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First published online April 1, 2005; 10.1105/tpc.105.031567 © 2005 American Society of Plant Biologists
DDM1 Binds Arabidopsis Methyl-CpG Binding Domain Proteins and Affects Their Subnuclear Localization
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INTRODUCTION |
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; Wan et al., 1999).
The importance of cytosine methylation for plant development was first demonstrated by treatment of plants with the hypomethylating agent 5-azacytidine and later by various genetic manipulations (Richards, 1997, and references therein). Phenotypic perturbations were reported in plants displaying a reduction in total genomic cytosine methylation, such as met1 mutants, where the DNA methyltransferase MET1 is misregulated (Finnegan et al., 1996; Ronemus et al., 1996; Kankel et al., 2003; Saze et al., 2003), or in mutants for the gene encoding the SWI2/SNF2 nucleosomal remodeling factor DDM1; in the ddm1-2 mutant, developmental abnormalities were progressively acquired during generations (Kakutani et al., 1996; Jeddeloh et al., 1999). On the other hand, plants carrying mutations in the gene coding for CHROMOMETHYLASE3 (CMT3), an enzyme required for maintenance of CpNpG methylation, displayed a wild-type phenotype (Lindroth et al., 2001).
Linkage between DNA methylation and histone methylation was demonstrated in Neurospora crassa and Arabidopsis thaliana. In N. crassa, the histone methyltransferase DIM5 is required for DNA methylation (Tamaru and Selker, 2001), whereas in Arabidopsis, the histone methyltransferase Kryptonite/SUVH4 controls CpNpG and CpNpN though not CpG methylation (Jackson et al., 2002; Malagnac et al., 2002). The interplay between DNA methylation and histone methylation in Arabidopsis is not clear. In ddm1 and met1 mutants, a reduction in H3K9 methylation was noted at certain heterochromatic regions while total H3K9 methylation remained unaffected, raising the question whether CpG methylation guides H3K9 methylation (Soppe et al., 2002; Tariq et al., 2003) or vice versa (Gendrel et al., 2002).
Recent data implicated mammalian MBD proteins in linking DNA methylation with histone methylation; MeCP2 was found to interact with histone methyltransferase and induce H3K9 methylation (Fuks et al., 2003), whereas MBD1 was found to interact with the Suv39h1-HP1 heterochromatic complex and induce DNA methylation-based transcriptional repression (Fujita et al., 2003). The finding that MBD proteins associate with histone deacetylases both in plants and animals (Hendrich and Tweedie, 2003; Zemach and Grafi, 2003) suggests that MBDs may induce heterochromatin formation by coordinating the activities of histone deacetylases and histone methyltransferases.
Several reports have characterized the MBD group of proteins in Arabidopsis and showed their capability to bind methylated CpG sites (Berg et al., 2003; Ito et al., 2003; Scebba et al., 2003; Zemach and Grafi, 2003). Although these reports demonstrated some differences among AtMBDs with respect to CpG binding activity, it becomes clear that the Arabidopsis MBD protein family is composed of at least two groups: one binds methylated CpG sites and the other does not. Binding to DNA independently of methylation was demonstrated for AtMBD11 (Scebba et al., 2003), whose downregulation by RNA interference induced developmental abnormalities (Berg et al., 2003). Using AtMBD fused to green fluorescent protein (GFP), Scebba et al. (2003) demonstrated that the heterochromatic distribution of AtMBD5 and AtMBD6 was affected by treatment with 5-azacytidine, thus confirming the importance of cytosine methylation for their subnuclear distribution.
Here, we employed genetic and biochemical approaches to study in planta the association of AtMBD proteins with methylated CpG sites and the molecular mechanism underlying their subnuclear distribution. To this end, AtMBDs were fused to GFP and either transiently expressed in Arabidopsis cells or stably transformed into Arabidopsis plants. Our results demonstrated differential subnuclear localization of AtMBDs in Arabidopsis cells, both heterochromatic and euchromatic; localization at specific nuclear domains may be facilitated by additional factors, such as the chromatin remodeling factor DDM1.
