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GENETICS, GENOMICS, AND MOLECULAR EVOLUTION

Epigenetic Switch from Posttranscriptional to Transcriptional Silencing Is Correlated with Promoter Hypermethylation¹

Institute of Biophysics, Academy of Sciences of the Czech Republic, Královopolská 135, 612 65 Brno, Czech Republic (M.F., A.K.); and Department of Plant Systems Biology, Flanders Interuniversity Institute for Biotechnology (V113), Ghent University, Technologiepark 927, B 9052 Gent, Belgium (H.V.H., A.D.)

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.

¹ This work was supported by the Grant Agency of the Czech Republic (grant nos. 521/01/0037 and Z5004920 to A.K. and 521/01/P042 to M.F.), by the Fund for Scientific Research (Flanders; visiting fellowship to A.K.), and by the Instituut voor de aanmoediging van Innovatie door Wetenschap en Technologie in Vlaanderen (postdoctoral fellowship to H.V.H.).

^* Corresponding author; e-mail kovarik{at}ibp.cz; fax 420–541211293.

Received March 18, 2003; returned for revision May 14, 2003; accepted July 1, 2003.

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