A Green Link to RNA Silencing

W Aufsatz, T Stoiber, B Rakic and K Naumann

Gregor Mendel Institute of Molecular Plant Biology, Austrian Academy of Sciences, Vienna, Austria

Epigenetic reprogramming is at the base of cancer together result in a transcriptionally repressive chroma- initiation and progression. Generally, genome-wide tin state and silencing of gene expression. Usually, both reduction in cytosine methylation contrasts with the alleles of a tumor suppressor gene have to be defective hypermethylation of control regions of functionally well- or inactivated for the full expression of a transformed established tumor suppressor genes and many other genes phenotype. Thus, epigenetic silencing of one tumor whose role in cancer biology is not yet clear. While insight suppressor allele does not result in disease, but predis- into mechanisms that induce aberrant cytosine methyla- poses cells to undergo malignant growth that occurs if tion in cancer cells is just beginning to emerge, the the second allele is disabled by mutation, chromosome initiating signals for analogous promoter methylation in rearrangements resulting in loss of heterozygosity or, plants are well documented. In Arabidopsis, the silencing again, by transcriptional gene silencing via epigenetic of promoters requires components of the RNA inter- mechanisms. The list of cancer-related genes that are ference machinery and promoter double-stranded RNA affected by aberrant de novo methylation in cancer (dsRNA) to induce a repressive chromatin state that is patients is growing steadily and may already outnumber characterized by cytosine methylation and histone deace- reported mutation events at those genes (Jones and tylation catalysed by the RPD3-type histone deacetylase Baylin, 2002). However, even some of the reported AtHDA6. Similar mechanisms have been shown to occur mutations might have been triggered by initial cytosine in fission yeast and mammals. This review focuses on the methylation. 5-methylcytosine is sensitive to hydrolytic connections between cytosine methylation, dsRNA and deamination that results in a cytosine to thymine AtHDA6-controlled histone deacetylation during promo- transition (Coulondre et al., 1978). Indeed, many ter silencing in Arabidopsis and discusses potential mutations in p53 found in cancer patients are positioned mechanistic similarities of these silencing events in cancer at sites of initial methylation (Rideout et al., 1990). and plant cells. Since epigenetic changes are often present already before Oncogene (2007) 26, 5477–5488; doi:10.1038/sj.onc.1210615 the onset of tumor progression in predisposed cells (Belinsky et al., 1998; Holst et al., 2003), their Keywords: RNA silencing; cytosine methylation; histone identification provides an early diagnostic mark for deacetylation; epigenetics; Arabidopsis; transcriptional cancer risk. In addition, resetting epigenetic states of gene silencing silent tumor suppressor genes by nucleoside analog drugs that interfere with the maintenance of cytosine methylation and by histone deacetylase inhibitors that reverse repressive histone marks represents a rather new line of therapy for cancer patients (Cress and Seto, 2000; Introduction Jones and Baylin, 2002). Cancer cells undergo drastic alterations in their It has become increasingly clear over the past few years overall cytosine methylation pattern: genome-wide that both the initiation as well as the progression of hypomethylation is contrasted with local hypermethyla- many types of cancer have a strong epigenetic compo- tion in the regulatory regions of many genes whose role nent. By yet poorly understood mechanisms, tumor in cancer biology is not yet understood (Jones and suppressor genes can acquire de novo cytosine methyla- Laird, 1999; Jones and Baylin, 2002; Herman and tion within CpG-rich regions that are typically located Baylin, 2003). Recent evidence suggests that de novo around their transcriptional start sites (Jones and Laird, methylation of at least some CpG islands in cancer cells 1999; Baylin and Herman, 2000; Jones and Baylin, is determined by an instructive mechanism that also 2002). This local epigenetic transition from DNA hypo- operates in healthy cells (Keshet et al., 2006; Schlesinger to hypermethylation is frequently accompanied by et al., 2006; Widschwendter et al., 2007). These regu- changes in histone modifications, such as histone latory regions are targeted by Polycomb complexes deacetylation and repressive histone methylation, which during development and, thus, exhibit trimethylation of lysine 27 of histone H3 (H3K27me3), a chromatin mark typical of Polycomb-mediated suppression. Importantly, Correspondence: Dr W Aufsatz, Gregor Mendel Institute of Molecular Plant Biology, Austrian Academy of Sciences, Dr Bohr-Gasse 3, although transcriptionally repressed, these genes are not A-1030 Vienna, Austria. associated with cytosine methylation within their CpG E-mail: [email protected] islands in normal tissue, but are efficiently targeted by AtHDA6 controls dsRNA-induced repressive chromatin W Aufsatz et al 5478 the DNA methyltransferases (DMTs) DNMT1, Aufsatz et al., 2002a; Melquist and Bender, 2003; DNMT3A and DNMT3B during neoplastic transfor- Mathieu and Bender, 2004; Cigan et al., 2005). In mation, which results in de novo methylation (Schle- contrast to the promoters of cancer genes, however, the singer et al., 2006). The Polycomb group protein EZH2 repressive signal for this silencing of plant promoters is is a histone methyltransferase capable of methylating