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). To study the interaction of AtMBDs with methylated DNA in vivo, AtMBDs were tagged at their C termini with GFP and placed downstream from the 35S promoter. The GFP fusion constructs were transformed into Arabidopsis leaf protoplasts by the polyethylene glycol methodology. In Arabidopsis, methylated DNA is found mainly at heterochromatic chromocenters (Figure 1A; see also Fransz et al., 2002; Soppe et al., 2002). In transient expression assays, AtMBD7-GFP was evenly distributed at all chromocenters (Figure 1B), whereas AtMBD5 and 6 showed preference for two large domains adjacent to the nucleolus (Figure 1B; see Supplemental Figure 1A online). By contrast, AtMBD2, previously shown incapable of binding in vitro methylated CpG sites (Zemach and Grafi, 2003), was dispersed within the nucleus but excluded from chromocenters and the nucleolus (Figure 1B; see Supplemental Figure 1B online).
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AtMBD6 Is Tightly Bound to Chromatin and Is Associated with Centromeric Repeats and the 18S rDNA
We estimated the strength of AtMBD6-GFP binding with chromatin by salt extraction of nuclei prepared from transgenic plants expressing AtMBD6-GFP. Nuclei were incubated in the presence of increasing concentrations of NaCl after which the soluble and the insoluble pellet fractions were resolved by SDS-PAGE. Immunoblotting analysis using 
GFP showed that AtMBD6-GFP was composed of two fractions: a major fraction (
85%), which was tightly associated with chromatin and could not be released even under 500 mM NaCl, and a minor fraction (15%), which was loosely associated with chromatin and was released to the soluble fraction with as low as 200 mM NaCl (Figure 2A). As expected, a control histone H3 was strongly associated with chromatin as revealed with anti-K4–methylated histone H3 (-K4m2H3). We also analyzed whether the dispersal of AtMBD2-GFP within the nucleus reflects the presence of AtMBD2 in the soluble nuclear fraction. Nuclei prepared from transgenic Arabidopsis expressing AtMBD2-GFP were subjected to salt extraction. Results showed that AtMBD2-GFP is not found in the soluble fraction but rather strongly bound to chromatin (see Supplemental Figure 2 online).
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GFP), dimethylated H3K4 (K4m2H3), a known histone modification associated with transcriptionally active chromatin (Fischle et al., 2003; Lachner et al., 2003), as well as dimethylated H3K9 (K9m2H3), a known histone modification associated with chromatin compaction and gene silencing (Jenuwein and Allis, 2001; Zhang and Reinberg, 2001). Precipitated DNAs were subjected to PCR to amplify a set of DNA sequences, including centromeric 180-bp repeats (CEN180), the 18S rDNA, as well as actin-encoding sequence. Results showed that anti-GFP precipitated CEN180 and 18S rDNA but not the actin-encoding sequence (Figure 2B). The association of the 18S rDNA with both K4- and K9-dimethylated histone H3 reflects the diverse chromatin configurations of rRNA gene clusters, being either transcriptionally active or inactive. As expected, the actin-encoding sequence was precipitated mainly with K4m2H3, slightly with K9m2H3, but not with GFP (Figure 2B), confirming the association of AtMBD6 with the transcriptionally inactive chromatin. The two large perinucleolar domains to which AtMBD5 and 6 bind are likely to define chromocenters of the acrocentric chromosomes 2 and/or 4, which together with the nucleolar organizing region form a large domain of heterochromatin. To assess this possibility, fixed nuclei from transgenic Arabidopsis expressing AtMBD6-GFP were first immunolabeled with anti-GFP followed by fluorescence in situ hybridization (FISH) with tetramethylrhodamine-5-dUTP–labeled CEN180 or 18S rDNA. Results showed that AtMBD6-GFP colocalized with the intensely DAPI-stained chromocenters and was associated with CEN180, displaying preference for perinucleolar chromocenters adjacent to the 18S rDNA (Figure 2C). Thus, AtMBD5 and 6 preferentially bind chromocenters of chromosome 2 and/or 4, where they might regulate chromatin compaction and silencing of rRNA genes.
