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

Article metrics


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.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

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.

Accession codes


Gene Expression Omnibus


  1. 1

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

  2. 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).

  3. 3

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

  4. 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).

  5. 5

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

  6. 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).

  7. 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).

  8. 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).

  9. 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).

  10. 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).

  11. 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).

  12. 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).

  13. 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).

  14. 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).

  15. 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).

  16. 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).

  17. 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).

  18. 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).

  19. 19

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

  20. 20

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

  21. 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).

  22. 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).

  23. 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).

  24. 24

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

  25. 25

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

  26. 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).

  27. 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).

  28. 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).

  29. 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).

  30. 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).

  31. 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).

  32. 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).

  33. 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).

  34. 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).

  35. 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).

  36. 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).

  37. 37

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

  38. 38

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

  39. 39

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

  40. 40

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

  41. 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).

  42. 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).

  43. 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).

  44. 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).

  45. 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).

  46. 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).

  47. 47

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

  48. 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).

  49. 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).

  50. 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).

  51. 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).

  52. 52

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

  53. 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).

  54. 54

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

  55. 55

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

  56. 56

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

  57. 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).

  58. 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).

  59. 59

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

  60. 60

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

  61. 61

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

  62. 62

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

  63. 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).

  64. 64

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

  65. 65

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

  66. 66

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

  67. 67

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

  68. 68

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

Download references


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

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.

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) doi:10.1038/ncb2081

Download citation

Further reading