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Abstract
In plants, silencing is usually accompanied by DNA methylation and heterochromatic histone marks. We studied these epigenetic modifications in different epialleles of 35S promoter (P35S)-driven tobacco transgenes. In locus 1, the T-DNA was organized as an inverted repeat, and the residing neomycin phosphotransferase II reporter gene (P35S-nptII) was silenced at the posttranscriptional (PTGS) level. Transcriptionally silenced (TGS) epialleles were generated by trans-acting RNA signals in hybrids or in a callus culture. PTGS to TGS conversion in callus culture was accompanied by loss of the euchromatic H3K4me3 mark in the transcribed region of locus 1, but this change was not transmitted to the regenerated plants from these calli. In contrast, cytosine methylation that spread from the transcribed region into the promoter was maintained in regenerants. Also, the TGS epialleles generated by trans-acting siRNAs did not change their active histone modifications. Thus, both TGS and PTGS epialleles exhibit euchromatic (H3K4me3 and H3K9ac) histone modifications despite heavy DNA methylation in the promoter and transcribed region, respectively. However, in the TGS locus (271), abundant heterochromatic H3K9me2 marks and DNA methylation were present on P35S. Heterochromatic histone modifications are not automatically installed on transcriptionally silenced loci in tobacco, suggesting that repressive histone marks and cytosine methylation may be uncoupled. However, transient loss of euchromatic modifications may guide de novo DNA methylation leading to formation of stable repressed epialleles with recovered eukaryotic marks. Compilation of available data on epigenetic modification of inactivated P35S in different systems is provided.
Submitted
01/23/2013
Revised
03/28/2013
Accepted
04/08/2013
Disclosure of Potential Conflicts of Interest
No potential conflict of interest was disclosed.
Acknowledgments
We thank Dr Hervé Vaucheret (INRA, Versailles, France) for providing us with a tobacco 271 line. The comments on the work from Dr Ortrun Mittelsten-Scheid (GMI, Austrian Academy of Sciences, Vienna) have been much appreciated. We thank Mrs. Jana Kubí?ková for her technical assistance. This work has been supported by the Czech Academy of Sciences, the Czech Science Foundation (P501/11/P667, P501/12/G090 and P501/13/10057S), Research Foundation-Flanders (grant no. G.0211.06N and Belgium-Czech Exchange Program).