It is generally assumed that DNA methylation changes at genomic regions targeted by the de novo RNA-directed DNA methylation (RdDM) pathway are unstable. Here, we show that RdDM targets in Arabidopsis can be classified into two groups on the basis of whether there is remethylation following the restoration of NRPD1 function in nrpd1 mutant plants—remethylable loci and non-remethylable loci. In contrast to the remethylable loci, the non-remethylable loci contain higher levels of the euchromatic marks of trimethylation at Lys 4 of histone H3 (H3K4me3), which interferes with the recruitment of the RdDM molecular machinery, and acetylation at Lys 18 of histone H3 (H3K18ac), which helps to recruit the DNA demethylase ROS1 to antagonize RdDM. Here, using targeted methylation erasure by CRISPR–dCas9–TET1, we demonstrate that methylated CG (mCG) and mCHG (where H represents A, C or T) are memory marks that are required for targeting the RdDM machinery to remethylable loci. Our results show that histone and DNA methylation marks are critical in determining the ability of RdDM target loci to form stable epialleles, and contribute to understanding the formation and transmission of epialleles.
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Zhang, H., Lang, Z. & Zhu, J.-K. Dynamics and function of DNA methylation in plants. Nat. Rev. Mol. Cell Biol. 19, 489–506 (2018).
Matzke, M. A., Kanno, T. & Matzke, A. J. RNA-directed DNA methylation: the evolution of a complex epigenetic pathway in flowering plants. Annu. Rev. Plant Biol. 66, 243–267 (2015).
Haag, J. R. et al. In vitro transcription activities of Pol IV, Pol V, and RDR2 reveal coupling of Pol IV and RDR2 for dsRNA synthesis in plant RNA silencing. Mol. Cell 48, 811–818 (2012).
Xie, Z. et al. Genetic and functional diversification of small RNA pathways in plants. PLoS Biol. 2, e104 (2004).
Zilberman, D., Cao, X. & Jacobsen, S. E. ARGONAUTE4 control of locus-specific siRNA accumulation and DNA and histone methylation. Science 299, 716–719 (2003).
Gao, Z. et al. An RNA polymerase II-and AGO4-associated protein acts in RNA-directed DNA methylation. Nature 465, 106–109 (2010).
Zhong, X. et al. Molecular mechanism of action of plant DRM de novo DNA methyltransferases. Cell 157, 1050–1060 (2014).
Aufsatz, W., Mette, M. F., van der Winden, J., Matzke, M. & Matzke, A. J. HDA6, a putative histone deacetylase needed to enhance DNA methylation induced by double-stranded RNA. EMBO J. 21, 6832–6841 (2002).
Jackson, J. P. et al. Dimethylation of histone H3 lysine 9 is a critical mark for DNA methylation and gene silencing in Arabidopsis thaliana. Chromosoma 112, 308–315 (2004).
Ebbs, M. L. & Bender, J. Locus-specific control of DNA methylation by the Arabidopsis SUVH5 histone methyltransferase. Plant Cell 18, 1166–1176 (2006).
Jackson, J. P., Lindroth, A. M., Cao, X. & Jacobsen, S. E. Control of CpNpG DNA methylation by the KRYPTONITE histone H3 methyltransferase. Nature 416, 556–560 (2002).
Deleris, A. et al. Involvement of a Jumonji‐C domain‐containing histone demethylase in DRM2‐mediated maintenance of DNA methylation. EMBO Rep. 11, 950–955 (2010).
Searle, I. R., Pontes, O., Melnyk, C. W., Smith, L. M. & Baulcombe, D. C. JMJ14, a JmjC domain protein, is required for RNA silencing and cell-to-cell movement of an RNA silencing signal in Arabidopsis. Genes Dev. 24, 986–991 (2010).
Kim, J., Kim, J. H., Richards, E. J., Chung, K. M. & Woo, H. R. Arabidopsis VIM proteins regulate epigenetic silencing by modulating DNA methylation and histone modification in cooperation with MET1. Mol. Plant 7, 1470–1485 (2014).
Stroud, H. et al. Non-CG methylation patterns shape the epigenetic landscape in Arabidopsis. Nat. Struct. Mol. Biol. 21, 64–72 (2014).
