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Involvement of a GHKL ATPase in RNA-Directed DNA Methylation in Arabidopsis thaliana
Current Biology 22, 933–938, May 22, 2012 ª2012 Elsevier Ltd All rights reserved
DOI 10.1016/j.cub.2012.03.061
Report Involvement of a GHKL ATPase in RNA-Directed DNA Methylation in Arabidopsis thaliana ,1 Ulf Naumann,1 Antonius J.M. Matzke,1 Zdravko J. Lorkovic and Marjori Matzke1,* 1Gregor Mendel Institute of Molecular Plant Biology, Austrian Academy of Sciences, Dr. Bohr-Gasse 3, 1030 Vienna, Austria
Summary RNA-directed DNA methylation (RdDM) is a small interfering RNA (siRNA)-mediated epigenetic modification that contributes to transposon silencing in plants. RdDM requires a complex transcriptional machinery comprising two RNA polymerase II-related RNA polymerases, called Pol IV and Pol V, as well as chromatin remodelers, transcription factors, and other novel proteins whose roles in the RdDM mechanism remain poorly understood [1–4]. We have identified a new component of the RdDM machinery, DMS11 (defective in meristem silencing 11), which has a GHKL (gyrase, Hsp90, histidine kinase, MutL) ATPase domain [5]. siRNAs accumulate in the dms11 mutant, and repressive epigenetic modifications undergo only modest reductions at target sequences. DMS11 interacts physically with another RdDM component, DMS3, an unusual structural maintenance of chromosomes (SMC) hinge domain-containing protein that lacks the ATPase motifs of authentic SMC proteins [6, 7]. The hinge region of DMS3 resembles that of the mammalian epigenetic factor SMCHD1, which also has a GHKL-type ATPase [8]. In vitro, DMS11 has ATPase activity that is stimulated by DMS3. We suggest that DMS11 provides the missing ATPase function for DMS3 and that these proteins cooperate in the RdDM pathway to promote transcriptional repression. GHKL ATPases are thus emerging as new players in epigenetic regulation in plants and mammals. Results To identify components of the RNA-directed DNA methylation (RdDM) machinery, we carried out a forward genetic screen using a transgene silencing system comprising unlinked target (T) and silencer (S) loci in Arabidopsis thaliana [6] (see Figure S1A available online). In this system, a target enhancer that is active in shoot and root meristem regions drives expression of a downstream gene encoding green fluorescent protein (GFP) (Figure S1A, T). GFP expression is transcriptionally silenced by hairpin-derived small interfering RNAs (siRNAs) that direct de novo methylation of the target enhancer (Figure S1A, T+S). Following ethane methylsulfonate mutagenesis of the silenced line, RdDM-defective mutants were identified by screening for recovery of GFP expression in root meristems of newly germinated seedlings. So far, nine dms (defective in meristem silencing) mutants (dms1 to dms9; Table S1) have been isolated in this screen. Here we report on two new mutants, dms10 and dms11. DMS10 corresponds to the previously identified RdDM factor IDN2/RDM12 [7, 9]. IDN2 is a double-stranded RNA
*Correspondence: [email protected]
binding protein similar to SGS3, a protein required for posttranscriptional gene silencing [10]. The role of IDN2 in the mechanism of RdDM is not yet known, but it has been hypothesized to stabilize interactions between an ARGONAUTEbound siRNA and a scaffold RNA synthesized at a target locus by the Pol V complex [7]. In our transgene silencing system, GFP expression is derepressed and methylation of endogenous RdDM targets is reduced in dms10 mutants (Figures S1B–S1E). In the dms11 mutant, release of GFP silencing is most visible in the shoot apical meristem after rosette leaves appear (Figure 1A) and is somewhat less efficient than in a Pol V-defective mutant (nrpe1) (Figure 1B). As demonstrated by bisulfite sequencing, alleviation of silencing in the dms11 mutant is accompanied by partial loss of CHH methylation—the hallmark of RdDM—at the target enhancer. By contrast, CG and CHG methylation remain at roughly wild-type levels (Figures 2A and 2B). As observed for other dms mutants [6], loss of methylation was even more extensive in the downstream region, which undergoes methylation by Pol IV-dependent secondary siRNAs [11] (Figure S1A), than in the target enhancer (Figures 2A and 2B). Analysis of DNA methylation at several endogenous targets of RdDM by methylation-sensitive PCR revealed variable changes in DNA methylation in the dms11 mutant. Although several intergenic (IGN) targets of RdDM [12] showed some loss of methylation, AtSN1 and other retrotransposon-related sequences retained methylation to some degree in dms11. By contrast, most of these sequences displayed substantial reductions of methylation in an nrpe1 mutant (Figure 2C). Levels of transgene-encoded, hairpin-derived siRNAs were unaffected in the dms11 mutant (Figure S2A) whereas endogenous siRNAs accumulate at normal or slightly reduced levels (Figure S2B). Pol IV-dependent secondary siRNAs, which induce methylation in the downstream region [11], were reduced in dms11 (Figure S2A). The decreased amount of secondary siRNAs in dms11 is consistent with the substantial reduction of DNA methylation in the downstream region in this mutant (Figures 2A and 2B). Similar losses of specifically Pol IV-dependent secondary siRNAs and methylation in the downstream region have been observed with other dms mutants [6] and are believed to reflect diminished activity of Pol IV on the hypomethylated target enhancer [6, 11]. Given the incomplete and variable losses of DNA methylation in the dms11 mutant, we investigated whether changes in histone modifications were more strongly correlated with the release of GFP silencing in this mutant. We first analyzed histone H3 methylation and acetylation of the target transgene enhancer in lines containing the T locus either alone or in combination with the silencer locus (T+S) by chromatin immunoprecipitation. The target enhancer and downstream regions were analyzed separately (Figure 3A). In the T line, both the target enhancer and downstream region are in a transcriptionally active state characterized by H3 lysine 4 trimethylation (H3K4me3) and H3 lysine 9 and lysine 14 acetylation (H3ac), whereas only background levels of repressive H3 lysine 9 dimethylation (H3K9me2) are observed (Figure 3B, T panel, and Figure S3A). When the target enhancer is silenced in the
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Figure 1. Release of GFP Silencing in the dms11 Mutant (A) Release of GFP silencing in the shoot meristem region in the dms11 mutant is visible in soil-grown plants. For comparison, wild-type plants containing the target locus (T; GFP-positive) and the target and silencer loci (T+S; GFP-negative) as well as an nrpe1 mutant containing T+S (GFP-positive) are shown. (B) Western blot analysis of GFP expression in plants of the same age as in (A), in the indicated genotypes. Expression of GFP in dms11 is approximately five times lower than in an nrpe1 mutant. Analysis of tubulin levels (middle panel) and the Coomassie blue (CB) stained gel (bottom panel) loaded identically to the one used for western blotting were used as loading controls. nrpd1 and nrpe1: mutants defective in the largest subunits of Pols IV and V, respectively. As reported previously, the nrpd1 mutant does not release GFP silencing in this system [11]. See also Figure S1.
