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Special issue: Systems Biology
DNA methylation as a system of plant genomic immunity M. Yvonne Kim and Daniel Zilberman Department of Plant and Microbial Biology, University of California, Berkeley, CA 94720, USA
Transposons are selfish genetic sequences that can increase their copy number and inflict substantial damage on their hosts. To combat these genomic parasites, plants have evolved multiple pathways to identify and silence transposons by methylating their DNA. Plants have also evolved mechanisms to limit the collateral damage from the antitransposon machinery. In this review, we examine recent developments that have elucidated many of the molecular workings of these pathways. We also highlight the evidence that the methylation and demethylation pathways interact, indicating that plants have a highly sophisticated, integrated system of transposon defense that has an important role in the regulation of gene expression. Transposons are parasites of sexually reproducing species Transposable elements (transposons) are ancient parasitic genetic entities that are able to increase their copy number within a host genome [1]. Unlike viruses, transposons are transmitted vertically from parent to offspring. Therefore, transposons of primarily asexual species operate under essentially the same selection constraints as other genes [2,3] and, thus, are generally few in number and frequently have adaptations to minimize harm to the host, such as strong insertion site selectivity and the ability to self-splice out of mRNA [4,5]. In sexually outcrossing species, such as humans, transposons can spread in a population through mating of carriers with noncarriers, enabling transposons to become fixed despite damaging the reproductive fitness of the host [2,3]. Therefore, transposons in sexually outcrossing species tend to be numerous and aggressive, presenting a major challenge for their hosts [4]. This challenge is compounded by the high diversity of transposons: there are multiple distinct families with different structures and mechanisms of transposition [1]. The only feature that all transposons share is their high copy number relative to conventional genes.
Corresponding author: Zilberman, D. ([email protected]). 1360-1385/$ – see front matter ß 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tplants.2014.01.014
Flowering plants typically reproduce by sexual outcrossing, and are vulnerable to transposons. Although the smallest flowering plant genomes contain approximately 100 million base pairs, genomes 10 to 100 times larger are common [6], mainly because of the number of transposons present in these genomes [7]. In response to the transposon challenge, plants have evolved mechanisms to identify transposons, destroy their RNA, and permanently transcriptionally inactivate them through cytosine methylation of the transposon DNA [8–13]. In parallel, plants have developed the means of minimizing damage to other genes from the antitransposon weaponry [14–16]. Both sets of pathways have been intensively studied and, over the past few years, much progress has been made in understanding the underlying mechanisms. Excitingly, as a result of enhanced mechanistic understanding, the way in which different transposon-related pathways are connected has begun to be revealed, so that it is now possible to discuss an interlocking system of genomic immunity that identifies and inactivates transposons while preserving gene activity. This sophisticated system has also been coopted to regulate gene transcription and now has an essential role in plant development [17–21]. Although in this review we examine the transposon silencing systems of plants, most of the pathways in question, including RNAi, DNA methylation, and histone methylation and demethylation, are found throughout eukaryotes, including humans [12,14,18]. For example, DNA methylation is mediated by a single enzymatic superfamily, and is associated with transposon silencing in plants, fungi, vertebrates, and stramenopiles [5,22]. Thus, the plant system is a variation on an ancient theme. Establishment of transposon silencing The first step of transposon silencing is likely to be the recognition of an RNA polymerase II (Pol II)-synthesized transposon transcript as being somehow aberrant. Doublestranded RNA produces a strong signal [8], but RNA that is unlikely to form double-stranded structures can also initiate silencing [23,24], indicating that other RNA properties are interrogated by the cell. Upon recognition, the aberrant RNA is funneled into the RNAi system (the first line of defense against actively transcribed transposons and viruses), which is discussed at length in several excellent recent reviews [8–14,17,25]. Aberrant RNA is made double-stranded by RNA-dependent RNA polymerase (RDR) 6 and processed into 21- and 22-nucleotide (nt) small Trends in Plant Science xx (2014) 1–7
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Review RNA (sRNA) molecules by Dicer-like nucleases (DCL2 and DCL4) (Figure 1A). These sRNAs are then loaded into ARGONAUTE (AGO) proteins (mainly AGO1 in Arabidopsis), which constitute the catalytic cores of RNase complexes that destroy cognate mRNA (Figure 1A). However, compelling evidence indicates that these sRNAs can also be loaded into effector complexes based around other AGO proteins (AGO4 and AGO6 in most Arabidopsis tissues) [26–28], which instead recognize homologous nascent nuclear transcripts [29,30] (Figure 1A). This recognition leads to methylation of the encoding DNA in all sequence contexts by Domains Rearranged (DRM) methyltransferases through an unknown mechanism (Figure 1A). If the methylated region includes the promoter, the start of transcription, or other key regulatory sequences, the Pol II-driven transcription of the transposon is either attenuated or shut off [31]. Stabilization and maintenance of DNA methylation and silencing Abrogation of Pol II transcription disrupts sRNA-directed DNA methylation (RdDM) at two key points: the generation of aberrant RNA that is converted into sRNA, and the production of nascent scaffold RNA recognized by AGO effector complexes. Thus, as a process that shuts off its own signal, RdDM would be inherently unstable. This problem is avoided by a pair of plant-specific offshoots of Pol II: RNA polymerase IV (Pol IV), which takes over sRNA production, and Pol V, which synthesizes the scaffold transcripts [32] (Figure 1B). Unlike Pol II, both polymerases are adapted to transcribe methylated DNA [33–35]. Pol IV works in close collaboration with RDR2 [36], the products of which are processed into 24-nt sRNA by DCL3 (Figure 1B). These are the only sRNAs associated with most Arabidopsis transposons [37], which rarely exhibit detectable Pol II transcription in most tissues [38]. The few Arabidopsis transposons that are transcribed by Pol II tend to be associated with 21and/or 22-nt sRNAs and RDR6-dependent RdDM [28], as are highly transcribed methylated transgenes [39–41]. However, RdDM mediated by Pol II and RDR6 is likely to be more prevalent in mutants and cell types that exhibit substantial transposon activation [42,43]. Furthermore, Pol II-synthesized, RDR6-generated double-stranded RNA, if present at high levels, can be processed by DCL3 [24] (Figure 1A), and Pol IV-dependent sRNA can use Pol II-synthesized scaffold transcripts [30], indicating that the plant RNAi system is highly integrated. Nonetheless, from the point of view of the host, the desired end state is a transposon that is transcribed only by Pol IV and Pol V for the sole purpose of maintaining DNA methylation. The DNA methylation guided by RNAi interfaces with two other DNA methylation pathways. The VARIANT IN METHYLATION (VIM) proteins (Uhrf1 in vertebrates) recognize hemimethylated symmetrical cytosine-guanine (CG) dinucleotides generated by DNA replication and recruit DNA METHYLTRANSFERASE 1 (MET1) (Dnmt1 in vertebrates) to methylate these sites fully [14] (Figure 1B,C). Methylation by MET1 is highly efficient, so that CG sites are usually either nearly fully methylated or fully unmethylated in a population of cells [38,44]. Therefore, once initiated, this process can operate on its 2
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Figure 1. Progression of transposon DNA methylation and silencing from Pol IIand RDR6- mediated RdDM (A) to self-reinforcing Pol IV- and Pol V-mediated RdDM, which interfaces with the MET1-VIM and KYP-CMT3/CMT2 pathways (B) to a highly compacted, H1-enriched state that excludes Pol IV and Pol V and is maintained exclusively by MET1-VIM and KYP-CMT3/CMT2 with the help of DDM1 (C). Small red spheres represent DNA methylation and small blue spheres represent H3K9me2. Abbreviations: DCL, Dicer-like nuclease; DDM1, DECREASE IN DNA METHYLATION 1; H3K9me2, Histone H3 dimethyl Lys9; KYP, KRYPTONITE; MET1, METHYLTRANSFERASE 1; nt, nucleotide; Pol, RNA polymerase; RdDM, sRNA-directed DNA methylation; RDR6, RNA-dependent RNA polymerase; TSS, transcription start site; VIM, VARIANT IN METHYLATION.