Stroud, H., Greenberg, M. V., Feng, S., Bernatavichute, Y. V. & Jacobsen, S. E. Comprehensive analysis of silencing mutants reveals complex regulation of the Arabidopsis methylome. Cell 152, 352–364 (2013).
Du, J. et al. Dual binding of chromomethylase domains to H3K9me2-containing nucleosomes directs DNA methylation in plants. Cell 151, 167–180 (2012).
Zemach, A. et al. The Arabidopsis nucleosome remodeler DDM1 allows DNA methyltransferases to access H1-containing heterochromatin. Cell 153, 193–205 (2013).
Zhu, J.-K. Active DNA demethylation mediated by DNA glycosylases. Annu. Rev. Genet. 43, 143–166 (2009).
Gong, Z. et al. ROS1, a repressor of transcriptional gene silencing in Arabidopsis, encodes a DNA glycosylase/lyase. Cell 111, 803–814 (2002).
Gehring, M. et al. DEMETER DNA glycosylase establishes MEDEA polycomb gene self-imprinting by allele-specific demethylation. Cell 124, 495–506 (2006).
Ortega-Galisteo, A. P., Morales-Ruiz, T., Ariza, R. R. & Roldán-Arjona, T. Arabidopsis DEMETER-LIKE proteins DML2 and DML3 are required for appropriate distribution of DNA methylation marks. Plant Mol. Biol. 67, 671–681 (2008).
Lei, M. et al. Regulatory link between DNA methylation and active demethylation in Arabidopsis. Proc. Natl Acad. Sci. USA 112, 3553–3557 (2015).
Lang, Z. et al. The methyl-CpG-binding protein MBD7 facilitates active DNA demethylation to limit DNA hyper-methylation and transcriptional gene silencing. Mol. Cell 57, 971–983 (2015).
Duan, C. G. et al. A pair of transposon-derived proteins function in a histone acetyltransferase complex for active DNA demethylation. Cell Res. 27, 226–240 (2017).
Qian, W. et al. A histone acetyltransferase regulates active DNA demethylation in Arabidopsis. Science 336, 1445–1448 (2012).
Nie, W.-F. et al. Histone acetylation recruits the SWR1 complex to regulate active DNA demethylation in Arabidopsis. Proc. Natl Acad. Sci. USA 116, 16641–16650 (2019).
Tang, K., Lang, Z., Zhang, H. & Zhu, J.-K. The DNA demethylase ROS1 targets genomic regions with distinct chromatin modifications. Nat. Plants 2, 16169 (2016).
Jiang, C. et al. Environmentally responsive genome-wide accumulation of de novo Arabidopsis thaliana mutations and epimutations. Genome Res. 24, 1821–1829 (2014).
Zhang, H., Zhang, K. & Zhu, J.-K. A model for the aberrant DNA methylomes in aging cells and cancer cells. Biochem. Soc. Trans. 47, 997–1003 (2019).
Schmitz, R. J. et al. Transgenerational epigenetic instability is a source of novel methylation variants. Science 334, 369–373 (2011).
Zhang, Q. et al. Methylation interactions in Arabidopsis hybrids require RNA-directed DNA methylation and are influenced by genetic variation. Proc. Natl Acad. Sci. USA 113, E4248–E4256 (2016).
Hollick, J. B. Paramutation and related phenomena in diverse species. Nat. Rev. Genet 18, 5–23 (2017).
Vongs, A., Kakutani, T., Martienssen, R. A. & Richards, E. J. Arabidopsis thaliana DNA methylation mutants. Science 260, 1926–1928 (1993).
Saze, H., Mittelsten Scheid, O. & Paszkowski, J. Maintenance of CpG methylation is essential for epigenetic inheritance during plant gametogenesis. Nat. Genet. 34, 65–69 (2003).
Kankel, M. W. et al. Arabidopsis MET1 cytosine methyltransferase mutants. Genetics 163, 1109–1122 (2003).
Kakutani, T., Munakata, K., Richards, E. J. & Hirochika, H. Meiotically and mitotically stable inheritance of DNA hypomethylation induced by ddm1 mutation of Arabidopsis thaliana. Genetics 151, 831–838 (1999).
Teixeira, F. K. et al. A role for RNAi in the selective correction of DNA methylation defects. Science 323, 1600–1604 (2009).