T+S line, the levels of H3K4me3 and H3ac decrease while the level of H3K9me2 increases (Figure 3B, T+S panel, and Figure S3A). H3 lysine 27 monomethylation remains the same in the T and T+S lines (Figure 3B), indicating a minimal contribution of this mark to siRNA-mediated transcriptional regulation of the target enhancer. We next analyzed H3 modifications in the dms11 mutant compared to the T+S line. Although GFP silencing is partially released in dms11, we did not observe reestablishment of the active mark H3K4me3. However, H3K9me2 was significantly decreased and H3ac was significantly increased in dms11 compared to the T+S line in both target enhancer and downstream regions (Figures 3C and S3B). Thus, the combinatorial effects of decreases in DNA methylation and H3K9me2
and an increase in H3ac contribute to the release of GFP silencing in the dms11 mutant. Mapping with codominant markers and whole-genome sequencing of dms11 nuclear DNA allowed us to identify DMS11 as At1g19100, which encodes a previously uncharacterized plant-specific protein that is 663 amino acids in length. The dms11 mutation (G to A) results in a premature stop codon at amino acid 439 (Figures S4A and S4B). DMS11 belongs to a small gene family with seven members in Arabidopsis (Figure S4C). There are full-length homologs of DMS11 in other flowering plant species (Figure S4D). DMS11 and related proteins are characterized by the presence at the N terminus of a GHKL (gyrase, Hsp90, histidine kinase, MutL)-type ATPase domain [5]. This ATPase is embedded in an extended and highly conserved sequence that is found in metazoan MORC (microrchidia) proteins [5, 13–16] (Figures 4A, S4B, S4E, and S4F). The C-terminal part of DMS11 contains a short coiled-coil domain that could potentially be involved in formation of homodimers or interactions with other coiled-coil proteins (Figures 4A, S4B, and S4D). Complementation of the dms11 mutant with a construct expressing HA-tagged DMS11 cDNA under the control of its own promoter confirmed that release of GFP silencing in dms11 is indeed due to the mutation in At1g19100 (Figure S4G). A recent paper described Arabidopsis GMI1, a DNA repair protein that contains a GHKL-type ATPase and structural maintenance of chromosomes (SMC) hinge domain [17]. Through its SMC hinge domain, GMI1 is related to DMS3, a smaller SMC hinge domain-containing protein that was identified in the same forward genetic screen as DMS11 [6]. SmcHD1, which is involved in X chromosome inactivation in mice [8], also contains a GHKL-type ATPase and an SMC hinge domain similar to DMS3 [6, 8, 17] (Figure 4B). DMS3 lacks the ATPase domains found in authentic SMC proteins and in GMI1 and SmcHD1 (Figure 4B). In view of this information, and because coexpression of DMS3 and DMS11 is strongly correlated (http://bbc.botany.utoronto.ca/ntools/ cgi-bin/ntools_expression_angler.cgi), we reasoned that DMS11 might interact with DMS3 and provide the missing ATPase function for this protein. To detect physical interactions between DMS3 and DMS11, we first carried out a yeast two-hybrid assay; however, no DMS3-DMS11 interactions were detected (Figure S4H). Given that DMS3 forms homodimers [6] (Figure S4H), it was possible that dimerization and fusion to the GAL4 DNA-binding domain sterically inhibited its interaction with DMS11. We therefore tried an alternate approach by coexpressing epitope-tagged versions of DMS3 and DMS11 in bacteria. In these experiments, we were able to copurify His-tagged DMS3 with GSTtagged DMS11, but not with GST alone or with GST-DMS4 (Figures 4C and S4J). These results demonstrate a specific interaction between DMS11 and DMS3 in a cellular context. We also tested whether DMS11 has ATPase activity. Using a colorimetric assay, we detected ATPase activity of GSTtagged recombinant DMS11 protein in vitro, and this activity was approximately 3.5 times higher in the presence of coexpressed His-tagged DMS3 (Figure 4D). DMS3 acts in the so-called DDR complex, which is required for synthesis of Pol V scaffold transcripts and RdDM [18]. We therefore investigated whether DMS11, which interacts with DMS3, is also needed for synthesis of the Pol V transcript IGN5. Synthesis of IGN5 RNA was indeed reduced in the dms11 mutant, although not to the degree observed in an nrpe1 mutant (Figure 4E). This result is consistent with the
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Figure 2. DNA Methylation in the dms11 Mutant (A) Bisulfite sequencing analysis of DNA methylation of the target transgene enhancer (black bar) and the downstream region subject to methylation spreading (gray bar) [11]. Percent methylation of cytosines in CG (black lines), CHG (blue lines), and CHH (red lines) sequence contexts is shown for dms11 and wild-type plants (T+S). (B) Cumulative analysis of DNA methylation in the target transgene enhancer and downstream region in dms11 and T+S plants. (C) DNA methylation analysis of the RdDM targets AtSN1, IGN5, IGN23, IGN25, soloLTR, LTR1, LTR3, IGN26, and IGN22. DNA was digested with the indicated methylation-sensitive restriction enzymes and used for PCR amplification. DNA from wild-type T+S plants and an nrpe1 mutant was used as a control. Amplification of a region of an actin gene (Act) region lacking HaeIII and AvaII sites or undigested DNA was used as a loading control. See also Figure S2.
