RNA-directed DNA methylation (RdDM) is prevalent in flowering plants and induces transcriptional silencing at repetitive DNA, including all types of transposons.
During RdDM, RNA polymerase IV (Pol IV) initiates production of 24-nucleotide small interfering RNAs (siRNAs) that direct transcriptionally repressive DNA methylation to homologous Pol V-transcribed loci. Pol IV and Pol V are recruited to genomic regions that contain transcriptionally repressive epigenetic marks, thereby reinforcing and maintaining the silent state.
Recent research has uncovered variations on the canonical RdDM pathway, including the involvement of RNA-DEPENDENT RNA POLYMERASE 6 (RDR6) and NEEDED FOR RDR2-INDEPENDENT DNA METHYLATION (NERD), which might allow 'young' (that is, recently acquired) transposons to come under the control of RdDM.
In additional to transposon control, RdDM might help hosts to respond to biotic or abiotic challenges, or to faithfully transmit DNA methylation patterns to their offspring. RdDM might also affect germ cell specification and parent-specific gene expression.
There is increasing evidence that siRNAs are used to communicate epigenetic states between homologous sequences within a nucleus or indeed between nuclei.
RNA-directed DNA methylation (RdDM) is the major small RNA-mediated epigenetic pathway in plants. RdDM requires a specialized transcriptional machinery that comprises two plant-specific RNA polymerases — Pol IV and Pol V — and a growing number of accessory proteins, the functions of which in the RdDM mechanism are only partially understood. Recent work has revealed variations in the canonical RdDM pathway and identified factors that recruit Pol IV and Pol V to specific target sequences. RdDM, which transcriptionally represses a subset of transposons and genes, is implicated in pathogen defence, stress responses and reproduction, as well as in interallelic and intercellular communication.
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Castel, S. E. & Martienssen, R. A. RNA interference in the nucleus: roles for small RNAs in transcription, epigenetics and beyond. Nature Rev. Genet. 14, 100–112 (2013).
Luteijn, M. J. & Ketting, R. F. PIWI-interacting RNAs: from generation to transgenerational epigenetics. Nature Rev. Genet. 14, 523–534 (2013).
Jones, A. L., Thomas, C. L. & Maule, A. J. De novo methylation and co-suppression induced by a cytoplasmically replicating plant RNA virus. EMBO J. 17, 6385–6393 (1998).
Wassenegger, M., Heimes, S., Riedel, L. & Sanger, H. L. RNA-directed de novo methylation of genomic sequences in plants. Cell 76, 567–576 (1994).
Eun, C. et al. Use of forward genetic screens to identify genes required for RNA-directed DNA methylation in Arabidopsis thaliana. Cold Spring Harb. Symp. Quant. Biol. 77, 195–204 (2012).
Mette, M. F., Aufsatz, W., van der Winden, J., Matzke, M. A. & Matzke, A. J. M. Transcriptional silencing and promoter methylation triggered by double-stranded RNA. EMBO J. 19, 5194–5201 (2000).
Chan, S. W. et al. RNA silencing genes control de novo DNA methylation. Science 303, 1336 (2004).
Haag, J. R. & Pikaard, C. S. Multisubunit RNA polymerases IV and V: purveyors of non-coding RNA for plant gene silencing. Nature Rev. Mol. Cell Biol. 12, 483–492 (2011).
Yamanaka, S. et al. RNAi triggered by specialized machinery silences developmental genes and retrotransposons. Nature 493, 557–560 (2013).
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). This methylome analysis of 86 A. thaliana gene silencing mutants provides a resource for epigenetics researchers.
Schmitz, R. J. et al. Patterns of population epigenomic diversity. Nature 495, 193–198 (2013).
Mosher, R. A., Schwach, F., Studholme, D. & Baulcombe, D. C. PolIVb influences RNA-directed DNA-methylation independently of its role in siRNA biogenesis. Proc. Natl Acad. Sci. USA 105, 3145–3150 (2008).
Zhang, X., Henderson, I. R., Lu, C., Green, P. J. & Jacobsen, S. E. Role of RNA polymerase IV in plant small RNA metabolism. Proc. Natl Acad. Sci. USA 104, 4536–4541 (2007).
Law, J. A. et al. Polymerase IV occupancy at RNA-directed DNA methylation sites requires SHH1. Nature 498, 385–389 (2013). Using ChIP–seq, this study identifies 928 high-confidence Pol IV peaks. The Pol IV-interacting protein SHH1 is shown to interact with H3K9me and recruit Pol IV to chromatin at 44% of Pol IV target sites.
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).
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).
