Abstract
RNA-directed DNA methylation (RdDM) is an important de novo DNA methylation pathway in plants. Small interfering RNAs (siRNAs) generated by Dicers from RNA polymerase IV (Pol IV) transcripts are thought to guide sequence-specific DNA methylation. To gain insight into the mechanism of RdDM, we performed whole-genome bisulfite sequencing of a collection of Arabidopsis mutants, including plants deficient in Pol IV (nrpd1) or Dicer (dcl1/2/3/4) activity. Unexpectedly, of the RdDM target loci that required Pol IV and/or Pol V, only 16% were fully dependent on Dicer activity. DNA methylation was partly or completely independent of Dicer activity at the remaining Pol IV- and/or Pol V-dependent loci, despite the loss of 24-nt siRNAs. Instead, DNA methylation levels correlated with the accumulation of Pol IV-dependent 25-50 nt RNAs at most loci in Dicer mutant plants. Our results suggest that RdDM in plants is largely guided by a previously unappreciated class of Dicer-independent non-coding RNAs, and that siRNAs are required to maintain DNA methylation at only a subset of loci.
Similar content being viewed by others
Log in or create a free account to read this content
Gain free access to this article, as well as selected content from this journal and more on nature.com
or
Accession codes
References
He XJ, Chen T, Zhu JK . Regulation and function of DNA methylation in plants and animals. Cell Res 2011; 21:442–465.
Law JA, Jacobsen SE . Establishing, maintaining and modifying DNA methylation patterns in plants and animals. Nat Rev Genet 2010; 11:204–220.
Mathieu O, Reinders J, Caikovski M, Smathajitt C, Paszkowski J . Transgenerational stability of the Arabidopsis epigenome is coordinated by CG methylation. Cell 2007; 130:851–862.
Martienssen RA, Richards EJ . DNA methylation in eukaryotes. Curr Opin Genet Dev 1995; 5:234–242.
Kankel MW, Ramsey DE, Stokes TL, et al. Arabidopsis MET1 cytosine methyltransferase mutants. Genetics 2003; 163:1109–1122.
Finnegan EJ, Dennis ES . Isolation and identification by sequence homology of a putative cytosine methyltransferase from Arabidopsis thaliana. Nucleic Acids Res 1993; 21:2383–2388.
Johnson LM, Bostick M, Zhang X, et al. The SRA methyl-cytosine-binding domain links DNA and histone methylation. Curr Biol 2007; 17:379–384.
Du J, Johnson LM, Groth M, et al. Mechanism of DNA methylation-directed histone methylation by KRYPTONITE. Mol Cell 2014; 55:495–504.
Ebbs ML, Bender J . Locus-specific control of DNA methylation by the Arabidopsis SUVH5 histone methyltransferase. Plant Cell 2006; 18:1166–1176.
Lindroth AM, Cao X, Jackson JP, et al. Requirement of CHROMOMETHYLASE3 for maintenance of CpXpG methylation. Science 2001; 292:2077–2080.
Du J, Zhong X, Bernatavichute YV, et al. Dual binding of chromomethylase domains to H3K9me2-containing nucleosomes directs DNA methylation in plants. Cell 2012; 151:167–180.
Stroud H, Do T, Du J, et al. Non-CG methylation patterns shape the epigenetic landscape in Arabidopsis. Nat Struct Mol Biol 2014; 21:64–72.
Zemach A, Kim MY, Hsieh PH, et al. The Arabidopsis nucleosome remodeler DDM1 allows DNA methyltransferases to access H1-containing heterochromatin. Cell 2013; 153:193–205.
Cao X, Jacobsen SE . Locus-specific control of asymmetric and CpNpG methylation by the DRM and CMT3 methyltransferase genes. Proc Natl Acad Sci USA 2002; 99 (Suppl 4):16491–16498.
Matzke MA, Mosher RA . RNA-directed DNA methylation: an epigenetic pathway of increasing complexity. Nat Rev Genet 2014; 15:394–408.