less extensive loss of DNA methylation at the IGN5 locus in dms11 compared to nrpe1 (Figure 2C). Discussion We have identified a GHKL-type ATPase, DMS11, as a new constituent of the RdDM pathway. GHKL ATPase domains are found in wide variety of prokaryotic and eukaryotic proteins that function in DNA structure rearrangement, heat shock, signal transduction, and DNA mismatch repair [5, 16]. The metazoan proteins most similar to DMS11 are the MORC ATPases, which belong to the GHKL superfamily of ATPases [15]. The first MORC protein was identified as a factor required for completion of male meiosis and spermatogenesis in mice [13]. MORC proteins have a domain composition similar to that found in DMS11, although they contain a CW-type zinc finger [19] between the MORC and coiled-coil domains (Figure S4E). Despite the partial similarity to MORC proteins, DMS11 and its full-length homologs are found only in flowering plants. Two major types of ATPases have been implicated previously in chromatin dynamics [16]. SWI2/SNF2 ATPases are important for local chromatin remodeling and nucleosome positioning. SMC ATPases, which belong to the ABC superfamily of ATPases, typically participate in large-scale chromatin dynamics [16]. By contrast, little is known about GHKL or MORC ATPases in chromatin remodeling and assembly [15]. Based on the functions of related proteins in prokaryotes, it has been suggested that MORC ATPases are involved in manipulation of DNA superstructure in response to epigenetic marks such as DNA methylation or histone modifications [15]. A role for DMS11 in higher order chromatin structure may be consistent with the rather modest epigenetic alterations we observed in the dms11 mutant; however, we cannot rule out that the allele we have studied is not a null. Therefore, further work is needed to test whether DMS11 is involved in altering
large-scale chromatin organization. A further consideration is that DMS11 is a member of a gene family and hence potentially redundant, which could also account for the weak effects of the dms11 mutation on DNA methylation compared to other RdDM mutants such as dms3 and nrpe1 [6]. One epigenetic change we observed at the target transgene enhancer in the dms11 mutant was an increase in histone H3 acetylation, suggesting that DMS11-mediated transcriptional gene silencing involves deacetylation of histones. Histone deacetylation was also found to be involved in the downregulation of carbonic anhydrase IX by human MORC2 [14]. DMS11 interacts physically with the SMC hinge-domain containing protein DMS3, which lacks ATPase motifs, and displays ATPase activity in vitro that is stimulated by DMS3. Two known proteins, mammalian SmcHD1 [8] and Arabidopsis GMI1 [17], comprise both GHKL ATPase domains and SMC hinge regions that are similar to the hinge region in DMS3 [6, 17]. Although GMI1 has been implicated specifically in DNA repair [17], SmcHD1 is required for murine X chromosome inactivation, which—similarly to RdDM—is an epigenetic process involving noncoding RNAs and DNA methylation [8]. Given the genetic and physical interactions between DMS11 and DMS3, and the stimulation of the ATPase activity of DMS11 by DMS3, it is reasonable to propose that these two proteins associate in vivo to form a functional analog of SmcHD1 in Arabidopsis (Figure 4B). Thus, single proteins (or protein partners) comprising GHKL ATPases and SMC hinge domains are emerging as new players in epigenetic regulation, perhaps specifically in RNA-mediated epigenetic processes, in both plants and mammals. The specific role of DMS11 in the RdDM mechanism is still not known. It may be part of the previously identified DDR complex [18], which contains in addition to DMS3 the chromatin remodeler DRD1 and RDM1, a small, plant-specific protein that coimmunoprecipitates with ARGONAUTE4 and the de novo DNA methyltransferase DRM2 [20]. The DDR complex is needed for production of Pol V scaffold transcripts that are thought to base pair with AGO4-bound siRNAs to recruit the silencing effector complex to target loci [18, 21]. It is not yet known how the DDR complex facilitates Pol V transcription. However, our finding that DMS11 is
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Figure 3. Analysis of Histone Modifications in the dms11 Mutant (A) Schematic presentation of the GFP reporter gene. The target transgene enhancer and downstream region of methylation spreading are depicted by blue and red boxes, respectively. Positions of oligonucleotides used for PCR are shown above. (B) Analysis of histone modifications in wild-type T and T+S lines. In the T line, the target enhancer and downstream region are in a transcriptionally active state (high H3K4me3 and H3ac) and in the T+S line they are in an inactive state (low H3K4me3 and H3ac, high H3K9me2). (C) Comparison of histone modifications in dms11 with the wild-type T+S line. Significant changes in dms11 include a decrease in H3K9me2 and increase in H3ac. In (B) and (C), the data shown are average values from three and four independent biological repetitions, respectively, with the error bars representing the standard deviation. Statistically significant p values as determined by Student’s t test are indicated. Semiquantitative PCR analysis of data in (B) and (C) is shown in Figures S3A and S3B. Analyses of tubulin as a euchromatin control and LTR1 and LTR3, which do not reactivate in RdDM mutants [28], as heterochromatin controls did not reveal any changes between T, T+S, dms11, and nrpe1 lines (Figure S3C), demonstrating specificity of our chromatin immunoprecipitation assays. See also Figure S3.
also needed for production of the Pol V transcript IGN5 is consistent with the participation of this protein in the DDR complex. The only other member of the DMS11 gene family for which there is functional information is At4g36290 or CRT1 (compromised recognition of TCV). CRT1 has been reported to encode an endosome-associated protein that functions in early stages of R (Resistance) gene-mediated defense signaling [22–24].