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).
Smith, L. M. et al. An SNF2 protein associated with nuclear RNA silencing and the spread of a silencing signal between cells in Arabidopsis. Plant Cell 19, 1507–1521 (2007).
Ji, L. J. & Chen, X. M. Regulation of small RNA stability: methylation and beyond. Cell Res. 22, 624–636 (2012).
Eun, C. et al. AGO6 functions in RNA-mediated transcriptional gene silencing in shoot and root meristems in Arabidopsis thaliana. PLoS ONE 6, e25730 (2011).
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).
Olmedo-Monfil, V. et al. Control of female gamete formation by a small RNA pathway in Arabidopsis. Nature 464, 628–632 (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).
Zhong, X. et al. DDR complex facilitates global association of RNA polymerase V to promoters and evolutionarily young transposons. Nature Struct. Mol. Biol. 19, 870–875 (2012).
Wierzbicki, A. T. et al. Spatial and functional relationships among Pol V-associated loci, Pol IV-dependent siRNAs, and cytosine methylation in the Arabidopsis epigenome. Genes Dev. 26, 1825–1836 (2012). Using ChIP–seq, this study identifies 1,157 high-confidence Pol V peaks that are distributed throughout the genome. Most of these peaks are associated with 24-nucleotide siRNAs and CHH methylation, but a consensus sequence for Pol V binding could not be discerned.
Lee, T. F. et al. RNA polymerase V-dependent small RNAs in Arabidopsis originate from small, intergenic loci including most SINE repeats. Epigenetics 7, 781–795 (2012).
Zemach, A. et al. The Arabidopsis nucleosome remodeler DDM1 allows DNA methyltransferases to access H1-containing heterochromatin. Cell 153, 193–205 (2013). This paper shows that most transposons in A. thaliana are silenced by DDM1 and RdDM pathways that act preferentially in highly heterochromatic and more euchromatic regions, respectively. CMT2 is identified as a CHH methyltransferase that functions independently of siRNAs in heterochromatin.
Zheng, Q. et al. RNA polymerase V targets transcriptional silencing components to promoters of protein-coding genes. Plant J. 73, 179–189 (2013).
Schoft, V. K. et al. Induction of RNA-directed DNA methylation upon decondensation of constitutive heterochromatin. EMBO Rep. 10, 1015–1021 (2009).
Stroud, H. et al. Non-CG methylation patterns shape the epigenetic landscape in Arabidopsis. Nature Struct. Mol. Biol. 21, 64–72 (2014).
Johnson, L. M., Law, J. A., Khattar, A., Henderson, I. R. & Jacobsen, S. E. SRA-domain proteins required for DRM2-mediated de novo DNA methylation. PLoS Genet. 4, e1000280 (2008).
Kuhlmann, M. & Mette, M. F. Developmentally non-redundant SET domain proteins SUVH2 and SUVH9 are required for transcriptional gene silencing in Arabidopsis thaliana. Plant Mol. Biol. 79, 623–633 (2012).
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). References 33 and 34 show that Pol V is recruited to methylated DNA at some loci by methyl DNA-binding proteins SUVH2 and SUVH9.
Law, J. A. et al. A protein complex required for polymerase V transcripts and RNA-directed DNA methylation in Arabidopsis. Curr. Biol. 20, 951–956 (2010).
Wierzbicki, A. T., Ream, T. S., Haag, J. R. & Pikaard, C. S. RNA polymerase V transcription guides ARGONAUTE4 to chromatin. Nature Genet. 41, 630–634 (2009).
Gao, Z. H. et al. An RNA polymerase II- and AGO4-associated protein acts in RNA-directed DNA methylation. Nature 465, 106–109 (2010).
Huang, L. F. et al. An atypical RNA polymerase involved in RNA silencing shares small subunits with RNA polymerase II. Nature Struct. Mol. Biol. 16, 91–93 (2009).
Bies-Etheve, N. et al. RNA-directed DNA methylation requires an AGO4-interacting member of the SPT5 elongation factor family. EMBO Rep. 10, 649–654 (2009).
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).
Ausin, I. et al. INVOLVED IN DE NOVO 2-containing complex involved in RNA-directed DNA methylation in Arabidopsis. Proc. Natl Acad. Sci. USA 109, 8374–8381 (2012).
Lorkovic, Z. J., Naumann, U., Matzke, A. J. & Matzke, M. Involvement of a GHKL ATPase in RNA-directed DNA methylation in Arabidopsis thaliana. Curr. Biol. 22, 933–938 (2012).
