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.

Department of Experimental Plant Biology Charles University Faculty of Science 128 44 Prague Czech Republic

Zobrazit více v PubMed

Bennetzen JL. Transposable elements, gene creation and genome rearrangement in flowering plants. Curr Opin Genet Dev. 2005;15:621–627. doi: 10.1016/j.gde.2005.09.010. PubMed DOI

Du J, Johnson LM, Jacobsen SE, Patel DJ. DNA methylation pathways and their crosstalk with histone methylation. Nat Rev Mol Cell Biol. 2015;16:519–532. doi: 10.1038/nrm4043. PubMed DOI PMC

Zhang H, Lang Z, Zhu J-K. Dynamics and function of DNA methylation in plants. Nat Rev Mol Cell Biol. 2018;19:489. doi: 10.1038/s41580-018-0016-z. PubMed DOI

Quadrana L, Bortolini Silveira A, Mayhew GF, LeBlanc C, Martienssen RA, Jeddeloh JA, et al. The Arabidopsis thaliana mobilome and its impact at the species level. eLife. 2016;5:e15716. doi: 10.7554/eLife.15716. PubMed DOI PMC

Zhou S, Liu X, Zhou C, Zhou Q, Zhao Y, Li G, et al. Cooperation between the H3K27me3 chromatin mark and non-CG methylation in epigenetic regulation. Plant Physiol. 2016;172:1131–1141. PubMed PMC

Wierzbicki AT, Haag JR, Pikaard CS. Noncoding transcription by RNA polymerase Pol IVb/Pol V mediates transcriptional silencing of overlapping and adjacent genes. Cell. 2008;135:635–648. doi: 10.1016/j.cell.2008.09.035. PubMed DOI PMC

Fojtová M, Houdt HV, Depicker A, Kovarik A. Epigenetic switch from posttranscriptional to transcriptional silencing is correlated with promoter hypermethylation. Plant Physiol. 2003;133:1240–1250. doi: 10.1104/pp.103.023796. PubMed DOI PMC

Saze H, Kakutani T. Differentiation of epigenetic modifications between transposons and genes. Curr Opin Plant Biol. 2011;14:81–87. doi: 10.1016/j.pbi.2010.08.017. PubMed DOI

Finnegan EJ, Peacock WJ, Dennis ES. Reduced DNA methylation in Arabidopsis thaliana results in abnormal plant development. PNAS. 1996;93:8449–8454. doi: 10.1073/pnas.93.16.8449. PubMed DOI PMC

Jones L, Ratcliff F, Baulcombe DC. RNA-directed transcriptional gene silencing in plants can be inherited independently of the RNA trigger and requires Met1 for maintenance. Curr Biol. 2001;11:747–757. doi: 10.1016/S0960-9822(01)00226-3. PubMed DOI

Woo HR, Pontes O, Pikaard CS, Richards EJ. VIM1, a methylcytosine-binding protein required for centromeric heterochromatinization. Gene Dev. 2007;21:267–277. doi: 10.1101/gad.1512007. PubMed DOI PMC

Du J, Zhong X, Bernatavichute YV, Stroud H, Feng S, Caro E, et al. Dual binding of chromomethylase domains to H3K9me2-containing nucleosomes directs DNA methylation in plants. Cell. 2012;151:167–180. doi: 10.1016/j.cell.2012.07.034. PubMed DOI PMC

Zemach A, Kim MY, Hsieh P-H, Coleman-Derr D, Eshed-Williams L, Thao K, et al. The Arabidopsis nucleosome remodeler DDM1 allows DNA methyltransferases to access H1-containing heterochromatin. Cell. 2013;153:193–205. doi: 10.1016/j.cell.2013.02.033. PubMed DOI PMC

Jackson JP, Lindroth AM, Cao X, Jacobsen SE. Control of CpNpG DNA methylation by the KRYPTONITE histone H3 methyltransferase. Nature. 2002;416:556–560. doi: 10.1038/nature731. PubMed DOI

Malagnac F, Bartee L, Bender J. An Arabidopsis SET domain protein required for maintenance but not establishment of DNA methylation. EMBO J. 2002;21:6842–6852. doi: 10.1093/emboj/cdf687. PubMed DOI PMC

Ebbs ML, Bartee L, Bender J. H3 lysine 9 methylation is maintained on a transcribed inverted repeat by combined action of SUVH6 and SUVH4 methyltransferases. Mol Cell Biol. 2005;25:10507–10515. doi: 10.1128/MCB.25.23.10507-10515.2005. PubMed DOI PMC