Holoch D, Moazed D . RNA-mediated epigenetic regulation of gene expression. Nat Rev Genet 2015; 16:71–84.
Wassenegger M . The role of the RNAi machinery in heterochromatin formation. Cell 2005; 122:13–16.
Bernstein E, Allis CD . RNA meets chromatin. Genes Dev 2005; 19:1635–1655.
Matzke MA, Kanno T, Matzke AJ . RNA-directed DNA methylation: the evolution of a complex epigenetic pathway in flowering plants. Annu Rev Plant Biol 2015; 66:243–267.
Haag JR, Pikaard CS . Multisubunit RNA polymerases IV and V: purveyors of non-coding RNA for plant gene silencing. Nat Rev Mol Cell Biol 2011; 12:483–492.
Law JA, Du J, Hale CJ, et al. Polymerase IV occupancy at RNA-directed DNA methylation sites requires SHH1. Nature 2013; 498:385–389.
Zhang H, Ma ZY, Zeng L, 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 2013; 110:8290–8295.
Johnson LM, Du J, Hale CJ, et al. SRA- and SET-domain-containing proteins link RNA polymerase V occupancy to DNA methylation. Nature 2014; 507:124–128.
Liu ZW, Shao CR, Zhang CJ, 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.
Haag JR, Ream TS, Marasco M, 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 2012; 48:811–818.
Xie Z, Johansen LK, Gustafson AM, et al. Genetic and functional diversification of small RNA pathways in plants. PLoS Biol 2004; 2:E104.
Henderson IR, Zhang X, Lu C, et al. Dissecting Arabidopsis thaliana DICER function in small RNA processing, gene silencing and DNA methylation patterning. Nat Genet 2006; 38:721–725.
Gasciolli V, Mallory AC, Bartel DP, Vaucheret H . Partially redundant functions of Arabidopsis DICER-like enzymes and a role for DCL4 in producing trans-acting siRNAs. Curr Biol 2005; 15:1494–1500.
Xie Z, Allen E, Wilken A, Carrington JC . DICER-LIKE 4 functions in trans-acting small interfering RNA biogenesis and vegetative phase change in Arabidopsis thaliana. Proc Natl Acad Sci USA 2005; 102:12984–12989.
Kasschau KD, Fahlgren N, Chapman EJ, et al. Genome-wide profiling and analysis of Arabidopsis siRNAs. PLoS Biol 2007; 5:e57.
Stroud H, Greenberg MV, Feng S, Bernatavichute YV, Jacobsen SE . Comprehensive analysis of silencing mutants reveals complex regulation of the Arabidopsis methylome. Cell 2013; 152:352–364.
Li S, Vandivier LE, Tu B, 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.
Havecker ER, Wallbridge LM, Hardcastle TJ, et al. The Arabidopsis RNA-directed DNA methylation argonautes functionally diverge based on their expression and interaction with target loci. Plant Cell 2010; 22:321–334.
Duan CG, Zhang H, Tang K, et al. Specific but interdependent functions for Arabidopsis AGO4 and AGO6 in RNA-directed DNA methylation. EMBO J 2015; 34:581–592.
Zhong X, Du J, Hale CJ, et al. Molecular mechanism of action of plant DRM de novo DNA methyltransferases. Cell 2014; 157:1050–1060.
Panda K, Slotkin RK . Proposed mechanism for the initiation of transposable element silencing by the RDR6-directed DNA methylation pathway. Plant Signal Behav 2013; 8.
Chelysheva L, Vezon D, Chambon A, et al. The Arabidopsis HEI10 is a new ZMM protein related to Zip3. PLoS Genet 2012; 8:e1002799.
Feng Z, Zhang B, Ding W, et al. Efficient genome editing in plants using a CRISPR/Cas system. Cell Res 2013; 23:1229–1232.