well established. De novo methylation is triggered by H3K27 and has recently been shown to interact with long double-stranded RNAs (dsRNAs) with sequence DNA methyltransferases both in vitro and in vivo homology to the promoters, which are processed to (Muller et al., 2002; Vire´ et al., 2006). Aberrant 21–24nucleotide long small RNAs by a Dicer-like recruitment of cytosine methylation to Polycomb target activity. This RNA silencing pathway is termed RNA- genes specifically in cancer cells might be facilitated by directed DNA methylation (RdDM). Promoters elevated levels of Polycomb proteins or DNA methyl- silenced by RdDM are characterized by histone transferases, respectively, which is a common feature of deacetylation, which in Arabidopsis is controlled by the many tumors (De Marzo et al., 1999; Robertson et al., RPD3-type histone deacetylase AtHDA6 (Aufsatz et al., 1999; Varambally et al., 2002; Bracken et al., 2003). 2002b). The aim of this review is to highlight connec- Since most of the pre-marked genes are already tions between cytosine methylation, dsRNA-triggered transcriptionally repressed by Polycomb complexes in silencing and AtHDA6 during RdDM in Arabidopsis, healthy tissue, the significance of their de novo cytosine and to potentially relate promoter silencing in plants methylation for cancer biology remains disputable with that occurring in cancer cells. (Schlesinger et al., 2006). De novo DNA methylation of regulatory regions in cancer cells is usually accompanied by deacetylation of histones H3 and H4, and the acquisition of H3K9me2, Arabidopsis HDACs H3K9me3 as well as H3K27me3, marks typically associated with transcriptional repression (Nguyen AtHDA6 is a member of a large gene family consisting et al., 2001; Fahrner et al., 2002; Kondo et al., 2003; of 16 histone deacetylases in the Arabidopsis genome McGarvey et al., 2006) Several layers of crosstalk (Pandey et al., 2002). Among these, ten proteins belong between cytosine methylation and repressive histone to the RPD3/HDA1 superfamily: AtHDA6, AtHDA7, modifications are evident on the molecular level. First, AtHDA9 and AtHDA19 are representatives of class I, methylcytosine is targeted by methyl CpG-binding RPD3-type HDACs. Three of the class I proteins, along proteins (MBDs), which in turn can recruit transcrip- with HDACs from soybean, rice and maize, further tional corepressors and/or chromatin remodeling group into two branches which form clusters A proteins, histone deacetylases (HDACs) and histone (AtHDA19) and B (AtHDA6 and AtHDA7), suggesting methyltransferases (HMTs) (Bird and Wolffe, 1999; functional specialization among these RPD3-type pro- Wade et al., 1999; De Ruijter et al., 2003). MBDs are teins (Pandey et al., 2002). Specialization is supported conserved across kingdoms and there is evidence that a by the phenotypes of mutant plants defective in subset of them is involved in interpreting DNA AtHDA19 and AtHDA6, respectively. Antisense knock- methylation also in plants (Zemach and Grafi, 2007). down plants or stable insertion mutants of AtHda19 Second, DMTs can interact directly with either HDACs exhibit pleiotropic developmental phenotypes, such as or HMTs (Fuks et al., 2001; Datta et al., 2003; Fuks early senescence, reduced apical dominance, homeotic et al., 2003; Li et al., 2006a). Thus, cytosine methylation changes, heterochronic developmental shifts and flower can nucleate repressive histone modifications as a defects coupled with male and female sterility, thus primary signal, but can also be their consequence. highlighting an essential function of AtHDA19 in plant DNA methylation provides repressive stability by development (Tian and Chen, 2001; Tian et al., 2003). ‘locking in’ the silent state, since inhibition of HDAC Furthermore, AtHDA19 antagonizes the histone acet- activity alone is insufficient to induce re-expression of yltransferase AtGCN5 in regulating the maintenance many silenced cancer genes, but synergistically enhances of embryo axis formation and light-responsive gene effects of low doses of demethylation drugs (Cameron expression, respectively (Benhamed et al., 2006; Long et al., 1999; Suzuki et al., 2002). In addition, DNA et al., 2006). Besides development, AtHDA19 is required methylation provides a heritable memory of gene for jasmonic acid- and ethylene-mediated plant re- repression, because it is copied during DNA replication sponses to plant pathogens, for responses of cultured by the maintenance enzyme DNMT1 and might serve as cells to sucrose starvation and for transcriptional an initial platform for the re-establishment of the repression in response to abscisic acid, drought and salt parental repressive histone code (Bestor, 2000; Rountree stress (Song et al., 2005; Zhou et al., 2005; Nicolai et al., et al., 2000). 2006; Song and Galbraith, 2006). The latter response is While the reinforcing interplay between cytosine likely executed by an AtHDA19/SIN3 repressor com- methylation and histone modifications is evident, little plex that is recruited to target genes via ethylene- is known about the initiating signals triggering this responsive element binding factors (ERFs) (Song et al., process. A look across kingdom borders might help to 2005; Song and Galbraith, 2006). In contrast to develop concepts addressing this important aspect. Plant AtHDA19, the role of AtHDA6 in development appears promoters can also be targeted for de novo methylation, to be minor, since Athda6 mutants develop normally which results in their inactivation (Wassenegger, 2000; under standard growth conditions with the exception of