Zhang, C. J. et al. IDN2 and its paralogs form a complex required for RNA-directed DNA methylation. PLoS Genet. 8, e1002693 (2012).
Xie, M., Ren, G., Costa-Nunes, P., Pontes, O. & Yu, B. A subgroup of SGS3-like proteins act redundantly in RNA-directed DNA methylation. Nucleic Acids Res. 40, 4422–4431 (2012).
Finke, A., Kuhlmann, M. & Mette, M. F. IDN2 has a role downstream of siRNA formation in RNA-directed DNA methylation. Epigenetics 7, 950–960 (2012).
Zhu, Y., Rowley, M. J., Bohmdorfer, G. & Wierzbicki, A. T. A. SWI/SNF chromatin-remodeling complex acts in noncoding RNA-mediated transcriptional silencing. Mol. Cell 49, 298–309 (2013).
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).
Enke, R. A., Dong, Z. & Bender, J. Small RNAs prevent transcription-coupled loss of histone H3 lysine 9 methylation in Arabidopsis thaliana. PLoS Genet. 7, e1002350 (2011).
Liu, X. et al. HDA6 directly interacts with DNA methyltransferase MET1 and maintains transposable element silencing in Arabidopsis. Plant Physiol. 158, 119–129 (2012).
To, T. K. et al. Arabidopsis HDA6 regulates locus-directed heterochromatin silencing in cooperation with MET1. PLoS Genet. 7, e1002055 (2011).
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).
Greenberg, M. V. et al. Interplay between active chromatin marks and RNA-directed DNA methylation in Arabidopsis thaliana. PLoS Genet. 9, e1003946 (2013).
Sridhar, V. V. et al. Control of DNA methylation and heterochromatic silencing by histone H2B deubiquitination. Nature 447, 735–738 (2007).
Brabbs, T. R. et al. The stochastic silencing phenotype of Arabidopsis morc6 mutants reveals a role in efficient RNA-directed DNA methylation. Plant J. 75, 836–846 (2013).
Moissiard, G. et al. MORC family ATPases required for heterochromatin condensation and gene silencing. Science 336, 1448–1451 (2012).
Becker, C. et al. Spontaneous epigenetic variation in the Arabidopsis thaliana methylome. Nature 480, 245–249 (2011).
Schmitz, R. J. et al. Transgenerational epigenetic instability is a source of novel methylation variants. Science 334, 369–373 (2011).
Jones, L., Ratcliff, F. & Baulcombe, D. C. RNA-directed transcriptional gene silencing in plants can be inherited independently of the RNA trigger and requires Met1 for maintenance. Curr. Biol. 11, 747–757 (2001).
Teixeira, F. K. et al. A role for RNAi in the selective correction of DNA methylation defects. Science 323, 1600–1604 (2009).
Law, J. A. & Jacobsen, S. E. Establishing, maintaining and modifying DNA methylation patterns in plants and animals. Nature Rev. Genet. 11, 204–220 (2010).
Huettel, B. et al. Endogenous targets of RNA-directed DNA methylation and Pol IV in Arabidopsis. EMBO J. 25, 2828–2836 (2006).
Zhang, H. & Zhu, J. K. Active DNA demethylation in plants and animals. Cold Spring Harb. Symp. Quant. Biol. 77, 161–173 (2012).
Li, X. et al. Antisilencing role of the RNA-directed DNA methylation pathway and a histone acetyltransferase in Arabidopsis. Proc. Natl Acad. Sci. USA 109, 11425–11430 (2012).
Wu, L. et al. DNA methylation mediated by a microRNA pathway. Mol. Cell 38, 465–475 (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).
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).
Mari-Ordonez, A. et al. Reconstructing de novo silencing of an active plant retrotransposon. Nature Genet. 45, 1029–1039 (2013). This study follows a newly activated retrotransposon through many generations. Initially silenced through PTGS, saturation of DCL2 and DCL4 activity leads to 24-nucleotide siRNA production by DCL3 and initiates canonical RdDM.
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). This paper identifies NERD as an AGO hook protein and, together with reference 68, defines more fully the non-canonical RDR6-dependent RdDM pathway that is independent of Pol IV and that relies instead on Pol II.
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).
You, W., Lorkovic, Z. J., Matzke, A. J. & Matzke, M. Interplay among RNA polymerases II, IV and V in RNA-directed DNA methylation at a low copy transgene locus in Arabidopsis thaliana. Plant Mol. Biol. 82, 85–96 (2013).
Perez-Hormaeche, J. et al. Invasion of the Arabidopsis genome by the tobacco retrotransposon Tnt1 is controlled by reversible transcriptional gene silencing. Plant Physiol. 147, 1264–1278 (2008).
