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Hypomethylating drugs efficiently decrease cytosine methylation in telomeric DNA and activate telomerase without affecting telomere lengths in tobacco cells

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Abstract

Telomere homeostasis is regulated at multiple levels, including the local chromatin structure of telomeres and subtelomeres. Recent reports demonstrated that a decrease in repressive chromatin marks, such as levels of cytosine methylation in subtelomeric regions, results in telomere elongation in mouse cells. Here we show that a considerable fraction of cytosines is methylated not only in subtelomeric, but also in telomeric DNA of tobacco BY-2 cells. Drug-induced hypomethylation (demonstrated at subtelomeric, telomeric, and global DNA levels) results in activation of telomerase. However, in contrast to mouse cells, the decrease in 5-methylcytosine levels and upregulation of telomerase do not result in any changes of telomere lengths. These results demonstrate the involvement of epigenetic mechanisms in the multilevel process of regulation of telomerase activity in plant cells and, at the same time, they indicate that changes in telomerase activity can be overridden by other factors governing telomere length stability.

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References

  • Baubec T, Pecinka A, Rozhon W, Mittelsten Scheid O (2009) Effective, homogeneous and transient interference with cytosine methylation in plant genomic DNA by zebularine. Plant J 57(3):542–554. doi:10.1111/j.1365-313X.2008.03699.x

    Article  PubMed  CAS  Google Scholar 

  • Beisler JA (1978) Isolation, characterization, and properties of a labile hydrolysis product of the antitumor nucleoside, 5-azacytidine. J Med Chem 21(2):204–208

    Article  PubMed  CAS  Google Scholar 

  • Benetti R, Garcia-Cao M, Blasco MA (2007) Telomere length regulates the epigenetic status of mammalian telomeres and subtelomeres. Nat Genet 39(2):243–250. doi:10.1038/ng1952

    Article  PubMed  CAS  Google Scholar 

  • Cao X, Jacobsen SE (2002a) Locus-specific control of asymmetric and CpNpG methylation by the DRM and CMT3 methyltransferase genes. Proc Natl Acad Sci USA 99(Suppl 4):16491–16498. doi:10.1073/pnas.162371599

    Article  PubMed  CAS  Google Scholar 

  • Cao X, Jacobsen SE (2002b) Role of the Arabidopsis DRM methyltransferases in de novo DNA methylation and gene silencing. Curr Biol 12(13):1138–1144. doi:S0960982202009259

    Article  PubMed  CAS  Google Scholar 

  • Cao X, Springer NM, Muszynski MG, Phillips RL, Kaeppler S, Jacobsen SE (2000) Conserved plant genes with similarity to mammalian de novo DNA methyltransferases. Proc Natl Acad Sci USA 97(9):4979–4984. doi:97/9/4979

    Article  PubMed  CAS  Google Scholar 

  • Chinnusamy V, Zhu JK (2009) RNA-directed DNA methylation and demethylation in plants. Sci China C Life Sci 52(4):331–343. doi:10.1007/s11427-009-0052-1

    Article  PubMed  CAS  Google Scholar 

  • Cihak A (1974) Biological effects of 5-azacytidine in eukaryotes. Oncology 30(5):405–422

    Article  PubMed  CAS  Google Scholar 

  • Cokus SJ, Feng S, Zhang X, Chen Z, Merriman B, Haudenschild CD, Pradhan S, Nelson SF, Pellegrini M, Jacobsen SE (2008) Shotgun bisulphite sequencing of the Arabidopsis genome reveals DNA methylation patterning. Nature 452(7184):215–219. doi:10.1038/nature06745

    Article  PubMed  CAS  Google Scholar 

  • De Clercq E, Descamps J, De Somer P, Holy A (1978) (S)-9-(2, 3-Dihydroxypropyl)adenine: an aliphatic nucleoside analog with broad-spectrum antiviral activity. Science 200(4341):563–565