Blevins, T. et al. A two-step process for epigenetic inheritance in Arabidopsis. Mol. Cell 54, 30–42 (2014).
Catoni, M. et al. DNA sequence properties that predict susceptibility to epiallelic switching. EMBO J. 36, 617–628 (2017).
Law, J. A. et al. Polymerase IV occupancy at RNA-directed DNA methylation sites requires SHH1. Nature 498, 385–389 (2013).
Matzke, M. A. & Mosher, R. A. RNA-directed DNA methylation: an epigenetic pathway of increasing complexity. Nat. Rev. Genet. 15, 394–408 (2014).
Zhang, X., Bernatavichute, Y. V., Cokus, S., Pellegrini, M. & Jacobsen, S. E. Genome-wide analysis of mono-, di- and trimethylation of histone H3 lysine 4 in Arabidopsis thaliana. Genome Biol. 10, R62 (2009).
Gendrel, A. V., Lippman, Z., Yordan, C., Colot, V. & Martienssen, R. A. Dependence of heterochromatic histone H3 methylation patterns on the Arabidopsis gene DDM1. Science 297, 1871–1873 (2002).
Tariq, M. et al. Erasure of CpG methylation in Arabidopsis alters patterns of histone H3 methylation in heterochromatin. Proc. Natl Acad. Sci. USA 100, 8823–8827 (2003).
Barman, S. Construction and Senescence Phenotype Analysis of Double Mutants Encoding H3K4me3 Methyltransferases in Arabidopsis thaliana. MSc thesis, California State Univ. (2017).
Chen, L.-Q. et al. ATX3, ATX4, and ATX5 encode putative H3K4 methyltransferases and are critical for plant development. Plant Physiol. 174, 1795–1806 (2017).
Saleh, A. et al. The highly similar Arabidopsis homologs of trithorax ATX1 and ATX2 encode proteins with divergent biochemical functions. Plant Cell 20, 568–579 (2008).
Guo, L., Yu, Y., Law, J. A. & Zhang, X. SET DOMAIN GROUP2 is the major histone H3 lysine 4 trimethyltransferase in Arabidopsis. Proc. Natl Acad. Sci. USA 107, 18557–18562 (2010).
Reinders, J. et al. Compromised stability of DNA methylation and transposon immobilization in mosaic Arabidopsis epigenomes. Genes Dev. 23, 939–950 (2009).
Mathieu, O., Reinders, J., Caikovski, M., Smathajitt, C. & Paszkowski, J. Transgenerational stability of the Arabidopsis epigenome is coordinated by CG methylation. Cell 130, 851–862 (2007).
Wu, X. & Zhang, Y. TET-mediated active DNA demethylation: mechanism, function and beyond. Nat. Rev. Genet. 18, 517–534 (2017).
Ji, L. et al. TET-mediated epimutagenesis of the Arabidopsis thaliana methylome. Nat. Commun. 9, 895 (2018).
Gallego-Bartolome, J. et al. Targeted DNA demethylation of the Arabidopsis genome using the human TET1 catalytic domain. Proc. Natl Acad. Sci. USA 115, E2125–E2134 (2018).
Jinek, M. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816–821 (2012).
Becker, C. et al. Spontaneous epigenetic variation in the Arabidopsis thaliana methylome. Nature 480, 245–249 (2011).
Schmitz, R. J. et al. Patterns of population epigenomic diversity. Nature 495, 193–198 (2013).
Zhang, H. et al. DTF1 is a core component of RNA-directed DNA methylation and may assist in the recruitment of Pol IV. Proc. Natl Acad. Sci. USA 110, 8290–8295 (2013).
Stewart, K. R. et al. Dynamic changes in histone modifications precede de novo DNA methylation in oocytes. Genes Dev. 29, 2449–2462 (2015).
Morselli, M. et al. In vivo targeting of de novo DNA methylation by histone modifications in yeast and mouse. eLife 4, e06205 (2015).
Greenberg, M. V. et al. Interplay between active chromatin marks and RNA-directed DNA methylation in Arabidopsis thaliana. PLoS Genet. 9, e1003946 (2013).
Onodera, Y. et al. Plant nuclear RNA polymerase IV mediates siRNA and DNA methylation-dependent heterochromatin formation. Cell 120, 613–622 (2005).