Ito, H. et al. An siRNA pathway prevents transgenerational retrotransposition in plants subjected to stress. Nature 472, 115–119 (2011). This study shows that heat stress triggers transcription of the ONSEN retrotransposon, but such transposition is only possible in some RdDM mutant backgrounds. Insertion of ONSEN near genes can confer heat-responsive transcription.
Ruiz-Ferrer, V. & Voinnet, O. Roles of plant small RNAs in biotic stress responses. Annu. Rev. Plant Biol. 60, 485–510 (2009).
Buchmann, R. C., Asad, S., Wolf, J. N., Mohannath, G. & Bisaro, D. M. Geminivirus AL2 and L2 proteins suppress transcriptional gene silencing and cause genome-wide reductions in cytosine methylation. J. Virol. 83, 5005–5013 (2009).
Yang, L. P. et al. C2-mediated decrease in DNA methylation, accumulation of siRNAs, and increase in expression for genes involved in defense pathways in plants infected with beet severe curly top virus. Plant J. 73, 910–917 (2013).
Zhang, Z. et al. BSCTV C2 attenuates the degradation of SAMDC1 to suppress DNA methylation-mediated gene silencing in Arabidopsis. Plant Cell 23, 273–288 (2011).
Romanel, E. et al. Global alteration of microRNAs and transposon-derived small RNAs in cotton (Gossypium hirsutum) during Cotton leafroll dwarf polerovirus (CLRDV) infection. Plant Mol. Biol. 80, 443–460 (2012).
Lopez, A., Ramirez, V., Garcia-Andrade, J., Flors, V. & Vera, P. The RNA silencing enzyme RNA polymerase V is required for plant immunity. PLoS Genet. 7, e1002434 (2011).
Dowen, R. H. et al. Widespread dynamic DNA methylation in response to biotic stress. Proc. Natl Acad. Sci. USA 109, E2183–E2191 (2012). This genome-wide analysis of DNA methylation during pathogen infection reveals transposon- associated demethylation that affects expression of neighbouring genes.
Luna, E., Bruce, T. J., Roberts, M. R., Flors, V. & Ton, J. Next-generation systemic acquired resistance. Plant Physiol. 158, 844–853 (2012).
Yu, A. et al. Dynamics and biological relevance of DNA demethylation in Arabidopsis antibacterial defense. Proc. Natl Acad. Sci. USA 110, 2389–2394 (2013).
Gohlke, J. et al. DNA methylation mediated control of gene expression is critical for development of crown gall tumors. PLoS Genet. 9, e1003267 (2013).
Ou, X. et al. Transgenerational inheritance of modified DNA methylation patterns and enhanced tolerance induced by heavy metal stress in rice (Oryza sativa L.). PLoS ONE 7, e41143 (2012).
Tricker, P. J., Gibbings, J. G., Rodriguez Lopez, C. M., Hadley, P. & Wilkinson, M. J. Low relative humidity triggers RNA-directed de novo DNA methylation and suppression of genes controlling stomatal development. J. Exp. Bot. 63, 3799–3813 (2012).
Popova, O. V., Dinh, H. Q., Aufsatz, W. & Jonak, C. The RdDM pathway is required for basal heat tolerance in Arabidopsis. Mol. Plant 6, 396–410 (2013).
Yao, Y., Bilichak, A., Golubov, A., Blevins, T. & Kovalchuk, I. Differential sensitivity of Arabidopsis siRNA biogenesis mutants to genotoxic stress. Plant Cell Rep. 29, 1401–1410 (2010).
Verhoeven, K. J., Jansen, J. J., van Dijk, P. J. & Biere, A. Stress-induced DNA methylation changes and their heritability in asexual dandelions. New Phytol. 185, 1108–1118 (2010).
Cubas, P., Vincent, C. & Coen, E. An epigenetic mutation responsible for natural variation in floral symmetry. Nature 401, 157–161 (1999).
Manning, K. et al. A naturally occurring epigenetic mutation in a gene encoding an SBP-box transcription factor inhibits tomato fruit ripening. Nature Genet. 38, 948–952 (2006).
Suter, L. & Widmer, A. Environmental heat and salt stress induce transgenerational phenotypic changes in Arabidopsis thaliana. PLoS ONE 8, e60364 (2013).
Luna, E. & Ton, J. The epigenetic machinery controlling transgenerational systemic acquired resistance. Plant Signal. Behav. 7, 615–618 (2012).
Calarco, J. P. et al. Reprogramming of DNA methylation in pollen guides epigenetic inheritance via small RNA. Cell 151, 194–205 (2012).