    Article  Google Scholar 

  • Dellaporta LS, Wood J, Hicks BJ (1983) A plant DNA minipreparation: version II. Plant Mol Biol Reptr 1(4):19–21

    Article  CAS  Google Scholar 

  • Doskocil J, Sorm F (1962) Distribution of 5-methylcytosine in pyrimidine sequences of deoxyribonucleic acids. Biochim Biophys Acta 55:953–959

    Article  PubMed  CAS  Google Scholar 

  • Fajkus J, Trifonov EN (2001) Columnar packing of telomeric nucleosomes. Biochem Biophys Res Commun 280(4):961–963. doi:10.1006/bbrc.2000.4208

    Article  PubMed  CAS  Google Scholar 

  • Fajkus J, Vyskot B, Bezdek M (1992) Changes in chromatin structure due to hypomethylation induced with 5-azacytidine or DL-ethionine. Febs Lett 314(1):13–16. doi:0014-5793(92)81449-V

    Article  PubMed  CAS  Google Scholar 

  • Fajkus J, Kovarik A, Kralovics R, Bezdek M (1995a) Organization of telomeric and subtelomeric chromatin in the higher plant Nicotiana tabacum. Mol Gen Genet 247(5):633–638

    Article  PubMed  CAS  Google Scholar 

  • Fajkus J, Kralovics R, Kovarik A, Fajkusova L (1995b) The telomeric sequence is directly attached to the HRS60 subtelomeric tandem repeat in tobacco chromosomes. Febs Lett 364(1):33–35. doi:0014-5793(95)00347-C

    Article  PubMed  CAS  Google Scholar 

  • Fajkus J, Kovarik A, Kralovics R (1996) Telomerase activity in plant cells. Febs Lett 391(3):307–309. doi:0014-5793(96)00757-0

    Article  PubMed  CAS  Google Scholar 

  • Fajkus J, Fulneckova J, Hulanova M, Berkova K, Riha K, Matyasek R (1998) Plant cells express telomerase activity upon transfer to callus culture, without extensively changing telomere lengths. Mol Gen Genet 260(5):470–474

    Article  PubMed  CAS  Google Scholar 

  • Fajkus J, Simickova M, Malaska J (2002) Tiptoeing to chromosome tips: facts, promises and perils of today’s human telomere biology. Philos Trans R Soc Lond B Biol Sci 357(1420):545–562. doi:10.1098/rstb.2001.1053

    Article  PubMed  CAS  Google Scholar 

  • Feng S, Jacobsen SE (2011) Epigenetic modifications in plants: an evolutionary perspective. Curr Opin Plant Biol 14(2):179–186. doi:10.1016/j.pbi.2010.12.002

    Article  PubMed  CAS  Google Scholar 

  • Feuerhahn S, Iglesias N, Panza A, Porro A, Lingner J (2010) TERRA biogenesis, turnover and implications for function. Febs Lett 584(17):3812–3818

    Article  PubMed  CAS  Google Scholar 

  • Finnegan EJ, Dennis ES (1993) Isolation and identification by sequence homology of a putative cytosine methyltransferase from Arabidopsis thaliana. Nucleic Acids Res 21(10):2383–2388

    Article  PubMed  CAS  Google Scholar 

  • Fitzgerald MS, McKnight TD, Shippen DE (1996) Characterization and developmental patterns of telomerase expression in plants. Proc Natl Acad Sci USA 93(25):14422–14427

    Article  PubMed  CAS  Google Scholar 

  • Fojtova M, Kovarik A, Votruba I, Holy A (1998) Evaluation of the impact of S-adenosylhomocysteine metabolic pools on cytosine methylation of the tobacco genome. Eur J Biochem 252(3):347–352

    Article  PubMed  CAS  Google Scholar 

  • Fojtova M, Fulneckova J, Fajkus J, Kovarik A (2002) Recovery of tobacco cells from cadmium stress is accompanied by DNA repair and increased telomerase activity. J Exp Bot 53(378):2151–2158

