siRNA-mediated DNA methylation and H3K9 dimethylation in plants

Abstract

Heterochromatic siRNAs regulate transcriptional gene silencing by inducing DNA methylation and histone H3K9 dimethylation. Recent advances have revealed the distinct phases involved in siRNA mediated silencing pathway, although the precise functions of a number of factors remain undesignated, putative mechanisms for the connection between DNA and histone methylation have been investigated, and much effort has been invested to understand the biological functions of siRNA-mediated epigenetic modification. In this review, we summarize the mechanism of siRNA-mediated epigenetic modification, which involves the production of siRNA and the recruitments of DNA and histone methytransferases to the target sequences assisted by complementary pairing between 24-nt siRNAs and nascent scaffold RNAs, the roles of siRNA-mediated epigenetic modification in maintaining genome stability and regulating gene expression have been discussed, newly identified players of the siRNA mediated silencing pathway have also been introduced.

Graphical abstract

Keywords

epigenetic / DNA methylation / histone methylation / heterochromatic siRNA / argonaute

INTRODUCTION

The genomic DNA of eukaryotes is tightly wrapped on histone octamers and packaged into chromatin (Fig. 1). Nucleosome is the smallest structural unit of chromatin, which consists of an octamer of core histones comprised of two copies each of the four histone proteins H2A, H2B, H3, and H4 associated with 146 base pairs of double-stranded DNA. The covalent modification of chromatin, which determines the structure of chromatin and plays a crucial role in maintaining genomic stability and controlling gene expression include: DNA methylation at the cytosine nucleotide (Espada and Esteller, 2010; Inagaki and Kakutani, 2010) and post-translational histone modifications, such as methylation and acetylation (Fig. 1) (Lachner et al., 2003; Barth and Imhof, 2010). Significant progress has been made in recent years in understanding the functional roles of these chromatin modification marks in transcriptional regulation (Wang et al., 2008; Espada and Esteller, 2010; Inagaki and Kakutani, 2010); numerous noncoding (nc) transcripts, which control the structure and function of chromatin by chromatin modification, have been discovered (Buhler et al., 2007; Johnson et al., 2007; Zaratiegui et al., 2007). Noncoding (nc) transcripts are derived from heterochromatic loci, which are enriched for transposons and are thought to be transcriptionally inactive. Recent studies have found that siRNAs derived from transposon can mediate DNA methylation and histone H3 lysine 9 dimethylation (H3K9me2), and thus not only repress transposons, but also affects the expression of genes that are located next to them (Liu et al., 2004; Jia et al., 2009). In this review, we will focus on this siRNA mediated epigenetic modification and their functions in plants.

DNA METHYLATION

DNA methylation is a major epigenetic modification in the genomes of eukaryotes, which involves the addition of a methyl group to the 5 position of the cytosine pyrimidine ring (5meC) by DNA methyltransferase enzymes. Compared with animals, in which cytosine is methylated exclusively at CpG sequences (Bird, 1986), DNA methylation in plants occurs in all sequence contexts (CpG, CpHpG, and CpHpH, where H is adenine, cytosine, or thymine). In the Arabidopsis genome, about 24% of CG, 6.7% of CHG, and 1.7% of CHH are methylated (Cokus et al., 2008). DNA methylation mainly occurs in gene-coding regions (Tran et al., 2005; Zhang et al., 2006) and transposon-rich heterochromatic regions (Lippman et al., 2004; Zhang et al., 2006; Zilberman and Henikoff, 2007). In Arabidopsis, the maintenance of DNA methylation is primarily dependent on three methyltransferases: The DOMAIN REARRANGED METHYLTRANSFERASE 2 (DRM2), a DNMT3 homolog, catalyzes de novo DNA methylation (Zhang et al., 2006; Zilberman and Henikoff, 2007; Saze and Kakutani, 2011; Takuno and Gaut, 2012); METHLYTRANSFERASE1 (MET1), a DNMT1 homolog, is responsible for maintaining CG methylation after DNA replication (Saze et al., 2003; Zilberman and Henikoff, 2007); and CHROMOMETHYLASE3 (CMT3), a plant-specific DNA methyltransferase, functions in CHG (H is A, T, or C) (Fig. 1) methylation maintenance (Cao and Jacobsen, 2002a, 2002b). DNA methylation is a reversible process. A family of 5-methylcytosine DNA glycosylases catalyzes active DNA demethylation in plants (Barreto et al., 2007). Recent studies find that IDM1, a histone acetyltransferase, regulates DNA demethylation by binding methylated DNA thus creating a chromatin environment permissible for 5-methylcytosine DNA glycosylases to function (Qian et al., 2012).

