Changes in the distribution of methylcytosine residues along a transgene locus of tobacco (Nicotiana tabacum) in relation to the type of gene silencing were studied in parental plant leaves, calli, and regenerated plants derived thereof. Parental-silenced HeLo1 (hemizygous for locus 1) plants show posttranscriptional silencing of the residing nptII (neomycin phosphotransferase II) transgene and cytosine methylation restricted to the 3' end and center part of the transcribed region. Here, we report that with an increasing number of cell cycles, DNA methylation changes gradually, and methylation is introduced into the promoter during cell culture and more slowly in vegetatively propagated plants. After 24 months of callus in vitro cultivation, an epigenetic variant, designated locus 1E, was obtained in which cytosine methylation of symmetrical (CG and CNG) sites was almost complete within the 5' end of the nptII-transcribed region and the 35S promoter. Further, methylation of nonsymmetrical sites appeared de novo in the promoter, whereas this type of methylation was significantly reduced in the 3' end of the transcribed region when compared with locus 1. The newly established epigenetic patterns were stably transmitted from calli into regenerated plants and their progeny. The protein and steady-state RNA levels remained low in locus 1E, whereas with nuclear run-on assays, no detectable amounts of primary transcripts were found along the nptII gene, indicating that the methylated promoter became inactivated. The results suggest that a switch between posttranscriptional and transcriptional gene silencing could be a mechanism leading to irrevocable shut down of gene expression within a finite number of generations.

Institute of Biophysics Academy of Sciences of the Czech Republic Královopolská 135 612 65 Brno Czech Republic

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Arnaud P, Goubely C, Pélissier T, Deragon JM (2000) SINE retroposons can be used in vivo as nucleation centers for de novo methylation. Mol Cell Biol 20: 3434–3441 PubMed PMC

Aufsatz W, Mette MF, van der Winden J, Matzke AJ, Matzke M (2002) RNA-directed DNA methylation in Arabidopsis. Proc Natl Acad Sci USA Suppl 99: 16499–16506 PubMed PMC

Bartee L, Bender J (2001) Two Arabidopsis methylation-deficiency mutations confer only partial effects on a methylated endogenous gene family. Nucleic Acids Res 29: 2127–2134 PubMed PMC

Boyes J, Bird A (1991) DNA methylation inhibits transcription indirectly via a methyl-CpG binding protein. Cell 64: 1123–1134 PubMed

Christman JK, Sheikhnejad G, Marasco CJ, Sufrin JR (1995) 5-Methyl-2′-deoxycytidine in single-stranded DNA can act in cis to signal de novo DNA methylation. Proc Natl Acad Sci USA 92: 7347–7351 PubMed PMC

De Buck S, Depicker A (2001) Disruption of their palindromic arrangement leads to selective loss of DNA methylation in inversely repeated gus transgenes in Arabidopsis. Mol Genet Genomics 265: 1060–1068 PubMed

Depicker A, Van Montagu M (1997) Posttranscriptional gene silencing in plants. Curr Opin Cell Biol 9: 373–382 PubMed

Dieguez MJ, Vaucheret H, Paszkowski J, Mittelsten Scheid O (1998) Cytosine methylation at CG and CNG sites is not a prerequisite for the initiation of transcriptional gene silencing in plants, but it is required for its maintenance. Mol Gen Genet 259: 207–215 PubMed

Dorer DR, Henikoff S (1994) Expansions of transgene repeats cause heterochromatin formation and gene silencing in Drosophila. Cell 77: 993–1002 PubMed

Fulnecek J, Matyasek R, Kovarik A, Bezdek M (1998) Mapping of 5-methylcytosine residues in Nicotiana tabacum 5S rRNA genes by genomic sequencing. Mol Gen Genet 259: 133–141 PubMed

Ingelbrecht I, Van Houdt H, Van Montagu M, Depicker A (1994) Posttranscriptional silencing of reporter transgenes in tobacco correlates with DNA methylation. Proc Natl Acad Sci USA 91: 10502–10506 PubMed PMC

Kaeppler SM, Kaeppler HF, Rhee Y (2000) Epigenetic aspects of somaclonal variation in plants. Plant Mol Biol 43: 179–188 PubMed

Kilby NJ, Leyser HM, Furner IJ (1992) Promoter methylation and progressive transgene inactivation in Arabidopsis. Plant Mol Biol 20: 103–112 PubMed

Kloti A, He X, Potrykus I, Hohn T, Futterer J (2002) Tissue-specific silencing of a transgene in rice. Proc Natl Acad Sci USA 99: 10881–10886 PubMed PMC

Kovarik A, Koukalova B, Bezdek M, Opatrny Z (1997) Hypermethylation of tobacco heterochromaic loci in response to osmotic stress. Theor Appl Genet 95: 301–306

Kovarik A, Van Houdt H, Holy A, Depicker A (2000) Drug-induced hypomethylation of a posttranscriptionally silenced transgene locus of tobacco leads to partial release of silencing. FEBS Lett 467: 47–51 PubMed

Kunz C, Narangajavana J, Jakowitsch J, Park YD, Rene Delon T, Kovarik A, Koukalova B, van der Winden J, Aufsatz W, Mette F et al. (2003) Studies on the effects of a flanking repetitive sequence on the expression of single copy transgenes in Nicotiana sylvestris and in N. sylvestris-N. tomentosiformis hybrids. Plant Mol Biol 52: 203–215 PubMed

