Small RNA-directed DNA methylation (RdDM) has been extensively studied in plants, resulting in a deep understanding of a major ‘canonical RdDM’ mechanism. However, current models of canonical RdDM cannot explain how this self-perpetuating mechanism is initiated. Recent investigations into the initiation of epigenetic silencing have determined that there are several alternative ‘non-canonical RdDM’ pathways that function through distinct mechanisms to modify chromatin. This Review aims to illustrate the diversity of non-canonical RdDM mechanisms described to date, recognize common themes within this dizzying array of interconnected pathways, and identify the key unanswered questions remaining in this field.
Subscribe to Journal
Get full journal access for 1 year
only $4.92 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Wassenegger, M., Heimes, S., Riedel, L. & Sänger, H. L. RNA-directed de novo methylation of genomic sequences in plants. Cell 76, 567–576 (1994).
Volpe, T. A. et al. Regulation of heterochromatic silencing and histone H3 lysine-9 methylation by RNAi. Science 297, 1833–1837 (2002).
Hamilton, A., Voinnet, O., Chappell, L. & Baulcombe, D. Two classes of short interfering RNA in RNA silencing. EMBO J. 21, 4671–4679 (2002).
Castel, S. E. & Martienssen, R. A. RNA interference in the nucleus: roles for small RNAs in transcription, epigenetics and beyond. Nat. Rev. Genet. 14, 100–112 (2013).
Heard, E. & Martienssen, R. A. Transgenerational epigenetic inheritance: myths and mechanisms. Cell 157, 95–109 (2014).
Hamilton, A. J. & Baulcombe, D. C. A species of small antisense RNA in posttranscriptional gene silencing in plants. Science 286, 950–952 (1999).
Matzke, M. A. & Mosher, R. A. RNA-directed DNA methylation: an epigenetic pathway of increasing complexity. Nat. Rev. Genet. 15, 394–408 (2014).
Law, J. A., Vashisht, A. A., Wohlschlegel, J. A. & Jacobsen, S. E. SHH1, a homeodomain protein required for DNA methylation, as well as RDR2, RDM4, and chromatin remodeling factors, associate with RNA polymerase IV. PLoS Genet. 7, e1002195 (2011).
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).
Blevins, T. et al. Identification of Pol IV and RDR2-dependent precursors of 24 nt siRNAs guiding de novo DNA methylation in Arabidopsis. Elife 4, e09591 (2015).
Zhai, J. et al. A one precursor one siRNA model for Pol IV-dependent siRNA biogenesis. Cell 163, 445–455 (2015).
Havecker, E. R. et al. The Arabidopsis RNA-directed DNA methylation Argonautes functionally diverge based on their expression and interaction with target loci. Plant Cell 22, 321–334 (2010).
Wierzbicki, A. T., Haag, J. R. & Pikaard, C. S. Noncoding transcription by RNA polymerase Pol IVb/Pol V mediates transcriptional silencing of overlapping and adjacent genes. Cell 135, 635–648 (2008).
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).
Ebbs, M. L. & Bender, J. Locus-specific control of DNA methylation by the Arabidopsis SUVH5 histone methyltransferase. Plant Cell 18, 1166–1176 (2006).
Herr, A. J., Jensen, M. B., Dalmay, T. & Baulcombe, D. C. RNA polymerase IV directs silencing of endogenous DNA. Science 308, 118–120 (2005).
Liu, L. & Chen, X. RNA quality control as a key to suppressing RNA silencing of endogenous genes in plants. Mol. Plant 9, 826–836 (2016).
Raja, P., Sanville, B. C., Buchmann, R. C. & Bisaro, D. M. Viral genome methylation as an epigenetic defense against geminiviruses. J. Virol. 82, 8997–9007 (2008).
Stroud, H., Greenberg, M. V. C., 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).
Panda, K. et al. Full-length autonomous transposable elements are preferentially targeted by expression-dependent forms of RNA-directed DNA methylation. Genome Biol. 17, 170 (2016).
Henderson, I. R. et al. Dissecting Arabidopsis thaliana DICER function in small RNA processing, gene silencing and DNA methylation patterning. Nat. Genet. 38, 721–725 (2006).
Wu, L. et al. DNA methylation mediated by a microRNA pathway. Mol. Cell 38, 465–475 (2010).
Slotkin, R. K., Freeling, M. & Lisch, D. Heritable transposon silencing initiated by a naturally occurring transposon inverted duplication. Nat. Genet. 37, 641–644 (2005).
Vazquez, F., Blevins, T., Ailhas, J., Boller, T. & Meins, F. Evolution of Arabidopsis MIR genes generates novel microRNA classes. Nucleic Acids Res. 36, 6429–6438 (2008).
Chellappan, P. et al. siRNAs from miRNA sites mediate DNA methylation of target genes. Nucleic Acids Res. 38, 6883–6894 (2010).
Khraiwesh, B. et al. Transcriptional control of gene expression by microRNAs. Cell 140, 111–122 (2010).
Wu, L., Mao, L. & Qi, Y. Roles of DICER-LIKE and ARGONAUTE proteins in TAS-derived small interfering RNA-triggered DNA methylation. Plant Physiol. 160, 990–999 (2012).
