Arabidopsis CMT3 activity is positively regulated by AtSIZ1-mediated sumoylation

Plant Science 239 (2015) 209–215

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Arabidopsis CMT3 activity is positively regulated by AtSIZ1-mediated sumoylation Do Youn Kim a , Yun Jung Han a , Sung-Il Kim a , Jong Tae Song b , Hak Soo Seo a,c,∗ a b c

Department of Plant Science and Research Institute of Agriculture and Life Sciences, Seoul National University, Seoul 151-921, Republic of Korea School of Applied Biosciences, Kyungpook National University, Daegu 702-701, Republic of Korea Bio-MAX Institute, Seoul National University, Seoul 151-818, Republic of Korea

a r t i c l e

i n f o

Article history: Received 11 June 2015 Received in revised form 4 August 2015 Accepted 4 August 2015 Available online 5 August 2015 Keywords: AtSIZ1 SUMO Sumoylation CMT3 DNA methylation DNA methyltransferase

a b s t r a c t The activities of mammalian DNA and histone methyltransferases are regulated by post-translational modifications such as phosphorylation and sumoylation; however, it is unclear how the activities of these enzymes are regulated at the post-translational level in plants. Here, we demonstrate that the DNA methylation activity of Arabidopsis CHROMOMETHYLASE 3 (CMT3) is positively regulated by the E3 SUMO ligase AtSIZ1. The methylation level of the Arabidopsis genome, including transposons, was significantly lower in the siz1-2 mutant than in wild-type plants. CMT3 was sumoylated by the E3 ligase activity of AtSIZ1 through a direct interaction, and the DNA methyltransferase activity of CMT3 was enhanced by this modification. In addition, the methylation levels of a large number of genes, including the nitrate reductase gene NIA2, were lower in siz1-2 and cmt3 plants than in wild-type plants. Furthermore, the CHG methylation activity of CMT3 was specific for NIA2and not NIA1 (the other nitrate reductase gene in Arabidopsis), indicating that CMT3 selectively regulates the CHG methylation levels of target genes. Taken together, our results indicate that the sumoylation of CMT3 is critical for its role in the control of gene expression and that AtSIZ1 positively controls the epigenetic repression of CMT3-mediated gene expression. © 2015 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Protein modification is an important mechanism for regulating protein stability and function. At least 200 types of post-translational modifications occur in eukaryotic cells [1], including sumoylation by small ubiquitin-related modifier (SUMO) proteins. The mechanism of the sumoylation reaction is similar to that of ubiquitination. In brief, the maturation of precursor SUMO protein occurs via the removal of its carboxyl terminus through SUMO protease activity. Next, the C-terminal glycine of mature SUMO forms a high-energy thioester bond at a cysteine residue of activating enzyme E1. Activated SUMO is then transferred to a cysteine residue of conjugating enzyme E2 and again forms a highenergy thioester bond. Finally, SUMO is transferred to the target lysine residue by E3 ligases [2]. However, the biological roles and consequences of these two modification types are very different [2]. For example, sumoylation modulates the stability and several important functions of target proteins, such as protein–protein and

∗ Corresponding author. Fax: +82 2 873 2056. E-mail address: [email protected] (H.S. Seo). http://dx.doi.org/10.1016/j.plantsci.2015.08.003 0168-9452/© 2015 Elsevier Ireland Ltd. All rights reserved.

protein-DNA interactions, as well as subcellular localization [3]. In addition, a distinct feature of sumoylation is its ability to antagonize ubiquitin-mediated protein degradation. DNA methylation is the transfer of a methyl group to a cytosine nucleotide in DNA through DNA methyltransferase activity to form 5-methylcytosine. This transfer commonly occurs in plants and animals and is a very important epigenetic mechanism for regulating gene expression and genome stability [4]. In plants, DNA methylation occurs at symmetric CG and CHG sequences and also at non-symmetric CHH (H is A, C, T) sequences. However, it only occurs in symmetric CG sequences in animals, although nonsymmetric methylation occurs in embryonic stem cells [5,6]. In animals, three types of DNA methyltransferases (DNMTs) have been characterized. The first type is DNMT1, which is responsible for both de novo and maintenance methylation; the second type is DNMT3, which is a family of DNMTs that could methylate both hemimethylated and unmethylated CpG sequences de novo [7]. The third type is the DNMT3-like (DNMT3L) enzyme, which has DNA methyltransferase motifs and only acts as a functional regulator of DNMT3A and DNMT3B [8,9]. In plants, three DNMTs have been identified to date, namely methyltransferase 1 (MET1), chromomethylase 3 (CMT3) and domains rearranged methyltransferase