Whether there is any relationship between this function of CRT1 and the role of DMS11 in RdDM is unknown. However, two recent papers have implicated two components of the Pol V pathway, AGO4 and NRPD2a, in plant immunity [25, 26]. Further investigations of DMS11 and CRT1 as well as other members of the CRT1/DMS11 gene family may reveal additional links between GHKL ATPases, RdDM, and plant defense responses. Figure 4. DMS11 Is a GHKL ATPase
(A) Schematic presentation of DMS11 domains. The position of the dms11-1 mutation is indicated (W439*). The ATPase domain is represented by a yellow oval embedded in a red box representing the MORC homology region. CC, coiled coil. (B) DMS11 may provide the missing ATPase activity for DMS3. Domain arrangements of SMC proteins, DMS11, DMS3, and two related proteins, Arabidopsis GMI1 and murine SmcHD1, are shown. SMC proteins have an ABC-type ATPase, whereas DMS11 and GMI1/SmcHD1 have GHKL-type ATPases. DMS3 is an SMC hinge domain-containing protein with short coiled coils at N and C termini, but it lacks the ATPase domain found in bona fide SMC proteins. We propose that interactions of DMS11 with DMS3 could form the Arabidopsis functional analog of murine SmcHD1. (C) Interaction of DMS11 with DMS3. Full-length DMS11 fused to GST was coexpressed with His6-DMS3 in E. coli. Proteins were purified over glutathione Sepharose beads (Figure S4J), and bound proteins were analyzed by western blotting with anti-His antibody. Coexpression and purification of GST with His6-DMS3 or GSTDMS4 with His6-DMS3 were used as negative controls. DMS4 is a putative transcription factor isolated in the same genetic screen as DMS3 and DMS11 (Table S1). Lanes I (input): 1/10 of volume of protein extracts used for purification over glutathione Sepharose beads. Lanes B (bound): 1/5 of proteins retained on glutathione Sepharose beads after washing. (D) ATPase activity of purified recombinant GST-DMS11 or GST-DMS11 coexpressed with His-DMS3. GST alone was used as a control. The data presented are mean values of at least three measurements, with the error bars representing the standard deviation. (E) Pol V transcript IGN5 is reduced in dms11. RT-PCR analysis of IGN5 and soloLTR transcripts in wild-type (T+S), nrpe1, and dms11 lines is shown. soloLTR serves as a positive control for target locus reactivation in RdDM-defective mutants [28]. Actin RT-PCR reactions were used as loading controls. PCRs without RT were used as controls for DNA contamination. See also Figure S4.
GHKL ATPase Acts in RNA-Directed DNA Methylation 937
Experimental Procedures
References
Plant Material All mutants lines used were in the Col-0 background. Plants were grown in a growth chamber set at 21 C with a long-day light regime (16 hr light/8 hr dark). Seedlings were grown under sterile conditions on solid Murashige and Skoog medium at 23 C with a long-day light regime. For complementation analyses, wild-type DMS11 cDNA and an alternatively spliced mutant version, both under the control of the native DMS11 promoter, were introduced into the dms11 mutant using the floral dip method [27]. For each construct, we obtained several independent lines containing single copies of the respective construct. More than 50% of the plants containing the wild-type construct were GFP negative, demonstrating restoration of silencing. By contrast, all of the plants containing the mutant version were GFP positive (see Figure S4G). The complementation data thus confirm that DMS11 is At1g19100.
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Cloning Details on construction of plasmids used for protein overexpression in Escherichia coli, yeast two-hybrid assays, and complementation of dms11 mutant are available in Supplemental Experimental Procedures. DNA Methylation Methylation of the target enhancer by bisulfite sequencing (average of 20 independent clones) was carried out as described previously [6, 11]. Methylation analysis of LTRs, IGNs, and AtSN1 was carried out by methylation-sensitive PCR [28, 29]. Oligonucleotides used in these experiments are listed in Supplemental Experimental Procedures. Chromatin Immunoprecipitation Chromatin immunoprecipitation analyses were conducted as described by W. Aufsatz (http://mescaline.igh.cnrs.fr/EpiGeneSys/images/stories/ protocols/pdf/20111025150640_p13.pdf). Real-time PCR analysis was performed with a Bio-Rad iQ5 machine using SensiFAST mix from Bioline. All data are expressed relative to input. Antibodies used for immunoprecipitations were from Abcam (H3, H3K4me3, and H3K9me2) and Millipore (H3K27me and H3ac). Yeast Two-Hybrid Assay, Recombinant Protein Analysis, and Western Blotting Yeast two-hybrid assays were carried out according to the manufacturer’s instructions (Clontech). Detailed protocols for recombinant protein expression and purification are available in Supplemental Experimental Procedures. For western blotting, proteins from seedlings or rosettes were extracted as described previously [30]. SDS-PAGE and western blotting were performed according to standard procedures. The level of GFP in dms11 was determined by comparison to serial dilutions of extracts from the target (T) line, which constitutively expresses GFP. Monoclonal antiGFP (Roche), anti-tubulin (Sigma-Aldrich), and anti-His (Sigma-Aldrich) antibodies were used at 1:1,000 dilutions. ATPase Assays and siRNA Analysis See Supplemental Experimental Procedures.