Allen, E., Xie, Z., Gustafson, A. M. & Carrington, J. C. microRNA-directed phasing during trans-acting siRNA biogenesis in plants. Cell 121, 207–221 (2005).
Nuthikattu, S. et al. The initiation of epigenetic silencing of active transposable elements is triggered by RDR6 and 21–22 nucleotide small interfering RNAs. Plant Physiol. 162, 116–131 (2013).
McCue, A. D. et al. ARGONAUTE 6 bridges transposable element mRNA-derived siRNAs to the establishment of DNA methylation. EMBO J. 34, 20–35 (2015).
Marí-Ordóñez, A. et al. Reconstructing de novo silencing of an active plant retrotransposon. Nat. Genet. 45, 1029–1039 (2013).
Gasciolli, V., Mallory, A. C., Bartel, D. P. & Vaucheret, H. Partially redundant functions of Arabidopsis DICER-like enzymes and a role for DCL4 in producing trans-acting siRNAs. Curr. Biol. 15, 1494–1500 (2005).
Pontier, D. et al. NERD, a plant-specific GW protein, defines an additional RNAi-dependent chromatin-based pathway in Arabidopsis. Mol. Cell 48, 121–132 (2012).
Garcia, D. et al. Ago hook and RNA helicase motifs underpin dual roles for SDE3 in antiviral defense and silencing of nonconserved intergenic regions. Mol. Cell 48, 109–120 (2012).
Wei, W. et al. A role for small RNAs in DNA double-strand break repair. Cell 149, 101–112 (2012).
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 2, 16049 (2016).
Ye, R. et al. A Dicer-independent route for biogenesis of siRNAs that direct DNA methylation in Arabidopsis. Mol. Cell 61, 222–235 (2016).
Yang, D.-L. et al. Dicer-independent RNA-directed DNA methylation in Arabidopsis. Cell Res. 26, 66–82 (2016).
Zemach, A. et al. The Arabidopsis nucleosome remodeler DDM1 allows DNA methyltransferases to access H1-containing heterochromatin. Cell 153, 193–205 (2013).
Li, Q. et al. RNA-directed DNA methylation enforces boundaries between heterochromatin and euchromatin in the maize genome. Proc. Natl Acad. Sci. USA 112, 14728–14733 (2015).
Jackel, J. N., Storer, J. M., Coursey, T. & Bisaro, D. M. Arabidopsis RNA polymerases IV and V are required to establish H3K9 methylation, but not cytosine methylation, on geminivirus chromatin. J. Virol. 90, 7529–7540 (2016).
Law, J. A. et al. Polymerase IV occupancy at RNA-directed DNA methylation sites requires SHH1. Nature 498, 385–389 (2013).
Blevins, T. et al. A two-step process for epigenetic inheritance in Arabidopsis. Mol. Cell 54, 30–42 (2014).
Johnson, L. M. et al. SRA- and SET-domain-containing proteins link RNA polymerase V occupancy to DNA methylation. Nature 507, 124–128 (2014).
Liu, Z.-W. et al. The SET domain proteins SUVH2 and SUVH9 are required for Pol V occupancy at RNA-directed DNA methylation loci. PLoS Genet. 10, e1003948 (2014).
Bond, D. M. & Baulcombe, D. C. Epigenetic transitions leading to heritable, RNA-mediated de novo silencing in Arabidopsis thaliana. Proc. Natl Acad. Sci. USA 112, 917–922 (2015).
Fultz, D., Choudury, S. G. & Slotkin, R. K. Silencing of active transposable elements in plants. Curr. Opin. Plant Biol. 27, 67–76 (2015).
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).
Du, J., Johnson, L. M., Jacobsen, S. E. & Patel, D. J. DNA methylation pathways and their crosstalk with histone methylation. Nat. Rev. Mol. Cell Biol. 16, 519–532 (2015).
Creasey, K. M. et al. miRNAs trigger widespread epigenetically activated siRNAs from transposons in Arabidopsis. Nature 508, 411–415 (2014).
Bousios, A. et al. A role for palindromic structures in the cis-region of maize Sirevirus LTRs in transposable element evolution and host epigenetic response. Genome Res. 26, 226–237 (2016).
D.C.-G. is supported by a Nuevo Leon state fellowship from the Mexico National Council of Science and Technology. The Slotkin lab is supported by U.S. National Science Foundation grants MCB-1252370 and MCB-1608392.
The authors declare no competing interests.
About this article
Cite this article
Cuerda-Gil, D., Slotkin, R. Non-canonical RNA-directed DNA methylation. Nature Plants 2, 16163 (2016). https://doi.org/10.1038/nplants.2016.163
Base-Pairing Requirements for Small RNA-Mediated Gene Silencing of Recessive Self-Incompatibility Alleles in Arabidopsis halleri
Trends in Genetics (2020)
Silencing of Mutator Elements in Maize Involves Distinct Populations of Small RNAs and Distinct Patterns of DNA Methylation
PLOS Genetics (2020)
Genome Research (2020)