Ebbs ML, Bender J. Locus-specific control of DNA methylation by the Arabidopsis SUVH5 histone methyltransferase. Plant Cell. 2006;18:1166–1176. doi: 10.1105/tpc.106.041400. PubMed DOI PMC

Johnson LM, Bostick M, Zhang X, Kraft E, Henderson I, Callis J, et al. The SRA methyl-cytosine-binding domain links DNA and histone methylation. Curr Biol. 2007;17:379–384. doi: 10.1016/j.cub.2007.01.009. PubMed DOI PMC

Du J, Johnson LM, Groth M, Feng S, Hale CJ, Li S, et al. Mechanism of DNA methylation-directed histone methylation by KRYPTONITE. Mol Cell. 2014;55:495–504. doi: 10.1016/j.molcel.2014.06.009. PubMed DOI PMC

Stoddard CI, Feng S, Campbell MG, Liu W, Wang H, Zhong X, et al. A nucleosome bridging mechanism for activation of a maintenance DNA methyltransferase. Mol Cell. 2019;73:73–83.e6. doi: 10.1016/j.molcel.2018.10.006. PubMed DOI PMC

Lindroth AM, Cao X, Jackson JP, Zilberman D, McCallum CM, Henikoff S, et al. Requirement of CHROMOMETHYLASE3 for maintenance of CpXpG methylation. Science. 2001;292:2077–2080. doi: 10.1126/science.1059745. PubMed DOI

Stroud H, Greenberg MVC, Feng S, Bernatavichute YV, Jacobsen SE. Comprehensive analysis of silencing mutants reveals complex regulation of the Arabidopsis methylome. Cell. 2013;152:352–364. doi: 10.1016/j.cell.2012.10.054. PubMed DOI PMC

Bologna NG, Voinnet O. The diversity, biogenesis, and activities of endogenous silencing small RNAs in Arabidopsis. Annu Rev Plant Biol. 2014;65:473–503. doi: 10.1146/annurev-arplant-050213-035728. PubMed DOI

Elvira-Matelot E, Martínez ÁE. Diversity of RNA silencing pathways in plants. Plant Gene Silenc Mech Appl. 2017;5:1–31.

Zhong X, Du J, Hale CJ, Gallego-Bartolome J, Feng S, Vashisht AA, et al. Molecular mechanism of action of plant DRM de novo DNA methyltransferases. Cell. 2014;157:1050–1060. doi: 10.1016/j.cell.2014.03.056. PubMed DOI PMC

Herr AJ, Jensen MB, Dalmay T, Baulcombe DC. RNA polymerase IV directs silencing of endogenous DNA. Science. 2005;308:118–120. doi: 10.1126/science.1106910. PubMed DOI

Onodera Y, Haag JR, Ream T, Nunes PC, Pontes O, Pikaard CS. Plant nuclear RNA polymerase IV mediates siRNA and DNA methylation-dependent heterochromatin formation. Cell. 2005;120:613–622. doi: 10.1016/j.cell.2005.02.007. PubMed DOI

Kanno T, Huettel B, Mette MF, Aufsatz W, Jaligot E, Daxinger L, et al. Atypical RNA polymerase subunits required for RNA-directed DNA methylation. Nat Genet. 2005;37:761–765. doi: 10.1038/ng1580. PubMed DOI

Pontier D, Yahubyan G, Vega D, Bulski A, Saez-Vasquez J, Hakimi M-A, et al. Reinforcement of silencing at transposons and highly repeated sequences requires the concerted action of two distinct RNA polymerases IV in Arabidopsis. Gene Dev. 2005;19:2030–2040. doi: 10.1101/gad.348405. PubMed DOI PMC

Law JA, Vashisht AA, Wohlschlegel JA, Jacobsen SE. SHH1, a homeodomain protein required for DNA methylation, as well as RDR2, RDM4, and chromatin remodeling factors, associate with RNA polymerase IV. PLoS Genet. 2011;7:e1002195. doi: 10.1371/journal.pgen.1002195. PubMed DOI PMC

Law JA, Du J, Hale CJ, Feng S, Krajewski K, Palanca AMS, et al. Polymerase-IV occupancy at RNA-directed DNA methylation sites requires SHH1. Nature. 2013;498:385–389. doi: 10.1038/nature12178. PubMed DOI PMC

