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Senescent cells harbour features of the cancer epigenome

Nature Cell Biology 15, 1495–1506 (2013) | Download Citation

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Abstract

Altered DNA methylation and associated destabilization of genome integrity and function is a hallmark of cancer. Replicative senescence is a tumour suppressor process that imposes a limit on the proliferative potential of normal cells that all cancer cells must bypass. Here we show by whole-genome single-nucleotide bisulfite sequencing that replicative senescent human cells exhibit widespread DNA hypomethylation and focal hypermethylation. Hypomethylation occurs preferentially at gene-poor, late-replicating, lamin-associated domains and is linked to mislocalization of the maintenance DNA methyltransferase (DNMT1) in cells approaching senescence. Low-level gains of methylation are enriched in CpG islands, including at genes whose methylation and silencing is thought to promote cancer. Gains and losses of methylation in replicative senescence are thus qualitatively similar to those in cancer, and this ‘reprogrammed’ methylation landscape is largely retained when cells bypass senescence. Consequently, the DNA methylome of senescent cells might promote malignancy, if these cells escape the proliferative barrier.

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References

  1. 1.

    , & The cancer epigenome–components and functional correlates. Genes Dev. 20, 3215–3231 (2006).

  2. 2.

    & Genomic insights into cancer-associated aberrant CpG island hypermethylation. Brief Funct. Genomic. 12, 174–190 (2013).

  3. 3.

    et al. Global DNA hypomethylation coupled to repressive chromatin domain formation and gene silencing in breast cancer. Genome. Res. 22, 246–258 (2012).

  4. 4.

    , , & The essence of senescence. Genes Dev. 24, 2463–2479 (2010).

  5. 5.

    et al. BRAFE600-associated senescence-like cell cycle arrest of human naevi. Nature 436, 720–724 (2005).

  6. 6.

    et al. Crucial role of p53-dependent cellular senescence in suppression of Pten-deficient tumorigenesis. Nature 436, 725–730 (2005).

  7. 7.

    et al. Oncogene-induced senescence as an initial barrier in lymphoma development. Nature 436, 660–665 (2005).

  8. 8.

    & Short telomeres limit tumour progression in vivo by inducing senescence. Cancer Cell 11, 461–469 (2007).

  9. 9.

    et al. Telomere dysfunction suppresses spontaneous tumorigenesis in vivo by initiating p53-dependent cellular senescence. EMBO Rep. 8, 497–503 (2007).

  10. 10.

    et al. Genome-scale DNA methylation pattern profiling of human bone marrow mesenchymal stem cells in long-term culture. Exp. Mol. Med. 44, 503–512 (2012).

  11. 11.

    et al. Rb-mediated heterochromatin formation and silencing of E2F target genes during cellular senescence. Cell 113, 703–716 (2003).

  12. 12.

    et al. Formation of MacroH2A-containing senescence-associated heterochromatin foci and senescence driven by ASF1a and HIRA. Dev. Cell 8, 19–30 (2005).

  13. 13.

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

  14. 14.

    & DNA methylation decreases in ageing but not in immortal cells. Science 220, 1055–1057 (1983).

  15. 15.

    et al. Replicative senescence of mesenchymal stem cells causes DNA-methylation changes which correlate with repressive histone marks. Aging 3, 873–888 (2011).

  16. 16.

    et al. Independence of repressive histone marks and chromatin compaction during senescent heterochromatic layer formation. Mol. Cell 47, 203–214 (2012).

  17. 17.

    et al. Redistribution of the Lamin B1 genomic binding profile affects rearrangement of heterochromatic domains and SAHF formation during senescence. Genes Dev. 27, 1800–1808 (2013).

  18. 18.

    et al. Lamin B1 depletion in senescent cells triggers large-scale changes in gene expression and the chromatin landscape. Genes Dev. 27, 1787–1799 (2013).

  19. 19.

    , , & Developmental differences in the immortalization of lung fibroblasts by telomerase. Aging Cell 2, 235–243 (2003).

  20. 20.

    , , & The senescence-associated secretory phenotype: the dark side of tumour suppression. Annu. Rev. Pathol. 5, 99–118 (2010).

  21. 21.

