Epigenetic switches of tobacco transgenes associate with transient redistribution of histone marks in callus culture

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.

Detailed insight into the dynamics of the initial phases of de novo RNA-directed DNA methylation in plant cells

“…Therefore, either the Pol IV occupancy was prevented by another chromatin/histone modification (e.g., H3K4me3) [31], or the presence of CHG methylation (the activity of CMT3) is not necessarily connected with H3K9me2. This situation has already been documented in tobacco by ChIP, where high CHG methylation in transcriptionally silenced P35S was unexpectedly accompanied by H3K9 acetylation and H3K4me3 marks [61]. H3K4me3 activation marks coexisted with methylated cytosines also in many human promoters, whose activity was frequently unaffected by the introduction of methylation marks [62].…”

Assessing the Response of Small RNA Populations to Allopolyploidy Using Resynthesized Brassica napus Allotetraploids

“…2013): EVD reactivated transcripts were first subject to the PTGS pathway with a high accumulation of 21-nt siRNAs, and then EVD transcript levels declined in response to TGS corresponding to an accumulation of 24-nt LTR-derived siRNAs, thus maintaining epigenetic silencing by RNA-directed DNA Methylation (RdDM) of the active retrotransposon. It can be noticed that PTGS-to-TGS conversion was also demonstrated to ensure transgene silencing, indicating a major role of siRNAs in controlling transcription of other targets than TEs during genome remodeling (Křížová et al. 2013).…”

Importance of DNA and histone methylation in in vitro plant propagation for crop improvement: a review

“…endosperm, tassel, seedlings) in maize. In callus culture of Nicotiana tabacum, Křížová et al (2013) reported that conversion of post-transcriptional gene silencing to TGS was associated with the reduction of euchromatic H3K4me3, but this modification was not observed in the regenerated plants. During de novo shoot regeneration in A. thaliana, Li et al (2011) also reported that both H3K4me3 and H3K9me2 contribute to regulate the expression of the transcription factor WUSCHEL.…”