HISTONE METHYLATION

The covalent modification of core histones is the other primary mode of chromatin modification. All four core histones contain a well-structured folded globular domain that is in close contact with the DNA and a less well-structured amino-terminal tail domain (Luger et al., 1997). The N-terminal tail of the core histone octamers protrudes out from the nucleosome particles and is susceptible to a variety of covalent modifications, including methylation, acetylation, and phosphorylation (Bernstein et al., 2007). It is important to point out that covalent modifications of histones, especially methylation is not limited in the N-terminal tails, they occur throughout all regions of the histones. Of the various modifications made to histones, methylation is one of the most complicated, both in terms of the nature of the signal and its biological consequences (Lachner and Jenuwein, 2002). Histone methylation can occur at different residues (lysine and arginine), distinct sites, and at different degrees, It is of note that not only the types and positions of residues, but also the numbers of methyl groups on the same residue affect molecular consequences of gene expression. In Arabidopsis, histone lysine methylation occurs mainly at Lys4 (K4), Lys9 (K9), Lys27 (K27), and Lys36 (K36) of histone H3 (Fig. 1) (Grossniklaus et al., 1998; Jackson et al., 2002; Ebbs et al., 2005; Ebbs and Bender, 2006; Cartagena et al., 2008; Saleh et al., 2008; Jacob et al., 2009). Histone methylation can be associated with either gene expression or repression. For example, methylation of histone H3 at K4 and K36 is associated with actively transcribed genes, whereas H3 methylation at K9 and K27 is enriched at constitutively condensed chromatin and developmentally inactive global genes (Berger, 2007). Mechanisms that control these various histone methylations are also different. For example, both H3K27 and H3K9 are associated with gene repression, but H3K27 is deposited at genes by the Polycomb group (PcG) proteins (Turck et al., 2007), while H3K9 is induced by heterochromatic siRNA, therefore, the histone methylation pattern we discuss in this paper will be focused on H3K9. Histone H3 lysine methylation is catalyzed by different histone methyltransferases with evolutionarily conserved SET domains (Liu et al., 2010); in Arabidopsis, three SET-domain methyltransferases-SU (VAR) 3–9 HOMOLOGUE4 (SUV4, also known as KYP ) SUV5 and SUV6 are responsible for H3K9 dimethylation (Fig. 1) (Jackson et al., 2002). Like DNA methylation, histone methylations are not permanent marks either. A large number of enzymes have been discovered with the ability to demethylate methylated histone lysine residues via amine oxidation, hydroxylation, or deaimination (Li et al., 2010).

MECHANISMS OF siRNA DIRECTED DNA METHYLATION AND H3K9 DIMETHYLATION

The mechanism of siRNA directed DNA methylation and H3K9 dimethylation can be divided into the following phases: biogenesis of small interfering RNAs (siRNAs), production of scaffold RNAs, and recruit of silencing complex to target sequences (Fig. 2).

Biogenesis of siRNAs

Plants have several types of endogenous siRNAs, such as: trans-acting siRNAs, natural antisense siRNAs, and heterochromatic siRNAs, with different biogenesis pathways and functions. Heterochromatic siRNAs are the type of siRNAs responsible for mediating gene silencing by directing DNA and histone methylation machineries (Xie et al., 2004; Lu et al., 2005; Kasschau et al., 2007; Zhang et al., 2007; Mosher et al., 2008), therefore, we refer to heterochromatic siRNA as siRNA for the remainder of this paper. siRNAs are usually 24 nt in length and tend to be produced from repeats and transposable elements, although many are also found in intergenic regions. siRNA production, which requires a potential chromatin remodeling protein, CLASSY1 (Herr et al., 2005; Onodera et al., 2005), is initiated by a plant-specific DNA-dependent RNA polymerase: Pol IV, generating single stranded RNAs from a transposon or repeats-containing region (Herr et al., 2005; Onodera et al., 2005; Erhard et al., 2009; Sidorenko et al., 2009; Havecker et al., 2010). It has been recently discovered that the recruitment of Pol IV to target loci requires the assistance of certain proteins, such as SHH1 (Law et al., 2013) and DTF1 (Zhang et al., 2013). The Pol IV transcripts are converted by RNA-dependent RNA polymerase 2 (RDR2) into double-stranded RNAs, which are subsequently cleaved by DICER-LIKE3 (DCL3) followed by the HEN1 methylation (Xie et al., 2004; Alleman et al., 2006). The resulting 24-nt siRNAs are then loaded onto AGO4 (argonaute 4) or its closely related argonaute proteins and are recruited back to homologous genomic loci (Chan et al., 2004; Ye et al., 2012). These argonaute-bound 24-nt siRNAs can then serve as sequence specific guides for methylation by pairing with complementary DNA or nascent scaffold RNA (Wierzbicki et al., 2009). It is important to point out that heterochromatic silencing may require two rounds of siRNA production. The secondary siRNAs are produced from methylated loci, which are associated predominately with short repetitive sequences in intergenic regions (Lee et al., 2012), for reinforcement and possibly spreading of methylation (Pontier et al., 2005; Matzke et al., 2009; Kanno et al., 2010). RDM1 was found to bind single-stranded methylated DNA and help the recruitment of the silencing complex to methylated DNA (Gao et al., 2010). AGO4 in the complex cuts Pol II and Pol V transcripts that are complementary to the initial trigger siRNAs. RDR2 copies the cleaved transcript fragments into double-strand RNAs, and DCL3 cleavage of double-strand RNAs leads to the production of secondary siRNAs at the methylated loci (Gao et al., 2010).