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Review own almost indefinitely, replicating CG methylation semiconservatively, much like DNA sequence. DNA methylation in contexts other than CG is recognized by the KRYPTONITE (KYP) family of histone methyltransferases, which dimethylates lysine nine of histone H3 (H3K9me2) [14] (Figure 1B,C); these enzymes may also be guided by RNAi without intermediating DNA methylation [45]. H3K9me2 is in turn recognized by a family of chromomethylases defined by its founding Arabidopsis member, CMT3. CMT3 binds H3K9me2 via its chromo and BAH domains to methylate cytosines in the CNG context (where N is any nucleotide) [46] (Figure 1B,C). The efficiency of CMT3 is more variable than that of MET1 [38,44] and, therefore, its methylation specificity is usually termed ‘CHG’ (where H is any nucleotide but G) to exclude CGG sites that are also methylated by MET1. A homologous enzyme family, CMT2, methylates cytosines in any sequence context [47], a specificity commonly referred to as ‘CHH’ to avoid overlap with MET1 and CMT3. Similar to CMT3, CMT2 is recruited via H3K9me2 [48], and the target loci of the two enzymes are similar [47] (Figure 1B,C). There is substantial evidence that RdDM is a self-reinforcing process. Pol IV and Pol V are both preferentially associated with methylated DNA in vivo [33–35], and RdDM is facilitated by proteins that bind methylated DNA [49–51]. Although Pol V functions downstream of sRNA biogenesis, it is nevertheless required for sRNA accumulation at many loci [37,52,53], indicating that DNA methylation promotes Pol IV activity. This conclusion is also supported by the observation of reduced sRNA accumulation in met1 [38] and drm1drm2cmt3 [48] mutant plants. DNA methylation may recruit Pol IV, at least in part, via H3K9me2, which is bound by SAWADEE HOMEODOMAIN HOMOLOG 1/DNABINDING TRANSCRIPTION FACTOR 1 (SHH1/DTF1) [35,54] (Figure 1B), a Pol IV-associated protein that is important for sRNA biogenesis [55,56]. As described above, KYP–CMT3/CMT2 and VIM–MET1 are also self-reinforcing pathways. Thus, the plant methylation system comprises three interlocking feed-forward loops that maintain DNA methylation and stable transcriptional silencing of transposons (Figure 1B,C). Progression to RdDM-independent transposon silencing Although most Arabidopsis transposons are methylated by the CMT3/CMT2 and MET1 pathways, the role of RdDM in maintaining DNA methylation is variable. Many transposons show little evidence of RdDM activity, or are targeted by RdDM, but do not require this pathway for efficient methylation [38,47,57]. Methylation of other transposon sequences is dependent on RdDM, which means that the CMT3/CMT2 and MET1 pathways cannot maintain methylation without RdDM activity [47,57]. The first group comprises longer transposons enriched for H3K9me2 and depleted of histone modifications associated with active genes [47]. Methylation of these elements is dependent on the nucleosome remodeling protein DECREASE IN DNA METHYLATION 1 (DDM1), which functions, to a large extent, to overcome the effects of linker histone H1 [47], a widely distributed chromatincompacting protein [58,59]. The second group comprises mostly short isolated transposons or sequences at the
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edges of longer ones that have relatively low levels of H3K9me2 and high levels of gene-associated histone modifications [47]. DDM1 has little effect on the methylation of these transposons [47]. Much of this variation is likely to be because of the intrinsic limitations on the stability of nucleosome-based silencing following DNA replication [60]. Nucleosomes are octamers of histones H2A, H2B, H3, and H4 wrapped by 146 base pairs (bp) of DNA [61]. When DNA is replicated, the nucleosomes are apportioned to the daughter molecules and the gaps filled with newly synthesized histones. Given that some short transposons or transposon remnants are barely long enough to complete a single nucleosome, features such as H3K9me2 cannot be stably inherited through cell division, and the KYP–CMT3/ CMT2 pathway cannot maintain DNA methylation. By contrast, longer transposons retain enough nucleosomes following DNA replication to inherit H3K9me2 and likely other important histone modifications [60]. Similarly, H1, which binds to the nucleosome and the linker DNA that separates nucleosomes, is likely to exert a stronger influence on arrays of many nucleosomes rather than on sequences that comprise just one or two. RdDM, which functions through base complementarity, does not have an inherent target size limitation beyond that required to base pair with the guiding sRNA molecules. Thus, this pathway is ideally suited to maintain methylation of smaller sequences. However, why does it target many larger transposons less effectively? This is unknown, but the requirement for RNA polymerase activity, that of either Pol II or Pol IV and Pol V, suggests a plausible explanation. Pol IV and Pol V may have evolved to transcribe methylated DNA that precludes Pol II activity, but as transposon chromatin becomes more compacted, even these polymerases may be inhibited. Depending on its size, chromatin environment, and likely intrinsic sequence features, a transposon that has progressed from an active, Pol II-transcribed state (Figure 1A) to an inactive Pol IVand Pol V-transcribed state (Figure 1B) may proceed to a state in which it is no longer transcribed at all, and its methylation is entirely dependent on the CMT3/CMT2 and MET1 pathways (Figure 1C). Protection of genes from methylation-induced silencing The DNA methylation pathways described above give plants powerful tools for silencing transposons. As with any system of immunity, it is crucial that these pathways are not misdirected against endogenous genes [8]. The initial, and still poorly understood, assessment of RNA structure must make an important contribution. However, even if the RNA recognition mechanisms function perfectly, some genes present special challenges. One of these is that many active flowering plant genes exhibit DNA methylation of the transcribed region (gene body) that does not interfere with gene expression [62,63]. Several functions, including attenuation of aberrant transcription and regulation of splicing, have been proposed for gene body methylation, which is preferentially found in moderately and constitutively expressed genes with important housekeeping activities, and is conserved across substantial evolutionary distance [64,65]. Whatever its function, gene 3
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Figure 2. Maintenance of gene activity by IBM1 and IBM2. IBM1 removes H3K9me2 from gene bodies that exhibit CG methylation maintained by MET1, thereby precluding the accumulation of nonCG methylation mediated by CMT2 and CMT3. IBM2 allows transcription through heterochromatic introns by an unknown mechanism. Small red spheres represent DNA methylation and small blue spheres represent H3K9me2. Abbreviations: AGO, ARGONAUTE; CG, cytosine–guanine; CMT, CHROMOMETHYLASE; H3K9me2, Histone H3 dimethyl Lys9; IBM, INCREASE IN BONSAI METHYLATION; MET1, METHYLTRANSFERASE 1; Pol II, RNA polymerase II; VIM, VARIANT IN METHYLATION.