    , & Telomerase expression in normal human fibroblasts stabilizes DNA 5-methylcytosine transferase I. J. Biol. Chem. 278, 19904–19908 (2003).

  22. 22.

    et al. Human DNA methylomes at base resolution show widespread epigenomic differences. Nature 462, 315–322 (2009).

  23. 23.

    , , & Discovering high-resolution patterns of differential DNA methylation that correlate with gene expression changes. Nucleic Acids Res. 41, 6816–6827 (2013).

  24. 24.

    , , & A targeting sequence directs DNA methyltransferase to sites of DNA replication in mammalian nuclei. Cell 71, 865–873 (1992).

  25. 25.

    et al. Differential maintenance and de novo methylating activity by three DNA methyltransferases in ageing and immortalized fibroblasts. J. Cell Biochem. 84, 324–334 (2002).

  26. 26.

    , & Demethylation of classical satellite 2 and 3 DNA with chromosomal instability in senescent human fibroblasts. Exp. Gerontol. 37, 1005–1014 (2002).

  27. 27.

    , , & Human chromosome 1 satellite 3 DNA is decondensed, demethylated and transcribed in senescent cells and in A431 epithelial carcinoma cells. Cytogenet. Genome. Res. 118, 42–54 (2007).

  28. 28.

    et al. Real-time in vivo imaging of p16Ink4a reveals cross talk with p53. J. Cell Biol. 186, 393–407 (2009).

  29. 29.

    & DNA methyltransferase inhibition in normal human fibroblasts induces a p21-dependent cell cycle withdrawal. J. Biol. Chem. 276, 19610–19616 (2001).

  30. 30.

    , , & Replication timing-related and gene body-specific methylation of active human genes. Hum. Mol. Genet. 20, 670–680 (2011).

  31. 31.

    et al. Sequencing newly replicated DNA reveals widespread plasticity in human replication timing. Proc. Natl Acad. Sci. USA 107, 139–144 (2010).

  32. 32.

    , & Dynamic properties of nuclear lamins: lamin B is associated with sites of DNA replication. J. Cell Biol. 125, 1201–1212 (1994).

  33. 33.

    et al. Molecular maps of the reorganization of genome-nuclear lamina interactions during differentiation. Mol. Cell 38, 603–613 (2010).

  34. 34.

    et al. Comparative analysis of DNA replication timing reveals conserved large-scale chromosomal architecture. PLoS Genet. 6, e1001011 (2010).

  35. 35.

    et al. Regions of focal DNA hypermethylation and long-range hypomethylation in colorectal cancer coincide with nuclear lamina-associated domains. Nat. Genet. 44, 40–46 (2012).

  36. 36.

    et al. Increased methylation variation in epigenetic domains across cancer types. Nat. Genet. 43, 768–775 (2011).

  37. 37.

    et al. Aberrant overexpression of satellite repeats in pancreatic and other epithelial cancers. Science 331, 593–596 (2011).

  38. 38.

    et al. CpG island methylator phenotype in colorectal cancer. Proc. Natl Acad. Sci. USA 96, 8681–8686 (1999).

  39. 39.

    et al. CpG island methylator phenotype underlies sporadic microsatellite instability and is tightly associated with BRAF mutation in colorectal cancer. Nat. Genet. 38, 787–793 (2006).

  40. 40.

    et al. The human colon cancer methylome shows similar hypo- and hypermethylation at conserved tissue-specific CpG island shores. Nat. Genet. 41, 178–186 (2009).

  41. 41.

    et al. DNA methylation screening identifies driver epigenetic events of cancer cell survival. Cancer Cell 21, 655–667 (2012).

  42. 42.

    et al. The role of nuclear lamin B1 in cell proliferation and senescence. Genes Dev. 25, 2579–2593 (2011).

  43. 43.

    , , & DNA methyltransferase 1 knockdown activates a replication stress checkpoint. Mol. Cell Biol. 26, 7575–7586 (2006).

  44. 44.

    et al. Epigenetic inactivation of SFRP genes allows constitutive WNT signalling in colorectal cancer. Nat. Genet. 36, 417–422 (2004).