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2 (DRM2) [10–12]. MET1 maintains symmetric CG methylation activity and is a homolog of mammalian DNMT1 [13], and CMT3 is a plant-specific DNMT that has DNA methylation activity at CHG sites [14,15]. DRM2 has de novo DNA methylation activity at the CG, CHG, and CHH sites [16]. In plants, SUMO and AtSIZ1-mediated sumoylation regulate responses to nutrient deficiency, hormones and environmental stress, and they also control vegetative growth and development [17]. To date, numerous SUMO conjugates have been identified in plants [17–20]. Interestingly, recent reports suggest that human DNMT3A, DNMT3B, and DNMT1 are modified by SUMO and that their sumoylation affects protein–protein interactions [21,22] and DNMT activity [23]. Furthermore, sumoylation also affects chromatin remodeling and gene expression. These findings suggest that the activities of plant DNMTs can also be modulated by sumoylation. Thus, to investigate this possibility, we chose the plant-specific DNMT CMT3 and the E3 SUMO ligase AtSIZ1, examining whether CMT3 is sumoylated by AtSIZ1. We also examined the DNMT activity of non-sumoylated and sumoylated CMT3. In addition, we investigated the effects of CMT3and At SIZ1 mutation on genome methylation. The results demonstrate that AtSIZ1 controls the methylation of a large number of genes by sumoylating CMT3, thus leading to an increase in its methyltransferase activity. This study is the first to demonstrate that DNMT activity can be regulated in plants by sumoylation through the activity of an E3 SUMO ligase.

1% Triton X-100, 1 mM imidazole, 5 mM DTT, 2 mM PMSF and proteinase inhibitor cocktail (Roche). The proteins were then purified using Ni2+ -nitrilotriacetate resins (Qiagen). To purify GST and GSTCMT3, the bacteria were lysed in PBS (pH 7.5) containing 1% Triton X-100, 2 mM PMSF and proteinase inhibitor cocktail (Roche). The proteins were then purified using glutathione resins (Pharmacia). To purify MBP and MBP-AtSIZ1-HA, the bacteria were lysed in buffer comprising 20 mM Tris-HCl (pH 7.4), 200 mM NaCl, 1 mM EDTA, 1% Triton X-100, 2 mM PMSF and proteinase inhibitor cocktail (Roche). The proteins were then purified using amylose resins (New England BioLabs). Protein concentrations were determined using the Bradford assay (Bio-Rad). 2.4. In vitro binding assay To examine their interaction in vitro, full-length MBP-AtSIZ1 (5 ␮g) and full-length His6 -CMT3 (5 ␮g) were added to 1 ml of binding buffer comprising 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 10 mM MgCl2 , 1% Triton X-100 and 0.5 mM ␤-mercaptoethanol. After incubation at 4 ◦ C for 2 h, the reaction mixtures were incubated with an amylose resin for 1 h, and then washed six times with buffer comprising 50 mM Tris–HCl (pH 7.5), 100 mM NaCl and 1% Triton X-100. The absorbed proteins were analysed by SDSPAGE and detected by western blotting with an anti-His antibody (0.4 ␮g/ml; Santa Cruz Biotechnology). 2.5. Sumoylation assays

2. Materials and methods 2.1. Plant materials and growth conditions The Arabidopsis thaliana Columbia-0 ecotype (wild-type) and two T-DNA insertion knockout mutants (siz1-2 and cmt3) were used in this study. For plants grown on plates, the seeds were surfacesterilized in commercial bleach containing 5% sodium hypochlorite and 0.1% Triton X-100 for 10 min, rinsed five times in sterilized water and then stratified at 4 ◦ C for 3 days in the dark. The seeds were then sown on agar plates (buffered to pH 5.7) containing Murashige and Skoog medium, 2% sucrose and 0.8% agar. For plants grown in soil, the seeds were sown in sterile vermiculite. All plants, including seedlings, were incubated in a growth chamber at 22 ◦ C under a 16 h light/8 h dark cycle. 2.2. Construction of recombinant plasmids To produce CMT3 containing a polyhistidine tag (His6 -CMT3), the cDNA encoding full-length CMT3 was amplified by PCR and inserted into the pET28a vector (Novagen). To generate the maltose-binding protein (MBP)-AtSIZ1-haemagglutinin (HA) fusion, the cDNA encoding full-length AtSIZ1 was amplified by PCR using a primer tagged with HA, and then inserted into the pMALc2x vector (New England Biolabs). To generate GST-CMT3, the cDNA encoding full-length CMT3 was amplified by PCR and inserted into the pGEX4T-1 vector. The full-length cDNA encoding SUMO1 was amplified by PCR with gene-specific primers and inserted into pET28a to produce His6 -AtSUMO1-GG. The SUMO E1 and E2 constructs were kindly provided by Colby et al. [24]. All constructs were transformed into Escherichia coli strain R2 cells. 2.3. Purification of recombinant proteins The transformed cells were treated with IPTG to induce expression of the fusion proteins. To purify His6 -AtSAE1b, His6 -AtSAE2, His6 -AtSCE1, His6 -AtSUMO1 and His6 -CMT3, the bacteria were lysed in buffer comprising 50 mM NaH2 PO4 (pH 8.0), 300 mM NaCl,