Supplemental Information Supplemental Information includes four figures, one table, and Supplemental Experimental Procedures and can be found with this article online at doi:10.1016/j.cub.2012.03.061. Acknowledgments This work was supported by the Austrian Academy of Sciences and the Austrian Fonds zur Fo¨rderung der wissenschaftlichen Forschung (grant SFB F4306-B09). We thank Johannes van der Winden for editorial assistance. Received: February 28, 2012 Revised: March 16, 2012 Accepted: March 19, 2012 Published online: May 3, 2012
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DOI 10.1016/j.cub.2012.03.061
Report Involvement of a GHKL ATPase in RNA-Directed DNA Methylation in Arabidopsis thaliana ,1 Ulf Naumann,1 Antonius J.M. Matzke,1 Zdravko J. Lorkovic and Marjori Matzke1,* 1Gregor Mendel Institute of Molecular Plant Biology, Austrian Academy of Sciences, Dr. Bohr-Gasse 3, 1030 Vienna, Austria
Summary RNA-directed DNA methylation (RdDM) is a small interfering RNA (siRNA)-mediated epigenetic modification that contributes to transposon silencing in plants. RdDM requires a complex transcriptional machinery comprising two RNA polymerase II-related RNA polymerases, called Pol IV and Pol V, as well as chromatin remodelers, transcription factors, and other novel proteins whose roles in the RdDM mechanism remain poorly understood [1–4]. We have identified a new component of the RdDM machinery, DMS11 (defective in meristem silencing 11), which has a GHKL (gyrase, Hsp90, histidine kinase, MutL) ATPase domain [5]. siRNAs accumulate in the dms11 mutant, and repressive epigenetic modifications undergo only modest reductions at target sequences. DMS11 interacts physically with another RdDM component, DMS3, an unusual structural maintenance of chromosomes (SMC) hinge domain-containing protein that lacks the ATPase motifs of authentic SMC proteins [6, 7]. The hinge region of DMS3 resembles that of the mammalian epigenetic factor SMCHD1, which also has a GHKL-type ATPase [8]. In vitro, DMS11 has ATPase activity that is stimulated by DMS3. We suggest that DMS11 provides the missing ATPase function for DMS3 and that these proteins cooperate in the RdDM pathway to promote transcriptional repression. GHKL ATPases are thus emerging as new players in epigenetic regulation in plants and mammals. Results To identify components of the RNA-directed DNA methylation (RdDM) machinery, we carried out a forward genetic screen using a transgene silencing system comprising unlinked target (T) and silencer (S) loci in Arabidopsis thaliana [6] (see Figure S1A available online). In this system, a target enhancer that is active in shoot and root meristem regions drives expression of a downstream gene encoding green fluorescent protein (GFP) (Figure S1A, T). GFP expression is transcriptionally silenced by hairpin-derived small interfering RNAs (siRNAs) that direct de novo methylation of the target enhancer (Figure S1A, T+S). Following ethane methylsulfonate mutagenesis of the silenced line, RdDM-defective mutants were identified by screening for recovery of GFP expression in root meristems of newly germinated seedlings. So far, nine dms (defective in meristem silencing) mutants (dms1 to dms9; Table S1) have been isolated in this screen. Here we report on two new mutants, dms10 and dms11. DMS10 corresponds to the previously identified RdDM factor IDN2/RDM12 [7, 9]. IDN2 is a double-stranded RNA
*Correspondence: [email protected]
binding protein similar to SGS3, a protein required for posttranscriptional gene silencing [10]. The role of IDN2 in the mechanism of RdDM is not yet known, but it has been hypothesized to stabilize interactions between an ARGONAUTEbound siRNA and a scaffold RNA synthesized at a target locus by the Pol V complex [7]. In our transgene silencing system, GFP expression is derepressed and methylation of endogenous RdDM targets is reduced in dms10 mutants (Figures S1B–S1E). In the dms11 mutant, release of GFP silencing is most visible in the shoot apical meristem after rosette leaves appear (Figure 1A) and is somewhat less efficient than in a Pol V-defective mutant (nrpe1) (Figure 1B). As demonstrated by bisulfite sequencing, alleviation of silencing in the dms11 mutant is accompanied by partial loss of CHH methylation—the hallmark of RdDM—at the target enhancer. By contrast, CG and CHG methylation remain at roughly wild-type levels (Figures 2A and 2B). As observed for other dms mutants [6], loss of methylation was even more extensive in the downstream region, which undergoes methylation by Pol IV-dependent secondary siRNAs [11] (Figure S1A), than in the target enhancer (Figures 2A and 2B). Analysis of DNA methylation at several endogenous targets of RdDM by methylation-sensitive PCR revealed variable changes in DNA methylation in the dms11 mutant. Although several intergenic (IGN) targets of RdDM [12] showed some loss of methylation, AtSN1 and other retrotransposon-related sequences retained methylation to some degree in dms11. By contrast, most of these sequences displayed substantial reductions of methylation in an nrpe1 mutant (Figure 2C). Levels of transgene-encoded, hairpin-derived siRNAs were unaffected in the dms11 mutant (Figure S2A) whereas endogenous siRNAs accumulate at normal or slightly reduced levels (Figure S2B). Pol IV-dependent secondary siRNAs, which induce methylation in the downstream region [11], were reduced in dms11 (Figure S2A). The decreased amount of secondary siRNAs in dms11 is consistent with the substantial reduction of DNA methylation in the downstream region in this mutant (Figures 2A and 2B). Similar losses of specifically Pol IV-dependent secondary siRNAs and methylation in the downstream region have been observed with other dms mutants [6] and are believed to reflect diminished activity of Pol IV on the hypomethylated target enhancer [6, 11]. Given the incomplete and variable losses of DNA methylation in the dms11 mutant, we investigated whether changes in histone modifications were more strongly correlated with the release of GFP silencing in this mutant. We first analyzed histone H3 methylation and acetylation of the target transgene enhancer in lines containing the T locus either alone or in combination with the silencer locus (T+S) by chromatin immunoprecipitation. The target enhancer and downstream regions were analyzed separately (Figure 3A). In the T line, both the target enhancer and downstream region are in a transcriptionally active state characterized by H3 lysine 4 trimethylation (H3K4me3) and H3 lysine 9 and lysine 14 acetylation (H3ac), whereas only background levels of repressive H3 lysine 9 dimethylation (H3K9me2) are observed (Figure 3B, T panel, and Figure S3A). When the target enhancer is silenced in the
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Figure 1. Release of GFP Silencing in the dms11 Mutant (A) Release of GFP silencing in the shoot meristem region in the dms11 mutant is visible in soil-grown plants. For comparison, wild-type plants containing the target locus (T; GFP-positive) and the target and silencer loci (T+S; GFP-negative) as well as an nrpe1 mutant containing T+S (GFP-positive) are shown. (B) Western blot analysis of GFP expression in plants of the same age as in (A), in the indicated genotypes. Expression of GFP in dms11 is approximately five times lower than in an nrpe1 mutant. Analysis of tubulin levels (middle panel) and the Coomassie blue (CB) stained gel (bottom panel) loaded identically to the one used for western blotting were used as loading controls. nrpd1 and nrpe1: mutants defective in the largest subunits of Pols IV and V, respectively. As reported previously, the nrpd1 mutant does not release GFP silencing in this system [11]. See also Figure S1.