  45. 45.

    et al. Comparison of global DNA methylation profiles in replicative versus premature senescence. Life Sci. 83, 475–480 (2008).

  46. 46.

    , , & Transcriptional control of the DNA methyltransferases is altered in ageing and neoplastically-transformed human fibroblasts. Mol. Cell Biochem. 252, 33–43 (2003).

  47. 47.

    , , & Chromosomal instability and tumours promoted by DNA hypomethylation. Science 300, 455 (2003).

  48. 48.

    et al. Induction of tumours in mice by genomic hypomethylation. Science 300, 489–492 (2003).

  49. 49.

    , , , & Hypermethylation trigger of the glutathione-S-transferase gene (GSTP1) in prostate cancer cells. Oncogene 21, 1048–1061 (2002).

  50. 50.

    et al. Epigenetic polymorphism and the stochastic formation of differentially methylated regions in normal and cancerous tissues. Nat. Genet. 44, 1207–1214 (2012).

  51. 51.

    , & Double strand breaks can initiate gene silencing and SIRT1-dependent onset of DNA methylation in an exogenous promoter CpG island. PLoS Genet. 4, e1000155 (2008).

  52. 52.

    et al. Sequential DNA methylation changes are associated with DNMT3B overexpression in colorectal neoplastic progression. Gut 60, 499–508 (2011).

  53. 53.

    et al. DNMT3B expression might contribute to CpG island methylator phenotype in colorectal cancer. Clin. Cancer Res. 15, 3663–3671 (2009).

  54. 54.

    et al. V600EBraf induces gastrointestinal crypt senescence and promotes tumour progression through enhanced CpG methylation of p16INK4a. EMBO Mol. Med. 2, 458–471 (2010).

  55. 55.

    , & Transcription of IAP endogenous retroviruses is constrained by cytosine methylation. Nat. Genet. 20, 116–117 (1998).

  56. 56.

    , & Undermethylation associated with retroelement activation and chromosome remodelling in an interspecific mammalian hybrid. Nature 393, 68–72 (1998).

  57. 57.

    et al. Landscape of somatic retrotransposition in human cancers. Science 337, 967–971 (2012).

  58. 58.

    et al. Genomes of replicatively senescent cells undergo global epigenetic changes leading to gene silencing and activation of transposable elements. Aging Cell 12, 247–256 (2013).

  59. 59.

    et al. A biomarker that identifies senescent human cells in culture and in ageing skin in vivo. Proc. Natl Acad. Sci. USA 92, 9363–9367 (1995).

  60. 60.

    , , , & Cellular senescence in ageing primates. Science 311, 1257 (2006).

  61. 61.

    et al. Senescing human cells and ageing mice accumulateDNA lesions with unrepairable double-strand breaks. Nat. Cell Biol. 6, 168–170 (2004).

  62. 62.

    , & The epigenetic progenitor origin of human cancer. Nat. Rev. Genet. 7, 21–33 (2006).

  63. 63.

    et al. Abrogation of BRAFV600E-induced senescence by PI3K pathway activation contributes to melanomagenesis. Genes. Dev. 26, 1055–1069 (2012).

  64. 64.

    et al. Activation of the PIK3CA/AKT pathway suppresses senescence induced by an activated RAS oncogene to promote tumorigenesis. Mol. Cell 42, 36–49 (2011).

  65. 65.

    et al. the HIRA histone chaperone complex in the chromatin landscape. Cell Rep. 3, 1012–1019 (2013).

  66. 66.

    , , & Protocols to detect senescence-associated beta-galactosidase (SA-betagal) activity, a biomarker of senescent cells in culture and in vivo. Nat. Protoc. 4, 1798–1806 (2009).

  67. 67.

    & Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1988).

  68. 68.

    , , & Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 10, R25 (2009).

  69. 69.

    & Bismark: a flexible aligner and methylation caller for Bisulfite-Seq applications. Bioinformatics 27, 1571–1572 (2011).

  70. 70.

    , & BSmooth: from whole genome bisulfite sequencing reads to differentially methylated regions. Genome Biol. 13, R83 (2012).