Zhang H, Ma Z-Y, Zeng L, Tanaka K, Zhang C-J, Ma J, et al. DTF1 is a core component of RNA-directed DNA methylation and may assist in the recruitment of Pol IV. PNAS. 2013;110:8290–8295. doi: 10.1073/pnas.1300585110. PubMed DOI PMC

Blevins T, Podicheti R, Mishra V, Marasco M, Wang J, Rusch D, et al. Identification of Pol IV and RDR2-dependent precursors of 24 nt siRNAs guiding de novo DNA methylation in Arabidopsis. eLife. 2015;4:e09591. doi: 10.7554/eLife.09591. PubMed DOI PMC

Li S, Vandivier LE, Tu B, Gao L, Won SY, Li S, et al. Detection of Pol IV/RDR2-dependent transcripts at the genomic scale in Arabidopsis reveals features and regulation of siRNA biogenesis. Genome Res. 2015;25:235–245. doi: 10.1101/gr.182238.114. PubMed DOI PMC

Zhai J, Bischof S, Wang H, Feng S, Lee T, Teng C, et al. A one precursor one siRNA model for Pol IV-dependent siRNA biogenesis. Cell. 2015;163:445–455. doi: 10.1016/j.cell.2015.09.032. PubMed DOI PMC

Zhang Z, Liu X, Guo X, Wang X-J, Zhang X. Arabidopsis AGO3 predominantly recruits 24-nt small RNAs to regulate epigenetic silencing. Nat Plants. 2016;2:16049. doi: 10.1038/nplants.2016.49. PubMed DOI

El-Shami M, Pontier D, Lahmy S, Braun L, Picart C, Vega D, et al. Reiterated WG/GW motifs form functionally and evolutionarily conserved ARGONAUTE-binding platforms in RNAi-related components. Gene Dev. 2007;21:2539–2544. doi: 10.1101/gad.451207. PubMed DOI PMC

Yang D-L, Zhang G, Tang K, Li J, Yang L, Huang H, et al. Dicer-independent RNA-directed DNA methylation in Arabidopsis. Cell Res. 2016;26:66–82. doi: 10.1038/cr.2015.145. PubMed DOI PMC

Kuhlmann M, Mette MF. Developmentally non-redundant SET domain proteins SUVH2 and SUVH9 are required for transcriptional gene silencing in Arabidopsis thaliana. Plant Mol Biol. 2012;79:623–633. doi: 10.1007/s11103-012-9934-x. PubMed DOI PMC

Johnson LM, Du J, Hale CJ, Bischof S, Feng S, Chodavarapu RK, et al. SRA- and SET-domain-containing proteins link RNA polymerase V occupancy to DNA methylation. Nature. 2014;507:124–128. doi: 10.1038/nature12931. PubMed DOI PMC

Liu Z-W, Shao C-R, Zhang C-J, Zhou J-X, Zhang S-W, Li L, et al. The SET domain proteins SUVH2 and SUVH9 are required for Pol V occupancy at RNA-directed DNA methylation loci. PLoS Genet. 2014;10:e1003948. doi: 10.1371/journal.pgen.1003948. PubMed DOI PMC

Marí-Ordóñez A, Marchais A, Etcheverry M, Martin A, Colot V, Voinnet O. Reconstructing de novo silencing of an active plant retrotransposon. Nat Genet. 2013;45:1029–1039. doi: 10.1038/ng.2703. PubMed DOI

Fultz D, Slotkin RK. Exogenous transposable elements circumvent identity-based silencing, permitting the dissection of expression-dependent silencing. Plant Cell. 2017;29:360–376. doi: 10.1105/tpc.16.00718. PubMed DOI PMC

Ye R, Chen Z, Lian B, Rowley MJ, Xia N, Chai J, et al. A Dicer-independent route for biogenesis of siRNAs that direct DNA methylation in Arabidopsis. Mol Cell. 2016;61:222–235. doi: 10.1016/j.molcel.2015.11.015. PubMed DOI PMC

Vaucheret H, Institut N de la RA. Promoter-dependent trans-inactivation in transgenic tobacco plants: kinetic aspects of gene silencing and gene reactivation. C R Acad Sci III. 1994:310–23.