In vitro sumoylation was performed in 30 ␮l of reaction buffer (20 mM HEPES (pH 7.5), 5 mM MgCl2 and 2 mM ATP) containing 1 ␮g of MBP-AtSIZ1, 200 ng of His6 -AtSAE1b, 200 ng of His6 -AtSAE2, 200 ng of His6 -AtSCE1, 5 ␮g of His6 -AtSUMO1-GG and 500 ng of GST-CMT3. After incubation at 30 ◦ C for 3 h, the reaction mixtures were separated by 7% SDS-PAGE. Sumoylated GST-CMT3 was detected by immunoblotting with an anti-GST antibody (0.2 ␮g/ml; Santa Cruz Biotechnology). 2.6. Effect of sumoylation on the DNMT activity of CMT3 The 40 ␮l in vitro DNA methylation reactions contained 2 ␮l of S-adenosyl-l-[methyl-3 H] methionine (SAM) (55–85 Ci/mmol; American Radiolabeled Chemicals), 4 mM H3K9me2 (64624-025; Anaspec), 200 ng of target DNA and the appropriate concentration of GST or GST-CMT3. The methylation reaction was performed in assay buffer (50 mM Tris–HCl (pH 8.0), 5 mM NaCl and 5% glycerol) for 3 h at 37 ◦ C, and then stopped by adding 2 ␮l of proteinase K (Promega). Each reaction was applied to DE81 paper (Whatmann) and washed twice with 200 mM ammonium bicarbonate, twice with water, and twice with 70% ethanol. The paper was then dried and placed into liquid scintillation cocktail (GE Healthcare). DNA methylation was determined by liquid scintillation counting (LSC). To examine the effect of sumoylation on the DNMT activity of CMT3, an in vitro sumoylation reaction was performed as described above. After the reaction, the components required for in vitro DNA methylation were added to the reaction products, which were incubated for a further 3 h at 37 ◦ C, as described above. Finally, the methylation levels of the target DNA were examined by LSC. The H3K9 peptide used in this study contained amino acids 1–21 of the N-terminal sequence of histone H3, including the di-methyl K9 residue. The following oligonucleotide contained methylated CHG: 5 -ATTCAGTCAGATCTGATCAGTACTGATT-3 . 2.7. Examination of DNA methylation by McrBC-PCR The procedure used for the 5-methylcytosine-specific restriction enzyme McrBC-based methylation analysis was based on a