Jullien, P. E., Susaki, D., Yelagandula, R., Higashiyama, T. & Berger, F. DNA methylation dynamics during sexual reproduction in Arabidopsis thaliana. Curr. Biol. 22, 1825–1830 (2012).
Singh, M. et al. Production of viable gametes without meiosis in maize deficient for an ARGONAUTE protein. Plant Cell 23, 443–458 (2011).
Garcia-Aguilar, M., Michaud, C., Leblanc, O. & Grimanelli, D. Inactivation of a DNA methylation pathway in maize reproductive organs results in apomixis-like phenotypes. Plant Cell 22, 3249–3267 (2010).
Garnier, O., Laoueille-Duprat, S. & Spillane, C. Genomic imprinting in plants. Epigenetics 3, 14–20 (2008).
Gehring, M., Bubb, K. L. & Henikoff, S. Extensive demethylation of repetitive elements during seed development underlies gene imprinting. Science 324, 1447–1451 (2009).
Mosher, R. A. et al. Uniparental expression of PolIV-dependent siRNAs in developing endosperm of Arabidopsis. Nature 460, 283–286 (2009).
Rodrigues, J. A. et al. Imprinted expression of genes and small RNA is associated with localized hypomethylation of the maternal genome in rice endosperm. Proc. Natl Acad. Sci. USA 110, 7934–7939 (2013).
Hsieh, T. F. et al. Genome-wide demethylation of Arabidopsis endosperm. Science 324, 1451–1454 (2009).
Ibarra, C. A. et al. Active DNA demethylation in plant companion cells reinforces transposon methylation in gametes. Science 337, 1360–1364 (2012).
Mosher, R. A. & Melnyk, C. W. siRNAs and DNA methylation: seedy epigenetics. Trends Plant Sci. 15, 204–210 (2010).
Mosher, R. A. et al. An atypical epigenetic mechanism affects uniparental expression of Pol IV-dependent siRNAs. PLoS ONE 6, e25756 (2011).
Vu, T. M. et al. RNA-directed DNA methylation regulates parental genomic imprinting at several loci in Arabidopsis. Development 140, 2953–2960 (2013).
Mosher, R. A. Maternal control of Pol IV-dependent siRNAs in Arabidopsis endosperm. New Phytol. 186, 358–364 (2010).
Erhard, K. F. Jr & Hollick, J. B. Paramutation: a process for acquiring trans-generational regulatory states. Curr. Opin. Plant Biol. 14, 210–216 (2011).
Arteaga-Vazquez, M. et al. RNA-mediated trans-communication can establish paramutation at the b1 locus in maize. Proc. Natl Acad. Sci. USA 107, 12986–12991 (2010).
Slotkin, R. K. et al. Epigenetic reprogramming and small RNA silencing of transposable elements in pollen. Cell 136, 461–472 (2009).
Dunoyer, P. et al. An endogenous, systemic RNAi pathway in plants. EMBO J. 29, 1699–1712 (2010).
Molnar, A. et al. Small silencing RNAs in plants are mobile and direct epigenetic modification in recipient cells. Science 328, 872–875 (2010).
Melnyk, C. W., Molnar, A., Bassett, A. & Baulcombe, D. C. Mobile 24 nt small RNAs direct transcriptional gene silencing in the root meristems of Arabidopsis thaliana. Curr. Biol. 21, 1678–1683 (2011).
Groszmann, M. et al. Changes in 24-nt siRNA levels in Arabidopsis hybrids suggest an epigenetic contribution to hybrid vigor. Proc. Natl Acad. Sci. USA 108, 2617–2622 (2011).
Li, Y., Varala, K., Moose, S. P. & Hudson, M. E. The inheritance pattern of 24 nt siRNA clusters in Arabidopsis hybrids is influenced by proximity to transposable elements. PLoS ONE 7, e47043 (2012).
He, G. et al. Global epigenetic and transcriptional trends among two rice subspecies and their reciprocal hybrids. Plant Cell 22, 17–33 (2010).
Barber, W. T. et al. Repeat associated small RNAs vary among parents and following hybridization in maize. Proc. Natl Acad. Sci. USA 109, 10444–10449 (2012).
Greaves, I. K. et al. Trans chromosomal methylation in Arabidopsis hybrids. Proc. Natl Acad. Sci. USA 109, 3570–3575 (2012). This paper analyses DNA methylation in Arabidopsis intraspecific hybrids and reveals interactions between chromosomes that result in non-additive methylation. This methylation can alter gene expression and potentially contributes to heterosis.