Philips JG, Dudley KJ, Waterhouse PM, Hellens RP. The rapid methylation of T-DNAs upon agrobacterium inoculation in plant leaves. Front Plant Sci. 2019 doi: 10.3389/fpls.2019.00312. PubMed DOI PMC

Xu C, Corces VG. Nascent DNA methylome mapping reveals inheritance of hemimethylation at CTCF/cohesin sites. Science. 2018;359:1166–1170. doi: 10.1126/science.aan5480. PubMed DOI PMC

Nagata T, Nemoto Y, Hasezawa S. Tobacco BY-2 cell line as the “HeLa” cell in the cell biology of higher plants. In: Jeon KW, Friedlander M, editors. International review of cytology. Cambridge: Academic Press; 1992. pp. 1–30.

Srba M, Černíková A, Opatrný Z, Fischer L. Practical guidelines for the characterization of tobacco BY-2 cell lines. Biol Plant. 2016;60:13–24. doi: 10.1007/s10535-015-0573-3. DOI

Nocarova E, Fischer L. Cloning of transgenic tobacco BY-2 cells; an efficient method to analyse and reduce high natural heterogeneity of transgene expression. BMC Plant Biol. 2009;9:44. doi: 10.1186/1471-2229-9-44. PubMed DOI PMC

Jupe F, Rivkin AC, Michael TP, Zander M, Motley ST, Sandoval JP, et al. The complex architecture and epigenomic impact of plant T-DNA insertions. PLoS Genet. 2019;15:e1007819. doi: 10.1371/journal.pgen.1007819. PubMed DOI PMC

Wroblewski T, Matvienko M, Piskurewicz U, Xu H, Martineau B, Wong J, et al. Distinctive profiles of small RNA couple inverted repeat-induced post-transcriptional gene silencing with endogenous RNA silencing pathways in Arabidopsis. RNA. 2014;20:1987–1999. doi: 10.1261/rna.046532.114. PubMed DOI PMC

Grandbastien M-A, Spielmann A, Caboche M. Tnt1, a mobile retroviral-like transposable element of tobacco isolated by plant cell genetics. Nature. 1989;337:376. doi: 10.1038/337376a0. PubMed DOI

Hirochika H, Sugimoto K, Otsuki Y, Tsugawa H, Kanda M. Retrotransposons of rice involved in mutations induced by tissue culture. PNAS. 1996;93:7783–7788. doi: 10.1073/pnas.93.15.7783. PubMed DOI PMC

Kankel MW, Ramsey DE, Stokes TL, Flowers SK, Haag JR, Jeddeloh JA, et al. Arabidopsis MET1 cytosine methyltransferase mutants. Genetics. 2003;163:1109–1122. PubMed PMC

Zabet NR, Catoni M, Prischi F, Paszkowski J. Cytosine methylation at CpCpG sites triggers accumulation of non-CpG methylation in gene bodies. Nucleic Acids Res. 2017;45:3777–3784. PubMed PMC

Panda K, Ji L, Neumann DA, Daron J, Schmitz RJ, Slotkin RK. Full-length autonomous transposable elements are preferentially targeted by expression-dependent forms of RNA-directed DNA methylation. Genome Biol. 2016;17:170. doi: 10.1186/s13059-016-1032-y. PubMed DOI PMC

Parent J-S, Bouteiller N, Elmayan T, Vaucheret H. Respective contributions of Arabidopsis DCL2 and DCL4 to RNA silencing. Plant J. 2015;81:223–232. doi: 10.1111/tpj.12720. PubMed DOI

Sijen T, Vijn I, Rebocho A, van Blokland R, Roelofs D, Mol JN, et al. Transcriptional and posttranscriptional gene silencing are mechanistically related. Curr Biol. 2001;11:436–440. doi: 10.1016/S0960-9822(01)00116-6. PubMed DOI

Čermák V, Fischer L. Pervasive read-through transcription of T-DNAs is frequent in tobacco BY-2 cells and can effectively induce silencing. BMC Plant Biol. 2018;18:252. doi: 10.1186/s12870-018-1482-3. PubMed DOI PMC

Křížová K, Depicker A, Kovařík A. Epigenetic switches of tobacco transgenes associate with transient redistribution of histone marks in callus culture. Epigenetics. 2013;8:666–676. doi: 10.4161/epi.24613. PubMed DOI PMC

Ford E, Grimmer MR, Stolzenburg S, Bogdanovic O, Mendoza A de, Farnham PJ, et al. Frequent lack of repressive capacity of promoter DNA methylation identified through genome-wide epigenomic manipulation. 2017. http://bioRxiv.org/170506.