Feng Z, Mao Y, Xu N, et al. Multigeneration analysis reveals the inheritance, specificity, and patterns of CRISPR/Cas-induced gene modifications in Arabidopsis. Proc Natl Acad Sci USA 2014; 111:4632–4637.
Laubinger S, Sachsenberg T, Zeller G, et al. Dual roles of the nuclear cap-binding complex and SERRATE in pre-mRNA splicing and microRNA processing in Arabidopsis thaliana. Proc Natl Acad Sci USA 2008; 105:8795–8800.
Luo C, Sidote DJ, Zhang Y, Kerstetter RA, Michael TP, Lam E . Integrative analysis of chromatin states in Arabidopsis identified potential regulatory mechanisms for natural antisense transcript production. Plant J 2013; 73:77–90.
Qian W, Miki D, Zhang H, et al. A histone acetyltransferase regulates active DNA demethylation in Arabidopsis. Science 2012; 336:1445–1448.
Zhang X, Clarenz O, Cokus S, et al. Whole-genome analysis of histone H3 lysine 27 trimethylation in Arabidopsis. PLoS Biol 2007; 5:e129.
Thorvaldsdottir H, Robinson JT, Mesirov JP . Integrative Genomics Viewer (IGV): high-performance genomics data visualization and exploration. Brief Bioinform 2013; 14:178–192.
Robinson JT, Thorvaldsdottir H, Winckler W, et al. Integrative genomics viewer. Nat Biotechnol 2011; 29:24–26.
Mi S, Cai T, Hu Y, et al. Sorting of small RNAs into Arabidopsis argonaute complexes is directed by the 5′ terminal nucleotide. Cell 2008; 133:116–127.
Baulcombe D . RNA silencing in plants. Nature 2004; 431:356–363.
Aufsatz W, Mette MF, van der Winden J, Matzke AJ, Matzke M . RNA-directed DNA methylation in Arabidopsis. Proc Natl Acad Sci USA 2002; 99 (Suppl 4):16499–16506.
Wassenegger M . RNA-directed DNA methylation. Plant Mol Biol 2000; 43:203–220.
Volpe TA, Kidner C, Hall IM, Teng G, Grewal SI, Martienssen RA . Regulation of heterochromatic silencing and histone H3 lysine-9 methylation by RNAi. Science 2002; 297:1833–1837.
Grewal SI, Moazed D . Heterochromatin and epigenetic control of gene expression. Science 2003; 301:798–802.
Dalakouras A, Dadami E, Wassenegger M . Viroid-induced DNA methylation in plants. Biomol Concepts 2013; 4:557–565.
Dalakouras A, Wassenegger M . Revisiting RNA-directed DNA methylation. RNA Biol 2013; 10:453–455.
Chan SW, Zhang X, Bernatavichute YV, Jacobsen SE . Two-step recruitment of RNA-directed DNA methylation to tandem repeats. PLoS Biol 2006; 4:e363.
Arteaga-Vazquez M, Sidorenko L, Rabanal FA, et al. RNA-mediated trans-communication can establish paramutation at the b1 locus in maize. Proc Natl Acad Sci USA 2010; 107:12986–12991.
Nagano H, Fukudome A, Hiraguri A, Moriyama H, Fukuhara T . Distinct substrate specificities of Arabidopsis DCL3 and DCL4. Nucleic Acids Res 2014; 42:1845–1856.
Thomas MJ, Platas AA, Hawley DK . Transcriptional fidelity and proofreading by RNA polymerase II. Cell 1998; 93:627–637.
Zhai J, Bischof S, Wang H, et al. A one precursor one siRNA model for Pol IV-dependent siRNA biogenesis. Cell 2015; 163:445–455.
Blevins T, Podicheti R, Mishra V, et al. Identification of Pol IV and RDR2-dependent precursors of 24 nt siRNAs guiding de novo DNA methylation in Arabidopsis. eLife 2015; 4.
Di Ruscio A, Ebralidze AK, Benoukraf T, et al. DNMT1-interacting RNAs block gene-specific DNA methylation. Nature 2013; 503:371–376.