Oncogene AtHDA6 controls dsRNA-induced repressive chromatin W Aufsatz et al 5479 slightly late flowering (Probst et al., 2004). While which are called sirtuins. The founding member of this knockdown of AtHDA19 causes striking global histone family, yeast Sir2p, controls gene silencing at telomeric, hyperacetylation (Tian and Chen, 2001), Athda6 mu- rDNA and silent-mating type loci and has further roles tants show only subtle effects, indicating that AtHDA60s in cell cycle progression, DNA-damage repair and aging silencing function is specialized for very specific (Gartenberg, 2000; Guarente, 2000). In Arabidopsis, the target genes, at least under normal growth conditions sirtuin family is represented by two proteins, SRT1 and (Probst et al., 2004). In agreement with this hypothesis, 2 (Pandey et al., 2002). Treatment of Arabidopsis plants endogenous regulatory functions of AtHDA6 have so with sirtinol, a small molecule inhibitor of sirtuins, far only been linked to the regulation of chromatin at inhibits body-axis formation and blocks the develop- recombinant DNA (rDNA) loci, where Athda6 alleles ment of the vascular system (Grozinger et al., 2001). have been shown to cause decreased CpG methylation However, these phenotypes are partially caused by as well as localized enhanced histone acetylation sirtinol targets different from sirtuins (Zhao et al., (Probst et al., 2004). Furthermore, AtHDA6 has been 2003) and it remains to be determined which role the characterized as a broad range HDAC that is necessary Arabidopsis SRT proteins have in these developmental for nucleolar dominance, a long-range repression of processes. rDNA specifically from one parent in allopolyploid hybrids (Earley et al., 2006). AtHDA5, AtHDA15 and AtHDA18 are members of the class II, HDA1-related group of HDACs. In AtHDA6: an intimate relationship with transcriptional animals, a subgroup of class II proteins possesses RNA silencing two tandem HDAC domains, whereas in Arabidopsis double-domain proteins are absent (Pandey et al., 2002). Among all the Arabidopsis HDACs, AtHDA6 is the Except for AtHDA18, nothing is known about the only one for which a genetic and molecular connection function of class II HDACs in Arabidopsis. AtHDA18 to a distinct silencing pathway has been made. Its has been recently reported to control the position- identification as an essential component in RdDM dependent expression of genes involved in defining the (Aufsatz et al., 2002b) links it to epigenetic regulation pattern of the root epidermis (Xu et al., 2005). through small RNAs that trigger the covalent modifica- AtHDA2 represents a member of class III HDACs, tion of histones and/or DNA in a sequence-specific which is a new group of proteins within the RPD3/ manner (Matzke and Birchler, 2005). These modifica- HDA1 family that involves enzymes from Drosophila tions result in a transcriptionally repressed state that can (HDA403), Caenorhabditis (HDA308) and humans be stably inherited, but is still potentially reversible. (HDAC11), but is absent from fungal genomes (Pandey In Arabidopsis, two pathways exist that utilize small et al., 2002). Class III proteins are part of a cluster that RNAs to modify chromatin. RNA interference (RNAi)- also comprises bacterial acetoin utilization proteins, mediated heterochromatin formation refers to the which provides support for a bacterial origin of class III targeting of repressive histone methylation to centro- enzymes. AtHDA8 and 14, the residual proteins of the mere sequences and other repeats by short inter- Arabidopsis RPD3/HDA1 family, fall into the same fering RNAs (siRNAs) and components of the RNAi major clade as class II proteins, but do not cluster with machinery, such as Dicer, Argonaute (AGO) and RNA- them. At present, the function of AtHDA2, AtHDA8 dependent RNA polymerases. This process was origin- and AtHDA14proteins is obscure. ally detected and extensively analysed in fission yeast HD2-type HDACs have only been detected in plants (Martienssen et al., 2005; Verdel and Moazed, 2005), and are distantly related to peptidyl-prolyl cis-trans and is conserved in plants, insects and vertebrates isomerases from insects, yeast and parasitic apicomplex- (Lippman and Martienssen, 2004; Kanellopoulou ans (Lusser et al., 1997; Aravind et al., 1998). The et al., 2005; Matzke and Birchler, 2005). Importantly, Arabidopsis genome encodes four members of this heterochromatin can spread from nucleation sites family, AtHDT1-4(Pandey et al., 2002). AtHDT1 is initially targeted by the RNAi machinery. Small RNAs required for reproductive development (Wu et al., 2000; can also induce de novo cytosine methylation at Zhou et al., 2004) and, together with AtHDT2, for the sequence-homologous DNA in a process called RdDM establishment of leaf polarity, possibly by controlling (Wassenegger, 2000; Aufsatz et al., 2002a; Chan et al., the levels or distribution of two micro RNAs (miRNAs) 2004; Mathieu and Bender, 2004). Cytosines in all involved in abaxial/adaxial axis formation (Ueno et al., sequence contexts are modified by RdDM, and methy- 2007). AtHDT1 further controls rRNA gene dosage in lation is restricted to the site of RNA/DNA identity, Arabidopsis and modulates rRNA transcription in suggesting the formation of an RNA/DNA hybrid genetic hybrids of different Arabidopsis species, which structure that is in turn recognized by the DNA is consistent with its nucleolar localization (Lawrence methylation machinery. RdDM results in transcrip- et al., 2004). AtHDT3 has recently been identified as a tional repression when promoters are targeted (Aufsatz regulator of abiotic stress responses that are controlled et al., 2002a). Some of the proteins involved in RdDM by the plant stress hormone abscisic acid (Sridha and seem to be plant-specific (see below). However, similar Wu, 2006). mechanisms apparently exist in mammalian cells, The last family of HDAC proteins is comprised of suggesting the presence of functionally related compo- nucleotide adenine dinucleotide-dependent enzymes, nents also in mammals. Short dsRNAs or siRNAs