Shen, H. et al. Genome-wide analysis of DNA methylation and gene expression changes in two Arabidopsis ecotypes and their reciprocal hybrids. Plant Cell 24, 875–892 (2012).
Chodavarapu, R. K. et al. Transcriptome and methylome interactions in rice hybrids. Proc. Natl Acad. Sci. USA 109, 12040–12045 (2012).
Ha, M. et al. Small RNAs serve as a genetic buffer against genomic shock in Arabidopsis interspecific hybrids and allopolyploids. Proc. Natl Acad. Sci. USA 106, 17835–17840 (2009).
Kenan-Eichler, M. et al. Wheat hybridization and polyploidization results in deregulation of small RNAs. Genetics 188, 263–272 (2011).
Josefsson, C., Dilkes, B. & Comai, L. Parent-dependent loss of gene silencing during interspecies hybridization. Curr. Biol. 16, 1322–1328 (2006).
Kashkush, K., Feldman, M. & Levy, A. A. Transcriptional activation of retrotransposons alters the expression of adjacent genes in wheat. Nature Genet. 33, 102–106 (2003).
Shivaprasad, P. V., Dunn, R. M., Santos, B. A., Bassett, A. & Baulcombe, D. C. Extraordinary transgressive phenotypes of hybrid tomato are influenced by epigenetics and small silencing RNAs. EMBO J. 31, 257–266 (2012).
Henderson, I. R. et al. Dissecting Arabidopsis thaliana DICER function in small RNA processing, gene silencing and DNA methylation patterning. Nature Genet. 38, 721–725 (2006).
Kozieradzka-Kiszkurno, M. & Plachno, B. J. Are there symplastic connections between the endosperm and embryo in some angiosperms?—a lesson from the Crassulaceae family. Protoplasma 249, 1081–1089 (2012).
Huang, Y., Kendall, T. & Mosher, R. Pol IV-Dependent siRNA production is reduced in Brassica rapa. Biology 2, 1210–1223 (2013).
Tucker, S. L., Reece, J., Ream, T. S. & Pikaard, C. S. Evolutionary history of plant multisubunit RNA polymerases IV and V: subunit origins via genome-wide and segmental gene duplications, retrotransposition, and lineage-specific subfunctionalization. Cold Spring Harb. Symp. Quant. Biol. 75, 285–297 (2010). This bioinformatic analysis of transcriptomes across the plant kingdom reveals stepwise evolution of Pol IV- and Pol V-specific subunits.
Ream, T. S. et al. Subunit compositions of the RNA-silencing enzymes Pol IV and Pol V reveal their origins as specialized forms of RNA polymerase II. Mol. Cell 33, 192–203 (2009).
Landick, R. Functional divergence in the growing family of RNA polymerases. Structure 17, 323–325 (2009).
Bellaoui, M. & Gruissem, W. Altered expression of the Arabidopsis ortholog of DCL affects normal plant development. Planta 219, 819–826 (2004).
Li, C. F. et al. An ARGONAUTE4-containing nuclear processing center colocalized with Cajal bodies in Arabidopsis thaliana. Cell 126, 93–106 (2006).
Tan, E. H., Blevins, T., Ream, T. S. & Pikaard, C. S. Functional consequences of subunit diversity in RNA polymerases II and V. Cell Rep. 1, 208–214 (2012).
Lahmy, S. et al. PolV(PolIVb) function in RNA-directed DNA methylation requires the conserved active site and an additional plant-specific subunit. Proc. Natl Acad. Sci. USA 106, 941–946 (2009).
Czeko, E., Seizl, M., Augsberger, C., Mielke, T. & Cramer, P. Iwr1 directs RNA polymerase II nuclear import. Mol. Cell 42, 261–266 (2011).
He, X. J. et al. A conserved transcriptional regulator is required for RNA-directed DNA methylation and plant development. Genes Dev. 23, 2717–2722 (2009).
Kanno, T. et al. RNA-directed DNA methylation and plant development require an IWR1-type transcription factor. EMBO Rep. 11, 65–71 (2010).
Luo, J. & Hall, B. D. A multistep process gave rise to RNA polymerase IV of land plants. J. Mol. Evol. 64, 101–112 (2007).
Dolgosheina, E. V. et al. Conifers have a unique small RNA silencing signature. RNA 14, 1508–1515 (2008).
Morin, R. D. et al. Comparative analysis of the small RNA transcriptomes of Pinus contorta and Oryza sativa. Genome Res. 18, 571–584 (2008).
Nystedt, B. et al. The Norway spruce genome sequence and conifer genome evolution. Nature 497, 579–584 (2013).