    Article  PubMed  CAS  Google Scholar 

  • Fulnecek J, Matyasek R, Votruba I, Holy A, Krizova K, Kovarik A (2011) Inhibition of SAH-hydrolase activity during seed germination leads to deregulation of flowering genes and altered flower morphology in tobacco. Mol Genet Genomics 285(3):225–236. doi:10.1007/s00438-011-0601-8

    Article  PubMed  CAS  Google Scholar 

  • Fulneckova J, Fajkus J (2000) Inhibition of plant telomerase by telomere-binding proteins from nuclei of telomerase-negative tissues. Febs Lett 467(2–3):305–310. doi:S0014-5793(00)01178-9

    Article  PubMed  CAS  Google Scholar 

  • Garcia-Cao M, O’Sullivan R, Peters AH, Jenuwein T, Blasco MA (2004) Epigenetic regulation of telomere length in mammalian cells by the Suv39h1 and Suv39h2 histone methyltransferases. Nat Genet 36(1):94–99. doi:10.1038/ng1278

    Article  PubMed  CAS  Google Scholar 

  • Geiman TM, Muegge K (2010) DNA methylation in early development. Mol Reprod Dev 77(2):105–113. doi:10.1002/mrd.21118

    PubMed  CAS  Google Scholar 

  • Ghoshal K, Bai S (2007) DNA methyltransferases as targets for cancer therapy. Drugs Today (Barc) 43(6):395–422. doi:10.1358/dot.2007.43.6.1062666

    Article  CAS  Google Scholar 

  • Gonzalez-Suarez E, Samper E, Ramirez A, Flores JM, Martin-Caballero J, Jorcano JL, Blasco MA (2001) Increased epidermal tumors and increased skin wound healing in transgenic mice overexpressing the catalytic subunit of telomerase, mTERT, in basal keratinocytes. EMBO J 20(11):2619–2630. doi:10.1093/emboj/20.11.2619

    Article  PubMed  CAS  Google Scholar 

  • Gonzalo S, Jaco I, Fraga MF, Chen T, Li E, Esteller M, Blasco MA (2006) DNA methyltransferases control telomere length and telomere recombination in mammalian cells. Nat Cell Biol 8(4):416–424. doi:10.1038/ncb1386

    Article  PubMed  CAS  Google Scholar 

  • Greider CW, Blackburn EH (1985) Identification of a specific telomere terminal transferase activity in Tetrahymena extracts. Cell 43(2 Pt 1):405–413. doi:0092-8674(85)90170-9

    Article  PubMed  CAS  Google Scholar 

  • Greider CW, Blackburn EH (1987) The telomere terminal transferase of Tetrahymena is a ribonucleoprotein enzyme with two kinds of primer specificity. Cell 51(6):887–898. doi:0092-8674(87)90576-9

    Article  PubMed  CAS  Google Scholar 

  • Gruenbaum Y, Stein R, Cedar H, Razin A (1981) Methylation of CpG sequences in eukaryotic DNA. Febs Lett 124(1):67–71. doi:0014-5793(81)80055-5

    Article  PubMed  CAS  Google Scholar 

  • Havlis J, Madden JE, Revilla AL, Havel J (2001) High-performance liquid chromatographic determination of deoxycytidine monophosphate and methyldeoxycytidine monophosphate for DNA demethylation monitoring: experimental design and artificial neural networks optimisation. J Chromatogr B Biomed Sci Appl 755(1–2):185–194

    Article  PubMed  CAS  Google Scholar 

  • Henikoff S, Comai L (1998) A DNA methyltransferase homolog with a chromodomain exists in multiple polymorphic forms in Arabidopsis. Genetics 149(1):307–318

    PubMed  CAS  Google Scholar 

  • Hiyama E, Hiyama K (2002) Clinical utility of telomerase in cancer. Oncogene 21(4):643–649. doi:10.1038/sj.onc.1205070