Oncogene AtHDA6 controls dsRNA-induced repressive chromatin W Aufsatz et al 5480 directed against promoters in mammalian cells cause plants completely lacking AtHDA6 (rts1-1 mutants) transcriptional repression, but this is not always exhibit transcriptional reactivation of the RdDM target associated with DNA methylation (Morris et al., 2004; promoter despite the continuous presence of the Castanotto et al., 2005; Ting et al., 2005; Weinberg silencing-inducing RNA signal. Silencing, however, is et al., 2006). Although RNAi-mediated heterochromatin readily re-established by backcrossing rts1-1 mutant formation and RdDM have similar genetic requirements plants to wild-type plants or by genetic complementa- in Arabidopsis, suggesting a considerable overlap be- tion with wild-type AtHDA6, which suggests that tween these pathways, they can result in qualitatively histone deacetylation acts as a fast switch in repressing distinct repressive chromatin at their targets (see below). transcriptional activity in response to silencing RNA Endogenous siRNAs capable of epigenetically modi- signals (Aufsatz et al., 2002b). Despite the remarkable fying the genome are typically 24nt long in Arabidopsis. saturation of the forward genetic screens, which resulted Their biogenesis and function is controlled by a in the recovery of 18 Athda6 alleles, AtHDA6 was the specialized set of RNAi proteins, which are DICER- only histone-modifying enzyme identified, indicating LIKE 3 (DCL3), the RNA-dependent RNA polymerase that histone hypoacetylation might be a main character- RDR2 and the Argonaute protein AGO4(Zilberman izing feature of the chromatin of RdDM-silenced et al., 2003; Xie et al., 2004). In addition, siRNA promoters. Indeed, exposure of a target promoter to generation and function is dependent on RNA poly- the RNA signal results in deacetylation of histone H3 merase IV, which is unique to plants (Herr et al., 2005; lysines 9 and 14, while histone marks defining constitu- Kanno et al., 2005; Onodera et al., 2005; Pontier et al., tive heterochromatin in plants, such as H3K9me2, 2005) and a plant-specific SNF2-like protein, DRD1 remain absent (Figure 1a; compare uppermost panel to (Kanno et al., 2004). Pol IV exists as two distinct middle panel). In rts1-1 mutant plants, the target complexes in Arabidopsis, which are functionally diverse promoter is derepressed despite the continuous presence and specified by their unique largest subunits, respec- of the RNA signal and the transcriptional activity is tively. PolIVa is required for biogenesis of siRNAs, associated with the re-acetylation of H3K9 and K14, whereas PolIVb and DRD1 act downstream of siRNAs which is in agreement with the prominent role of to mediate de novo DNA methylation by a yet unknown AtHDA6 in controlling histone deacetylation during mechanism (Kanno et al., 2005; Pontier et al., 2005). RdDM (Figure 1a, lower panel). Thus, in summary, The nuclear-acting siRNAs are processed from their neither DNA methylation nor the presence of sequence- long dsRNA precursors in the nucleolus, where they homologous siRNAs is sufficient to convert promoters colocalize with the proteins mentioned above except for targeted by RdDM to heterochromatin, regardless of PolIVa subunits and DRD1. Currently, it is hypothe- whether they represent endogenous targets or reside sized that the latter proteins associate with loci that on integrated transgenes (Huettel et al., 2006). This either generate precursor RNAs or that are targets for clearly discriminates promoter targets of RdDM the mature siRNAs, respectively (Pontes et al., 2006; from targets of RNAi-mediated heterochromatin forma- Li et al., 2006b). tion, which in Arabidopsis are typically characterized Genetic forward screens based on two-component by their association with siRNAs, cytosine hyper- transgene RdDM model systems in Arabidopsis, with a methylation and H3K9me2 (Lippman and Martienssen, promoter/reporter gene as silencing target and a source 2004). of siRNAs homologous to the promoter, identified AtHDA6 also has a role in RNAi-mediated hetero- AtHDA6 as an important factor for the maintenance of chromatin formation. In Arabidopsis, this pathway is the silenced state (Aufsatz et al., 2002a, b). Mutant involved in controlling heterochromatin mainly at

Figure 1 Chromatin status of targets for RNA-directed DNA methylation (a) or RNA interference-mediated heterochromatin formation (b) in wild-type (WT) or Athda6 (rts1-1) mutant plants analysed by chromatin immunoprecipitation (ChIP). ChIP was performed with antibodies speciﬁc for H3K4me3, H3K9me2, H3K27me1 and histone H3 diacetylated at lysines 9 and 14 or without antibody (–). IN, input; T, active target promoter in the absence of the RNA source; T þ S, silent (WT) or reactivated (rts1-1) target promoter in the presence of the RNA source. At4g04040 represents a control for a transcriptionally active, euchromatic gene, which is associated with the active histone mark H3K4me3.