Zhang, J. et al. Dynamic expression of small RNA populations in larch (Larix leptolepis). Planta 237, 89–101 (2013).
Wan, L. C. et al. Identification and characterization of small non-coding RNAs from Chinese fir by high throughput sequencing. BMC Plant Biol. 12, 146 (2012).
Molnar, A., Schwach, F., Studholme, D. J., Thuenemann, E. C. & Baulcombe, D. C. miRNAs control gene expression in the single-cell alga Chlamydomonas reinhardtii. Nature 447, 1126–U1115 (2007).
Axtell, M. J., Snyder, J. A. & Bartel, D. P. Common functions for diverse small RNAs of land plants. Plant Cell 19, 1750–1769 (2007).
Cho, S. H. et al. Physcomitrella patens DCL3 is required for 22–24 nt siRNA accumulation, suppression of retrotransposon-derived transcripts, and normal development. PLoS Genet. 4, e1000314 (2008).
Pikaard, C. S., Haag, J. R., Pontes, O. M., Blevins, T. & Cocklin, R. A transcription fork model for Pol IV and Pol V-dependent RNA-directed DNA methylation. Cold Spring Harb. Symp. Quant. Biol. 77, 205–212 (2012).
Panda, K. & Slotkin, R. K. Proposed mechanism for the initiation of transposable element silencing by the RDR6-directed DNA methylation pathway. Plant Signal. Behav. 8, e25206 (2013).
Herr, A. J., Jensen, M. B., Dalmay, T. & Baulcombe, D. RNA polymerase IV directs silencing of endogenous DNA. Science 308, 118–120 (2005).
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).
Kanno, T. et al. Atypical RNA polymerase subunits required for RNA-directed DNA methylation. Nature Genet. 37, 761–765 (2005).
He, X. J. et al. NRPD4, a protein related to the RPB4 subunit of RNA polymerase II, is a component of RNA polymerases IV and V and is required for RNA-directed DNA methylation. Genes Dev. 23, 318–330 (2009).
Daxinger, L. et al. A stepwise pathway for biogenesis of 24-nt secondary siRNAs and spreading of DNA methylation. EMBO J. 28, 48–57 (2009).
Greenberg, M. V. et al. Identification of genes required for de novo DNA methylation in Arabidopsis. Epigenetics 6, 344–354 (2011).
Kanno, T. et al. Involvement of putative SNF2 chromatin remodeling protein DRD1 in RNA-directed DNA methylation. Curr. Biol. 14, 801–805 (2004).
Ausin, I., Mockler, T. C., Chory, J. & Jacobsen, S. E. IDN1 and IDN2 are required for de novo DNA methylation in Arabidopsis thaliana. Nature Struct. Mol. Biol. 16, 1325–1327 (2009).
Kanno, T. et al. A structural-maintenance-of-chromosomes hinge domain-containing protein is required for RNA-directed DNA methylation. Nature Genet. 40, 670–675 (2008).
Naumann, U. et al. Genetic evidence that DNA methyltransferase DRM2 has a direct catalytic role in RNA-directed DNA methylation in Arabidopsis thaliana. Genetics 187, 977–979 (2011).
M.A.M. thanks Academia Sinica for financial support and C. Ying for editorial assistance. R.A.M. is supported by the US National Science Foundation under grant MCB-1243608. The authors apologize to colleagues whose publications are not cited owing to space limitations.
The authors declare no competing financial interests.
(DCR). A ribonuclease III enzyme that cleaves double-stranded RNA precursors into small RNAs of 20–30 nucleotides. In plants, homologues of Dicer are referred to as DICER-LIKE (DCL). Of the four DCL enzymes in Arabidopsis thaliana, DCL3 produces 24-nucleotide small interfering RNAs (siRNAs) that act in the canonical RNA-directed DNA methylation pathway.
(AGO). A family of effector proteins of RNA interference that bind to small interfering RNAs (siRNAs) through their PAZ (PIWI–AGO–ZWILLE) and MID (middle) domains and, in some cases, slice RNA through their PIWI domain. Of the ten AGOs in Arabidopsis thaliana, AGO4, AGO6 and AGO9 act in canonical RNA-directed DNA methylation and/or transcriptional gene silencing.
Invasive genetic elements that move within a genome and that are sometimes associated with replicative movement which produces many copies. Transposons include retrotransposons, DNA transposons and helitrons.
- de novo methylation
Methylation of a previously unmodified DNA sequence. Small interfering RNAs (siRNAs) in the RNA-directed DNA methylation pathway are well-known triggers of sequence-specific de novo methylation of cytosines in all sequence contexts.