Mutations in DDM1 and MET1, but Not in CMT3, Disrupt the Localization of AtMBD Proteins
To confirm the preference of AtMBD5, 6, and 7 for methylated CpG sites, we examined the distribution of AtMBD-GFPs in three Arabidopsis DNA methylation mutants: ddm1-2, a mutation in the DDM1 gene encoding the SWI2/SNF2 chromatin remodeling factor; met1-1, a mutation in MET1 DNA methyltransferase gene—both of which show reduction in CpG methylation (Kakutani et al., 1996; Kankel et al., 2003); cmt3, a mutation in the CMT3 gene that reduces CpNpG and CpNpN methylation (Bartee et al., 2001; Lindroth et al., 2001). Reduced DNA methylation in ddm1-2 and met1-1 was demonstrated by the sensitivity of the centromeric 180-bp repeats as well as the 18S rDNA to digestion by the methylation-sensitive HpaII enzyme (data not shown). In ddm1-2 and met1-1 cells, the subnuclear localization of AtMBD5, 6, and 7 was disrupted (Figure 3A). A greater effect was observed in ddm1-2 cells where AtMBD5 and 6 were evenly dispersed within the nucleus. In met1-1 (Figure 3A), as well as in met1-3 cells, where CpG methylation is thought to be almost completely lacking (Tariq et al., 2003), AtMBD5 and 6 showed patchy distribution with a certain fraction of these proteins still associated with chromocenters (see Supplemental Figure 3A online). To quantify these differences, we compared the magnitude of fluctuation in fluorescence intensity by determining the coefficient of variation for each population of nuclei (shown in Supplemental Figure 3A online) using the NIH Image program (Htun et al., 1999; see Methods). In wild-type cells, the coefficient of variation (see Supplemental Figure 3B online) was the highest (0.772 ± 0.18), indicating very high fluctuation in fluorescence intensity of AtMBD6-GFP within the nucleus. The coefficient of variation for met1-1 (0.239 ± 0.06) and met1-3 (0.225 ± 0.062) was significantly higher (approximately twofold) than that of ddm1-2 (0.114 ± 0.029), thus confirming higher fluctuation in fluorescence intensity for AtMBD6-GFP in met1 mutants compared with ddm1-2. In the cmt3 mutant, subnuclear localization of AtMBD-GFP proteins was similar to that found in wild-type plants (Figure 3A). These results verified the importance of CpG methylation in controlling AtMBD5, 6, and 7 subnuclear localization. Interestingly, in ddm1-2 and met1-1 mutant cells, AtMBD2-GFP was accumulated at chromocenters as confirmed by immunolabeling/FISH assay on nuclei derived from transgenic ddm1-2 expressing AtMBD2-GFP (Figures 3A and 3B). This redistribution, however, cannot be accounted for by direct binding of AtMBD2 to unmethylated CpG sites inasmuch as glutathione S-transferase (GST)-AtMBD2 failed to form complexes with unmethylated sites in electrophoretic mobility shift assays (data not shown).
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). We therefore analyzed possible interaction between AtMBDs and the C-terminal region of DDM1 (DDM1/511-764) using the N-terminal region of DDM1 (DDM1/1-205) as a control. Both AtMBD2 and AtMBD6 were capable of binding to the C-terminal region of DDM1, though at a reduced affinity compared with the full-length DDM1. No significant interaction of AtMBDs with the DDM1 N-terminal region (DDM1/1-205) could be detected (Figure 4B). To confirm this interaction, we performed reciprocal assays and analyzed the capability of the full-length DDM1 fused to GST (GST-DDM1) to precipitate the AtMBD6 from nuclear extract derived from transgenic plants expressing AtMBD6-GFP. To this end, GST alone, GST-DDM1, as well as GST-AtMBD2 and GST-AtMBD5 immobilized onto glutathione sepharose were mixed with nuclear extract, and after incubation (4°C, 12 h) and extensive washing precipitated proteins were resolved on SDS-PAGE and immunoblotted using anti-GFP. Results showed that GST-DDM1 and GST-AtMBD5, but not GST alone or GST-AtMBD2, were capable of precipitating AtMBD6-GFP from nuclear extract. These results further support physical interaction between AtMBD6 and the chromatin remodeling factor DDM1.
Association of AtMBDs with Chromocenters Is Not Altered in the kyp-2 Mutant
In ddm1-2 and met1 mutants, H3K9 dimethylation at chromocenters is reduced (Soppe et al., 2002; Tariq et al., 2003). To assess the involvement of H3K9 dimethylation in recruiting AtMBDs to chromocenters, we transiently expressed AtMBD-GFP constructs in kyp-2 cells where H3K9 dimethylation at chromocenters is significantly reduced (Figure 5A; see also Jasencakova et al., 2003; Jackson et al., 2004), whereas CpG methylation at centromeres is unaffected (Jackson et al., 2002; Malagnac et al., 2002). Results showed that the distribution of AtMBD-GFP proteins in kyp-2 was similar to that of wild-type plants (Figure 5B), suggesting that H3K9 dimethylation is dispensable for the localization of AtMBDs at chromocenters.