Oncogene AtHDA6 controls dsRNA-induced repressive chromatin W Aufsatz et al 5481 centromeric repeats, other repetitive elements and depending on the experimental system and the transposons and also at intergenic regions. However, repressed loci, sometimes even within a given organism. there is redundancy with other silencing pathways that Pharmacological experiments with the HDAC inhibitor involve methyltransferase 1 (MET1), the Arabidopsis Trichostatin A (TSA) and the inhibitor of DNA ortholog of the mammalian CpG-specific maintenance methylation, 5-aza(deoxy)cytidine (5AZA) revealed, DMT DNMT1, and the chromatin remodeling ATPase for example, that histone deacetylation and DNA DDM1 (Lippman and Martienssen, 2004). Due to this methylation act synergistically to repress genes in redundancy, only a few transposons are reactivated in Neurospora and in mammalian tumor cells (Selker, RNAi mutants (Lippman and Martienssen, 2004). One 1998; Momparler, 2003). Similar findings have been of those, ATLANTYS 2-1, is a gypsy-class retroelement, reported for silent plant rDNA genes in Arabidopsis or which is associated with many siRNA hits in databases Brassica hybrids, where application of either inhibitor that have been obtained from large-scale cloning and results in erasure of both repressive marks and gene sequencing of endogenous small RNAs in Arabidopsis reactivation (Chen and Pikaard, 1997; Lawrence et al., (Lu et al., 2005; http://asrp.cgrb.oregonstate.edu/db/). 2004). These reports are consistent with a model in In wild-type plants, ATLANTYS 2-1 chromatin is which cytosine methylation and histone deacetylation characterized by histone marks that are typical for plant crosstalk and are part of a self-reinforcing repressive heterochromatin, such as H3K9me2 and H3K27me1 cycle. Crosstalk between these modifications is well (Figure 1b, top, upper panel). In plants devoid of supported on the level of protein interactions, which AtHDA6, H3K9me2 is drastically reduced, indicating indicate either direct interactions between HDACs and that functional AtHDA6 is required to control siRNA- DMTs (Fuks et al., 2001; Datta et al., 2003) or the dependent heterochromatin and that deacetylation is a recruitment of HDACs to methylated DNA via MBPs prerequisite for subsequent methylation by HMTs (De Ruijter et al., 2003). On the other hand, recent (Figure 1b, top, lower panel). In fact, silencing of transcript profiling of inhibitor-treated Arabidopsis many heterochromatic transposons depends on AtH- plants revealed distinct sets of up- and downregulated DA6 (Lippman et al., 2003; W Aufsatz, unpublished genes in response to TSA, 5AZA or both, and in some results). cases even opposite effects of the inhibitor treatments A role for RNA in the silencing of other identified were observed (Chang and Pikaard, 2005). Thus, DNA AtHDA6 targets is highly suggestive. The first alleles of methylation and histone deacetylation are often AtHDA6 were identified in screens resulting in the intimately linked in gene silencing, but can as well act activation of complex transgenes (Furner et al., 1998; autonomously or even antagonistically. Murfett et al., 2001). These transgenes were character- Cytosine methylation and histone deacetylation also ized by either having intrinsic inverted promoter repeats cooperate during silencing of promoters by RdDM. In or by harboring scrambled, reiterated copies of identical response to dsRNA, cytosines in all sequence contexts sequences involving promoters, which renders them a are de novo methylated by the domain-rearranged class potential source for the generation of promoter dsRNA of de novo DMTs, DRM1 and DRM2 (Cao and and subsequent production of promoter siRNAs. Jacobsen, 2002; Cao et al., 2003). In contrast to Finally, siRNAs derived from rDNA repeats have asymmetric cytosine methylation in a CpHpH (H is A, genetic properties consistent with functional siRNAs T or C) context, which depends on the continuous (Xie et al., 2004; Lu et al., 2005; http://asrp.cgrb. presence of the inducing signal and DRM function, oregonstate.edu/db/). The roles of these siRNAs in methylation of symmetric cytosines in CpG or CpHpG AtHDA6-controlled regional or long-range silencing of contexts can be maintained by the plant maintenance rDNA repeats have not been addressed so far (Probst methylation machinery even when the inducing signal is et al., 2004; Lawrence et al., 2004; Earley et al., 2006). In no longer present (Aufsatz et al., 2002a). In Arabidopsis, summary, compared to other Arabidopsis RPD3-type the DNMT1 ortholog MET1 maintains CpG methyla- HDACs, AtHDA6 shows preponderance for the tion, whereas the plant-specific chromomethylase CMT3 involvement in silencing processes that are likely maintains CpHpG methylation (Chan et al., 2005). controlled by dsRNA, which is in agreement with the MET1 has an additional role in CpG de novo methyla- proposed functional diversification within this family tion, but so far this function is restricted to methylation (Pandey et al., 2002). induced by RNA signals (Aufsatz et al., 2004). In addition to cytosine methylation, the chromatin of RdDM-silenced promoters is characterized by histone deacetylation, which is controlled by AtHDA6 (see AtHDA6: a bridge between histone acetylation and above). DNA methylation Complete loss-of-function mutants for AtHDA6 exhibit reactivation of RdDM-silenced promoters, In many organisms, silent genes are DNA methylated despite the continuous presence of the RNA-silencing and deacetylated at histones H3 and H4(Dobosy and signal. Moreover, cytosine methylation in symmetric Selker, 2001). The relative importance of each modifica- sequence contexts, predominantly CpG methylation, tion for inducing and maintaining the repressed state is reduced, highlighting a function for AtHDA6 in and the mutual dependence of both modifications, maintenance methylation (Aufsatz et al., 2002b). however, are not yet clear, since they seem to vary Although not proven yet, this function of AtHDA6 in