T+S line, the levels of H3K4me3 and H3ac decrease while the level of H3K9me2 increases (Figure 3B, T+S panel, and Figure S3A). H3 lysine 27 monomethylation remains the same in the T and T+S lines (Figure 3B), indicating a minimal contribution of this mark to siRNA-mediated transcriptional regulation of the target enhancer. We next analyzed H3 modifications in the dms11 mutant compared to the T+S line. Although GFP silencing is partially released in dms11, we did not observe reestablishment of the active mark H3K4me3. However, H3K9me2 was significantly decreased and H3ac was significantly increased in dms11 compared to the T+S line in both target enhancer and downstream regions (Figures 3C and S3B). Thus, the combinatorial effects of decreases in DNA methylation and H3K9me2
and an increase in H3ac contribute to the release of GFP silencing in the dms11 mutant. Mapping with codominant markers and whole-genome sequencing of dms11 nuclear DNA allowed us to identify DMS11 as At1g19100, which encodes a previously uncharacterized plant-specific protein that is 663 amino acids in length. The dms11 mutation (G to A) results in a premature stop codon at amino acid 439 (Figures S4A and S4B). DMS11 belongs to a small gene family with seven members in Arabidopsis (Figure S4C). There are full-length homologs of DMS11 in other flowering plant species (Figure S4D). DMS11 and related proteins are characterized by the presence at the N terminus of a GHKL (gyrase, Hsp90, histidine kinase, MutL)-type ATPase domain [5]. This ATPase is embedded in an extended and highly conserved sequence that is found in metazoan MORC (microrchidia) proteins [5, 13–16] (Figures 4A, S4B, S4E, and S4F). The C-terminal part of DMS11 contains a short coiled-coil domain that could potentially be involved in formation of homodimers or interactions with other coiled-coil proteins (Figures 4A, S4B, and S4D). Complementation of the dms11 mutant with a construct expressing HA-tagged DMS11 cDNA under the control of its own promoter confirmed that release of GFP silencing in dms11 is indeed due to the mutation in At1g19100 (Figure S4G). A recent paper described Arabidopsis GMI1, a DNA repair protein that contains a GHKL-type ATPase and structural maintenance of chromosomes (SMC) hinge domain [17]. Through its SMC hinge domain, GMI1 is related to DMS3, a smaller SMC hinge domain-containing protein that was identified in the same forward genetic screen as DMS11 [6]. SmcHD1, which is involved in X chromosome inactivation in mice [8], also contains a GHKL-type ATPase and an SMC hinge domain similar to DMS3 [6, 8, 17] (Figure 4B). DMS3 lacks the ATPase domains found in authentic SMC proteins and in GMI1 and SmcHD1 (Figure 4B). In view of this information, and because coexpression of DMS3 and DMS11 is strongly correlated (http://bbc.botany.utoronto.ca/ntools/ cgi-bin/ntools_expression_angler.cgi), we reasoned that DMS11 might interact with DMS3 and provide the missing ATPase function for this protein. To detect physical interactions between DMS3 and DMS11, we first carried out a yeast two-hybrid assay; however, no DMS3-DMS11 interactions were detected (Figure S4H). Given that DMS3 forms homodimers [6] (Figure S4H), it was possible that dimerization and fusion to the GAL4 DNA-binding domain sterically inhibited its interaction with DMS11. We therefore tried an alternate approach by coexpressing epitope-tagged versions of DMS3 and DMS11 in bacteria. In these experiments, we were able to copurify His-tagged DMS3 with GSTtagged DMS11, but not with GST alone or with GST-DMS4 (Figures 4C and S4J). These results demonstrate a specific interaction between DMS11 and DMS3 in a cellular context. We also tested whether DMS11 has ATPase activity. Using a colorimetric assay, we detected ATPase activity of GSTtagged recombinant DMS11 protein in vitro, and this activity was approximately 3.5 times higher in the presence of coexpressed His-tagged DMS3 (Figure 4D). DMS3 acts in the so-called DDR complex, which is required for synthesis of Pol V scaffold transcripts and RdDM [18]. We therefore investigated whether DMS11, which interacts with DMS3, is also needed for synthesis of the Pol V transcript IGN5. Synthesis of IGN5 RNA was indeed reduced in the dms11 mutant, although not to the degree observed in an nrpe1 mutant (Figure 4E). This result is consistent with the
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Figure 2. DNA Methylation in the dms11 Mutant (A) Bisulfite sequencing analysis of DNA methylation of the target transgene enhancer (black bar) and the downstream region subject to methylation spreading (gray bar) [11]. Percent methylation of cytosines in CG (black lines), CHG (blue lines), and CHH (red lines) sequence contexts is shown for dms11 and wild-type plants (T+S). (B) Cumulative analysis of DNA methylation in the target transgene enhancer and downstream region in dms11 and T+S plants. (C) DNA methylation analysis of the RdDM targets AtSN1, IGN5, IGN23, IGN25, soloLTR, LTR1, LTR3, IGN26, and IGN22. DNA was digested with the indicated methylation-sensitive restriction enzymes and used for PCR amplification. DNA from wild-type T+S plants and an nrpe1 mutant was used as a control. Amplification of a region of an actin gene (Act) region lacking HaeIII and AvaII sites or undigested DNA was used as a loading control. See also Figure S2.
