In plants, posttranscriptional gene silencing (PTGS) is induced by small RNAs (sRNAs) generated from various dsRNA precursors. To assess the impact of dsRNA origin, we compared downregulation of GFP expression triggered by inverted repeat (IR), antisense (AS) and unterminated sense (UT) transcripts transiently expressed from the estradiol-inducible promoter. The use of homogeneously responding tobacco BY-2 cell lines allowed monitoring the onset of silencing and its reversibility. In this system, IR induced the strongest and fastest silencing accompanied by dense DNA methylation. At low induction, silencing in individual cells was binary (either strong or missing), suggesting that a certain threshold sRNA level had to be exceeded. The AS variant specifically showed a deviated sRNA-strand ratio shifted in favor of antisense orientation. In AS lines and weakly induced IR lines, only the silencer DNA was methylated, but the same target GFP sequence was not, showing that DNA methylation accompanying PTGS was influenced both by the level and origin of sRNAs, and possibly also by the epigenetic state of the locus. UT silencing appeared to be the least effective and resembled classical sense PTGS. The best responding UT lines behaved relatively heterogeneously possibly due to complexly arranged T-DNA insertions. Unlike IR and AS variants that fully restored GFP expression upon removal of the inducer, only partial reactivation was observed in some UT lines. Our results pointed out several not yet described phenomena and differences between the long-known silencer variants that may direct further research and affect selection of proper silencer variants for specific applications.
- MeSH
- DNA bakterií MeSH
- dvouvláknová RNA genetika MeSH
- metylace DNA MeSH
- posttranskripční úpravy RNA * MeSH
- regulace genové exprese u rostlin * MeSH
- reportérové geny MeSH
- RNA interference * MeSH
- RNA rostlin genetika MeSH
- tabák genetika MeSH
- umlčovací elementy transkripční MeSH
- umlčování genů * MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
BACKGROUND: Methylation of cytosines is an evolutionarily conserved epigenetic mark that is essential for the control of chromatin activity in many taxa. It acts mainly repressively, causing transcriptional gene silencing. In plants, de novo DNA methylation is established mainly by RNA-directed DNA-methylation pathway. Even though the protein machinery involved is relatively well-described, the course of the initial phases remains covert. RESULTS: We show the first detailed description of de novo DNA-methylation dynamics. Since prevalent plant model systems do not provide the possibility to collect homogenously responding material in time series with short intervals, we developed a convenient system based on tobacco BY-2 cell lines with inducible production of siRNAs (from an RNA hairpin) guiding the methylation machinery to the CaMV 35S promoter controlling GFP reporter. These lines responded very synchronously, and a high level of promoter-specific siRNAs triggered rapid promoter methylation with the first increase observed already 12 h after the induction. The previous presence of CG methylation in the promoter did not affect the methylation dynamics. The individual cytosine contexts reacted differently. CHH methylation peaked at about 80% in 2 days and then declined, whereas CG and CHG methylation needed more time with CHG reaching practically 100% after 10 days. Spreading of methylation was only minimal outside the target region in accordance with the absence of transitive siRNAs. The low and stable proportion of 24-nt siRNAs suggested that Pol IV was not involved in the initial phases. CONCLUSIONS: Our results show that de novo DNA methylation is a rapid process initiated practically immediately with the appearance of promoter-specific siRNAs and independently of the prior presence of methylcytosines at the target locus. The methylation was precisely targeted, and its dynamics varied depending on the cytosine sequence context. The progressively increasing methylation resulted in a smooth, gradual inhibition of the promoter activity, which was entirely suppressed in 2 days.
- MeSH
- Caulimovirus genetika MeSH
- estradiol farmakologie MeSH
- malá interferující RNA genetika metabolismus MeSH
- metylace DNA * účinky léků MeSH
- plazmidy genetika metabolismus MeSH
- promotorové oblasti (genetika) MeSH
- RNA interference MeSH
- rostlinné buňky metabolismus MeSH
- tabák cytologie MeSH
- zelené fluorescenční proteiny antagonisté a inhibitory genetika metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