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DISCUSSION |
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). Also, in mammalian cells, MBD proteins are localized mainly to nuclear foci enriched in methyl CpG; in mouse cells deficient in CpG methylation, most MBDs are dispersed within the nucleus (Hendrich and Bird, 1998). In this work, we showed that AtMBD proteins capable of binding methylated CpG sites displayed differential subnuclear localization in Arabidopsis cells. Whereas AtMBD5 and 6 preferentially bound to perinucleolar chromocenters adjacent to the rRNA gene clusters, AtMBD7, a unique member of the AtMBD family containing three putative MBD motifs (Berg et al., 2003), displayed even distribution at all chromocenters. These localization patterns imply that CpG methylation, while essential, may not be sufficient to determine the subnuclear localization of AtMBD proteins; other factors may be involved in determining the localization of AtMBDs at specific nuclear domains. The findings that DDM1 and AtMBDs share common nuclear sites and both proteins interact with each other suggest that DDM1 may play an active role in determining AtMBDs subnuclear localization. Consistent with our finding, Harikrishnan et al. (2005) demonstrated an interaction between the chromatin remodeling factor Brahma and the human MBD protein MeCP2 and raised the hypothesis that chromatin remodeling activity at methylated sites is required for the assembly of MeCP2 to establish heterochromatin.
Notably, similarly to DDM1, the Lsh gene, a mouse homolog of DDM1, is required for genome-wide methylation (Dennis et al., 2001). In Lsh-deficient tissues as well as in ddm1 mutant plants, the activity of DNA methyltransferases is not altered significantly compared with wild-type tissues (Kakutani et al., 1995; Dennis et al., 2001), suggesting that Lsh and DDM1 do not act in enhancing the DNA methylation machinery. Instead, DDM1 and Lsh have been suggested to function as chromatin remodeling factors regulating the accessibility of chromatin to DNA methyltransferases (Jeddeloh et al., 1999; Dennis et al., 2001). However, no effect on localization of the DNA methyltransferase 1 (Dnmt1) was observed in Lsh–/– cells, suggesting that Lsh is not essential for proper subnuclear localization of Dnmt1 (Yan et al., 2003). An alternative explanation for DDM1 function emerged from the finding that in N. crassa and Arabidopsis DNA methylation is tightly associated with histone H3K9 methylation (Tamaru and Selker, 2001; Jackson et al., 2002; Malagnac et al., 2002; Tamaru et al., 2003). In the ddm1 mutant, a shift from H3K9 methylation toward H3K4 methylation was noted in the heterochromatic knob of chromosome 4, raising the hypothesis that the primary effect of DDM1 is to facilitate H3K9 methylation, which in turn induces cytosine methylation (Gendrel et al., 2002). In the Arabidopsis kyp-2 mutant, however, reduction in H3K9 methylation at chromocenters had no effect on CpG methylation (Jackson et al., 2002), indicating that in Arabidopsis, CpG methylation is not necessarily dependent on H3K9 methylation (Richards, 2002). Our results point to the possibility that the effect of DDM1 on DNA methylation is mediated, at least partly, by MBD proteins. Accordingly, the ATP-dependent chromatin remodeling activity of DDM1 (Brzeski and Jerzmanowski, 2003) is required for making CpG-methylated sites accessible for binding AtMBD proteins. Alternatively, DDM1 may function as an assembly platform guiding AtMBDs to their binding sites, thus protecting CpG-methylated sites from demethylating activities. It remains to be determined whether such a relationship also exists between mammalian MBDs and the chromatin remodeling factor Lsh.
Transient expression of AtMBD2-GFP in wild-type Arabidopsis cells showed a nearly uniform distribution within the nucleus, excluding the heterochromatic, CpG-methylated chromocenters and the nucleolus. However, in ddm1-2 and met1-1 cells, AtMBD2 was found at chromocenters, suggesting that reduction in cytosine methylation plays a major role in the accumulation of AtMBD2 at these sites. Presently, we do not know what brings AtMBD2 to chromocenters in these mutants. Because AtMBD2 did not bind in vitro–unmethylated DNA, it is likely that its association with chromocenters is mediated through interaction with an as yet unknown factor(s).
