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

Abstract

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.

RdDM pathway components differentially modulate Tobamovirus symptom development

“…We also observed that the abundance of the 24-nt sRNAs that mapped to promoter regions was lower in infected plants in comparison to non-infected plants and transcription level of the genes mapped by these sRNAs was higher in Col-0 ecotype (when we compare infected versus mock inoculated plants). This finding is consistent with the knowledge that DNA methylation in regulatory regions inhibits transcription; therefore, a negative correlation between the amount of sRNAs (associated to a reduced level of methylation) and the level of mRNA is expected (Mette et al 2000;Přibylová et al 2019). On the other hand, controversy surrounds the effect of methylation of gene body regions.…”

Barley somatic embryogenesis-an attempt to modify variation induced in tissue culture

“…Interestingly, the CHG context is the most affected one by DMV. Possibly, the cell reprogramming step [ 85 ] involving active [ 86 , 87 ] and passive [ 88 , 89 ] DNA demethylation followed by different mechanisms of de novo methylation (depending on sequence contexts) [ 90 , 91 ] and variations in the preferences towards methylation of specific cytosines compared to methylation density of some genomic regions [ 92 ] and correctness of pattern reestablishment due to (epi)genetic mechanisms [ 93 ] may be the most probable explanation both for the increased values of DMV and preferential demethylation of the CHG sequences.…”

DNA methylation in plants: mechanisms and tools for targeted manipulation

“…A recent study reported new insights into the speed by which an IR construct triggered TGS. By using an inducible IR construct, they observed targeted methylation and significant silencing of a reporter transgene just a few hours after the accumulation of IR‐derived siRNAs (Pribylova et al , 2019). Another recent study employed IR constructs to target methylation to distal enhancers of the flowering master regulator FLOWERING LOCUS T ( FT ) and trigger its down‐regulation (Zicola et al , 2019).…”