less extensive loss of DNA methylation at the IGN5 locus in dms11 compared to nrpe1 (Figure 2C). Discussion We have identified a GHKL-type ATPase, DMS11, as a new constituent of the RdDM pathway. GHKL ATPase domains are found in wide variety of prokaryotic and eukaryotic proteins that function in DNA structure rearrangement, heat shock, signal transduction, and DNA mismatch repair [5, 16]. The metazoan proteins most similar to DMS11 are the MORC ATPases, which belong to the GHKL superfamily of ATPases [15]. The first MORC protein was identified as a factor required for completion of male meiosis and spermatogenesis in mice [13]. MORC proteins have a domain composition similar to that found in DMS11, although they contain a CW-type zinc finger [19] between the MORC and coiled-coil domains (Figure S4E). Despite the partial similarity to MORC proteins, DMS11 and its full-length homologs are found only in flowering plants. Two major types of ATPases have been implicated previously in chromatin dynamics [16]. SWI2/SNF2 ATPases are important for local chromatin remodeling and nucleosome positioning. SMC ATPases, which belong to the ABC superfamily of ATPases, typically participate in large-scale chromatin dynamics [16]. By contrast, little is known about GHKL or MORC ATPases in chromatin remodeling and assembly [15]. Based on the functions of related proteins in prokaryotes, it has been suggested that MORC ATPases are involved in manipulation of DNA superstructure in response to epigenetic marks such as DNA methylation or histone modifications [15]. A role for DMS11 in higher order chromatin structure may be consistent with the rather modest epigenetic alterations we observed in the dms11 mutant; however, we cannot rule out that the allele we have studied is not a null. Therefore, further work is needed to test whether DMS11 is involved in altering
large-scale chromatin organization. A further consideration is that DMS11 is a member of a gene family and hence potentially redundant, which could also account for the weak effects of the dms11 mutation on DNA methylation compared to other RdDM mutants such as dms3 and nrpe1 [6]. One epigenetic change we observed at the target transgene enhancer in the dms11 mutant was an increase in histone H3 acetylation, suggesting that DMS11-mediated transcriptional gene silencing involves deacetylation of histones. Histone deacetylation was also found to be involved in the downregulation of carbonic anhydrase IX by human MORC2 [14]. DMS11 interacts physically with the SMC hinge-domain containing protein DMS3, which lacks ATPase motifs, and displays ATPase activity in vitro that is stimulated by DMS3. Two known proteins, mammalian SmcHD1 [8] and Arabidopsis GMI1 [17], comprise both GHKL ATPase domains and SMC hinge regions that are similar to the hinge region in DMS3 [6, 17]. Although GMI1 has been implicated specifically in DNA repair [17], SmcHD1 is required for murine X chromosome inactivation, which—similarly to RdDM—is an epigenetic process involving noncoding RNAs and DNA methylation [8]. Given the genetic and physical interactions between DMS11 and DMS3, and the stimulation of the ATPase activity of DMS11 by DMS3, it is reasonable to propose that these two proteins associate in vivo to form a functional analog of SmcHD1 in Arabidopsis (Figure 4B). Thus, single proteins (or protein partners) comprising GHKL ATPases and SMC hinge domains are emerging as new players in epigenetic regulation, perhaps specifically in RNA-mediated epigenetic processes, in both plants and mammals. The specific role of DMS11 in the RdDM mechanism is still not known. It may be part of the previously identified DDR complex [18], which contains in addition to DMS3 the chromatin remodeler DRD1 and RDM1, a small, plant-specific protein that coimmunoprecipitates with ARGONAUTE4 and the de novo DNA methyltransferase DRM2 [20]. The DDR complex is needed for production of Pol V scaffold transcripts that are thought to base pair with AGO4-bound siRNAs to recruit the silencing effector complex to target loci [18, 21]. It is not yet known how the DDR complex facilitates Pol V transcription. However, our finding that DMS11 is
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Figure 3. Analysis of Histone Modifications in the dms11 Mutant (A) Schematic presentation of the GFP reporter gene. The target transgene enhancer and downstream region of methylation spreading are depicted by blue and red boxes, respectively. Positions of oligonucleotides used for PCR are shown above. (B) Analysis of histone modifications in wild-type T and T+S lines. In the T line, the target enhancer and downstream region are in a transcriptionally active state (high H3K4me3 and H3ac) and in the T+S line they are in an inactive state (low H3K4me3 and H3ac, high H3K9me2). (C) Comparison of histone modifications in dms11 with the wild-type T+S line. Significant changes in dms11 include a decrease in H3K9me2 and increase in H3ac. In (B) and (C), the data shown are average values from three and four independent biological repetitions, respectively, with the error bars representing the standard deviation. Statistically significant p values as determined by Student’s t test are indicated. Semiquantitative PCR analysis of data in (B) and (C) is shown in Figures S3A and S3B. Analyses of tubulin as a euchromatin control and LTR1 and LTR3, which do not reactivate in RdDM mutants [28], as heterochromatin controls did not reveal any changes between T, T+S, dms11, and nrpe1 lines (Figure S3C), demonstrating specificity of our chromatin immunoprecipitation assays. See also Figure S3.
also needed for production of the Pol V transcript IGN5 is consistent with the participation of this protein in the DDR complex. The only other member of the DMS11 gene family for which there is functional information is At4g36290 or CRT1 (compromised recognition of TCV). CRT1 has been reported to encode an endosome-associated protein that functions in early stages of R (Resistance) gene-mediated defense signaling [22–24].