body methylation is normally confined almost exclusively to the CG context and mediated by MET1 [38,44] (Figure 2). What, then, prevents the self-reinforcing RdDM and KYP– CMT3/CMT2 loops from operating in these genes? One part of the answer is that the SRA domains of the KYP H3K9 methyltransferases, which bind to methylated DNA, have weak affinity for CG methylation that contrasts with their strong binding to DNA methylated in the CHG and CHH contexts [66]. Thus, the KYP methyltransferases would be expected to recognize efficiently the methylation products of DRM, CMT2, and CMT3, but not MET1. Nonetheless, the KYP enzymes can bind to CG-methylated DNA, and methylated gene bodies exhibit low levels of CHG methylation [47]. CHG methylation is kept low by INCREASE IN BONSAI METHYLATION 1 (IBM1), a member of a large histone demethylase family that removes H3K9 methylation (Figure 2) [67–69]. In the absence of IBM1, genes with methylated bodies exhibit extensive and nearly universal CHG and CHH hypermethylation [47,68,70,71]. This hypermethylation is insensitive to mutations in the RdDM pathway [47], indicating that only the KYP–CMT3/CMT2 system is responsible, potentially because H3K4 methylation, which is found in genes, interferes with SHH1 binding [35]. Nevertheless, the removal of H3K9me2 is also likely to disfavor RdDM over the long term. IBM1 appears to be specifically recruited to actively transcribed genes by an unknown mechanism [68,70]. Given that body-methylated genes are generally constitutively transcribed, this would ensure that they are kept free of nonCG methylation. This arrangement suggests a model where methylated genes exist in one of two stable states: body methylated at CG sites and transcriptionally active, and methylated in all sequence contexts and inactive. If sequences in the first state produce sufficient aberrant RNA to trigger strong RdDM, the resulting nonCG methylation and increased H3K9me2 would overwhelm IBM1 and transition the sequence into a stable silent state that no longer attracts IBM1 activity. 4
IBM1 may protect gene bodies from methylation by RdDM and CMT3/CMT2, but what about those genes that have undergone a transposon insertion within an intron? These transposons are methylated by the full weight of the silencing machinery, just like elements elsewhere [38,44]. Two recent papers have provided a tantalizing clue for how genes deal with such transposons [72,73]. Both studies identified the same gene, IBM2/ANTI-SILENCING 1 (ASI1), inactivation of which causes transcripts of genes with methylated intronic transposons to terminate within the elements. IBM2 is preferentially associated with intronic transposons [72,73], indicating that these have distinctive chromatin features, perhaps as a result of readthrough transcription. IBM2 contains an RNA-binding domain and may facilitate proper mRNA processing, but exactly how this protein aids transcription through intronic transposons remains unknown (Figure 2). Attenuation of gene-adjacent DNA methylation Even if genes are correctly distinguished from transposons, the methylation and silencing of transposons that are inserted near genes can have deleterious effects on gene expression [74], particularly when the transposons are targeted by RdDM [75] (Figure 3A). Therefore, regulation of gene-adjacent transposon methylation would be expected to balance the tradeoff between transposon inactivation and gene repression. In particular, a pathway that attenuates transposon methylation, acting in opposition to RdDM and other methylation-promoting mechanisms, would be needed to achieve the optimum balance (Figure 3). The best-understood pathway in plants centers around the DEMETER/REPRESSOR OF SILENCING 1 (DME/ROS1) family of DNA glycosylases that function as DNA demethylases by excising methylated cytosine from DNA [16]. The activity of the demethylases is facilitated by the protein INCREASED DNA METHYLATION 1 (IDM1), which mediates histone acetylation [76,77] (Figure 3B), a feature of active chromatin, and consistently these
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Figure 3. The balance between RdDM and DNA demethylation regulates gene activity. Expression of a gene is negatively affected by RdDM-mediated DNA methylation of a nearby transposon (A) and positively affected by DME/ROS1mediated DNA demethylation, which is facilitated by IDM1-catalyzed histone acetylation (B). Small red spheres represent DNA methylation and small green spheres represent histone acetylation. Abbreviations: DME/ROS1, DEMETER/ REPRESSOR OF SILENCING 1; DRM, DOMAINS REARRANGED METHYLTRANSFERASE; IDM1, INCREASED DNA METHYLATION 1; RdDM, sRNA-directed DNA methylation; TSS, transcription start site.