Oncogene AtHDA6 controls dsRNA-induced repressive chromatin W Aufsatz et al 5482 cytosine methylation might be mediated by the physical association with the maintenance DMTs, MET1 and CMT3. Two additional alleles of AtHDA6, rts1-3 and rts1-4, which have changes of amino acids that are invariant in RPD3-type enzymes from yeast to humans, also cause a reduction in symmetric cytosine methylation at RdDM-silenced promoters similar to the null allele (Naumann et al., manuscript in preparation). The rts1-4 mutation changes a residue involved in binding of the cofactor zinc and might, therefore, lack catalytic activity (Finnin et al., 1999), suggesting that the functional integrity of histone deacetylase activity is important for the crosstalk with the DNA methylation machinery. The rts1-3 mutation resides within a SANT- like domain within the C-terminal part of the HDAC domain. SANT domains can mediate DNA binding as well as protein–protein interactions (Aasland et al., 1996). Interestingly, SANT-like domains also occur in DNMT1 and the methyl CpG-binding proteins, MBD2 and MECP2 (Battaglioli et al., 2002). Thus, it is tempting to speculate that mutations within this domain in AtHDA6 block communication with the methylation and/or methylation-readout machinery, which would be in accordance with the rts1-3 phenotype of reduced DNA methylation. However, the rts1-5 allele of AtHDA6 results in a different methylation phenotype. The corresponding mutation is located at a conserved Figure 2 Potential mechanisms of AtHDA6 recruitment during position within the HDAC domain of AtHDA6, just promoter silencing. The target promoter is shown as a black bar. N-terminal to the SANT-like domain (Naumann et al., Open, half filled and solid lollipop symbols indicate no, incomplete manuscript in preparation). Mutant plants homozygous or complete cytosine methylation in the specified contexts, for the rts1-5 allele are characterized by strong respectively. Silencing RNAs are shown as dashed lines. The reactivation of an RdDM-silenced target promoter, circularized arrows indicate maintenance methylation performed by methyltransferase 1 (CG) or CMT3 (CHG), respectively. despite maintaining wild-type levels of cytosine methy- Potential protein complexes containing AtHDA6 are pointed out lation. Thus, AtHDA6 functions in transcriptional as boxes. RBP, RNA-binding protein. Please refer to the main text repression can be uncoupled from those required for for details. maintenance of cytosine methylation. Moreover, the rts1-5 phenotype clearly demonstrates that DNA methylation initiated during RdDM is insufficient to induce transcriptional repression in the absence of would be similar to the reported interactions between functional AtHDA6 protein. In summary, these Athda6 HDACs and DNMT1 in mammals (Fuks et al., 2000; alleles with very distinct effects on DNA methylation Figure 2, top). An alternative mechanism would be provide an excellent starting point to closer investigate recruitment by MBD proteins, which read out CpG the relationships between the two major repressive methylation induced by RdDM and could connect chromatin modifications that characterize promoters DNA methylation and corepressor complexes that silenced by dsRNA. contain HDAC activity (Dobosy and Selker, 2001; De Ruijter et al., 2003). Some human MBD proteins exhibit a high sensitivity to methylated DNA in vitro and can efficiently bind to single methylated CpG sites (Bird and Multiple ways of recruiting AtHDA6? Wolffe, 1999). However, in vivo, the strength of MBD- mediated repression likely depends on a delicate balance Although cytosine methylation alone is not sufficient to between CpG methylation density and promoter repress promoters targeted by the RdDM pathway, it strength, with a high density of CpG methylation still might be necessary and serve as an important signal correlating with strong and stable repression (Boyes for the recruitment of AtHDA6, which finally executes and Bird, 1992). The transgene target promoter in the silencing. In support of this conclusion, met1 mutants genetic screens detecting Athda6 mutant alleles is fail to repress RdDM targets despite the presence of characterized by a high number of cytosines in a CpG functional AtHDA6 protein (Aufsatz et al., 2002a, context, which would make it a good target for MBD- 2004). Since many mutant alleles of AtHDA6 affect mediated repression (Figure 2, top) (Aufsatz et al., symmetric cytosine methylation, HDAC recruitment 2002a, b). could potentially be achieved via association with the AtHDA6, however, is also involved in the repression of major maintenance DMTs, MET1 and CMT3, which targets with a low CpG content. Recently, endogenous

Oncogene AtHDA6 controls dsRNA-induced repressive chromatin W Aufsatz et al 5483 targets of the RdDM repression pathway in Arabidopsis formation in fission yeast that trans-acting siRNAs that were identified (Huettel et al., 2006). They are typically originate from centromeric repeats can target ectopic located in euchromatic regions of the genome and are repeats, but are much less efficient than siRNAs acting associated with siRNA and high levels of cytosine in cis (Martienssen et al., 2005). Thus, for promoters methylation in non-CpG contexts. They are repressed in silenced by RdDM in trans, the importance of AtHDA6 wild-type plants, where they are associated primarily recruitment by RNA signals might be minor for with deacetylated histones, but not with H3K9me2. silencing compared to recruitment by cytosine methyla- These targets are hypomethylated and derepressed in tion. It will be important in the future to evaluate the mutants that affect the generation or function of significance of this novel potential HDAC recruitment siRNAs, indicating that their repression