Whether there is any relationship between this function of CRT1 and the role of DMS11 in RdDM is unknown. However, two recent papers have implicated two components of the Pol V pathway, AGO4 and NRPD2a, in plant immunity [25, 26]. Further investigations of DMS11 and CRT1 as well as other members of the CRT1/DMS11 gene family may reveal additional links between GHKL ATPases, RdDM, and plant defense responses. Figure 4. DMS11 Is a GHKL ATPase
(A) Schematic presentation of DMS11 domains. The position of the dms11-1 mutation is indicated (W439*). The ATPase domain is represented by a yellow oval embedded in a red box representing the MORC homology region. CC, coiled coil. (B) DMS11 may provide the missing ATPase activity for DMS3. Domain arrangements of SMC proteins, DMS11, DMS3, and two related proteins, Arabidopsis GMI1 and murine SmcHD1, are shown. SMC proteins have an ABC-type ATPase, whereas DMS11 and GMI1/SmcHD1 have GHKL-type ATPases. DMS3 is an SMC hinge domain-containing protein with short coiled coils at N and C termini, but it lacks the ATPase domain found in bona fide SMC proteins. We propose that interactions of DMS11 with DMS3 could form the Arabidopsis functional analog of murine SmcHD1. (C) Interaction of DMS11 with DMS3. Full-length DMS11 fused to GST was coexpressed with His6-DMS3 in E. coli. Proteins were purified over glutathione Sepharose beads (Figure S4J), and bound proteins were analyzed by western blotting with anti-His antibody. Coexpression and purification of GST with His6-DMS3 or GSTDMS4 with His6-DMS3 were used as negative controls. DMS4 is a putative transcription factor isolated in the same genetic screen as DMS3 and DMS11 (Table S1). Lanes I (input): 1/10 of volume of protein extracts used for purification over glutathione Sepharose beads. Lanes B (bound): 1/5 of proteins retained on glutathione Sepharose beads after washing. (D) ATPase activity of purified recombinant GST-DMS11 or GST-DMS11 coexpressed with His-DMS3. GST alone was used as a control. The data presented are mean values of at least three measurements, with the error bars representing the standard deviation. (E) Pol V transcript IGN5 is reduced in dms11. RT-PCR analysis of IGN5 and soloLTR transcripts in wild-type (T+S), nrpe1, and dms11 lines is shown. soloLTR serves as a positive control for target locus reactivation in RdDM-defective mutants [28]. Actin RT-PCR reactions were used as loading controls. PCRs without RT were used as controls for DNA contamination. See also Figure S4.
GHKL ATPase Acts in RNA-Directed DNA Methylation 937
Experimental Procedures
References
Plant Material All mutants lines used were in the Col-0 background. Plants were grown in a growth chamber set at 21 C with a long-day light regime (16 hr light/8 hr dark). Seedlings were grown under sterile conditions on solid Murashige and Skoog medium at 23 C with a long-day light regime. For complementation analyses, wild-type DMS11 cDNA and an alternatively spliced mutant version, both under the control of the native DMS11 promoter, were introduced into the dms11 mutant using the floral dip method [27]. For each construct, we obtained several independent lines containing single copies of the respective construct. More than 50% of the plants containing the wild-type construct were GFP negative, demonstrating restoration of silencing. By contrast, all of the plants containing the mutant version were GFP positive (see Figure S4G). The complementation data thus confirm that DMS11 is At1g19100.
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Cloning Details on construction of plasmids used for protein overexpression in Escherichia coli, yeast two-hybrid assays, and complementation of dms11 mutant are available in Supplemental Experimental Procedures. DNA Methylation Methylation of the target enhancer by bisulfite sequencing (average of 20 independent clones) was carried out as described previously [6, 11]. Methylation analysis of LTRs, IGNs, and AtSN1 was carried out by methylation-sensitive PCR [28, 29]. Oligonucleotides used in these experiments are listed in Supplemental Experimental Procedures. Chromatin Immunoprecipitation Chromatin immunoprecipitation analyses were conducted as described by W. Aufsatz (http://mescaline.igh.cnrs.fr/EpiGeneSys/images/stories/ protocols/pdf/20111025150640_p13.pdf). Real-time PCR analysis was performed with a Bio-Rad iQ5 machine using SensiFAST mix from Bioline. All data are expressed relative to input. Antibodies used for immunoprecipitations were from Abcam (H3, H3K4me3, and H3K9me2) and Millipore (H3K27me and H3ac). Yeast Two-Hybrid Assay, Recombinant Protein Analysis, and Western Blotting Yeast two-hybrid assays were carried out according to the manufacturer’s instructions (Clontech). Detailed protocols for recombinant protein expression and purification are available in Supplemental Experimental Procedures. For western blotting, proteins from seedlings or rosettes were extracted as described previously [30]. SDS-PAGE and western blotting were performed according to standard procedures. The level of GFP in dms11 was determined by comparison to serial dilutions of extracts from the target (T) line, which constitutively expresses GFP. Monoclonal antiGFP (Roche), anti-tubulin (Sigma-Aldrich), and anti-His (Sigma-Aldrich) antibodies were used at 1:1,000 dilutions. ATPase Assays and siRNA Analysis See Supplemental Experimental Procedures.
Supplemental Information Supplemental Information includes four figures, one table, and Supplemental Experimental Procedures and can be found with this article online at doi:10.1016/j.cub.2012.03.061. Acknowledgments This work was supported by the Austrian Academy of Sciences and the Austrian Fonds zur Fo¨rderung der wissenschaftlichen Forschung (grant SFB F4306-B09). We thank Johannes van der Winden for editorial assistance. Received: February 28, 2012 Revised: March 16, 2012 Accepted: March 19, 2012 Published online: May 3, 2012
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