- Silencing effector complex
A multiprotein complex that elicits RNA interference and related small RNA-mediated gene silencing pathways. It is composed of an Argonaute protein (which binds to the small RNA guide) and, in the case of RNA-directed DNA methylation, cofactors that aid in directing DNA methylation to the small RNA-targeted region of the genome.
Pertaining to the region surrounding the centromere, which is the chromosomal region where two sister chromatids are joined.
- RNA-dependent RNA polymerase
(RDR). A cellular enzyme that copies single-stranded RNAs to produce double-stranded RNA precursors, which are processed by Dicer-like proteins to generate small interfering RNAs (siRNAs). Of the six RDRs in Arabidopsis thaliana, RDR2 is associated with the canonical RdDM pathway.
- Structural maintenance of chromosomes
(SMC). A large family of ATPases that can manipulate chromosome-sized molecules and that contribute to higher-order chromatin structure and dynamics.
- Symmetrical methylation
Cytosine methylation at CG:GC and CHG:GHC nucleotide groups in both DNA strands. As a result of complementary base pairing, CG and CHG are base-paired to GC and GHC, respectively, on the opposite DNA strand and hence considered symmetrical.
- Maintenance methylation
The preservation of pre-existing methylation at symmetrical CG and CHG sites after DNA replication by the DNA methyltransferases MET1 and CMT3, which recognize hemimethylated substrates (that is, those methylated on one strand but not the other).
(miRNAs). Small non-coding RNAs (~21–23 nucleotides) that silence gene expression by mRNA degradation or translational repression through complementarity with the target transcripts.
- Trans-acting siRNAs
(tasiRNAs). A class of small interfering RNAs (siRNAs) that silences gene expression in land plants by targeting complementary mRNAs for cleavage. Their biogenesis depends on microRNA (miRNA)-mediated cleavage of longer TAS RNA precursors that are further acted on by RNA-DEPENDENT RNA POLYMERASE 6 (RDR6) and DICER-LIKE 4 (DCL4). The miRNA-triggered initiation followed by DCL4 cleavage results in a phased pattern of accumulation, in which small RNAs are in an exact head-to-tail arrangement. tasiRNAs are one category of 'phased' siRNA (phasiRNA).
The process of mobilizing a retrotransposon. It involves transcription, processing of the RNA, translation, reverse transcription of the transposon RNA and integration of the reverse-transcribed DNA into a new genomic location.
Alleles that differ in transcriptional level from other genetically identical alleles, frequently owing to DNA methylation. Some epialleles are faithfully transmitted to the progeny.
- Lamarckian inheritance
The hypothesis that an organism can pass on traits acquired during its lifetime to its progeny.
The multicellular structure formed through mitosis from a single haploid spore. Male and female gametophytes contain sperm and egg cells, respectively.
- Diplosporous apomixis
A process of reproduction whereby failure of meiosis produces an unreduced female gametophyte. An embryo then develops from the diploid egg cell and forms a clone of the maternal plant.
Pertaining to an evolutionary change in the timing of a developmental process so that a character or process occurs earlier or later in ontogeny, or grows at a different rate.
- Genomic imprinting
A phenomenon whereby differential epigenetic marks on maternally and paternally derived alleles result in uniparental gene expression.
A process whereby a transcriptionally silent allele confers meiotically heritable silencing on an active sister allele.
Crossing of two different plant varieties to combine valuable traits from each variety.
- Vegetative cell
A haploid cell in the male gametophyte (that is, the pollen grain) that assists fertilization but that does not directly contribute to the zygote.
A tissue in the seed that supports the growth of the embryo. Endosperm is produced after fertilization of the diploid (2N) central cell by a haploid (1N) sperm cell, which creates a maternal:paternal genome ratio of 2:1.
Regions of undifferentiated cells at the shoot or root apex that is responsible for cell division and organogenesis. All aerial tissues, including the germ line, arise from the shoot meristem, and all root tissues arise from the root meristem.
- Additive gene expression
Gene expression in a hybrid that is the average of the expression levels in the two parental lines.
- Interspecific hybrids
Crosses between two closely related but distinct species.
- Hybrid vigour
Increase in fitness associated with crosses between distinct inbred strains.
- Introgression lines
Lines into which defined DNA segments have been introduced from a different line through backcrossing.
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Matzke, M., Mosher, R. RNA-directed DNA methylation: an epigenetic pathway of increasing complexity. Nat Rev Genet 15, 394–408 (2014). https://doi.org/10.1038/nrg3683
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