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-direc...
| Main Authors: | , , , |
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| Format: | Article |
| Language: | English |
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BMC
2019-09-01
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| Series: | Epigenetics & Chromatin |
| Subjects: | |
| Online Access: | http://link.springer.com/article/10.1186/s13072-019-0299-0 |
| _version_ | 1817984416092258304 |
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| author | Adéla Přibylová Vojtěch Čermák Dimitrij Tyč Lukáš Fischer |
| author_facet | Adéla Přibylová Vojtěch Čermák Dimitrij Tyč Lukáš Fischer |
| author_sort | Adéla Přibylová |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-04-13T23:45:12Z |
| format | Article |
| id | doaj.art-4596f56b6251492998a58129f9352d31 |
| institution | Directory Open Access Journal |
| issn | 1756-8935 |
| language | English |
| last_indexed | 2024-04-13T23:45:12Z |
| publishDate | 2019-09-01 |
| publisher | BMC |
| record_format | Article |
| series | Epigenetics & Chromatin |
| spelling | doaj.art-4596f56b6251492998a58129f9352d312022-12-22T02:24:22ZengBMCEpigenetics & Chromatin1756-89352019-09-0112111410.1186/s13072-019-0299-0Detailed insight into the dynamics of the initial phases of de novo RNA-directed DNA methylation in plant cellsAdéla Přibylová0Vojtěch Čermák1Dimitrij Tyč2Lukáš Fischer3Department of Experimental Plant Biology, Charles University, Faculty of ScienceDepartment of Experimental Plant Biology, Charles University, Faculty of ScienceDepartment of Experimental Plant Biology, Charles University, Faculty of ScienceDepartment of Experimental Plant Biology, Charles University, Faculty of ScienceAbstract 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.http://link.springer.com/article/10.1186/s13072-019-0299-0EpigeneticsRdDMRNA interferencesRNA sequencingTranscriptional gene silencing |
| spellingShingle | Adéla Přibylová Vojtěch Čermák Dimitrij Tyč Lukáš Fischer Detailed insight into the dynamics of the initial phases of de novo RNA-directed DNA methylation in plant cells Epigenetics & Chromatin Epigenetics RdDM RNA interference sRNA sequencing Transcriptional gene silencing |
| title | Detailed insight into the dynamics of the initial phases of de novo RNA-directed DNA methylation in plant cells |
| title_full | Detailed insight into the dynamics of the initial phases of de novo RNA-directed DNA methylation in plant cells |
| title_fullStr | Detailed insight into the dynamics of the initial phases of de novo RNA-directed DNA methylation in plant cells |
| title_full_unstemmed | Detailed insight into the dynamics of the initial phases of de novo RNA-directed DNA methylation in plant cells |
| title_short | Detailed insight into the dynamics of the initial phases of de novo RNA-directed DNA methylation in plant cells |
| title_sort | detailed insight into the dynamics of the initial phases of de novo rna directed dna methylation in plant cells |
| topic | Epigenetics RdDM RNA interference sRNA sequencing Transcriptional gene silencing |
| url | http://link.springer.com/article/10.1186/s13072-019-0299-0 |
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