    Article  PubMed  CAS  Google Scholar 

  • Holy A (1975) Aliphatic analogues of nucleosides, nucleotides and oligonucleotides. Collect Czech Chem Commun 40:187–214

    CAS  Google Scholar 

  • Jelinek R, Holy A, Votruba I (1981) Embryotoxicity of 9-(S)-(2, 3-dihydroxypropyl)adenine. Teratology 24(3):267–275. doi:10.1002/tera.1420240304

    Article  PubMed  CAS  Google Scholar 

  • Kannan K, Nelson AD, Shippen DE (2008) Dyskerin is a component of the Arabidopsis telomerase RNP required for telomere maintenance. Mol Cell Biol 28(7):2332–2341. doi:MCB.01490-07

    Article  PubMed  CAS  Google Scholar 

  • Kim CH, Marquez VE, Mao DT, Haines DR, McCormack JJ (1986) Synthesis of pyrimidin-2-one nucleosides as acid-stable inhibitors of cytidine deaminase. J Med Chem 29(8):1374–1380

    Article  PubMed  CAS  Google Scholar 

  • Koukalova B, Reich J, Matyasek R, Kuhrova V, Bezdek M (1989) A BamHI family of highly repetitive DNA sequences of Nicotiana Tabacum. Theor Appl Genet 78:77–80

    Article  CAS  Google Scholar 

  • Koukalova B, Votruba I, Fojtova M, Holy A, Kovarik A (2002) Hypomethylation of CNG targets induced with dihydroxypropyladenine is rapidly reversed in the course of mitotic cell division in tobacco. Theor Appl Genet 105(6–7):1097–1098. doi:10.1007/s00122-002-1152-5

    PubMed  Google Scholar 

  • Kovarik A, Koukalova B, Holy A, Bezdek M (1994) Sequence-specific hypomethylation of the tobacco genome induced with dihydroxypropyladenine, ethionine and 5-azacytidine. Febs Lett 353(3):309–311

    Article  PubMed  CAS  Google Scholar 

  • Kovarik A, Koukalova B, Lim KY, Matyasek R, Lichtenstein CP, Leitch AR, Bezdek M (2000) Comparative analysis of DNA methylation in tobacco heterochromatic sequences. Chromosom Res 8(6):527–541

    Article  CAS  Google Scholar 

  • Kunicka Z, Mucha I, Fajkus J (2008) Telomerase activity in head and neck cancer. Anticancer Res 28(5B):3125–3129

    PubMed  CAS  Google Scholar 

  • Lee YW, Kim WT (2010) Tobacco GTBP1, a homolog of human heterogeneous nuclear ribonucleoprotein, protects telomeres from aberrant homologous recombination. Plant Cell 22(8):2781–2795. doi:tpc.110.076778

    Article  PubMed  CAS  Google Scholar 

  • Meyer P, Niedenhof I, ten Lohuis M (1994) Evidence for cytosine methylation of non-symmetrical sequences in transgenic Petunia hybrida. EMBO J 13(9):2084–2088

    PubMed  CAS  Google Scholar 

  • Nagata T, Nemoto Y, Hasezawa S (1992) Tobacco BY-2 cell line as the “HeLa” cell in the cell biology of higher plants. Int Rev Cytol 112:1–30

    Article  Google Scholar 

  • Ng LJ, Cropley JE, Pickett HA, Reddel RR, Suter CM (2009) Telomerase activity is associated with an increase in DNA methylation at the proximal subtelomere and a reduction in telomeric transcription. Nucleic Acids Res 37(4):1152–1159. doi:10.1093/nar/gkn1030

    Article  PubMed  CAS  Google Scholar 

  • Richards EJ, Elgin SC (2002) Epigenetic codes for heterochromatin formation and silencing: rounding up the usual suspects. Cell 108(4):489–500. doi:S009286740200644X

    Article  PubMed  CAS  Google Scholar 

  • Riha K, Fajkus J, Siroky J, Vyskot B (1998) Developmental control of telomere lengths and telomerase activity in plants. Plant Cell 10(10):1691–1698