The capability of GST-AtMBD5 to precipitate AtMBD6-GFP from nuclear extract suggests either that these proteins interact physically with each other or they are present in the same protein complex. GST pull-down assay using in vitro–translated, 35S-Met–labeled AtMBD6 showed that neither GST-AtMBD5 nor GST-AtMBD6 binds 35S-labeled AtMBD6 protein (data not shown). Taken together, our results imply that the plant MBD protein complex possesses the chromatin remodeling factor DDM1 and at least two AtMBD molecules. This is similar to mammalian cells where the main multiprotein repressory complex MeCP1 contains the SNF2 chromatin remodeling factor Mi2, MBD2, MBD3, and histone deacetylases as major components (Wade et al., 1999; Feng and Zhang, 2001).
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMETHODSREFERENCES |
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) and met1-3 by PCR as described (Saze et al., 2003). Wild-type and Arabidopsis mutants (all in the background of Columbia ecotype) were grown under short-day conditions at 20°C. At 4 to 6 weeks after germination, rosette leaves were collected for the isolation and transformation of protoplasts essentially as described (Sheen, 2002). The GFP signal was detected 24 h after transfection using a laser confocal microscope (Olympus IX70; Hamburg, Germany). Images were obtained using an excitation wavelength of 488 nm, and images for GFP and chlorophyll signals were collected through 505 to 525- and 630-nm filters, respectively.
Immunolabeling and FISH
Nuclei were isolated from leaves as previously described (Fass et al., 2002) and fixed in 4% paraformaldehyde dissolved in PBS for 15 min at room temperature followed by washing twice with PBS. Nuclei were placed on slides, air dried, permeabilized in cold acetone (100%) for 7 min in –20°C, and washed twice with PBS. Slides were blocked in 2% BSA in PBS for 2 h at room temperature followed by overnight incubation at 4°C with 100 µL of primary antibody mixture containing 2% BSA and 2 µg of antidimethylated Lys 9 histone H3 (Upstate Biotechnology, Lake Placid, NY) or anti-GFP (Roche, Indianapolis, IN). Slides were washed three times, 5 min each, in PBS, followed by incubation at room temperature for 2 h with the appropriate secondary antibody tagged with fluorescein (Sigma-Aldrich, St. Louis, MO). For FISH assays, slides washed as above were fixed with 1% paraformaldehyde for 5 min, denatured with formamide, and probed with the 180-bp repeats (CEN180) and the 18S rDNA, both labeled with tetramethylrhodamine-5-dUTP (Roche) as described (Avivi et al., 2004). After hybridization, slides were washed, stained with 10 µg/mL of DAPI, washed twice, and mounted in Vectashield (Vector Laboratories, Burlingame, CA). Hybridization signals were visualized by a fluorescence microscope (Olympus) equipped with a CCD camera (Imago; TILL Photonics, Hamburg, Germany) using Olympus filters U-MNU, U-MWIBA2, and U-MNG to detect DAPI, fluorescein, and rhodamine, respectively. Images were pseudocolored and merged using TILL Vision version 3.3 software (TILL Photonics). All images were processed using Adobe Photoshop software (Mountain View, CA).
Immunolabeling with polyclonal anti-5-methyl cytosine (Megabase Research Products, Lincoln, NE) was performed on nuclei fixed with ethanol/acetic acid (3:1) essentially as described above except that before blocking with BSA, nuclei were processed to DNA denaturation as previously described for FISH (Avivi et al., 2004).
Construction of AtMBD-GFP Plasmids and Generation of Transgenic Plants
Fusion of AtMBD to GFP was performed by subcloning each of the indicated AtMBD cDNA into pUC19-35S-GFP (a gift from A. Levitan and A. Danon), downstream from the 35S promoter and in frame with the GFP. Each AtMBD cDNA was amplified by PCR to eliminate the stop codon using the following primers: AtMBD2-S, 5'-GAGAGGATCCATGCCTTCAATGCAGAAGTATGAA-3'; AtMBD2-AS, 5'-TCTCCCCGGGTCTATCAGCAAGTTCGTCGTTGG-3'; AtMBD5-S, 5'-TGATATCAGATCTATGTCGAACGGCACGGATCAG-3'; AtMBD5-AS, 5'-TCTCCCCGGGGAACATCGTTTTTCCAGCGTT-3'; AtMBD6-S, 5'-GAGATCTAGAATGTCAGATTCTGTGGCCGGC-3'; AtMBD6-AS, 5'-TCTCCCCGGGAGCCGACACTTTACTAGGG-3'; AtMBD7-S, 5'-GAGAAGATCTAGAATGCAGACGAGATCCTCTTCCTCTCC-3'; AtMBD7-AS, 5'-GAGACCCGGGAATTCTTAAGAGCGGTCTTCGATCAGTG-3'.