Production of scaffold RNAs

The DNA and histone H3K9 dimethylation requires that the AGO-siRNA complex and the chromatin methylation enzymes recruited to target loci. Production of long non-coding RNAs (lncRNAs), which act as scaffold RNAs, is a key step in this recruitment, which is independent of siRNA biogenesis shown in Arabidopsis mutants defective in NRPD1, RDR2, or DCL3 (Wierzbicki et al., 2008). Pol V plays predominant role in scaffold RNAs production, Pol II is also needed at certain loci (Wierzbicki et al., 2008; Zheng et al., 2009; Wierzbicki et al., 2012). The Pol V-dependent scaffold RNAs production requires a so-called DDR complex, which is composed of a chromatin-remodeling protein DRD1, a chromosome hinge domain protein DMS3 and a protein that binds single-stranded methylated DNA in vitro RDM1, to facilitate Pol V transcription (Kanno et al., 2005; Kanno et al., 2008; Law and Jacobsen, 2010). A GHKL-type ATPase, DMS11, is also needed for production of the Pol V transcript (Lorkovic et al., 2012).

Recruit of silencing machinery to target sequences

Mechanism about how the silencing machinery, including DNA methyltransferases and histone methyltransferases, is recruited to target sequences is not fully understood. The recruitment is assisted by complementary pairing between 24-nt siRNAs and nascent scaffold RNAs. Several models have been proposed to explain the recruitment mechanism (Wierzbicki et al., 2008). According to one of the models, after DCL3-generated 24-nt siRNAs being incorporated into AGO4, scaffold RNAs interact with the argonaute-bound 24-nt siRNAs complex by basepairing with siRNAs. IDN2, a recently identified component of the RdDM pathway with the ability to bind dsRNA with 5’ overhangs (Zhang et al., 2012) may play a role in this siRNA-scaffold RNA interaction to stabilize interactions between an ARGONAUTE bound siRNA and a scaffold RNA synthesized at a target locus by the Pol V complex (Finke et al., 2012). The interaction of the siRNA with the scaffold RNAs might then direct the silencing machinery, including the de novo cytosine methyltransferase DRM2 and/or histone modifying activities, to the target loci. An alternative possibility is that scaffold RNAs may stabilize siRNA-DNA interactions by binding directly to AGO4. The third possibility is that scaffold RNAs may increase the interaction efficiency of AGO4 with target sequence by influencing structural features of heterochromatin (Wierzbicki et al., 2008). Recruitment of silencing machinery including DNA methyltransferases and histone methyltransferases to the target sequences results in the silencing of specific genomic loci.

A CONNECTION BETWEEN H3K9 AND DNA METHYLATION

A genome-wide analysis has showed a high level of co-localization of H3K9me2 and CHG methylation (Bernatavichute et al., 2008), suggesting a connection between these epigenetic marks. Possible explanations for the connection have been proposed as a result of the combined action of recruitment of CMT3-KYP to the target regions (Law and Jacobsen, 2010) and exclusion of H3K9me2 from genic regions by INCREASE IN BONSAI METHYLATION1 (IBM1) (Miura et al., 2009; Inagaki et al., 2010).

The connection between H3K9 and DNA methylation could be due to protein modules recognizing methylated cytosine and H3K9 residues respectively. KYP has a SRA domain, which is one of the three protein domains that recognize methylated cytosines, preferentially binding double-stranded oligonucleotides containing methylated cytosines in a non-CG context. This suggests that this histone modifier can read DNA methylation marks directly (Johnson et al., 2007). Furthermore, CMT3 carrying a chromodomain can recognize and bind K9 dimethylated H3 tails (Lindroth et al., 2004; Du et al., 2012). The interdependent recruitment of CMT3 and KYP couples the regulation of DNA methylation and histone H3K9 dimethylation by a positive feedback loop (Lindroth et al., 2004; Johnson et al., 2007).

The other putative mechanism includes removal of H3K9me2 by IBM1 (Saze et al., 2008). IBM1 encodes a histone demethylase that catalyzes the removal of H3K9 monomethylation and dimethylation in Arabidopsis (Inagaki et al., 2010). A loss-of-function of IBM1 causes ectopic H3K9 methylation at the BONSAI locus, leading to KYP- and CMT3-dependent non-CG DNA hypermethylation and gene silencing (Saze et al., 2008). Genome-wide profiling has shown that a large number of genes are hypermethylated at non-CG cytosines and histone H3K9 in IBM1 (Miura et al., 2009; Inagaki et al., 2010), suggesting that IBM1 protects protein-coding genes from repression via H3K9 and non-CG DNA methylation (Saze et al., 2008). Recent study suggested that IBM1 can also regulate gene expression by indirect silencing of gene expression through siRNA-dependent repression, in addition to direct silencing of gene expression through preventing the coupling of histone and DNA methylation (Fan et al., 2012).