enzymes preferentially demethylate small, gene-adjacent transposable elements low in H3K9me2 and high in H3K4me2 and other gene-associated histone modifications [38,78,79]. Long H3K9me2-rich transposons are rarely demethylated, except at their edges [79]. Thus, the demethylases act on the same kinds of sequences targeted by RdDM, and the ones likely to influence gene expression. There is intriguing evidence that the balance between RdDM and active demethylation is fine-tuned through the regulation of gene expression, at least in Arabidopsis. ROS1, the main somatic DNA demethylase, is strongly downregulated in RdDM pathway mutants and plants lacking MET1 [77,80]. By contrast, ROS1 expression is increased in the idm1 mutant, which impairs ROS1-catalyzed demethylation [76]. Given that RdDM and active demethylation generally converge on the same sequences, these observations suggest that ROS1 expression is calibrated through adjustment of DNA methylation at a regulatory sequence: increased RdDM leads to higher levels of ROS1, which in turn reduces methylation, thereby attenuating its own expression. Gene regulation by DNA methylation The ability of the balance between RdDM and DNA demethylation to influence gene expression (Figure 3) naturally lends itself to gene-regulatory purposes. The most dramatic known example of this occurs during sexual development. Unlike in animals, the products of meiosis in flowering plants undergo further mitotic divisions and differentiation, which produce the gametes (egg and
sperm) and their companion cells. DNA demethylation is hyperactivated in the male companion cell (the vegetative cell) and in one of the female companion cells (the central cell) [79,81]; RdDM may also be attenuated, at least on the female side [82]. The result is strong, often complete, demethylation that affects thousands of transposons and either activates or represses multiple genes [79,81,83–85]. The central cell, after fusion with one of the two sperm cells in the pollen (the other fuses with egg), gives rise to the endosperm, a placenta-like nutritive tissue. Given that the sperm does not undergo unusual demethylation, the genes affected by central cell demethylation are expressed differently from the maternally and paternally inherited chromosomes [86]. This process, known as imprinted expression (the methylation is the imprint), is absolutely essential, because lack of a functional DME protein (responsible for companion cell demethylation) in the central cell causes seed abortion [87]. Thus, the pathways that have evolved to limit the impact of transposons on the genome have become co-opted to regulate gene expression and plant development. Concluding remarks Many gaps remain in our knowledge of plant silencing and antisilencing pathways that immunize the genome against transposons. For example, we do not fully understand which RNA features are recognized as aberrant and how this recognition occurs, neither do we know how recognition of a nascent transcript by AGO proteins causes DNA methylation. We also do not know which features of Pol IV and Pol V enable transcription of methylated DNA and, perhaps surprisingly, we have at best a partial understanding of how DNA methylation interferes with Pol II activity. It is unclear how IBM1 is recruited to active genes and how IBM2/ASI1 facilitates transcription through silenced transposons. Although the propensity of DME and related enzymes to demethylate relatively euchromatic sequences appears to be general, we do not know how these enzymes interrogate chromatin or the specific chromatin features that are sought. There are also components of transposon silencing, such as monomethylation of lysine 27 of histone H3 [88,89] and proteins that function in parallel to or downstream of DNA methylation [90–92], the interaction of which with the DNA methylation pathways discussed here is not understood. If the recent past is a reliable guide, further investigation of these and other open questions should reveal more points of interaction between plant silencing mechanisms, deepening understanding of the complex and fascinating system of pathways that mediate transposon silencing and regulate gene expression. References 1 Wicker, T. et al. (2007) A unified classification system for eukaryotic transposable elements. Nat. Rev. Genet. 8, 973–982 2 Hickey, D.A. (1982) Selfish DNA: a sexually-transmitted nuclear parasite. Genetics 101, 519–531 3 Orgel, L.E. and Crick, F.H. (1980) Selfish DNA: the ultimate parasite. Nature 284, 604–607 4 Bestor, T.H. (1999) Sex brings transposons and genomes into conflict. Genetica 107, 289–295 5 Zemach, A. and Zilberman, D. (2010) Evolution of eukaryotic DNA methylation and the pursuit of safer sex. Curr. Biol. 20, R780–R785 5
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34 Zhong, X. et al. (2012) DDR complex facilitates global association of RNA polymerase V to promoters and evolutionarily young transposons. Nat. Struct. Mol. Biol. 19, 870–875 35 Law, J.A. et al. (2013) Polymerase IV occupancy at RNA-directed DNA methylation sites requires SHH1. Nature 498, 385–389 36 Haag, J.R. et al. (2012) 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 37 Lee, T.F. et al. (2012) RNA polymerase V-dependent small RNAs in Arabidopsis originate from small, intergenic loci including most SINE repeats. Epigenetics 7, 781–795 38 Lister, R. et al. (2008) Highly integrated single-base resolution maps of the Arabidopsis genome. Cell 133, 523–536 39 Hamilton, A. et al. (2002) Two classes of short interfering RNA in RNA silencing. EMBO J. 21, 4671–4679 40 Aufsatz, W. et al. (2002) RNA-directed DNA methylation in Arabidopsis. Proc. Natl. Acad. Sci. U.S.A. 99 (Suppl. 4), 16499–16506 41 Zilberman, D. (2004) Role of Arabidopsis ARGONAUTE4 in RNAdirected DNA methylation triggered by inverted repeats. Curr. Biol. 14, 1214–1220 42 Schoft, V.K. et al. (2009) Induction of RNA-directed DNA methylation upon decondensation of constitutive heterochromatin. EMBO Rep. 10, 1015–1021 43 Slotkin, R.K. et al. (2009) Epigenetic reprogramming and small RNA silencing of transposable elements in pollen. Cell 136, 461–472 44 Cokus, S.J. et al. (2008) Shotgun bisulphite sequencing of the Arabidopsis genome reveals DNA methylation patterning. Nature 452, 215–219 45 Enke, R.A. et al. (2011) Small RNAs prevent transcription-coupled loss of histone H3 lysine 9 methylation in Arabidopsis thaliana. PLoS Genet. 