in wild-type pathway for loci, which are both producers and targets plants is under primary, potentially exclusive control of of silencing-inducing RNA signals. RNA silencing. One of the identified targets, a solo LTR Multiple recruitment mechanisms of AtHDA6 to derived from a previously uncharacterized, small Copia- RNA-silenced targets might not be mutually exclusive. like retrotransposon family, was analysed for its For example, targeting by RNAs with silencing capacity transcriptional status in Athda6 mutant plants and (that is, small RNAs, long dsRNA or as yet unidentified found to be derepressed in all mutant alleles tested so single-stranded intermediates) would provide sequence- far (Naumann and Aufsatz, unpublished results). The specific HDAC recruitment before fully established solo LTR harbors only two CpG sites contrasted with a DNA methylation. Later, DNA methylation might multitude of CpHpH sites, which are heavily methylated serve as a memory mark to allow continuous HDAC in wild-type plants. The solo LTR is transcriptionally recruitment via MBDs (Figure 2). not reactivated in plants mutant for the CpG-specific DNMT MET1, suggesting that recruitment of AtHDA6 by methyl-CpG-binding proteins in this case is not De novo methylation of promoters in plant and cancer relevant for silencing (Huettel et al., 2006). One cells: are there common principles? alternative possibility would be AtHDA6 recruitment by proteins that can bind to methylated cytosines in a In Arabidopsis, de novo cytosine methylation and non-CpG context (Figure 2, top). In accordance with transcriptional inactivation can be clearly triggered by this hypothesis, a subset of the 13 MBD proteins of RNA signals. On the histone level, RNA silenced Arabidopsis has been shown to bind cytosines in a promoters are characterized by histone deacetylation CpHpC or CpHpH context in vitro, although in some rather than by repressive histone methylation. Histone cases binding was not dependent on cytosine methyla- deacetylation is executed by the specialized RPD3-type tion (Zemach and Grafi, 2007). Moreover, plant MBD HDAC AtHDA6, which crosstalks with the DNA proteins lack the transcriptional repressor domain methylation machinery and ‘locks in’ the silenced state. (TRD) identified in their human homologs that recruits Are there common underlying principles of the well- corepressor complexes. Thus, it is currently unknown by documented hypermethylation and inactivation of plant which molecular mechanism plant MBDs can actually promoters and the de novo methylation and silencing of attract HDAC activity to methylated sequences and CpG control regions during neoplastic development in whether they possess a yet unidentified, potentially various types of cancer? plant-specific TRD motif. A second possibility of Many tumor cells are characterized by the para- HDAC recruitment would be directly via the silencing doxical situation in which localized hypermethylation of RNA molecules, for example via the association of CpG islands is initiated in the background of global, AtHDA6 with RNA binding proteins (RBP; Figure 2, genome-wide hypomethylation (Jones and Laird, 1999; middle), by a mechanism that would act in parallel to Jones and Baylin, 2002; Herman and Baylin, 2003). and independent of DNA methylation. Interestingly, an Similarly, Arabidopsis mutants that show genome-wide extensive search for AtHDA6-interacting proteins by hypomethylation, acquire ectopic aberrant hypermethy- yeast two-hybrid analyses identified two RNA-binding lation of certain flower-specific genes at high frequency, proteins and an RNA helicase (Stoiber and Aufsatz, which can account for at least a subset of phenotypes of unpublished results). All three proteins are annotated to hypomethylation mutants (Jacobsen et al., 2000). localize to the nucleolus, and thus potentially colocalize Interestingly, one of these genes, Superman, has been in planta with AtHDA6, for which nucleolar localization shown by genetic screens to be a potential target of has been reported (Earley et al., 2006), as well as with RNA-instructed silencing (Zilberman et al., 2003). components of the processing machinery of nuclear- Peculiarities of DNA structure, such as pyrimidine acting siRNAs (Pontes et al., 2006; Li et al., 2006b). richness coupled to the formation of hairpin structures, Although the repression of a transgene RdDM target have been discussed as potential features that might promoter can not be maintained in mutants affecting attract aberrant methylation at these genes, but this does DNA methylation (see above), which challenges the not explain why methylation is only induced in the hypothesis of methylation-independent recruitment situation of global hypomethylation. One alternative pathways, it should be kept in mind that silencing in mechanism is possibly the reduction of genic CpG the two-component RdDM model system is triggered methylation, which occurs within transcribed sequences by RNA signals, which are supplied in trans. It has at an unexpectedly high frequency in Arabidopsis and been reported for RNAi-mediated heterochromatin has been discussed to suppress the activity of cryptic

