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.
Transient 5-azacytidine treatment of leaf explants from potato plants with transcriptionally silenced transgenes allows de novo regeneration of plants with restored transgene expression at the whole plant level. Transgenes introduced into plant genomes frequently become silenced either at the transcriptional or the posttranscriptional level. Transcriptional silencing is usually associated with DNA methylation in the promoter region. Treatments with inhibitors of maintenance DNA methylation were previously shown to allow reactivation of transcriptionally silenced transgenes in single cells or tissues, but not at the whole plant level. Here we analyzed the effect of DNA methylation inhibitor 5-azacytidine (AzaC) on the expression of two silenced reporter genes encoding green fluorescent protein (GFP) and neomycin phosphotransferase (NPTII) in potato plants. Whereas no obvious reactivation was observed in AzaC-treated stem cuttings, transient treatment of leaf segments with 10 μM AzaC and subsequent de novo regeneration of shoots on the selective medium with kanamycin resulted in the production of whole plants with clearly reactivated expression of previously silenced transgenes. Reactivation of nptII expression was accompanied by a decrease in cytosine methylation in the promoter region of the gene. Using the plants with reactivated GFP expression, we found that re-silencing of this transgene can be accidentally triggered by de novo regeneration. Thus, testing the incidence of transgene silencing during de novo regeneration could be a suitable procedure for negative selection of transgenic lines (insertion events) which have an inclination to be silenced. Based on our analysis of non-specific inhibitory effects of AzaC on growth of potato shoots in vitro, we estimated that AzaC half-life in the culture media is approximately 2 days.
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