    Article  PubMed  CAS  Google Scholar 

  • Ruckova E, Friml J, Prochazkova Schrumpfova P, Fajkus J (2008) Role of alternative telomere lengthening unmasked in telomerase knock-out mutant plants. Plant Mol Biol 66(6):637–646. doi:10.1007/s11103-008-9295-7

    Article  PubMed  CAS  Google Scholar 

  • Šebestová L, Votruba I, Holy A (1984) S-adenosyl-L-homcysteine hydrolase from Nicotiana tabacum L.: isolation and properties. Collect Czech Chem Commun 49:1543–1551

    Article  Google Scholar 

  • Shay JW, Wright WE (2010) Telomeres and telomerase in normal and cancer stem cells. Febs Lett 584(17):3819–3825. doi:10.1016/j.febslet.2010.05.026

    Article  PubMed  CAS  Google Scholar 

  • Vaillant I, Paszkowski J (2007) Role of histone and DNA methylation in gene regulation. Curr Opin Plant Biol 10(5):528–533. doi:10.1016/j.pbi.2007.06.008

    Article  PubMed  CAS  Google Scholar 

  • Vrbsky J, Akimcheva S, Watson JM, Turner TL, Daxinger L, Vyskot B, Aufsatz W, Riha K (2010) siRNA-mediated methylation of Arabidopsis telomeres. PLoS Genet 6(6):e1000986. doi:10.1371/journal.pgen.1000986

    Article  PubMed  Google Scholar 

  • Walder RY, Langtimm CJ, Chatterjee R, Walder JA (1983) Cloning of the MspI modification enzyme. The site of modification and its effects on cleavage by MspI and HpaII. J Biol Chem 258(2):1235–1241

    PubMed  CAS  Google Scholar 

  • Wingard JR, Hess AD, Stuart RK, Saral R, Burns WH (1983) Effect of several antiviral agents on human lymphocyte functions and marrow progenitor cell proliferation. Antimicrob Agents Chemother 23(4):593–597

    PubMed  CAS  Google Scholar 

  • Yang SW, Kim SK, Kim WT (2004) Perturbation of NgTRF1 expression induces apoptosis-like cell death in tobacco BY-2 cells and implicates NgTRF1 in the control of telomere length and stability. Plant Cell 16(12):3370–3385. doi:10.1105/tpc.104.026278

    Article  PubMed  CAS  Google Scholar 

  • Yoo CB, Cheng JC, Jones PA (2004) Zebularine: a new drug for epigenetic therapy. Biochem Soc Trans 32(Pt 6):910–912. doi:10.1042/BST0320910

    PubMed  CAS  Google Scholar 

  • Zhang M, Kimatu JN, Xu K, Liu B (2010) DNA cytosine methylation in plant development. J Genet Genomics 37(1):1–12. doi:10.1016/S1673-8527(09)60020-5

    Article  PubMed  Google Scholar 

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Acknowledgments

This work was supported by the Czech Science Foundation (P501/11/0289, P501/11/0596) the Czech Ministry of Education (MSM0021622415, LC06004 and LC 06035) and by the project “CEITEC—Central European Institute of Technology” (CZ.1.05/1.1.00/02.0068) from European Regional Development Fund. We thank Jana Kapustová for technical assistance, Dr. Ivan Votruba, Institute of Organic Chemistry and Biochemistry ASCR v.v.i., Prague, for providing us with DHPA and Dr. Aleš Kovařík, Institute of Biophysics, ASCR v.v.i., Brno, for providing us with a bacterial clone containing the subtelomeric repeat HRS60.

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Majerová, E., Fojtová, M., Mozgová, I. et al. Hypomethylating drugs efficiently decrease cytosine methylation in telomeric DNA and activate telomerase without affecting telomere lengths in tobacco cells. Plant Mol Biol 77, 371 (2011). https://doi.org/10.1007/s11103-011-9816-7

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