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protocol published previously [25]. Genomic DNA (500 ng) isolated from 4-week-old wild-type, siz1-2 and cmt3 mutant plants was digested with 30 units of McrBC endonuclease (New England Biolabs) for 3 h at 37 ◦ C. Subsequently, quantitative PCR (ABI Prism 79000 HT) analyses were performed using Vivagen DNA polymerase and 50 ng of the digested genomic DNA as template. The cycling conditions were as follows: 95 ◦ C for 5 min, followed by 28 cycles of 95 ◦ C for 1 min and 61 ◦ C for 30 s, and then a final incubation at 72 ◦ C for 30 s. The PCR products were confirmed by agarose gel electrophoresis. The primers used for quantitative PCR were as follows: ATGP1, 5 -ACAGTGCCACAGTTGAGCAG-3 and 5 -CAGAAAAATACTCGGTGCCAAT-3 ; ATCOPIA4, 5   CCTTTCCCTTCTCCAACCTC-3 and 5 -TTGTCGGCTGTGATGAATGTATLINE1-4, 5 -GTGGCACAGAAAGCAGAACA-3 3 ; and 5 -ACGGAGTATCCAACCTGTGC-3 ; NIA1, 5 -GCTAGTAAGCATAAGGAGAG-3 and 5 CCTTCACGTTGTAACCCATCTTCT-3 ; NIA2, 5 -TGTCTCAGTACCTAGACTCTTTGC-3 and NRT2.6, 5 5 -GACGTACATTTCAGTCTCAT-3 ; GGAGCTCTCTTTGGTGTTGC-3 and 5 -CTCTCTGACCGGCTGTTTTCVIP1, 5 -GAAACCTCATCGAACGGTGT-3 and 3 ; 5 -GGATCAAGCAAAGCAAGCTC-3 ; PHYD, 5   GGCTCTATTGCGTCGTTAGC-3 and 5 -TGCCGCACCATTACATTTTA3 ; IAA7, 5 -TCGGCCAACTTATGAACCTC-3 and and tubulin, 5 5 -CTGCTCCTCCACCAAAGTTC-3 ;   GTGAGCGAACAGTTCACAGC-3 and 5 -TTATTGCTCCTCCTGCACTT -3 . 2.8. Bisulphite sequencing Genomic DNAs were isolated from rosette leaves of 4-weekold wild-type, siz1-2 and cmt3 mutant plants. Bisulphite treatment and recovery of samples were carried out using the EpiTect Bisulfite Kit (59124; Qiagen), according to the manufacturer’s instructions. Briefly, the 20 ␮l reaction containing 2 ␮g of DNA was mixed with 85 ␮l of bisulphite mix and 35 ␮l of DNA protect buffer. Bisulphite conversion was performed by incubating the mixture at 99 ◦ C for 5 min, followed by 60 ◦ C for 25 min, 99 ◦ C for 5 min, 60 ◦ C for 85 min, 99 ◦ C for 5 min, 60 ◦ C for 175 min and then 20 ◦ C indefinitely. The bisulphite-treated DNA was recovered using EpiTect spin columns and sequenced to confirm the efficiency of bisulphite conversion. The primers were designed using MethPrimer software (http://www.urogene. org/methprimer/). The percentage methylation (% C) was calculated as 100 × C/(C + T). Cytosine methylation in the CG, CHG and CHH contexts was analysed and displayed using CyMATE [26]. 2.9. Methylated DNA immunoprecipitation Genomic DNA was sonicated to produce random fragments of 200–600 bp. The fragmented DNA (4 mg) was used in a standard methylated DNA immunoprecipitation (MeDIP) assay, as described previously [27]. Briefly, following denaturation at 95 ◦ C for 10 min, immunoprecipitation (IP) was performed at 4 ◦ C for 2 h using 10 ␮g of a monoclonal antibody against 5-methylcytidine (315-80541; Diagenode) in a final volume of 500 ␮l of IP buffer (10 mM sodium phosphate (pH 7.0), 140 mM NaCl and 0.05% Triton X-100). The mixture was incubated with 40 ␮l of Dynabeads and an M-280 sheep antibody against mouse IgG (Dynal Biotech) for 12 h at 4 ◦ C, and then washed with 700 ␮l of IP buffer seven times. The beads were then treated with proteinase K for 4 h at 50 ◦ C, and the methylated DNA was recovered by phenol-chloroform extraction, followed by ethanol precipitation.

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2.10. Illumina Genome Analyzer sequencing To perform second strand synthesis of MeDIP-enriched ssDNA fragments, samples containing approximately 200 ng of DNA and 500 ng of random primer in a final volume of 57.9 ␮l were incubated at 70 ◦ C for 10 min, and then cooled gradually for 40 min. Subsequently, 2 ml of 2.5 mM dNTPs, 20 ␮l of 5× second strand buffer (100 mM Tris–HCl (pH 7.5), 500 mM KCl, 25 mM MgCl2 , 50 mM (NH4 )2 SO4 and 250 mg/ml BSA), 10 ␮l of 100 mM DTT, 3 ␮l of 5 mM beta-NAD+ , 0.5 ␮l (5 U) of E. coli DNA ligase (TaKaRa Bio) and 6.6 ␮l (25 units) of E. coli DNA polymerase I (TaKaRa Bio) were added to the sample (100 ␮l final volume). The reaction was incubated at 14 ◦ C for 12 h, and the dsDNA fragments were then purified using a PCR purification kit (Qiagen). End-repair of the DNA fragments, addition of an adenine residue to the 3’ ends of the fragments, adaptor ligation and PCR amplification using Illumina paired-end primers were performed as described previously [27]. After agarose gel electrophoresis, the bands were excised to produce libraries with insert sizes of 250–350 bp, which were quantified using Quant-iT PicoGreen dsDNA Reagent and Kits (Invitrogen). Flow cells were prepared with 8 pM DNA according to the manufacturer’s recommended protocol, and were sequenced for 36 cycles on an Illumina Genome Analyzer II. The obtained images were analysed and basecalled using GA pipeline software with default settings (version 1.3; Illumina). 2.11. Mapping reads The Arabidopsis genome sequence was downloaded, and the reads were mapped onto the Arabidopsis genome reference sequence (BSgenome.Athaliana.TAIR.TAIR9) using Bowtie2. The peaks for each sample were called using Peak Analyzer with TAIR10 GTF information (false discovery rate < 0.05). The sequenced reads were used as a control for each type and categorized methylation sequence coverage gene. 3. Results 3.1. Genome methylation is reduced in siz1-2 mutant plants A recent study demonstrated that the CpG methylation activity of human DNMT1 is enhanced by SUMO conjugation in vitro and vivo [23], suggesting that sumoylation may regulate the catalytic activities of other DNA and histone methyltransferases. In addition, AtSIZ1 has an E3 SUMO ligase activity towards various proteins [2], suggesting that it may function as an E3 SUMO ligase of some DNA and histone methyltransferases. Hence, we examined the methylation levels of three transposable elements, ATGP1, ATCOPIA4 and ATLINE1-4, in siz1-2 mutant plants. The cmt3 mutant was also used as a positive control because CMT3 is a plant-specific DNMT [15]. For this experiment, the samples were grown in soil for 4 weeks (Fig. 1A), and then genomic DNAs isolated from each sample were treated with the 5-methylcytosine-specific restriction enzyme McrBC. PCR amplification of the digested samples using gene-specific primers revealed that the methylation levels of the transposable elements were lower in the siz1-2 and cmt3 mutants than the wild-type plants (Fig. 1B). Next, a MeDIP sequencing analysis was performed to determine whether the methylation level of the whole genome was downregulated in the siz1-2 and cmt3 mutants. As expected, enrichments of methylated CpG and C regions were lower in the siz1-2 and cmt3 mutants than the wild-type samples (Table 1). Genomic coverage was also quantified by counting of the number of reads that overlapped with CpG islands. This analysis showed that the CpG coverage and sequence pattern coverage were lower in the