7, e1002350 46 Du, J. et al. (2012) Dual binding of chromomethylase domains to H3K9me2-containing nucleosomes directs DNA methylation in plants. Cell 151, 167–180 47 Zemach, A. et al. (2013) The Arabidopsis nucleosome remodeler DDM1 allows DNA methyltransferases to access H1-containing heterochromatin. Cell 153, 193–205 48 Stroud, H. et al. (2014) Non-CG methylation patterns shape the epigenetic landscape in Arabidopsis. Nat. Struct. Mol. Biol. 21, 64–72 49 Johnson, L.M. et al. (2008) SRA-domain proteins required for DRM2mediated de novo DNA methylation. PLoS Genet. 4, e1000280 50 Gao, Z. et al. (2010) An RNA polymerase II- and AGO4-associated protein acts in RNA-directed DNA methylation. Nature 465, 106–109 51 Johnson, L.M. et al. (2014) SRA- and SET-domain-containing proteins link RNA polymerase V occupancy to DNA methylation. Nature http:// dx.doi.org/10.1038/nature12931 52 Zhang, X. et al. (2007) Role of RNA polymerase IV in plant small RNA metabolism. Proc. Natl. Acad. Sci. U.S.A. 104, 4536–4541 53 Mosher, R.A. et al. (2008) PolIVb influences RNA-directed DNA methylation independently of its role in siRNA biogenesis. Proc. Natl. Acad. Sci. U.S.A. 105, 3145–3150 54 Zhang, H. 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 55 Law, J.A. et al. (2011) 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 56 Liu, J. et al. (2011) An atypical component of RNA-directed DNA methylation machinery has both DNA methylation-dependent and independent roles in locus-specific transcriptional gene silencing. Cell Res. 21, 1691–1700 57 Stroud, H. et al. (2013) Comprehensive analysis of silencing mutants reveals complex regulation of the Arabidopsis methylome. Cell 152, 352–364 58 Woodcock, C.L. et al. (2006) Role of linker histone in chromatin structure and function: H1 stoichiometry and nucleosome repeat length. Chromosome Res. 14, 17–25 59 Robinson, P.J. and Rhodes, D. (2006) Structure of the ‘30 nm’ chromatin fibre: a key role for the linker histone. Curr. Opin. Struct. Biol. 16, 336–343 60 Zilberman, D. and Henikoff, S. (2004) Silencing of transposons in plant genomes: kick them when they’re down. Genome Biol. 5, 249
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76 Qian, W. et al. (2012) A histone acetyltransferase regulates active DNA demethylation in Arabidopsis. Science 336, 1445–1448 77 Li, X. et al. (2012) Antisilencing role of the RNA-directed DNA methylation pathway and a histone acetyltransferase in Arabidopsis. Proc. Natl. Acad. Sci. U.S.A. 109, 11425–11430 78 Penterman, J. et al. (2007) DNA demethylation in the Arabidopsis genome. Proc. Natl. Acad. Sci. U.S.A. 104, 6752–6757 79 Ibarra, C.A. et al. (2012) Active DNA demethylation in plant companion cells reinforces transposon methylation in gametes. Science 337, 1360–1364 80 Huettel, B. et al. (2006) Endogenous targets of RNA-directed DNA methylation and Pol IV in Arabidopsis. EMBO J. 25, 2828–2836 81 Calarco, J.P. et al. (2012) Reprogramming of DNA methylation in pollen guides epigenetic inheritance via small RNA. Cell 151, 194–205 82 Vu, T.M. et al. (2013) RNA-directed DNA methylation regulates parental genomic imprinting at several loci in Arabidopsis. Development 140, 2953–2960 83 Zemach, A. et al. (2010) Local DNA hypomethylation activates genes in rice endosperm. Proc. Natl. Acad. Sci. U.S.A. 107, 18729–18734 84 Waters, A.J. et al. (2011) Parent-of-origin effects on gene expression and DNA methylation in the maize endosperm. Plant Cell 23, 4221– 4233 85 Rodrigues, J.A. et al. (2013) Imprinted expression of genes and small RNA is associated with localized hypomethylation of the maternal genome in rice endosperm. Proc. Natl. Acad. Sci. U.S.A. 110, 7934– 7939 86 Huh, J.H. et al. (2008) Cellular programming of plant gene imprinting. Cell 132, 735–744 87 Choi, Y. et al. (2002) DEMETER, a DNA glycosylase domain protein, is required for endosperm gene imprinting and seed viability in Arabidopsis. Cell 110, 33–42 88 Jacob, Y. et al. (2009) ATXR5 and ATXR6 are H3K27 monomethyltransferases required for chromatin structure and gene silencing. Nat. Struct. Mol. Biol. 16, 763–768 89 Jacob, Y. et al. (2010) Regulation of heterochromatic DNA replication by histone H3 lysine 27 methyltransferases. Nature 466, 987–991 90 Amedeo, P. et al. (2000) Disruption of the plant gene MOM releases transcriptional silencing of methylated genes. Nature 405, 203–206 91 Moissiard, G. et al. (2012) MORC family ATPases required for heterochromatin condensation and gene silencing. Science 336, 1448–1451 92 Lorkovic, Z.J. et al. (2012) Involvement of a GHKL ATPase in RNAdirected DNA methylation in Arabidopsis thaliana. Curr. Biol. 22, 933–938
7
Review
Special issue: Systems Biology
DNA methylation as a system of plant genomic immunity M. Yvonne Kim and Daniel Zilberman Department of Plant and Microbial Biology, University of California, Berkeley, CA 94720, USA
Transposons are selfish genetic sequences that can increase their copy number and inflict substantial damage on their hosts. To combat these genomic parasites, plants have evolved multiple pathways to identify and silence transposons by methylating their DNA. Plants have also evolved mechanisms to limit the collateral damage from the antitransposon machinery. In this review, we examine recent developments that have elucidated many of the molecular workings of these pathways. We also highlight the evidence that the methylation and demethylation pathways interact, indicating that plants have a highly sophisticated, integrated system of transposon defense that has an important role in the regulation of gene expression. Transposons are parasites of sexually reproducing species Transposable elements (transposons) are ancient parasitic genetic entities that are able to increase their copy number within a host genome [1]. Unlike viruses, transposons are transmitted vertically from parent to offspring. Therefore, transposons of primarily asexual species operate under essentially the same selection constraints as other genes [2,3] and, thus, are generally few in number and frequently have adaptations to minimize harm to the host, such as strong insertion site selectivity and the ability to self-splice out of mRNA [4,5]. In sexually outcrossing species, such as humans, transposons can spread in a population through mating of carriers with noncarriers, enabling transposons to become fixed despite damaging the reproductive fitness of the host [2,3]. Therefore, transposons in sexually outcrossing species tend to be numerous and aggressive, presenting a major challenge for their hosts [4]. This challenge is compounded by the high diversity of transposons: there are multiple distinct families with different structures and mechanisms of transposition [1]. The only feature that all transposons share is their high copy number relative to conventional genes.