EFFECTS OF siRNA-MEDIATED EPIGENETIC MODIFICATION

Effects of siRNA-mediated epigenetic modification have been well documented. It is important for genome stability over generations, as well as for regulation of gene expression. Maintaining genome integrity by silencing transposable elements is the major function of siRNA-mediated epigenetic modification. DCL3-generated 24-nt siRNAs are first incorporated into AGO4 and form a complex, which assist the recruit of DNA methyltransferases and histone H3K9 methyltransferases to the target sequence (Qi et al., 2006). Loss-of-function mutations in AGO4 can supress DNA methylation and histone H3K9 methylation, which results in reactivation of heterochromatic loci (Zilberman et al., 2003; Xie et al., 2004).

Although siRNA-mediated epigenetic modification typically participates in the suppression of transposable and repetitive sequences and in genome stability, it also plays an important role in the expression of a large number of endogenous genes. In Arabidopsis, variation in flowering time is due to an epiallele of FLOWERING LOCUS C (FLC) generated by siRNA mediated gene silencing. A transposon inserted into an intron of the FLC, from which siRNAs are generated. These siRNAs direct methylation of the transposon region, which spreads into the adjacent FLC gene to result in low levels of FLC expression and early flowering (Liu et al., 2004). Another example is the regulation of the FWA gene by a SINE transposon insertion in the promoter. These transposons give rise to siRNAs that trigger DNA methylation of the promoter, which lead to the repression of FWA gene (Soppe et al., 2000; Chan et al., 2006). When drm2 mutant plants are transformed with FWA, the introduced transgenes remain unmethylated, which results in FWA expression and late flowering (Chan et al., 2006). Regulation of SDC, a gene encodes an F-box protein and possesses seven direct repeats in its promoter, also displays the function of siRNA-mediated methylation. SDC repeats generate siRNA that trigger DNA methylation and transcriptional gene silencing (Henderson and Jacobsen, 2008). Loss of SDC silencing results in downward-curled leaves instead of flat leaves that are produced by wildtype Arabidopsis.

CONCLUSIONS AND PERSPECTIVE

It is clear that specific genomic loci are targeted for silencing through 24-nt heterochromatic siRNAs. siRNAs can mediate DNA methylation and/or H3K9 dimethylation of target sequences and thus affects the transcription of the target loci. siRNA-mediated epigenetic modification mechanism involves the biogenesis of small interfering RNAs (siRNAs), production of scaffold RNAs, and recruit of silencing complex to target sequences, these two siRNA-mediated epigenetic marks are conncected by the combined action of active and passive systems. Numerous evidences have proved the importance of siRNAs mediated DNA methylation and/or H3K9 dimethylation in regulating gene expression and genome stability. Despite all these accomplishments, several as yet unanswered questions remain in the siRNA-mediated silencing pathway. For example: How DNA methyltransferases and histone H3K9 methyltransferases are recruited by AGO4/siRNAs? How particular sequences are recognized or eliminated as substrates of DNA methyltransferases and histone methyltranfereases? what factors provide specificity to CMT3-KYP for recognition of methylated cytosines in specific regions? Are there more key players and targets involved in siRNA-mediated epigenetic modification? Future studies that address these questions will lead to a better understanding of siRNA-mediated silencing pathway.

References

[1] Alleman, M., Sidorenko, L., McGinnis, K., Seshadri, V., Dorweiler, J.E., White, J., Sikkink, K., and Chandler, V.L. (2006). An RNA-dependent RNA polymerase is required for paramutation in maize. Nature 442, 295-298.

[2] Barreto, G., Schafer, A., Marhold, J., Stach, D., Swaminathan, S.K., Handa, V., Doderlein, G., Maltry, N., Wu, W., Lyko, F., et al. (2007). Gadd45a promotes epigenetic gene activation by repair-mediated DNA demethylation. Nature 445, 671-675.

[3] Barth, T.K., and Imhof, A. (2010). Fast signals and slow marks: the dynamics of histone modifications. Trends Biochem Sci 35, 618-626.

[4] Berger, S.L. (2007). The complex language of chromatin regulation during transcription. Nature 447, 407-412.

[5] Bernatavichute, Y.V., Zhang, X., Cokus, S., Pellegrini, M., and Jacobsen, S.E. (2008). Genome-wide association of histone H3 lysine nine methylation with CHG DNA methylation in Arabidopsis thaliana. PLoS One 3, e3156.

[6] Bernstein, B.E., Meissner, A., and Lander, E.S. (2007). The mammalian epigenome. Cell 128, 669-681.

[7] Bird, A.P. (1986). CpG-rich islands and the function of DNA methylation. Nature 321, 209-213.

[8] Buhler, M., Haas, W., Gygi, S.P., and Moazed, D. (2007). RNAi-dependent and -independent RNA turnover mechanisms contribute to heterochromatic gene silencing. Cell 129, 707-721.

[9] Cao, X., and Jacobsen, S.E. (2002a). Locus-specific control of asymmetric and CpNpG methylation by the DRM and CMT3 methyltransferase genes. Proc Natl Acad Sci U S A 99 Suppl 4, 16491-16498.

[10] Cao, X., and Jacobsen, S.E. (2002b). Role of the arabidopsis DRM methyltransferases in de novo DNA methylation and gene silencing. Curr Biol 12, 1138-1144.

[11] Cartagena, J.A., Matsunaga, S., Seki, M., Kurihara, D., Yokoyama, M., Shinozaki, K., Fujimoto, S., Azumi, Y., Uchiyama, S., and Fukui, K. (2008). The Arabidopsis SDG4 contributes to the regulation of pollen tube growth by methylation of histone H3 lysines 4 and 36 in mature pollen. Dev Biol 315, 355-368.

[12] Chan, S.W., Zhang, X., Bernatavichute, Y.V., and Jacobsen, S.E. (2006). Two-step recruitment of RNA-directed DNA methylation to tandem repeats. PLoS Biol 4, e363.

[13] Chan, S.W., Zilberman, D., Xie, Z., Johansen, L.K., Carrington, J.C., and Jacobsen, S.E. (2004). RNA silencing genes control de novo DNA methylation. Science 303, 1336.

[14] Cokus, S.J., Feng, S., Zhang, X., Chen, Z., Merriman, B., Haudenschild, C.D., Pradhan, S., Nelson, S.F., Pellegrini, M., and Jacobsen, S.E. (2008). Shotgun bisulphite sequencing of the Arabidopsis genome reveals DNA methylation patterning. Nature 452, 215-219.

[15] Du, J., Zhong, X., Bernatavichute, Y.V., Stroud, H., Feng, S., Caro, E., Vashisht, A.A., Terragni, J., Chin, H.G., Tu, A., et al. (2012). Dual binding of chromomethylase domains to H3K9me2-containing nucleosomes directs DNA methylation in plants. Cell 151, 167-180.

[16] Ebbs, M.L., Bartee, L., and Bender, J. (2005). H3 lysine 9 methylation is maintained on a transcribed inverted repeat by combined action of SUVH6 and SUVH4 methyltransferases. Mol Cell Biol 25, 10507-10515.

[17] Ebbs, M.L., and Bender, J. (2006). Locus-specific control of DNA methylation by the Arabidopsis SUVH5 histone methyltransferase. Plant Cell 18, 1166-1176.

[18] Erhard, K.F., Stonaker, J.L., Parkinson, S.E., Lim, J.P., Hale, C.J., and Hollick, J.B. (2009). RNA polymerase IV functions in paramutation in Zea mays. Science 323, 1201-1205.

[19] Espada, J., and Esteller, M. (2010). DNA methylation and the functional organization of the nuclear compartment. Semin Cell Dev Biol 21, 238-246.

[20] Fan, D., Dai, Y., Wang, X., Wang, Z., He, H., Yang, H., Cao, Y., Deng, X.W., and Ma, L. (2012). IBM1, a JmjC domain-containing histone demethylase, is involved in the regulation of RNA-directed DNA methylation through the epigenetic control of RDR2 and DCL3 expression in Arabidopsis. Nucleic Acids Res 40, 8905-8916.

[21] Finke, A., Kuhlmann, M., and Mette, M.F. (2012). IDN2 has a role downstream of siRNA formation in RNA-directed DNA methylation. Epigenetics 7, 950-960.

[22] Gao, Z., Liu, H.L., Daxinger, L., Pontes, O., He, X., Qian, W., Lin, H., Xie, M., Lorkovic, Z.J., Zhang, S., et al. (2010). An RNA polymerase II- and AGO4-associated protein acts in RNA-directed DNA methylation. Nature 465, 106-109.

[23] Grossniklaus, U., Vielle-Calzada, J.P., Hoeppner, M.A., and Gagliano, W.B. (1998). Maternal control of embryogenesis by MEDEA, a polycomb group gene in Arabidopsis. Science 280, 446-450.

[24] Havecker, E.R., Wallbridge, L.M., Hardcastle, T.J., Bush, M.S., Kelly, K.A., Dunn, R.M., Schwach, F., Doonan, J.H., and Baulcombe, D.C. (2010). The Arabidopsis RNA-directed DNA methylation argonautes functionally diverge based on their expression and interaction with target loci. Plant Cell 22, 321-334.

[25] Henderson, I.R., and Jacobsen, S.E. (2008). Tandem repeats upstream of the Arabidopsis endogene SDC recruit non-CG DNA methylation and initiate siRNA spreading. Genes Dev 22, 1597-1606.

[26] Herr, A.J., Jensen, M.B., Dalmay, T., and Baulcombe, D.C. (2005). RNA polymerase IV directs silencing of endogenous DNA. Science 308, 118-120.

[27] Inagaki, S., and Kakutani, T. (2010). Control of genic DNA methylation in Arabidopsis. J Plant Res 123, 299-302.

[28] Inagaki, S., Miura-Kamio, A., Nakamura, Y., Lu, F., Cui, X., Cao, X., Kimura, H., Saze, H., and Kakutani, T. (2010). Autocatalytic differentiation of epigenetic modifications within the Arabidopsis genome. EMBO J 29, 3496-3506.

[29] Jackson, J.P., Lindroth, A.M., Cao, X., and Jacobsen, S.E. (2002). Control of CpNpG DNA methylation by the KRYPTONITE histone H3 methyltransferase. Nature 416, 556-560.

[30] Jacob, Y., Feng, S., LeBlanc, C.A., Bernatavichute, Y.V., Stroud, H., Cokus, S., Johnson, L.M., Pellegrini, M., Jacobsen, S.E., and Michaels, S.D. (2009). ATXR5 and ATXR6 are H3K27 monomethyltransferases required for chromatin structure and gene silencing. Nat Struct Mol Biol 16, 763-768.

[31] Jia, Y., Lisch, D.R., Ohtsu, K., Scanlon, M.J., Nettleton, D., and Schnable, P.S. (2009). Loss of RNA-dependent RNA polymerase 2 (RDR2) function causes widespread and unexpected changes in the expression of transposons, genes, and 24-nt small RNAs. PLoS Genet 5, e1000737.

[32] Johnson, L.M., Bostick, M., Zhang, X., Kraft, E., Henderson, I., Callis, J., and Jacobsen, S.E. (2007). The SRA methyl-cytosine-binding domain links DNA and histone methylation. Curr Biol 17, 379-384.

[33] Kanno, T., Aufsatz, W., Jaligot, E., Mette, M.F., Matzke, M., and Matzke, A.J. (2005). A SNF2-like protein facilitates dynamic control of DNA methylation. EMBO Rep 6, 649-655.

[34] Kanno, T., Bucher, E., Daxinger, L., Huettel, B., Bohmdorfer, G., Gregor, W., Kreil, D.P., Matzke, M., and Matzke, A.J. (2008). A structural-maintenance-of-chromosomes hinge domain-containing protein is required for RNA-directed DNA methylation. Nat Genet 40, 670-675.

[35] Kanno, T., Bucher, E., Daxinger, L., Huettel, B., Kreil, D.P., Breinig, F., Lind, M., Schmitt, M.J., Simon, S.A., Gurazada, S.G., et al. (2010). RNA-directed DNA methylation and plant development require an IWR1-type transcription factor. EMBO Rep 11, 65-71.

[36] Kasschau, K.D., Fahlgren, N., Chapman, E.J., Sullivan, C.M., Cumbie, J.S., Givan, S.A., and Carrington, J.C. (2007). Genome-wide profiling and analysis of Arabidopsis siRNAs. PLoS Biol 5, e57.

[37] Lachner, M., and Jenuwein, T. (2002). The many faces of histone lysine methylation. Curr Opin Cell Biol 14, 286-298.

[38] Lachner, M., O’Sullivan, R.J., and Jenuwein, T. (2003). An epigenetic road map for histone lysine methylation. J Cell Sci 116, 2117-2124.

[39] Law, J.A., Du, J., Hale, C.J., Feng, S., Krajewski, K., Palanca, A.M., Strahl, B.D., Patel, D.J., and Jacobsen, S.E. (2013). Polymerase IV occupancy at RNA-directed DNA methylation sites requires SHH1. Nature 498, 385-389.

[40] Law, J.A., and Jacobsen, S.E. (2010). Establishing, maintaining and modifying DNA methylation patterns in plants and animals. Nat Rev Genet 11, 204-220.

[41] Lee, T.F., Gurazada, S.G., Zhai, J., Li, S., Simon, S.A., Matzke, M.A., Chen, X., and Meyers, B.C. (2012). RNA polymerase V-dependent small RNAs in Arabidopsis originate from small, intergenic loci including most SINE repeats. Epigenetics 7, 781-795.

[42] Li, K.K., Luo, C., Wang, D., Jiang, H., and Zheng, Y.G. (2010). Chemical and biochemical approaches in the study of histone methylation and demethylation. Med Res Rev 32, 815-867.

[43] Lindroth, A.M., Shultis, D., Jasencakova, Z., Fuchs, J., Johnson, L., Schubert, D., Patnaik, D., Pradhan, S., Goodrich, J., Schubert, I., et al. (2004). Dual histone H3 methylation marks at lysines 9 and 27 required for interaction with CHROMOMETHYLASE3. EMBO J 23, 4286-4296.

[44] Lippman, Z., Gendrel, A.V., Black, M., Vaughn, M.W., Dedhia, N., McCombie, W.R., Lavine, K., Mittal, V., May, B., Kasschau, K.D., et al. (2004). Role of transposable elements in heterochromatin and epigenetic control. Nature 430, 471-476.

[45] Liu, C., Lu, F., Cui, X., and Cao, X. (2010). Histone methylation in higher plants. Ann Rev Plant Biol 61, 395-420.

[46] Liu, J., He, Y., Amasino, R., and Chen, X. (2004). siRNAs targeting an intronic transposon in the regulation of natural flowering behavior in Arabidopsis. Genes Dev 18, 2873-2878.

[47] Lorkovic, Z.J., Naumann, U., Matzke, A.J., and Matzke, M. (2012). Involvement of a GHKL ATPase in RNA-directed DNA methylation in Arabidopsis thaliana. Curr Biol 22, 933-938.

[48] Lu, C., Tej, S.S., Luo, S., Haudenschild, C.D., Meyers, B.C., and Green, P.J. (2005). Elucidation of the small RNA component of the transcriptome. Science 309, 1567-1569.

[49] Luger, K., Mader, A.W., Richmond, R.K., Sargent, D.F., and Richmond, T.J. (1997). Crystal structure of the nucleosome core particle at 2.8 A resolution. Nature 389, 251-260.

[50] Matzke, M., Kanno, T., Daxinger, L., Huettel, B., and Matzke, A.J. (2009). RNA-mediated chromatin-based silencing in plants. Curr Opin Cell Biol 21, 367-376.

[51] Miura, A., Nakamura, M., Inagaki, S., Kobayashi, A., Saze, H., and Kakutani, T. (2009). An Arabidopsis jmjC domain protein protects transcribed genes from DNA methylation at CHG sites. EMBO J 28, 1078-1086.

[52] Mosher, R.A., Schwach, F., Studholme, D., and Baulcombe, D.C. (2008). PolIVb influences RNA-directed DNA methylation independently of its role in siRNA biogenesis. Proc Natl Acad Sci U S A 105, 3145-3150.

[53] Onodera, Y., Haag, J.R., Ream, T., Costa Nunes, P., Pontes, O., and Pikaard, C.S. (2005). Plant nuclear RNA polymerase IV mediates siRNA and DNA methylation-dependent heterochromatin formation. Cell 120, 613-622.

[54] Pontier, D., Yahubyan, G., Vega, D., Bulski, A., Saez-Vasquez, J., Hakimi, M.A., Lerbs-Mache, S., Colot, V., and Lagrange, T. (2005). 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.

[55] Qi, Y., He, X., Wang, X.J., Kohany, O., Jurka, J., and Hannon, G.J. (2006). Distinct catalytic and non-catalytic roles of ARGONAUTE4 in RNA-directed DNA methylation. Nature 443, 1008-1012.

[56] Qian, W., Miki, D., Zhang, H., Liu, Y., Zhang, X., Tang, K., Kan, Y., La, H., Li, X., Li, S., et al. (2012). A histone acetyltransferase regulates active DNA demethylation in Arabidopsis. Science 336, 1445-1448.

[57] Saleh, A., Alvarez-Venegas, R., Yilmaz, M., Le, O., Hou, G., Sadder, M., Al-Abdallat, A., Xia, Y., Lu, G., Ladunga, I., et al. (2008). The highly similar Arabidopsis homologs of trithorax ATX1 and ATX2 encode proteins with divergent biochemical functions. Plant Cell 20, 568-579.

[58] Saze, H., and Kakutani, T. (2011). Differentiation of epigenetic modifications between transposons and genes. Curr Opin Plant Biol 14, 81-87.

[59] Saze, H., Mittelsten Scheid, O., and Paszkowski, J. (2003). Maintenance of CpG methylation is essential for epigenetic inheritance during plant gametogenesis. Nat Genet 34, 65-69.

[60] Saze, H., Shiraishi, A., Miura, A., and Kakutani, T. (2008). Control of genic DNA methylation by a jmjC domain-containing protein in Arabidopsis thaliana. Science 319, 462-465.

[61] Sidorenko, L., Dorweiler, J.E., Cigan, A.M., Arteaga-Vazquez, M., Vyas, M., Kermicle, J., Jurcin, D., Brzeski, J., Cai, Y., and Chandler, V.L. (2009). A dominant mutation in mediator of paramutation2, one of three second-largest subunits of a plant-specific RNA polymerase, disrupts multiple siRNA silencing processes. PLoS Genet 5, e1000725.

[62] Soppe, W.J., Jacobsen, S.E., Alonso-Blanco, C., Jackson, J.P., Kakutani, T., Koornneef, M., and Peeters, A.J. (2000). The late flowering phenotype of fwa mutants is caused by gain-of-function epigenetic alleles of a homeodomain gene. Mol Cell 6, 791-802.

[63] Takuno, S., and Gaut, B.S. (2012). Body-methylated genes in Arabidopsis thaliana are functionally important and evolve slowly. Mol Biol Evol 29, 219-227.

[64] Tran, R.K., Henikoff, J.G., Zilberman, D., Ditt, R.F., Jacobsen, S.E., and Henikoff, S. (2005). DNA methylation profiling identifies CG methylation clusters in Arabidopsis genes. Curr Biol 15, 154-159.

[65] Turck, F., Roudier, F., Farrona, S., Martin-Magniette, M.L., Guillaume, E., Buisine, N., Gagnot, S., Martienssen, R.A., Coupland, G., and Colot, V. (2007). Arabidopsis TFL2/LHP1 specifically associates with genes marked by trimethylation of histone H3 lysine 27. PLoS Genet 3, e86.

[66] Wang, Z., Zang, C., Rosenfeld, J.A., Schones, D.E., Barski, A., Cuddapah, S., Cui, K., Roh, T.Y., Peng, W., Zhang, M.Q., et al. (2008). Combinatorial patterns of histone acetylations and methylations in the human genome. Nat Genet 40, 897-903.

[67] Wierzbicki, A.T., Cocklin, R., Mayampurath, A., Lister, R., Rowley, M.J., Gregory, B.D., Ecker, J.R., Tang, H., and Pikaard, C.S. (2012). 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.

[68] Wierzbicki, A.T., Haag, J.R., and Pikaard, C.S. (2008). Noncoding transcription by RNA polymerase Pol IVb/Pol V mediates transcriptional silencing of overlapping and adjacent genes. Cell 135, 635-648.

[69] Wierzbicki, A.T., Ream, T.S., Haag, J.R., and Pikaard, C.S. (2009). RNA polymerase V transcription guides ARGONAUTE4 to chromatin. Nat Genet 41, 630-634.

[70] Xie, Z., Johansen, L.K., Gustafson, A.M., Kasschau, K.D., Lellis, A.D., Zilberman, D., Jacobsen, S.E., and Carrington, J.C. (2004). Genetic and functional diversification of small RNA pathways in plants. PLoS Biol 2, e104.

[71] Ye, R., Wang, W., Iki, T., Liu, C., Wu, Y., Ishikawa, M., Zhou, X., and Qi, Y. (2012). Cytoplasmic assembly and selective nuclear import of Arabidopsis Argonaute4/siRNA complexes. Mol Cell 46, 859-870.

[72] Zaratiegui, M., Irvine, D.V., and Martienssen, R.A. (2007). Noncoding RNAs and gene silencing. Cell 128, 763-776.

[73] Zhang, C.J., Ning, Y.Q., Zhang, S.W., Chen, Q., Shao, C.R., Guo, Y.W., Zhou, J.X., Li, L., Chen, S., and He, X.J. (2012). IDN2 and its paralogs form a complex required for RNA-directed DNA methylation. PLoS Genet 8, e1002693.

[74] Zhang, H., Ma, Z.Y., Zeng, L., Tanaka, K., Zhang, C.J., Ma, J., Bai, G., Wang, P., Zhang, S.W., Liu, Z.W., et al. (2013). DTF1 is a core component of RNA-directed DNA methylation and may assist in the recruitment of Pol IV. Proc Natl Acad Sci U S A 110, 8290-8295.

[75] Zhang, X., Henderson, I.R., Lu, C., Green, P.J., and Jacobsen, S.E. (2007). Role of RNA polymerase IV in plant small RNA metabolism. Proc Natl Acad Sci U S A 104, 4536-4541.

[76] Zhang, X., Yazaki, J., Sundaresan, A., Cokus, S., Chan, S.W., Chen, H., Henderson, I.R., Shinn, P., Pellegrini, M., Jacobsen, S.E., et al. (2006). Genome-wide high-resolution mapping and functional analysis of DNA methylation in arabidopsis. Cell 126, 1189-1201.

[77] Zheng, B., Wang, Z., Li, S., Yu, B., Liu, J.Y., and Chen, X. (2009). 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.

[78] Zilberman, D., Cao, X., and Jacobsen, S.E. (2003). ARGONAUTE4 control of locus-specific siRNA accumulation and DNA and histone methylation. Science 299, 716-719.

[79] Zilberman, D., and Henikoff, S. (2007). Genome-wide analysis of DNA methylation patterns. Development 134, 3959-3965.

RIGHTS & PERMISSIONS

Higher Education Press and Springer-Verlag Berlin Heidelberg 2013