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Fig. 1. DNA methylation of transposable elements was decreased in siz1-2 and cmt3 mutants. (A) Photographs of wild-type, siz1-2 and cmt3 plants grown in soil for 4 weeks. (B) The levels of methylation of three transposable elements (ATGP1, ATCOPIA4 and ATLINE1-4) in genomic DNA samples extracted from the plants shown in (A). PCRs were performed with gene-specific primers after treatment of the DNA samples with McrBC. The gene encoding tubulin was used as a control.

Table 1 CpG and C enrichment analysis of wild-type, siz1-2and cmt3 plants by MeDIP sequencing.

CG regions C regions Total base (bp) Alignment rate (%)

WT

siz1-2

cmt3

15,407,837 105,277,236 3,101,970,144 79.67

8,924,729 60,661,714 3,161,486,736 82.43

12,271,936 84,371,718 3,254,753,514 73.74

siz1-2 and cmt3 mutants than the wild-type samples (Supplementary Fig. 1A and B). Moreover, pairwise correlations of wild-type/wild-type, wild-type/ siz1-2 and wild-type/ cmt3 samples using scatter plots confirmed the difference in the genome methylation patterns between wild-type and siz1-2 or cmt3 mutant plants (Supplementary Fig. 2). 3.2. CMT3 is sumoylated by AtSIZ1 The results of the McrBC-PCR and MeDIP analyses described above suggest that genome methylation is affected by sumoylation mediated through the E3 ligase activity of AtSIZ1. To determine whether AtSIZ1 regulates the methylation activity of DNMTs, we chose to examine CMT3 because it has plant-specific methyltransferase activity for CHG sequences and contains three possible sumoylation sites at amino acid positions 431, 696, and 706 (Fig. 2A). To determine whether AtSIZ1 can interact with CMT3, two recombinant plasmids expressing MBP-tagged AtSIZ1 and His-tagged CMT3 were generated; overexpression of these tagged proteins in E. coli was induced by IPTG treatment, and the proteins were purified with amylose and nickel resins, respectively (Fig. 2B, left). After purification, in vitro pull-down assays were performed using MBP or MBP-AtSIZ1, and CMT3 was detected using an anti-His antibody. As expected, this experiment revealed a physical interaction between AtSIZ1 and CMT3 (Fig. 2B, right), suggesting that AtSIZ1 can act as an E3 SUMO ligase for CMT3. To confirm this hypothesis, an in vitro sumoylation assay was performed using the recombinant proteins MBP-AtSIZ1 and GSTCMT3. In this experiment, GST-CMT3 was sumoylated by AtSIZ1, and the reaction was dependent on E1 and E2 activity (Fig. 2C). By contrast, the control material (BSA) had no effect on CMT3 sumoylation. Previous studies have demonstrated that direct SUMO transfer from the SUMO-conjugating E2 enzyme to the target protein can occur through ligase-independent mechanisms. For example, Ubc9 can directly recognize the sumoylation motif -K-x-[D/E] (, an aliphatic branched amino acid; x, any amino acid) and conjugate the lysine residue [28]. In addition, some SUMO substrates contain SUMO-interacting motifs (SIMs). These SIMs bind to the SUMO