Matsunaga W, Shimura H, Shirakawa S, Isoda R, Inukai T, Matsumura T, et al. Transcriptional silencing of 35S driven-transgene is differentially determined depending on promoter methylation heterogeneity at specific cytosines in both plus- and minus-sense strands. BMC Plant Biol. 2019;19:24. doi: 10.1186/s12870-019-1628-y. PubMed DOI PMC

Johnson LM, Law JA, Khattar A, Henderson IR, Jacobsen SE. SRA-domain proteins required for DRM2-mediated de novo DNA methylation. PLoS Genet. 2008;4:e1000280. doi: 10.1371/journal.pgen.1000280. PubMed DOI PMC

Jackel JN, Storer J, Coursey T, Bisaro D. Arabidopsis RNA polymerases IV and V are required to establish H3K9 methylation, but not cytosine methylation, on geminivirus chromatin. J Virol. 2016;90:7529–7540. doi: 10.1128/JVI.00656-16. PubMed DOI PMC

Murashige T, Skoog F. A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant. 1962;15:473–497. doi: 10.1111/j.1399-3054.1962.tb08052.x. DOI

Davis SJ, Vierstra RD. Soluble, highly fluorescent variants of green fluorescent protein (GFP) for use in higher plants. Plant Mol Biol. 1998;36:521–528. doi: 10.1023/A:1005991617182. PubMed DOI

Duchoslav M, Fischer L. Parallel subfunctionalisation of PsbO protein isoforms in angiosperms revealed by phylogenetic analysis and mapping of sequence variability onto protein structure. BMC Plant Biol. 2015;15:133. doi: 10.1186/s12870-015-0523-4. PubMed DOI PMC

Motylová Š. The influence of RDR6 activity and mode of RNAi induction on dynamics and mechanism of silencing of the reporter GFP gene in tobacco cell line BY-2. Diploma thesis. Faculty of Science, Charles University. 2015. http://hdl.handle.net/20.500.11956/74403.

Zuo J, Niu Q-W, Chua N-H. An estrogen receptor-based transactivator XVE mediates highly inducible gene expression in transgenic plants. Plant J. 2000;24:265–273. doi: 10.1046/j.1365-313x.2000.00868.x. PubMed DOI

Ruijter JM, Ramakers C, Hoogaars WMH, Karlen Y, Bakker O, van den Hoff MJB, et al. Amplification efficiency: linking baseline and bias in the analysis of quantitative PCR data. Nucleic Acids Res. 2009;37:e45. doi: 10.1093/nar/gkp045. PubMed DOI PMC

Nicot N, Hausman J-F, Hoffmann L, Evers D. Housekeeping gene selection for real-time RT-PCR normalization in potato during biotic and abiotic stress. J Exp Bot. 2005;56:2907–2914. doi: 10.1093/jxb/eri285. PubMed DOI

Dvořáková L, Srba M, Opatrny Z, Fischer L. Hybrid proline-rich proteins: novel players in plant cell elongation? Ann Bot. 2012;109:453–462. doi: 10.1093/aob/mcr278. PubMed DOI PMC

Tyč D, Nocarová E, Sikorová L, Fischer L. 5-Azacytidine mediated reactivation of silenced transgenes in potato (Solanum tuberosum) at the whole plant level. Plant Cell Rep. 2017;36:1311–1322. doi: 10.1007/s00299-017-2155-7. PubMed DOI

Bond DM, Baulcombe DC. Epigenetic transitions leading to heritable, RNA-mediated de novo silencing in Arabidopsis thaliana. PNAS. 2015;112:917–922. doi: 10.1073/pnas.1413053112. PubMed DOI PMC

Fehlmann T, Reinheimer S, Geng C, Su X, Drmanac S, Alexeev A, et al. cPAS-based sequencing on the BGISEQ-500 to explore small non-coding RNAs. Clin Epigenetics. 2016;8:123. doi: 10.1186/s13148-016-0287-1. PubMed DOI PMC

Exploring the crop epigenome: a comparison of DNA methylation profiling techniques

Sulfadiazine and phosphinothricin selection systems optimised for the transformation of tobacco BY-2 cells

DNA methylation can alter CRISPR/Cas9 editing frequency and DNA repair outcome in a target-specific manner