Pontier, D. et al. Reinforcement of silencing at transposons and highly repeated sequences requires the concerted action of two distinct RNA polymerases IV in Arabidopsis. Genes Dev. 19, 2030–2040 (2005).
Berr, A. et al. Arabidopsis SET DOMAIN GROUP2 is required for H3K4 trimethylation and is crucial for both sporophyte and gametophyte development. Plant Cell 22, 3232–3248 (2010).
Pien, S. et al. ARABIDOPSIS TRITHORAX1 dynamically regulates FLOWERING LOCUS C activation via histone 3 lysine 4 trimethylation. Plant Cell 20, 580–588 (2008).
He, X. J. et al. An effector of RNA-directed DNA methylation in Arabidopsis is an ARGONAUTE 4- and RNA-binding protein. Cell 137, 498–508 (2009).
Mao, Y. et al. Application of the CRISPR–Cas system for efficient genome engineering in plants. Mol. Plant 6, 2008–2011 (2013).
Zhang, Z. et al. A multiplex CRISPR/Cas9 platform for fast and efficient editing of multiple genes in Arabidopsis. Plant Cell Rep. 35, 1519–1533 (2016).
Clough, S. J. & Bent, A. F. Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 16, 735–743 (1998).
Rowland, L. J. & Nguyen, B. Use of polyethylene glycol for purification of DNA from leaf tissue of woody plants. Biotechniques 14, 734–736 (1993).
Gruntman, E. et al. Kismeth: analyzer of plant methylation states through bisulfite sequencing. BMC Bioinform. 9, 371 (2008).
Xi, Y. & Li, W. BSMAP: whole genome bisulfite sequence MAPping program. BMC Bioinform. 10, 232 (2009).
Haynes, W. in Encyclopedia of Systems Biology (eds Dubitzky, W. et al.) 78–78 (Springer, 2013).
Yang, D. L. et al. Dicer-independent RNA-directed DNA methylation in Arabidopsis. Cell Res. 26, 66–82 (2016).
Schultz, M. D., Schmitz, R. J. & Ecker, J. R. ‘Leveling’ the playing field for analyses of single-base resolution DNA methylomes. Trends Genet. 28, 583–585 (2012).
Langmead, B., Trapnell, C., Pop, M. & Salzberg, S. L. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 10, R25 (2009).
Saleh, A., Alvarez-Venegas, R. & Avramova, Z. An efficient chromatin immunoprecipitation (ChIP) protocol for studying histone modifications in Arabidopsis plants. Nat. Protoc. 3, 1018–1025 (2008).
Luo, C. et al. Integrative analysis of chromatin states in Arabidopsis identified potential regulatory mechanisms for natural antisense transcript production. Plant J. 73, 77–90 (2013).
Tian, T. et al. agriGO v2.0: a GO analysis toolkit for the agricultural community, 2017 update. Nucleic Acids Res. 45, W122–W129 (2017).
Bernatavichute, Y. V., Zhang, X., Cokus, S., Pellegrini, M. & Jacobsen, S. E. Genome-wide association of histone H3 lysine nine methylation with CHG DNA methylation in Arabidopsis thaliana. PLoS ONE 3, e3156 (2008).
Zheng, B. et al. Intergenic transcription by RNA polymerase II coordinates Pol IV and Pol V in siRNA-directed transcriptional gene silencing in Arabidopsis. Genes Dev. 23, 2850–2860 (2009).
We thank Q. Zhang for suggestions. This research was financially supported by the Chinese Academy of Sciences, including the CAS Strategic Priority Research Program, grant number XDB27040101 (to J.-K.Z.).
The authors declare no competing interests.
Peer review information Nature Plants thanks Tzung-Fu Hsieh, Jean Molinier and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary Figs. 1–10.
NRPD1-CD, NRPD1-ND, NRPE1-ND and RDR2-ND lists.
Primers used in this study.
Derepressed genes/TEs in the nrpd1-3 mutant.
CD/ND gene lists.
Source data for supplementary figures.
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Li, J., Yang, D., Huang, H. et al. Epigenetic memory marks determine epiallele stability at loci targeted by de novo DNA methylation. Nat. Plants 6, 661–674 (2020). https://doi.org/10.1038/s41477-020-0671-x
Nature Plants (2020)