Fig. 2. CMT3 is sumoylated by AtSIZ1 in vitro. (A) The deduced amino acid sequence of the CMT3 protein. Putative sumoylation sites (␺KXE) identified using the SUMOplot Analysis Program are shown in bold and underlined type. (B) In vitro pull-down assays showing the interaction of AtSIZ1 with CMT3. MBP, MBP-AtSIZ1 and His6 CMT3 were overexpressed in E. coli and purified using amylose (MBP) or nickel (His) resins. The purified proteins were separated by 11% SDS-PAGE (left). The asterisks indicate MBP, MBP-AtSIZ1 and His6 -CMT3. CMT3 was pulled down with full-length AtSIZ1. His6 -CMT3 bound to MBP-AtSIZ1 was detected by western blotting with an anti-His antibody (right). (C) His6 -AtSAE1b, His6 -AtSAE2, His6 -AtUBC9, MBP-AtSIZ1, His6 -AtSUMO1-GG and GST-CMT3 were overexpressed in E. coli and purified with amylose (MBP), glutathione (GST) or nickel (His) resins. The E3 ligase activity of AtSIZ1 towards CMT3 was determined in the presence or absence of His6 -AtSAE1+2, His6 -AtUBC9, MBP-AtSIZ1, His6 -AtSUMO1-GG and GST-CMT3. After the reaction, sumoylated CMT3 was detected by western blotting with an anti-GST antibody.

moiety to which Ubc9 is attached, thereby facilitating sumoylation [29]. However, in the current study, if AtSIZ1 was not included in the reaction, CMT3 was not sumoylated, even in the presence of high concentrations of enzymes E1 (200 ng) and E2 (200 ng). Overall, these results indicate that CMT3 is sumoylated by the E3 ligase activity of AtSIZ1.

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Fig. 3. The methyltransferase activity of CMT3 is stimulated by sumoylation in vitro. (A) The GST-CMT3 protein was overexpressed in E. coli, purified using a glutathione column and then separated by 10% SDS-PAGE. (B) The in vitro methyltransferase activity of GST-CMT3. Either GST or GST-CMT3 was mixed with 1 or 2 ␮g of oligomer and the methyl donor SAM. After the reaction, the methylated oligomers were purified, and the incorporation of 3 H-methyl groups was determined by LSC. (C) Sumoylation reactions of GST-CMT3 performed without (reaction 1) or with (reaction 2) AtSIZ1. (D) The methyltransferase activities of sumoylated and unmodified GST-CMT3. One microgram of oligomer and SAM were added to reaction products 1 and 2. The incorporation of 3 H-methyl groups was determined by LSC.

3.3. Sumoylation enhances the DNMT activity of CMT3 It is well-known that sumoylation affects various aspects of target protein function [3]. Therefore, we examined the effect of sumoylation on the enzymatic activity of CMT3. To this end, GSTCMT3 was overexpressed in E. coli and purified using a glutathione column (Fig. 3A). The catalytic activity of GST-CMT3 was measured using a hemi-methylated oligonucleotide substrate and SAM as a methyl donor. GST alone was also used as a control. The level of 3 Hmethyl-incorporated oligonucleotide was increased by GST-CMT3, and the ratio of 3 H-methyl-incorporation was proportional to the amount of oligonucleotide added (Fig. 3B), indicating that purified GST-CMT3 has a DNMT activity. To investigate the effect of sumoylation on CMT3 activity, in vitro sumoylation reactions were performed with or without AtSIZ1 (Fig. 3C), and the DNMT activities of the reaction products were determined. The catalytic activity of the reaction containing sumoylated CMT3 was markedly higher than that of the reaction containing unmodified CMT3 (Fig. 3D), indicating that sumoylation stimulates the DNA methylation activity of CMT3. 3.4. Methylation of the NIA2gene is regulated by AtSIZ1 and CMT3 Comparisons of whole genome methylation using MeDIP sequencing analyses showed that, compared with those in wild-

type plants, the methylation levels of 18,010 and 16,056 gene loci were down-regulated in the siz1-2 and cmt3 mutants, respectively (Supplementary Tables 1 and 2). Of these, the methylation levels of 10,789 gene loci were commonly down-regulated in both mutants (Supplementary Table 3), suggesting that the methylation status of various genes can be regulated by AtSIZ1-mediated sumoylation of CMT3. To investigate this finding further, we examined the methylation statuses of several genes that are involved in nitrogen assimilation, flowering, light perception or auxin signalling. PCR analyses of McrBC-treated genomic DNA samples revealed that the methylation levels of the nitrate reductase gene NIA2, the nitrate transporter gene NRT2.6, the flowering repressor gene VIP1, the photoreceptor gene PHYD and the auxin-responsive protein gene IAA7 were lower in the siz1-2 and cmt3 mutants than in the wild-type plants (Fig. 4). Arabidopsis contains two nitrate reductase genes, NIA1 and NIA2; however, unlike that of the NIA2 gene, the methylation level of the NIA1 gene was not affected by knockout of SIZ1 or CMT3 (Fig. 4). Subsequent analyses using bisulphite sequencing to confirm the McrBC-PCR data revealed that the level of CHG methylation of the NIA2gene was lower in siz1-2 mutants than in wild-type plants (Table 2B), while there was no CHG methylation of the NIA1gene in either the wild-type, siz1-2 or cmt3 plants (Table 2A). In addition, CHG methylation of the NIA2 gene was not observed in the cmt3 mutants (Table 2B). These results indicate that CMT3 specifically controls CHG methylation of the NIA2 gene.