Corresponding author: Zilberman, D. ([email protected]). 1360-1385/$ – see front matter ß 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tplants.2014.01.014
Flowering plants typically reproduce by sexual outcrossing, and are vulnerable to transposons. Although the smallest flowering plant genomes contain approximately 100 million base pairs, genomes 10 to 100 times larger are common [6], mainly because of the number of transposons present in these genomes [7]. In response to the transposon challenge, plants have evolved mechanisms to identify transposons, destroy their RNA, and permanently transcriptionally inactivate them through cytosine methylation of the transposon DNA [8–13]. In parallel, plants have developed the means of minimizing damage to other genes from the antitransposon weaponry [14–16]. Both sets of pathways have been intensively studied and, over the past few years, much progress has been made in understanding the underlying mechanisms. Excitingly, as a result of enhanced mechanistic understanding, the way in which different transposon-related pathways are connected has begun to be revealed, so that it is now possible to discuss an interlocking system of genomic immunity that identifies and inactivates transposons while preserving gene activity. This sophisticated system has also been coopted to regulate gene transcription and now has an essential role in plant development [17–21]. Although in this review we examine the transposon silencing systems of plants, most of the pathways in question, including RNAi, DNA methylation, and histone methylation and demethylation, are found throughout eukaryotes, including humans [12,14,18]. For example, DNA methylation is mediated by a single enzymatic superfamily, and is associated with transposon silencing in plants, fungi, vertebrates, and stramenopiles [5,22]. Thus, the plant system is a variation on an ancient theme. Establishment of transposon silencing The first step of transposon silencing is likely to be the recognition of an RNA polymerase II (Pol II)-synthesized transposon transcript as being somehow aberrant. Doublestranded RNA produces a strong signal [8], but RNA that is unlikely to form double-stranded structures can also initiate silencing [23,24], indicating that other RNA properties are interrogated by the cell. Upon recognition, the aberrant RNA is funneled into the RNAi system (the first line of defense against actively transcribed transposons and viruses), which is discussed at length in several excellent recent reviews [8–14,17,25]. Aberrant RNA is made double-stranded by RNA-dependent RNA polymerase (RDR) 6 and processed into 21- and 22-nucleotide (nt) small Trends in Plant Science xx (2014) 1–7
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Review RNA (sRNA) molecules by Dicer-like nucleases (DCL2 and DCL4) (Figure 1A). These sRNAs are then loaded into ARGONAUTE (AGO) proteins (mainly AGO1 in Arabidopsis), which constitute the catalytic cores of RNase complexes that destroy cognate mRNA (Figure 1A). However, compelling evidence indicates that these sRNAs can also be loaded into effector complexes based around other AGO proteins (AGO4 and AGO6 in most Arabidopsis tissues) [26–28], which instead recognize homologous nascent nuclear transcripts [29,30] (Figure 1A). This recognition leads to methylation of the encoding DNA in all sequence contexts by Domains Rearranged (DRM) methyltransferases through an unknown mechanism (Figure 1A). If the methylated region includes the promoter, the start of transcription, or other key regulatory sequences, the Pol II-driven transcription of the transposon is either attenuated or shut off [31]. Stabilization and maintenance of DNA methylation and silencing Abrogation of Pol II transcription disrupts sRNA-directed DNA methylation (RdDM) at two key points: the generation of aberrant RNA that is converted into sRNA, and the production of nascent scaffold RNA recognized by AGO effector complexes. Thus, as a process that shuts off its own signal, RdDM would be inherently unstable. This problem is avoided by a pair of plant-specific offshoots of Pol II: RNA polymerase IV (Pol IV), which takes over sRNA production, and Pol V, which synthesizes the scaffold transcripts [32] (Figure 1B). Unlike Pol II, both polymerases are adapted to transcribe methylated DNA [33–35]. Pol IV works in close collaboration with RDR2 [36], the products of which are processed into 24-nt sRNA by DCL3 (Figure 1B). These are the only sRNAs associated with most Arabidopsis transposons [37], which rarely exhibit detectable Pol II transcription in most tissues [38]. The few Arabidopsis transposons that are transcribed by Pol II tend to be associated with 21and/or 22-nt sRNAs and RDR6-dependent RdDM [28], as are highly transcribed methylated transgenes [39–41]. However, RdDM mediated by Pol II and RDR6 is likely to be more prevalent in mutants and cell types that exhibit substantial transposon activation [42,43]. Furthermore, Pol II-synthesized, RDR6-generated double-stranded RNA, if present at high levels, can be processed by DCL3 [24] (Figure 1A), and Pol IV-dependent sRNA can use Pol II-synthesized scaffold transcripts [30], indicating that the plant RNAi system is highly integrated. Nonetheless, from the point of view of the host, the desired end state is a transposon that is transcribed only by Pol IV and Pol V for the sole purpose of maintaining DNA methylation. The DNA methylation guided by RNAi interfaces with two other DNA methylation pathways. The VARIANT IN METHYLATION (VIM) proteins (Uhrf1 in vertebrates) recognize hemimethylated symmetrical cytosine-guanine (CG) dinucleotides generated by DNA replication and recruit DNA METHYLTRANSFERASE 1 (MET1) (Dnmt1 in vertebrates) to methylate these sites fully [14] (Figure 1B,C). Methylation by MET1 is highly efficient, so that CG sites are usually either nearly fully methylated or fully unmethylated in a population of cells [38,44]. Therefore, once initiated, this process can operate on its 2
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Figure 1. Progression of transposon DNA methylation and silencing from Pol IIand RDR6- mediated RdDM (A) to self-reinforcing Pol IV- and Pol V-mediated RdDM, which interfaces with the MET1-VIM and KYP-CMT3/CMT2 pathways (B) to a highly compacted, H1-enriched state that excludes Pol IV and Pol V and is maintained exclusively by MET1-VIM and KYP-CMT3/CMT2 with the help of DDM1 (C). Small red spheres represent DNA methylation and small blue spheres represent H3K9me2. Abbreviations: DCL, Dicer-like nuclease; DDM1, DECREASE IN DNA METHYLATION 1; H3K9me2, Histone H3 dimethyl Lys9; KYP, KRYPTONITE; MET1, METHYLTRANSFERASE 1; nt, nucleotide; Pol, RNA polymerase; RdDM, sRNA-directed DNA methylation; RDR6, RNA-dependent RNA polymerase; TSS, transcription start site; VIM, VARIANT IN METHYLATION.