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Acknowledgements

Thanks to S. Pepper in the CRUK microarray facility and to S. Hansen for assistance with DNA replication timing data. Thanks to Beijing Genome Institute for bisulfite sequencing. Work in the laboratory of P.D.A. was funded by NIA Program Project P01 AG031862 and CRUK Program A10250. S.L.B.’s laboratory was funded by NIA Program Project P01 AG031862. R.R.M.’s laboratory was funded by the MRC and the BBSRC. P.D.A. thanks P. Cairns for critical formative discussions.

Author information

    • Hazel A. Cruickshanks
    •  & Taranjit Singh Rai

    Present addresses: MRC Human Genetics Unit at the Institute of Genetics and Molecular Medicine at the University of Edinburgh, Crewe Road, Edinburgh, EH4 2XU, UK; (H.A.C.); Institute of Biomedical and Environmental Health Research, University of the West of Scotland, Paisley, PA1 2BE, UK (T.S.R.)

    • Hazel A. Cruickshanks
    •  & Tony McBryan

    These authors contributed equally to this work

Affiliations

  1. Institute of Cancer Sciences, University of Glasgow and Beatson Institute for Cancer Research, Glasgow, G61 1BD, UK

    • Hazel A. Cruickshanks
    • , Tony McBryan
    • , David M. Nelson
    • , John van Tuyn
    • , Taranjit Singh Rai
    • , Claire Brock
    • , Mark E. Drotar
    •  & Peter D. Adams

  2. Center for Pharmacogenomics, Washington University School of Medicine, St Louis, Missouri 63110, USA

    • Nathan D. VanderKraats
    •  & John R. Edwards

  3. Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA

    • Parisha P. Shah
    • , Greg Donahue
    •  & Shelley L. Berger

  4. MRC Human Genetics Unit at the Institute of Genetics and Molecular Medicine at the University of Edinburgh, Crewe Road, Edinburgh, EH4 2XU, UK

    • Donncha S. Dunican
    •  & Richard R. Meehan

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Contributions

H.A.C. carried out the bulk of the experiments. D.M.N., P.P.S., J.v.T., T.S.R., C.B., M.E.D. and D.S.D. carried out further experiments. T.M. carried out the bulk of the data analysis. N.D.V. and G.D. carried out further data analyses. H.A.C. and T.M. provided substantial and critical intellectual input. R.R.M., J.R.E. and S.L.B. provided further intellectual input. P.D.A., H.A.C. and T.M. conceived the project and wrote the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Peter D. Adams.

Integrated supplementary information

Supplementary figures

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    Confirmation of senescence in IMR-90 cells.

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    Altered gene expression in senescence.

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    Concordance of replicates and methylation changes across all chromosomes.

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    Methylation changes relative to gene expression.

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    Promoters of repressed cell cycle genes are methylated in senescence.

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    Knock down of DNMT1 triggers cell senescence, expression of satellite 2 RNA and senescence-associated chromatin changes; and overlap of hypomethylated DMRs in senescence and cancer.

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    Increased methylation of CpG islands in senescence.

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    SV40-infected “bypass” cells proliferate and uncropped versions of Figures.

Supplementary tables

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    Sequence yield.

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    CpG Methylation.

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    Methylated CpG sites.

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    Pearson Correlation Coefficients.

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    CHG Methylation.

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    CHH Methylation.

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    Error rates.

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    Individual CpG differences.

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    Genes whose promoter methylation increases and expression decreases in senescence.

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    Overlap of sDMRs, LADs, CpG islands, early and late replicating regions of the genome.

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    Overlap of sDMRs with cDMRs described in Ref. 36.

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    Overlap of sDMRs with cDMRs described in Ref. 35.

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    Overlap of sDMRs with cDMRs described in Ref. 3.

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    Number (no.) of CpGs call classified as methylated and unmethylated in indicated regions (CpG islands) in proliferating cells (P) and senescent cells (S).

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    Overlap of sDMRs with bypass DMRs.

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    Overlap of sDMR and bypass DMR intersect with cDMRs described in Ref. 36.

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    Overlap of sDMR and bypass DMR intersect with cDMRs described in Ref. 35.

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    Overlap of sDMR and bypass DMR intersect with cDMRs described in Ref. 3.

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    Statistics source data.

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    Antibodies and shRNAs used in this study.

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