Oncogene AtHDA6 controls dsRNA-induced repressive chromatin W Aufsatz et al 5484 promoters (Tran et al., 2005; Zhang et al., 2006; enzymes, such as HDACs and HMTs, in mammalian Zilberman et al., 2007). Reduction of gene body cells (Ting et al., 2005). In summary, the analysis of methylation might result in activation of cryptic relationships between dsRNA signals, cytosine methyla- antisense promoters, whose activity would result in the tion and recruitment of histone-modifying enzymes in formation of regional dsRNA that, in turn, could trigger well-characterized plant model silencing systems has aberrant de novo methylation by an RdDM-like potential implications that extend far beyond the plant mechanism. Depending on the location of cryptic field. It might assist in detecting mechanisms involved in antisense promoters, the region of dsRNA formation reprogramming epigenetic states during cancer initiation could encompass extreme 50 regions of genes, from and development. which RNA-induced chromatin modifications might spread into proximal promoter regions. Although genome-wide analysis does not support a general Conclusions and perspectives correlation between loss of gene body methylation and antisense transcription, such a mechanism could be Plant silencing systems clearly demonstrate that pro- effective, however, for a limited number of genes (Zhang moters can be inactivated by sequence-homologous et al., 2006), which is supported by the existence of dsRNA. Promoter inactivation is associated with de several ‘hot-spots’ for hypermethylation in hypomethy- novo cytosine methylation and histone deacetylation. lated Arabidopsis genomic backgrounds (Jacobsen et al., There is emerging evidence that histone deacetylation 2000). Since vertebrate genes are also characterized by can be triggered either in response to dsRNA-induced extensive gene body methylation (Goll and Bestor, 2005; cytosine methylation or directly instructed by dsRNA. Klose and Bird, 2006), similar mechanisms could Cancer cells exhibit analogous reprogramming of potentially apply for genes that are hypermethylated in promoter activity associated with cytosine hypermethy- cancer cells. In mammalian cells, a mechanistic link lation and the acquisition of repressive histone modi- between siRNAs directed to promoters and induction of fications. Recent evidence suggests that one mechanism cytosine methylation has been convincingly demon- to recruit aberrant DNA methylation involves crosstalk strated, which supports the basic existence of RdDM- between Polycomb-silencing complexes and DNA like processes also in mammals (Morris et al., 2004; methyltransferases. The significance of this de novo Castanotto et al., 2005). The existence of cancer cell or methylation pathway for cancer biology, however, is still tumor-specific dsRNA is supported by a recent publica- unclear. Many of its target genes are already silenced by tion, which reports on natural antisense transcripts that Polycomb proteins in healthy precursor cells, indicating are differentially expressed in various tumors (Klimov that their encoded proteins are likely not important for et al., 2006). In mammals, however, long dsRNA the protection of cells against neoplastic transformation. induces an antiviral response mediated by interferon, This knowledge about alternative mechanisms from which results in a general inhibition of protein synthesis plant models might assist in the detection of control and nonspecific degradation of mRNA (Kawai and regions of validated or candidate tumor suppressor Akira, 2006). The interferon pathway usually impedes genes, which change epigenetic states in the transition gene-specific silencing induced by long dsRNA in most from healthy to cancer cells. It is likely that, analogous mammalian cell types. Thus, in order to trigger specific to the situation in plants, this transition is triggered by silencing effects, dsRNAs generated by cancer cell- dsRNA and components of the RNAi machinery. specific antisense transcription have either to be Specific dsRNAs could potentially arise as a conse- sufficiently short to avoid induction of the interferon quence of enhanced cancer-specific antisense transcrip- response or are effective only in cancer cell types with a tion due to the activation of cryptic promoters by the compromised or absent interferon response (Billy et al., genome-wide loss of cytosine methylation typically 2001), which would limit the conditions for such an associated with cancer initiation. Thus, it will be RNA-instructed de novo methylation pathway. RNA important to carefully analyse hypermethylated target signals, however, might only be one of multiple signals genes in cancer cells for evidence of antisense transcrip- that induce cancer-specific aberrant cytosine methylation and to explore whether the initiation and/or tion, since Polycomb-mediated silencing has recently maintenance of the hypermethylated state requires the been reported to premark many genes that acquire integrity of the RNAi pathway. Since plants can methylation during neoplastic transformation (Keshet potentially recruit HDACs directly via RNA signals et al., 2006; Schlesinger et al., 2006; Widschwendter and mammals can silence promoters by siRNAs and/or et al., 2007). There is emerging evidence of connections dsRNA without concomitant DNA methylation, RNA- between Polycomb silencing and RNAi (Grimaud et al., based gene silencing in cancer cells might not always 2006; Kim et al., 2006), indicating that dsRNA might be be accompanied by hypermethylation. Thus, many at the basis of multiple pathways that can potentially consequences of misregulated RNA silencing pathways result in ectopic DNA methylation in cancer develop- in cancer cells might have gone unnoticed so far. ment. However, siRNAs directed to promoter sequences in cancer cells can also induce transcriptional gene Acknowledgements silencing and repressive chromatin in the absence of cytosine methylation, which provides evidence for We thank Ortrun Mittelsten Scheid, Dieter Schweizer and two an RNA-triggered recruitment of histone-modifying anonymous reviewers for critical comments and Maria Siomos

Oncogene AtHDA6 controls dsRNA-induced repressive chromatin W Aufsatz et al 5485 for scientiﬁc editing of the manuscript. The authors are Aufsatz laboratory discussed in this review is supported by the grateful to Thomas Jenuwein for donating antibodies speciﬁc GEN-AU program of the Austrian Ministry for Science and for H3K9me2 and H3K27me1. Part of the work in the Research (BMWF).

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Oncogene