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4. Discussion

Fig. 4. DNA methylation of various genes, including NIA2, is reduced in siz1-2 and cmt3 mutants. The methylation statuses of the nitrate reductase genes NIA1 and NIA2, the nitrate transporter gene NRT2.6, the flowering repressor gene VIP1, the photoreceptor gene PHYD, and the auxin-responsive gene IAA7 in McrBC-treated genomic DNA samples, as assessed by PCR using gene-specific primers. The gene encoding tubulin was used as a control. Table 2 Methylation analysis of NIA1 and NIA2 genes by bisulfite sequencing. (A) NIA1 WT

CG CHG CHH All

Methylated (%)

Non-methylated (%)

10.00 0.00 0.00 1.89

90.00 100.00 100.00 98.11

Methylated (%) 5.55 0.00 3.89 3.85

Non-methylated (%) 94.44 100.00 96.10 96.15

Methylated (%) 0.00 0.00 2.56 1.92

Non-methylated (%) 100.00 100.00 97.43 93.08

siz1-2 CG CHG CHH All cmt3 CG CHG CHH All (B) NIA2 WT

CG CHG CHH All

Methylated (%)

Non-methylated (%)

11.11 11.11 16.66 14.81

88.88 88.88 83.33 85.19

Methylated (%) 15.78 5.55 9.72 10.09

Non-methylated (%) 84.21 94.44 90.27 89.91

Methylated (%) 11.11 0.00 3.03 4.05

Non-methylated (%) 88.88 100.00 96.96 95.95

siz1-2 CG CHG CHH All cmt3 CG CHG CHH All

The pleiotropic phenotypes of siz1-2 mutant plants suggest that AtSIZ1 participates in a large number of signal transduction and developmental pathways. In fact, accumulating evidence suggests that AtSIZ1 plays a role in regulating nutrient assimilation, stress responses, hormone responses, vegetative growth and development, and flowering [17–20]. The current results demonstrate that Arabidopsis CMT3 is sumoylated through the E3 ligase activity of AtSIZ1. Consistent with this modification, the sumoylation of CMT3 stimulates its methylation activity towards target DNA. Sumoylation regulates various cell-signaling processes in mammalian systems [3]. In addition, the sumoylation system modulates epigenetic gene expression; for example, sumoylated DNMT3A does not interact with histone deacetylases and is not able to repress transcription [22]. In addition, sumoylating DNMT1 increases its methylation activity in chromatin [23]. These findings suggest that epigenetic gene expression in plants can also be regulated by sumoylation. Indeed, in the current study, the siz1-2 mutants exhibited low levels of whole-genome methylation (Table 1), and sumoylation of SLY1 by AtSIZ1 increased the DNA methylation activity of CMT3 (Fig. 3), indicating that AtSIZ1 tightly controls CMT3-mediated DNA methylation and gene expression. However, the fact that whole-genome methylation levels were lower in the siz1-2 mutants than in the cmt3 mutant also suggests that AtSIZ1 may be able to directly or indirectly control the activity of other DNMTs, such as MET1 and DRM2 [10–12]. The observed reductions in the methylation levels of more than 10,000 genes in both siz1-2 and cmt3, including genes related to nitrate signaling, light perception, auxin signaling, and flowering (Fig. 4 and Supplementary Table 3), indicate that the modification of these genes is regulated by the AtSIZ1-mediated sumoylation of CMT3. In addition, the low level of whole-genome methylation in siz1-2 compared to cmt3 (Fig. 1) suggests that the DNA methylation of a large number of Arabidopsis genes can also be controlled by the AtSIZ1-mediated sumoylation of other DNMTs. Arabidopsis contains two nitrate reductase genes in its genome (NIA1and NIA2). We previously reported that the nitrate reductase activities of NIA1 and NIA2 are stimulated by sumoylation through the E3 ligase activity of AtSIZ1 [30]. However, the current results demonstrate that CMT3 has CHG methylation activity only for NIA2 but not for NIA1 (Fig. 4 and Table 2), and the CHG methylation of NIA2 is only regulated by the AtSIZ1-mediated sumoylation of CMT3 (Fig. 4 and Table 2). However, the mechanism by which CMT3 distinguishes between NIA1 and NIA2 and specifically methylates only NIA2 is currently unclear. An interesting finding was recently reported, i.e., DNMT3A is citrullinated by peptidylarginine deiminase (PADI4), and citrullination stimulates the DNA methyltransferase activity of DNMT3A, resulting in an increased methylation level of the target gene [31], although it is still unknown whether citrullination can induce the sumoylation of the target protein. Citrullination is the elimination of the positive charge on the arginine side chain, which can disrupt or promote the interaction of the target protein with neighboring proteins, thereby affecting protein structure and function [32–34]. CMT3 can also be citrullinated by a plant PADI4 homolog. Therefore, citrullination of CMT3 can modulate its ability to interact with partners involved in targeting CMT3 and DNA methylation to specific regions of the genome. Thus, the findings also suggest that citrullination can act as a factor that helps CMT3 distinguish between NIA1 and NIA2, as well as between CHG and CHH sequences. It is unclear how sumoylation stimulates the DNA methylation activity of CMT3. CMT3 is known to interact with heterochromatin protein 1 and histones [35], but our understanding of how this protein is structurally tuned to exert its function is currently limited. Notably, our data demonstrate that purified CMT3 is activated by

