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Nucleic Acids Research 2006 34(8):2280-2293; doi:10.1093/nar/gkl180
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Published online 2 May 2006

© The Author 2006. Published by Oxford University Press. All rights reserved
The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions@oxfordjournals.org

Article

The trans-silencing capacity of invertedly repeated transgenes depends on their epigenetic state in tobacco

Miloslava Fojtová, Annick Bleys1, Jana Bedrichová, Helena Van Houdt1, Katerina Krízová, Anna Depicker1 and Ales Kovarík*

Institute of Biophysics, Academy of Sciences of the Czech Republic CZ-612 65 Brno, Czech Republic 1 Department of Plant Systems Biology, Flanders Interuniversity Institute for Biotechnology, Ghent University B-9052 Ghent, Belgium

*To whom correspondence should be addressed at Institute of Biophysics, Academy of Sciences of the Czech Republic, Královopolská 135, CZ 612 65 Brno, Czech Republic. Tel: +420 541 517 178; Fax: +420 541 211 293; Email: kovarik{at}ibp.cz

Received January 27, 2006. Revised March 13, 2006. Accepted March 22, 2006.

We studied the in trans-silencing capacities of a transgene locus that carried the neomycin phosphotransferase II reporter gene linked to the 35S promoter in an inverted repeat (IR). This transgene locus was originally posttranscriptionally silenced but switched to a transcriptionally silenced epiallele after in vitro tissue culture. Here, we show that both epialleles were strongly methylated in the coding region and IR center. However, by genomic sequencing, we found that the 1.0 kb region around the transcription start site was heavily methylated in symmetrical and non-symmetrical contexts in transcriptionally but not in posttranscriptionally silenced epilallele. Also, the posttranscriptionally silenced epiallele could trans-silence and trans-methylate homologous transgene loci irrespective of their genomic organization. We demonstrate that this in trans-silencing was accompanied by the production of small RNA molecules. On the other hand, the transcriptionally silenced variant could neither trans-silence nor trans-methylate homologous sequences, even after being in the same genetic background for generations and meiotic cycles. Interestingly, 5-aza-2-deoxy-cytidine-induced hypomethylation could partially restore signaling from the transcriptionally silenced epiallele. These results are consistent with the hypothesis that non-transcribed highly methylated IRs are poor silencers of homologous loci at non-allelic positions even across two generations and that transcription of the inverted sequences is essential for their trans-silencing potential.


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