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Review own almost indefinitely, replicating CG methylation semiconservatively, much like DNA sequence. DNA methylation in contexts other than CG is recognized by the KRYPTONITE (KYP) family of histone methyltransferases, which dimethylates lysine nine of histone H3 (H3K9me2) [14] (Figure 1B,C); these enzymes may also be guided by RNAi without intermediating DNA methylation [45]. H3K9me2 is in turn recognized by a family of chromomethylases defined by its founding Arabidopsis member, CMT3. CMT3 binds H3K9me2 via its chromo and BAH domains to methylate cytosines in the CNG context (where N is any nucleotide) [46] (Figure 1B,C). The efficiency of CMT3 is more variable than that of MET1 [38,44] and, therefore, its methylation specificity is usually termed ‘CHG’ (where H is any nucleotide but G) to exclude CGG sites that are also methylated by MET1. A homologous enzyme family, CMT2, methylates cytosines in any sequence context [47], a specificity commonly referred to as ‘CHH’ to avoid overlap with MET1 and CMT3. Similar to CMT3, CMT2 is recruited via H3K9me2 [48], and the target loci of the two enzymes are similar [47] (Figure 1B,C). There is substantial evidence that RdDM is a self-reinforcing process. Pol IV and Pol V are both preferentially associated with methylated DNA in vivo [33–35], and RdDM is facilitated by proteins that bind methylated DNA [49–51]. Although Pol V functions downstream of sRNA biogenesis, it is nevertheless required for sRNA accumulation at many loci [37,52,53], indicating that DNA methylation promotes Pol IV activity. This conclusion is also supported by the observation of reduced sRNA accumulation in met1 [38] and drm1drm2cmt3 [48] mutant plants. DNA methylation may recruit Pol IV, at least in part, via H3K9me2, which is bound by SAWADEE HOMEODOMAIN HOMOLOG 1/DNABINDING TRANSCRIPTION FACTOR 1 (SHH1/DTF1) [35,54] (Figure 1B), a Pol IV-associated protein that is important for sRNA biogenesis [55,56]. As described above, KYP–CMT3/CMT2 and VIM–MET1 are also self-reinforcing pathways. Thus, the plant methylation system comprises three interlocking feed-forward loops that maintain DNA methylation and stable transcriptional silencing of transposons (Figure 1B,C). Progression to RdDM-independent transposon silencing Although most Arabidopsis transposons are methylated by the CMT3/CMT2 and MET1 pathways, the role of RdDM in maintaining DNA methylation is variable. Many transposons show little evidence of RdDM activity, or are targeted by RdDM, but do not require this pathway for efficient methylation [38,47,57]. Methylation of other transposon sequences is dependent on RdDM, which means that the CMT3/CMT2 and MET1 pathways cannot maintain methylation without RdDM activity [47,57]. The first group comprises longer transposons enriched for H3K9me2 and depleted of histone modifications associated with active genes [47]. Methylation of these elements is dependent on the nucleosome remodeling protein DECREASE IN DNA METHYLATION 1 (DDM1), which functions, to a large extent, to overcome the effects of linker histone H1 [47], a widely distributed chromatincompacting protein [58,59]. The second group comprises mostly short isolated transposons or sequences at the
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edges of longer ones that have relatively low levels of H3K9me2 and high levels of gene-associated histone modifications [47]. DDM1 has little effect on the methylation of these transposons [47]. Much of this variation is likely to be because of the intrinsic limitations on the stability of nucleosome-based silencing following DNA replication [60]. Nucleosomes are octamers of histones H2A, H2B, H3, and H4 wrapped by 146 base pairs (bp) of DNA [61]. When DNA is replicated, the nucleosomes are apportioned to the daughter molecules and the gaps filled with newly synthesized histones. Given that some short transposons or transposon remnants are barely long enough to complete a single nucleosome, features such as H3K9me2 cannot be stably inherited through cell division, and the KYP–CMT3/ CMT2 pathway cannot maintain DNA methylation. By contrast, longer transposons retain enough nucleosomes following DNA replication to inherit H3K9me2 and likely other important histone modifications [60]. Similarly, H1, which binds to the nucleosome and the linker DNA that separates nucleosomes, is likely to exert a stronger influence on arrays of many nucleosomes rather than on sequences that comprise just one or two. RdDM, which functions through base complementarity, does not have an inherent target size limitation beyond that required to base pair with the guiding sRNA molecules. Thus, this pathway is ideally suited to maintain methylation of smaller sequences. However, why does it target many larger transposons less effectively? This is unknown, but the requirement for RNA polymerase activity, that of either Pol II or Pol IV and Pol V, suggests a plausible explanation. Pol IV and Pol V may have evolved to transcribe methylated DNA that precludes Pol II activity, but as transposon chromatin becomes more compacted, even these polymerases may be inhibited. Depending on its size, chromatin environment, and likely intrinsic sequence features, a transposon that has progressed from an active, Pol II-transcribed state (Figure 1A) to an inactive Pol IVand Pol V-transcribed state (Figure 1B) may proceed to a state in which it is no longer transcribed at all, and its methylation is entirely dependent on the CMT3/CMT2 and MET1 pathways (Figure 1C). Protection of genes from methylation-induced silencing The DNA methylation pathways described above give plants powerful tools for silencing transposons. As with any system of immunity, it is crucial that these pathways are not misdirected against endogenous genes [8]. The initial, and still poorly understood, assessment of RNA structure must make an important contribution. However, even if the RNA recognition mechanisms function perfectly, some genes present special challenges. One of these is that many active flowering plant genes exhibit DNA methylation of the transcribed region (gene body) that does not interfere with gene expression [62,63]. Several functions, including attenuation of aberrant transcription and regulation of splicing, have been proposed for gene body methylation, which is preferentially found in moderately and constitutively expressed genes with important housekeeping activities, and is conserved across substantial evolutionary distance [64,65]. Whatever its function, gene 3
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Gene Heterochromac intron
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Figure 2. Maintenance of gene activity by IBM1 and IBM2. IBM1 removes H3K9me2 from gene bodies that exhibit CG methylation maintained by MET1, thereby precluding the accumulation of nonCG methylation mediated by CMT2 and CMT3. IBM2 allows transcription through heterochromatic introns by an unknown mechanism. Small red spheres represent DNA methylation and small blue spheres represent H3K9me2. Abbreviations: AGO, ARGONAUTE; CG, cytosine–guanine; CMT, CHROMOMETHYLASE; H3K9me2, Histone H3 dimethyl Lys9; IBM, INCREASE IN BONSAI METHYLATION; MET1, METHYLTRANSFERASE 1; Pol II, RNA polymerase II; VIM, VARIANT IN METHYLATION.