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sumoylation in vitro; this process appears to occur independently of other interacting partners. Presumably, sumoylation results in a conformational change that stimulates methylation or enhances the ability of CMT3 to interact with DNA. However, it is still possible that CMT3-interacting partners can affect CMT3 sumoylation and, therefore, its methyltransferase activity in plants. Further examination of the interactions between HPT1 and sumoylated CMT3 or non-sumoylated CMT3 may provide clues about how sumoylation regulates the DNA methylation activity of CMT3. Nonetheless, the observation that CMT3 is sumoylated provides new insights into the regulation of this protein and its role in various biological processes, such as protein–protein interactions, enzyme activity, and subcellular localization. AtSIZ1 participates in stress and hormone responses, as well as growth and development [17–20], indicating that the expression and activity of AtSIZ1 are regulated by endogenous signals and exogenous stimuli. The current data show that CMT3 activity is modulated by the E3 ligase activity of AtSIZ1 (Fig. 3) and is also involved in the methylation of various genes related to nitrate signaling, light perception, auxin signaling, and flowering (Fig. 4 and Supplementary Table 3). These data strongly suggest that, together, AtSIZ1 and CMT3 play important roles in the control of global genome methylation levels at various stages of growth and development. In conclusion, this study demonstrates that sumoylation has a direct effect on CMT3-mediated DNA methylation; however, the mechanism by which CMT3 selectively methylates target genes remains unknown. Future studies examining the interplay between sumoylated CMT3 and other interacting factors will provide further insights into this matter and will help elucidate the specific control of CMT3 activity. Acknowledgements Arabidopsis SUMO E1 and E2 enzyme-encoding constructs were kindly provided by Dr Hans-Peter Stuible, Department of Plant Microbe Interactions, Max Planck Institute for Plant Breeding Research, Germany. This work was supported by a grant from the Next-Generation BioGreen 21 Program (Plant Molecular Breeding Center no. PJ01108701), Rural Development Administration, Republic of Korea. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.plantsci.2015.08. 003 References [1] C. Walsh, Posttranslational Modification of Proteins: Expanding Nature’s Inventory, vol. Xxi, Roberts and Co. Publishers, Englewood, Colorado, 2006, pp. 2006. [2] H.J. Park, D.J. Yun, New insights into the role of the small ubiquitin-like modifier (SUMO) in plants, Int. Rev. Cell Mol. Biol. 300 (2013) 161–209. [3] G.J. Praefcke, K. Hofmann, R.J. Dohmen, SUMO playing tag with ubiquitin, Trends Biochem. Sci. 37 (2012) 23–31. [4] J.G. Herman, S.B. Baylin, Gene silencing in cancer in association with promoter hypermethylation, N. Engl. J. Med. 349 (2003) 2042–2054. [5] B. Ramsahoye, D. Biniszkiewicz, F. Lyko, V. Clark, A.P. Bird, et al., Non-CpG methylation is prevalent in embryonic stem cells and may be mediated by DNA methyltransferase 3a, Proc. Natl. Acad. Sci. U. S. A. 2000 (2000) 5237–5242. [6] R. Lister, M. Pelizzola, R.H. Dowen, R.D. Hawkins, G. Hon, et al., Human DNA methylomes at base resolution show widespread epigenomic differences, Nature 462 (2009) 315–322.

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