporting gene sets with application in gene set enrichment analysis. Nucleic Acids Res. 37, W329–W334 (2009).

    Article  CAS  Google Scholar 

  58. Subramanian, A. et al. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc. Natl Acad. Sci. USA 102, 15545–15550 (2005).

    Article  CAS  Google Scholar 

  59. Jiang, Z. & Gentleman, R. Extensions to gene set enrichment. Bioinformatics 23, 306–313 (2007).

    Article  Google Scholar 

  60. Al-Shahrour, F. et al. Babelomics: advanced functional profiling of transcriptomics, proteomics and genomics experiments. Nucleic Acids Res. 36, W341–W346 (2008).

    Article  CAS  Google Scholar 

  61. Quail, M. A. et al. A large genome center's improvements to the Illumina sequencing system. Nat. Methods 5, 1005–1010 (2008).

    Article  CAS  Google Scholar 

  62. Ji, H. et al. An integrated software system for analyzing ChIP-chip and ChIP-seq data. Nat. Biotechnol. 26, 1293–1300 (2008).

    Article  CAS  Google Scholar 

  63. Chen, C. C. et al. Acetylated lysine 56 on histone H3 drives chromatin assembly after repair and signals for the completion of repair. Cell 134, 231–243 (2008).

    Article  CAS  Google Scholar 

  64. Long, J. D. et al. Evidence review of technology and dietary assessment. Worldviews Evid. Based Nurs. (in the press) (2009).

  65. Mahony, S. & Benos, P. V. STAMP: a web tool for exploring DNA-binding motif similarities. Nucleic Acids Res. 35, W253–W258 (2007).

    Article  Google Scholar 

  66. Schneider, T. D. & Stephens, R. M. Sequence logos: a new way to display consensus sequences. Nucleic Acids Res. 18, 6097–6100 (1990).

    Article  CAS  Google Scholar 

  67. Rice, P., Longden, I. & Bleasby, A. EMBOSS: the European Molecular Biology Open Software Suite. Trends Genet. 16, 276–277 (2000).

    Article  CAS  Google Scholar 

  68. Kent, W. J. et al. The human genome browser at UCSC. Genome Res. 12, 996–1006 (2002).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We are indebted to L. Harrington for Tert-deficient MEFs; S. West for Rap1 antibodies; and R. Serrano for animal care. P.M. is funded by a 'Ramón y Cajal' grant from the Spanish Ministry of Innovation and Science. M.A.B.'s laboratory is funded by the Spanish Ministry of Innovation and Science, the European Union (FP7-Genica, Telomarker), the European Research Council (ERC Advance Grants), the Spanish Association Against Cancer (AECC) and a Körber European Science Award to M.A.B. The work in M.T.'s laboratory is funded by Cancer Research UK.

Author information

Authors and Affiliations

  1. Telomeres and Telomerase Group, Molecular Oncology Program, Spanish National Cancer Research Centre (CNIO), Melchor Fernández Almagro 3, Madrid, E-28029, Spain

    Paula Martinez, Agueda M. Tejera, Stefan Schoeftner & Maria A. Blasco

  2. Telomere and Genome Stability Group, The CR-UK/MRC Gray Institute for Radiation Oncology and Biology, Old Campus Road, OX3 7DQ, Oxford, UK

    Maria Thanasoula, Ana R. Carlos & Madalena Tarsounas

  3. Bioinformatics Core Unit, Structural Biology and Biocomputing Program, Spanish National Cancer Research Centre (CNIO), Melchor Fernández Almagro 3, Madrid, E-28029, Spain

    Gonzalo Gómez-López & David G. Pisano

  4. Genomics Core Unit, Biotechnology Program, Spanish National Cancer Research Centre (CNIO), Melchor Fernández Almagro 3, Madrid, E-28029, Spain

    Orlando Dominguez

Authors
  1. Paula Martinez
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Maria Thanasoula
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ana R. Carlos
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Gonzalo Gómez-López
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Agueda M. Tejera
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Stefan Schoeftner
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Orlando Dominguez
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. David G. Pisano
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Madalena Tarsounas
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Maria A. Blasco
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

M.A.B. conceived the original idea. M.A.B. and P.M. designed experiments and wrote the manuscript. P.M. performed most of the experiments. M.T.A., A.R.C. and M.T. contributed results in Figs 2a, b and 3a–f. A.T. performed experiments in Fig. 2c, d and Supplementary Information, Fig. S2e. S.S. designed the knockout allele. O.D. performed DNA methylation analysis, microarray and ChIP-seq experiments. G.G. and D.G.P. performed microarray and ChIP-seq data analysis.

Corresponding author

Correspondence to Maria A. Blasco.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

Supplementary Information (PDF 5148 kb)

Supplementary Information

Supplementary Information (PDF 1412 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Martinez, P., Thanasoula, M., Carlos, A. et al. Mammalian Rap1 controls telomere function and gene expression through binding to telomeric and extratelomeric sites. Nat Cell Biol 12, 768–780 (2010). https://doi.org/10.1038/ncb2081

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/ncb2081

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing