Targets of RNA-directed DNA methylation

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RNA-directed DNA methylation contributes substantially to epigenetic regulation of the plant genome. Methylation is guided to homologous DNA target sequences by 24 nt ‘heterochromatic’ small RNAs produced by nucleolar-localized components of the RNAi machinery and a plant-specific RNA polymerase, Pol IV. Plants contain unusually large and diverse populations of small RNAs, many of which originate from transposons and repeats. These sequences are frequent targets of methylation, and they are able to bring plant genes in their vicinity under small RNA-mediated control. RNA-directed DNA methylation can be removed by enzymatic demethylation, providing plants with a versatile system that facilitates epigenetic plasticity. In addition to subduing transposons, RNA-directed DNA methylation has roles in plant development and, perhaps, stress responses.

Introduction

RNA-directed DNA methylation (RdDM) in plants refers to a specific process in which small interfering RNAs (siRNAs) produced by the RNA interference (RNAi) pathway guide de novo methylation of cytosines in all sequence contexts (CG, CNG and CNN, where N is A, T or C) at the homologous DNA region [1, 2]. Promoter-directed siRNAs can induce promoter methylation and transcriptional gene silencing (TGS) of transgenes [1, 3] and endogenous genes [1, 4, 5] in different plant species.

Although the mechanism of RdDM remains incompletely understood, recent work has identified novel components of the silencing machinery and localized them to specialized subnuclear domains. Considerable information on DNA targets of RdDM has accumulated from analyses of silencing-defective mutants, the small RNA transcriptome, and the whole genome ‘methylome’ in Arabidopsis thaliana. It is increasingly apparent that RdDM contributes not only to transposon silencing but also to regulation of genes important for plant physiology and development. This often involves the antagonistic process of enzymatic demethylation, which allows dynamic control of DNA methylation in response to developmental and environmental cues. Here we describe recent research highlights pertaining to these aspects of this fast-moving field.

Section snippets

Components of the RdDM pathway

Forward and reverse genetics approaches in Arabidopsis have identified proteins acting in the RdDM pathway [1, 2] (Table 1). These can be grouped into nuclear ‘RNAi’ proteins required for producing, stabilizing and interacting with the siRNA trigger [RNA-dependent RNA polymerase2 (RDR2), DICER-like3 (DCL3), Argonaute4 (AGO4), AGO6, Hua enhancer1 (HEN1)]; DNA cytosine methyltransferases that catalyze de novo and maintenance methylation [domains rearranged methyltransferase (DRM1, DRM2),

Pol IV

Pol IV subunits were discovered during the analysis of the Arabidopsis genome sequence [8]. Subsequent work revealed a requirement for these plant-specific RNA polymerase subunits in RdDM and defined two functionally diversified forms, Pol IVa and Pol IVb [9•, 10•, 11•, 12•]. The two forms are distinguished by their unique largest subunits, NRPD1a and NRPD1b, which act together with the same second largest subunit, NRPD2a, at different steps of the RdDM pathway. In addition, each Pol IV isoform

AGO proteins in RdDM

AGO proteins are core components of silencing effector complexes in all RNAi-mediated pathways. In addition to binding siRNAs through their PAZ domain, AGO proteins have a catalytic (RNA ‘slicer’) activity conferred by their PIWI domain, which resembles RNase H [19]. Three of the ten AGO proteins in Arabidopsis have been implicated in RdDM. AGO4 binds siRNAs from a number of transposons and repeats [20••] and interacts with the CTD of NRPD1b [7••], which is consistent with the involvement of

Reversible methylation

An important feature of RdDM is its potential reversibility, which is due in part to the induction of CNN methylation. In contrast to symmetrical CG and CNG methylation, which can be maintained during DNA replication in the absence of the RNA trigger (Figure 1), asymmetrical CNN methylation requires the continuous presence of the inducing RNA. Therefore, CNN methylation can be lost passively in dividing cells if the RNA signal is withdrawn [1, 2].

In nondividing cells, DNA methylation can be

Small RNA transcriptome

Deep sequencing has revealed a vast world of small regulatory RNAs in Arabidopsis [28, 29, 30, 31••, 32••, 33], rice [34, 35], and maize [35] (Supplementary Table 1). Compared to other eukaryotes, flowering plants have extraordinarily large and complex populations of endogenous siRNAs. In an analysis of approximately 340 000 unique small RNAs from Arabidopsis [32••], more than half comprised distinct 24 nt ‘heterochromatic’ siRNAs, all of which can potentially trigger RdDM. Around a third of

Methylome

Arabidopsis mutants defective in DNA methyltransferases and other components of the RdDM pathway have been used to investigate DNA methylation and/or transcription [36, 37•, 38••, 39•] (Supplementary Table 2). An extensive analysis exploited Arabidopsis whole genome tiling arrays to examine genome-wide DNA methylation (the ‘methylome’) as well as transcription from both DNA strands in met1 mutants and drm1 drm2 cmt3 triple mutants [38••]. Small RNA-generating regions were usually methylated,

Targets of RdDM

Data on the methylome and transcript profiling can be combined with information on the small RNA transcriptome to provide a genome-wide perspective of RdDM-generated methylation patterns and their impact on gene expression in wild type and mutant plants. Major targets of RdDM include transposons and repeats that are present in constitutive and facultative heterochromatin [9•, 10•, 11•, 12•, 41, 42]. Dispersed copies of these sequences that are present between and within genes in euchromatic

RdDM in development

FLOWERING WAGENINGEN (FWA) exemplifies a developmental gene that is regulated by RdDM and active demethylation. FWA, which encodes a homeobox transcription factor, is imprinted and expressed only from the maternal genome in endosperm [48]. The FWA promoter contains a pair of transposon-associated tandem repeats that give rise to siRNAs [17, 41]. These are sufficient to induce DNA methylation and silence FWA expression, which is the default state [47, 49•]. In the central cell of the female

RdDM in stress responses

In addition to the aforementioned examples, a role for RdDM in development is suggested by the pleiotropic developmental abnormalities observed in drm1 drm2 cmt3 triple mutants [58]. However, the relatively normal appearance of other RdDM-defective mutants, such as drd1, nrpd2a and nrpd1b (T Kanno, M Matzke, unpublished data), suggests that the primary function of this pathway is not to regulate development. There are several reasons to consider a role for reversible RdDM in stress responses. A

Conclusions and outlook

Although RdDM can silence transposons, the functions of this RNAi-mediated pathway extend beyond permanently suppressing these elements. For that purpose, mitotically heritable CG methylation would suffice, as it does in mammals. Even though a process identical to RdDM does not appear to exist in mammalian somatic cells [1], testes-specific 24–31 nt Piwi-interacting (pi)RNAs, which are produced independently of Dicer, may guide methylation of retrotransposons in the male germline [64, 65].

References and recommended reading

Papers of particular interest, published within the annual period of review, have been highlighted as:

  • • of special interest

  • •• of outstanding interest

Acknowledgements

Work in our lab is supported by the European Science Foundation under the EUROCORES Programme EuroDYNA, through contract No, ERAS-CT-980409 of the European Commission, DG Research, FP6, and the Austrian Fonds zur Förderung der wissenschaftlichen Forschung, grant I26-B03.

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