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Avian Sleep Group, Max Planck Institute forOrnithology, Eberhard-Gwinner-Strasse,82319 Seewiesen, Germany.E-mail: rattenborg@orn.mpg.de

DOI: 10.1016/j.cub.2012.03.036

RNA-Directed DNA Methylation:Getting a Grip on Mechanism

Small RNAs guide repressive chromatinmodifications to regions of the genomecontaining transposons and repeats. An Arabidopsis genetic screen revealsthat the guidancemachinery includes a novel ATPase complex that could act asa dynamic molecular gripper.

Judith Bender

In addition to genes and sequencesneeded to replicate chromosomes,eukaryotic genomes are riddled withpotentially destructive transposonsand transposon-derived repeats. Thetransposon agenda is to integratecopies of itself into new genomic sites,creating insertional mutations thatcan damage host cell genes. Eventransposon repeats that are notcompetent for movement can expressaberrant RNAs and proteins that saphost cell resources. A fundamentalmechanism by which eukaryotic cellsfight back against transposons is totarget them for chromatin-basedtranscriptional silencing. Abreakthrough in understanding howthe eukaryotic cell tells genomic ‘us’from ‘them’ came from studies in plantsand fission yeast showing that smallRNAs produced from transposon andrepeat sequences guide chromatinmodifications back to matchingsequences in the genome [1]. Morerecently, analogous pathways havebeen described for germline-generatedtransposon small RNAs in Drosophila

and mammals [2–4]. However, manyquestions remain about smallRNA-guided chromatin pathways,including how small RNAs areproduced, how the small RNAs areharnessed to detect matchingDNA sequences, and howchromatin-modifying enzymes arerecruited to the sites of detection.A new study in this issue of CurrentBiology [5], using a sensitive andcomprehensive genetic screeningsystem in Arabidopsis, has identifiedkey components of the guidancemachinery for small RNA-directed DNAmethylation of transposons andrepeats in the plant genome.

Factors previously recoveredfrom this genetic screening systeminclude RNA polymerase subunits,RNA-processing and -binding factors,a chromatin-remodeling protein, a DNAmethyltransferase, and an intriguingprotein called DMS3 [6–10]. DMS3contains a hinge dimerization domainsimilar to the hinge domains foundin structural maintenance ofchromosome (SMC) proteins thatcontrol chromosome organization, but,unlike other SMC proteins, DMS3 lacks

an ATPase domain [6]. In this newstudy, Lorkovic and colleagues presentthe most recent discovery from thegenetic screen, an ATPase DMS11 [5].They show that DMS11 interacts withDMS3 to constitute a complex withthe potential for driving a dynamiccomponent of the RNA-directed DNAmethylation machinery.Based on a combination of genetic

and biochemical approaches, thecurrent view is that RNA-directedDNA methylation involves tethering ofsmall RNA–protein complexes at thetarget region on the DNA by nascentnon-coding RNA transcripts that readthrough the target region (Figure 1) [11].Small RNA complexes couldbase-pair with nascent transcripts in asimilar interaction to the small RNAcomplex–messenger RNA interactionthat occurs during RNA interference, orthey could base-pair with unwoundsingle-stranded DNA in the transcribedregion. Small RNA complexes thenserve as platforms to recruit DNAmethyltransferases and otherchromatin-modifying factors to thetarget region. Plants have evolved anRNApolymerase variant—Pol V— thatis specifically dedicated to making thetethering transcripts for RNA-directedDNA methylation. How Pol V differsfrom RNA polymerase II to facilitatenon-coding transcription in DNAmethylated regions of the genomeis a key question in this field.For genetic dissection of the

small RNA-directed DNA methylationpathway, including Pol V function,

Pol V**

Current Biology

??

11ATPase

11ATPase

Hinge3

Hinge3

Figure 1. A mechanistic model for RNA-dependent DNA methylation.

RNA-dependent DNA methylation requirestranscription through the target region, medi-ated in plants by Pol V. DMS3, DMS11, andperhaps a partner protein (?) could form aring that promotes Pol V processivity by grip-ping together theRNA transcript and templateDNA. Asterisksmark potential targets for basepairing with a small RNA protein complex.

DispatchR401

Lorkovic and colleagues used atarget-plus-silencer double transgenesystem [5,6]. The target transgeneexpresses green fluorescent protein(GFP) in the growing shoot of the plant,and the silencer transgene expressessmall RNAs corresponding to thetarget promoter sequences. Whenthe two transgenes are combined, thetarget promoter region becomesDNA-methylated and transcriptionallysilenced, shutting off GFP. Throughmutations that reactivate GFPexpression, the double transgenescreening system has allowedidentification of eleven factors affectingevery step of the silencing pathway [5].Mutations in the DMS3 hinge domainprotein or the DMS11 ATPase reducepromoter DNA methylation on thetarget transgene, but do not affect thelevels of primary small RNAs producedby the silencer transgene [5,6].Furthermore, dms3 and dms11mutations prevent production of PolV-dependent transcripts atendogenous DNA methylation targetswhere such transcripts accumulate tohigh enough levels to be detectable[5,6,12]. Together, these findingsindicate that the DMS3–DMS11complex is required to promote Pol Vactivity. The dms11 mutation causesweak reactivation of GFP expressionrelative to dms3 and polVmutations [5].A potential explanation for the weakdms11 phenotype is that Arabidopsisencodes six DMS11-related ATPases,

at least some of which could act ina partially redundant manner withDMS11.

The DMS11 ATPase is in a distinctstructural class from the ATPasesfound in SMC hinge domain proteins[5]. However, the functionalorganization of the DMS11–DMS3complex with an ATPase at one endand a hinge domain at the other endis still analogous to the SMC hingedomain protein organization. SMChinge domain proteins form V-shapedheterodimers with the ATPase domainsat the ends of the V and the hinge–hingeinteraction at the point of the V [13,14].The ATPase ends of the SMC hingeheterodimers bind partner proteins toform ring structures that can encircleDNA strands. ATP hydrolysis providesa means to regulate the opening andthe closing of the ring. Pairs ofDMS11–DMS3 complexes could formanalogous dynamic ring structures thathold together DNA and/or RNA strandsduring Pol V transcription. For example,DMS11–DMS3 rings could holdnascent Pol V transcripts in associationwith the DNA template to promote Pol Vprocessivity, and to create a structurethat allows access of small RNAcomplexes to either unpaired RNAor DNA in the transcribed region(Figure 1). Whether DMS11–DMS3rings would include partner proteinsremains to be determined.

Lending support to the modelthat DMS11–DMS3 acts as a gripperfor RNA-directed DNA methylation,the structurally related mammalianATPase–hinge domain proteinSmcHD1 is required for maintainingDNA methylation on the silencedX-chromosome in femalemice — another process that involvesRNA–chromatin interactions [15].Compared with SmcHD1 and the SMChinge domain proteins, the separationof the ATPase and the hinge domainsinto two interacting proteins in theDMS11–DMS3 complex presents a newvariation on the theme of hinge domainprotein organization. This separationcould allow shuffling of DMS11-relatedATPase subunits for functionaldiversification. The discovery ofDMS11 and its interaction with DMS3in RNA-directed DNA methylationbroadens the spectrum of potentialATPase–hinge protein functions toinclude retention of nascentnon-coding transcripts on templateDNA at genomic regions where thesetranscripts serve as molecular tethers.

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Brown University, MCB Department, 185Meeting St, Providence, RI 02912, USA.E-mail: judith_bender@brown.edu

DOI: 10.1016/j.cub.2012.04.010