body methylation is normally confined almost exclusively to the CG context and mediated by MET1 [38,44] (Figure 2). What, then, prevents the self-reinforcing RdDM and KYP– CMT3/CMT2 loops from operating in these genes? One part of the answer is that the SRA domains of the KYP H3K9 methyltransferases, which bind to methylated DNA, have weak affinity for CG methylation that contrasts with their strong binding to DNA methylated in the CHG and CHH contexts [66]. Thus, the KYP methyltransferases would be expected to recognize efficiently the methylation products of DRM, CMT2, and CMT3, but not MET1. Nonetheless, the KYP enzymes can bind to CG-methylated DNA, and methylated gene bodies exhibit low levels of CHG methylation [47]. CHG methylation is kept low by INCREASE IN BONSAI METHYLATION 1 (IBM1), a member of a large histone demethylase family that removes H3K9 methylation (Figure 2) [67–69]. In the absence of IBM1, genes with methylated bodies exhibit extensive and nearly universal CHG and CHH hypermethylation [47,68,70,71]. This hypermethylation is insensitive to mutations in the RdDM pathway [47], indicating that only the KYP–CMT3/CMT2 system is responsible, potentially because H3K4 methylation, which is found in genes, interferes with SHH1 binding [35]. Nevertheless, the removal of H3K9me2 is also likely to disfavor RdDM over the long term. IBM1 appears to be specifically recruited to actively transcribed genes by an unknown mechanism [68,70]. Given that body-methylated genes are generally constitutively transcribed, this would ensure that they are kept free of nonCG methylation. This arrangement suggests a model where methylated genes exist in one of two stable states: body methylated at CG sites and transcriptionally active, and methylated in all sequence contexts and inactive. If sequences in the first state produce sufficient aberrant RNA to trigger strong RdDM, the resulting nonCG methylation and increased H3K9me2 would overwhelm IBM1 and transition the sequence into a stable silent state that no longer attracts IBM1 activity. 4
IBM1 may protect gene bodies from methylation by RdDM and CMT3/CMT2, but what about those genes that have undergone a transposon insertion within an intron? These transposons are methylated by the full weight of the silencing machinery, just like elements elsewhere [38,44]. Two recent papers have provided a tantalizing clue for how genes deal with such transposons [72,73]. Both studies identified the same gene, IBM2/ANTI-SILENCING 1 (ASI1), inactivation of which causes transcripts of genes with methylated intronic transposons to terminate within the elements. IBM2 is preferentially associated with intronic transposons [72,73], indicating that these have distinctive chromatin features, perhaps as a result of readthrough transcription. IBM2 contains an RNA-binding domain and may facilitate proper mRNA processing, but exactly how this protein aids transcription through intronic transposons remains unknown (Figure 2). Attenuation of gene-adjacent DNA methylation Even if genes are correctly distinguished from transposons, the methylation and silencing of transposons that are inserted near genes can have deleterious effects on gene expression [74], particularly when the transposons are targeted by RdDM [75] (Figure 3A). Therefore, regulation of gene-adjacent transposon methylation would be expected to balance the tradeoff between transposon inactivation and gene repression. In particular, a pathway that attenuates transposon methylation, acting in opposition to RdDM and other methylation-promoting mechanisms, would be needed to achieve the optimum balance (Figure 3). The best-understood pathway in plants centers around the DEMETER/REPRESSOR OF SILENCING 1 (DME/ROS1) family of DNA glycosylases that function as DNA demethylases by excising methylated cytosine from DNA [16]. The activity of the demethylases is facilitated by the protein INCREASED DNA METHYLATION 1 (IDM1), which mediates histone acetylation [76,77] (Figure 3B), a feature of active chromatin, and consistently these
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Euchromac transposon
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TSS Non-CG
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Figure 3. The balance between RdDM and DNA demethylation regulates gene activity. Expression of a gene is negatively affected by RdDM-mediated DNA methylation of a nearby transposon (A) and positively affected by DME/ROS1mediated DNA demethylation, which is facilitated by IDM1-catalyzed histone acetylation (B). Small red spheres represent DNA methylation and small green spheres represent histone acetylation. Abbreviations: DME/ROS1, DEMETER/ REPRESSOR OF SILENCING 1; DRM, DOMAINS REARRANGED METHYLTRANSFERASE; IDM1, INCREASED DNA METHYLATION 1; RdDM, sRNA-directed DNA methylation; TSS, transcription start site.
enzymes preferentially demethylate small, gene-adjacent transposable elements low in H3K9me2 and high in H3K4me2 and other gene-associated histone modifications [38,78,79]. Long H3K9me2-rich transposons are rarely demethylated, except at their edges [79]. Thus, the demethylases act on the same kinds of sequences targeted by RdDM, and the ones likely to influence gene expression. There is intriguing evidence that the balance between RdDM and active demethylation is fine-tuned through the regulation of gene expression, at least in Arabidopsis. ROS1, the main somatic DNA demethylase, is strongly downregulated in RdDM pathway mutants and plants lacking MET1 [77,80]. By contrast, ROS1 expression is increased in the idm1 mutant, which impairs ROS1-catalyzed demethylation [76]. Given that RdDM and active demethylation generally converge on the same sequences, these observations suggest that ROS1 expression is calibrated through adjustment of DNA methylation at a regulatory sequence: increased RdDM leads to higher levels of ROS1, which in turn reduces methylation, thereby attenuating its own expression. Gene regulation by DNA methylation The ability of the balance between RdDM and DNA demethylation to influence gene expression (Figure 3) naturally lends itself to gene-regulatory purposes. The most dramatic known example of this occurs during sexual development. Unlike in animals, the products of meiosis in flowering plants undergo further mitotic divisions and differentiation, which produce the gametes (egg and
sperm) and their companion cells. DNA demethylation is hyperactivated in the male companion cell (the vegetative cell) and in one of the female companion cells (the central cell) [79,81]; RdDM may also be attenuated, at least on the female side [82]. The result is strong, often complete, demethylation that affects thousands of transposons and either activates or represses multiple genes [79,81,83–85]. The central cell, after fusion with one of the two sperm cells in the pollen (the other fuses with egg), gives rise to the endosperm, a placenta-like nutritive tissue. Given that the sperm does not undergo unusual demethylation, the genes affected by central cell demethylation are expressed differently from the maternally and paternally inherited chromosomes [86]. This process, known as imprinted expression (the methylation is the imprint), is absolutely essential, because lack of a functional DME protein (responsible for companion cell demethylation) in the central cell causes seed abortion [87]. Thus, the pathways that have evolved to limit the impact of transposons on the genome have become co-opted to regulate gene expression and plant development. Concluding remarks Many gaps remain in our knowledge of plant silencing and antisilencing pathways that immunize the genome against transposons. For example, we do not fully understand which RNA features are recognized as aberrant and how this recognition occurs, neither do we know how recognition of a nascent transcript by AGO proteins causes DNA methylation. We also do not know which features of Pol IV and Pol V enable transcription of methylated DNA and, perhaps surprisingly, we have at best a partial understanding of how DNA methylation interferes with Pol II activity. It is unclear how IBM1 is recruited to active genes and how IBM2/ASI1 facilitates transcription through silenced transposons. Although the propensity of DME and related enzymes to demethylate relatively euchromatic sequences appears to be general, we do not know how these enzymes interrogate chromatin or the specific chromatin features that are sought. There are also components of transposon silencing, such as monomethylation of lysine 27 of histone H3 [88,89] and proteins that function in parallel to or downstream of DNA methylation [90–92], the interaction of which with the DNA methylation pathways discussed here is not understood. If the recent past is a reliable guide, further investigation of these and other open questions should reveal more points of interaction between plant silencing mechanisms, deepening understanding of the complex and fascinating system of pathways that mediate transposon silencing and regulate gene expression. References 1 Wicker, T. et al. (2007) A unified classification system for eukaryotic transposable elements. Nat. Rev. Genet. 8, 973–982 2 Hickey, D.A. (1982) Selfish DNA: a sexually-transmitted nuclear parasite. Genetics 101, 519–531 3 Orgel, L.E. and Crick, F.H. (1980) Selfish DNA: the ultimate parasite. Nature 284, 604–607 4 Bestor, T.H. (1999) Sex brings transposons and genomes into conflict. Genetica 107, 289–295 5 Zemach, A. and Zilberman, D. (2010) Evolution of eukaryotic DNA methylation and the pursuit of safer sex. Curr. Biol. 20, R780–R785 5
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