Bond DM, Baulcombe DC . Epigenetic transitions leading to heritable, RNA-mediated de novo silencing in Arabidopsis thaliana. Proc Natl Acad Sci USA 2015; 112:917–922.
Wang XB, Wu Q, Ito T, et al. RNAi-mediated viral immunity requires amplification of virus-derived siRNAs in Arabidopsis thaliana. Proc Natl Acad Sci USA 2010; 107:484–489.
Langmead B, Trapnell C, Pop M, Salzberg SL . Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol 2009; 10:R25.
Zhang H, Tang K, Qian W, et al. An Rrp6-like protein positively regulates noncoding RNA levels and DNA methylation in Arabidopsis. Mol Cell 2014; 54:418–430.
Groszmann M, Greaves IK, Albertyn ZI, Scofield GN, Peacock WJ, Dennis ES . Changes in 24-nt siRNA levels in Arabidopsis hybrids suggest an epigenetic contribution to hybrid vigor. Proc Natl Acad Sci USA 2011; 108:2617–2622.
Quinlan AR, Hall IM . BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics 2010; 26:841–842.
Acknowledgements
This work was supported by the Chinese Academy of Sciences and US National Institutes of Health (R01GM070795) to J-K Z.
Author information
Authors and Affiliations
Corresponding author
Additional information
( Supplementary information is linked to the online version of the paper on the Cell Research website.)
Supplementary information
Supplementary information, Figure S1
DNA methylation levels at the 5' UTR region of HEI10. (PDF 58 kb)
Supplementary information, Figure S2
DNA methylation and small RNA levels at DAE loci. (PDF 106 kb)
Supplementary information, Figure S3
DNA methylation and small RNA levels at DA loci. (PDF 74 kb)
Supplementary information, Figure S4
Introducing dcl1 mutations in the dcl2/3/4 mutant. (PDF 84 kb)
Supplementary information, Figure S5
Overlap between SHH1/DTF1-dependent loci and DCL-independent (ID), -partially dependent (PD) or -fully dependent (FD) RdDM loci. (PDF 44 kb)
Supplementary information, Figure S6
The distribution of 24-nt and >24-nt RNAs on the genome. (PDF 271 kb)
Supplementary information, Figure S7
Size distribution of 18–85 nt RNAs. (PDF 100 kb)
Supplementary information, Table S1
Whole genome bisulfite sequencing and RNA sequencing information (XLSX 13 kb)
Supplementary information, Table S2
Position information of DMRs (XLSX 1106 kb)
Supplementary information, Table S3
Statistic analysis results (XLSX 18 kb)
Supplementary information, Table S4
List of oligos used in this study (PDF 12 kb)
Supplementary information, Table S5
List of mutants used in this study (PDF 9 kb)
Rights and permissions
About this article
Cite this article
Yang, DL., Zhang, G., Tang, K. et al. Dicer-independent RNA-directed DNA methylation in Arabidopsis. Cell Res 26, 66–82 (2016). https://doi.org/10.1038/cr.2015.145
Received:
Revised:
Accepted:
Published:
Issue date:
DOI: https://doi.org/10.1038/cr.2015.145
Keywords
This article is cited by
-
Roles of RNA silencing in viral and non-viral plant immunity and in the crosstalk between disease resistance systems
Nature Reviews Molecular Cell Biology (2022)
-
Epigenetic memory marks determine epiallele stability at loci targeted by de novo DNA methylation
Nature Plants (2020)
-
Cryptic variation in RNA-directed DNA-methylation controls lateral root development when auxin signalling is perturbed
Nature Communications (2020)
-
Four putative SWI2/SNF2 chromatin remodelers have dual roles in regulating DNA methylation in Arabidopsis
Cell Discovery (2018)
-
Genome-wide bisulphite-sequencing reveals organ-specific methylation patterns in chickpea
Scientific Reports (2018)