Lam E, Benfey PN, Gilmartin PM, Fang RX, Chua NH (1989) Site-specific mutations alter in vitro factor binding and change promoter expression pattern in transgenic plants. Proc Natl Acad Sci USA 86: 7890–7894 PubMed PMC

Mette MF, Aufsatz W, van der Winden J, Matzke MA, Matzke AJ (2000) Transcriptional silencing and promoter methylation triggered by double-stranded RNA. EMBO J 19: 5194–5201 PubMed PMC

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

Mitsuhara I, Shirasawa-Seo N, Iwai T, Nakamura S, Honkura R, Ohashi Y (2002) Release from posttranscriptional gene silencing by cell proliferation in transgenic tobacco plants: possible mechanism for noninheritance of the silencing. Genetics 160: 343–352 PubMed PMC

Morel JB, Mourrain P, Beclin C, Vaucheret H (2000) DNA methylation and chromatin structure affect transcriptional and posttranscriptional transgene silencing in Arabidopsis. Curr Biol 10: 1591–1594 PubMed

Muller A, Marins M, Kamisugi Y, Meyer P (2002) Analysis of hypermethylation in the RPS element suggests a signal function for short inverted repeats in de novo methylation. Plant Mol Biol 48: 383–399 PubMed

Nelson M, Raschke E, McClelland M (1993) Effect of site-specific methylation on restriction endonucleases and DNA modification methyltransferases. Nucleic Acids Res 21: 3139–3154 PubMed PMC

Olhoft PM, Phillips RL (1999) Genetic and epigenetic instability in tissue culture and regenerated progenies. In HR Lerner, ed, Plant Responses to Environmental Stresses: From Phytohormones to Genome Reorganization. Marcel Decker, New York, pp 111–148

Park YD, Papp I, Moscone EA, Iglesias VA, Vaucheret H, Matzke AJ, Matzke MA (1996) Gene silencing mediated by promoter homology occurs at the level of transcription and results in meiotically heritable alterations in methylation and gene activity. Plant J 9: 183–194 PubMed

Pélissier T, Thalmeir S, Kempe D, Sanger HL, Wassenegger M (1999) Heavy de novo methylation at symmetrical and non-symmetrical sites is a hallmark of RNA-directed DNA methylation. Nucleic Acids Res 27: 1625–1634 PubMed PMC

Pradhan S, Esteve PO (2003) Allosteric activator domain of maintenance human DNA (Cytosine-5) methyltransferase and its role in methylation spreading. Biochemistry 42: 5321–5332 PubMed

Selker EU (1999) Gene silencing: repeats that count. Cell 97: 157–160 PubMed

Schmitt F, Oakeley EJ, Jost JP (1997) Antibiotics induce genome-wide hypermethylation in cultured Nicotiana tabacum plants. J Biol Chem 272: 1534–1540 PubMed

Stam M, Viterbo A, Mol JN, Kooter JM (1998) Position-dependent methylation and transcriptional silencing of transgenes in inverted T-DNA repeats: implications for posttranscriptional silencing of homologous host genes in plants. Mol Cell Biol 18: 6165–6177 PubMed PMC

Sugiura M, Shinozaki K, Zaita N, Kusuda M, Kumano M (1986) Clone bank of the tobacco (Nicotiana tabacum) chloroplast genome as a set of overlapping restriction endonuclease fragments: mapping of eleven ribosomal protein genes. Plant Sci 44: 211–216

Turker MS (2002) Gene silencing in mammalian cell and the spread of DNA methylation. Oncogene 21: 5388–5393 PubMed

Vaistij FE, Jones L, Baulcombe DC (2002) Spreading of RNA targeting and DNA methylation in RNA silencing requires transcription of the target gene and a putative RNA-dependent RNA polymerase. Plant Cell 14: 857–867 PubMed PMC

van Blokland R, van der Geest N, Mol JN, Kooter JM (1994) Transgene-mediated suppression of chalcone synthase expression in Petunia hybrida results from an increase in RNA turnover. Plant J 6: 861–877

Van Houdt H, Bleys A, Depicker A (2003) RNA target sequences promote spreading of RNA silencing. Plant Physiol 131: 245–253 PubMed PMC

Van Houdt H, Ingelbrecht I, Van Montagu M, Depicker A (1997) Posttranscriptional silencing of a neomycin phosphotransferase II transgene correlates with the accumulation of unproductive RNAs and with increased cytosine methylation of 3′ flanking regions. Plant J 12: 379–392

Van Houdt H, Kovarik A, Van Montagu M, Depicker A (2000) Cross-talk between posttranscriptionally silenced neomycin phosphotransferase II transgenes. FEBS Lett 467: 41–46 PubMed

Vaucheret H, Beclin C, Fagard M (2001) Posttranscriptional gene silencing in plants. J Cell Sci 114: 3083–3091 PubMed

Vaucheret H, Fagard M (2001) Transcriptional gene silencing in plants: targets, inducers and regulators. Trends Genet 17: 29–35 PubMed

Wassenegger M (2000) RNA-directed DNA methylation. Plant Mol Biol 43: 203–220 PubMed

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