The various AtMBD PCR products were digested either with BamHI and SmaI or with BglII and SmaI and subcloned into BglII-SmaI sites of pUC19-35S-GFP. The integrity of each construct was verified by sequencing. These constructs were used for protoplast transformation experiments using polyethylene glycol essentially as described (Sheen, 2002). To generate transgenic Arabidopsis plants expressing AtMBD fused to GFP, the 35S-AtMBD-GFP fragment was excised out using EcoRI and subcloned into the same site of the binary vector pPZP-111 to generate pPZP-35S-AtMBD-GFP followed by transformation into Agrobacterium tumefaciens and Arabidopsis plants (ecotypes Colombia and Wassilewskija as well as the ddm1-2 mutant).
Salt Extraction and ChIP
Salt extraction of nuclei was performed as described (Fass et al., 2002). ChIP was performed on nuclei prepared from Arabidopsis plants expressing AtMBD6-GFP essentially as described (Lawrence et al., 2004) using anti-GFP (Roche), anti-dimethylated K4 histone H3 (Upstate Biotechnology), as well as antidimethylated K9 histone H3 (Upstate Biotechnology). Precipitated DNAs were subjected to PCR using the following primers: CEN180-S, 5'-GAGAGGATCCCGTAAGAATTGTATCCTTGTTAG-3'; CEN180-AS, 5'-GAGAGAATTCCCTTTAAGATCCGGTTGTGG-3'; 18SrDNA-S, 5'-GCTACCTGGTTGATCCTGCCAGTAGTC-3'; 18SrDNA-AS, 5'-CGACCTTTTATCTAATAAATGCGTCCC-3'; Actin-S, 5'-GGTTTTGCTGGGGATGATGC-3'; Actin-AS, 5'-CATTGAATGTCTCAAACATGATTTGAGTC-3'.
ChIP PCR conditions were as follows: 94°C, 5 min; 30 cycles of 94°C, 30 s; 56°C, 30 s; 72°C, 45 s; 72°C, 10 min. PCR products were resolved on 1.2% agarose gel and stained with ethidium bromide.
DNA Extraction, DNA Gel Blot Analysis, and Electrophoretic Mobility Shift Assay
DNA was extracted from Arabidopsis leaves by modification of the C-elyltrimethyl ammonium bromide method (Wagner et al., 1987). To determine the DNA methylation status at chromocenters, genomic DNA was digested with the methylation-sensitive enzymes HpaII and MspI, and samples were run on 1% agarose gel, blotted onto nylon membrane, and hybridized with the 180-bp repeats labeled with [32P]dCTP using the Nick Translation kit (Roche) according to the manufacturer's protocol.
Electrophoretic mobility shift assay was performed as previously described using GST alone, GST-AtMBD2, and GST-AtMBD5 and 32P-labeled umCG or 1mCG double-stranded oligonucleotides as described (Zemach and Grafi, 2003).
Construction of DDM1 Plasmids and GST Pull-Down Assay
DDM1 and its truncated forms were constructed in pBluescript KS (Stratagene, La Jolla, CA) by PCR using the full length of DDM1 (kindly provided by Kazusa DNA Research Institute, Chiba, Japan) as a template and the following primers: D(1-205)-S, 5'-CACAGGATCCCCTTCGATGGTTAGTCTTCGCTCC-3'; D(1-205)-AS, 5'-CTCCCCGGGTCAATAAGACTTTAACTGTCC-3'; D(511-764)-S, 5'-GAGAGGATCCATGTATCTCTACCCTCCTGTTG-3'; D(511-764)-AS, 5'-TGTGGAATTCCTAACTGTTCAGGGAAGACAGC-3'.
The PCR products were digested either with BamHI and SmaI or BamHI and EcoRI and subcloned into the same sites of either pGEX2T or pBluescript KS+ downstream from the T7 promoter to generate pBs-DDM1(FL), pBs-DDM1(1-205), and pBs-DDM1(511-764). These plasmids were subjected to in vitro transcription–coupled translation in the presence of 35S-Met using a Promega kit (Madison, WI) and according to the supplied protocol. Labeled proteins were subjected to GST pull-down assay using GST-AtMBDs (Zemach and Grafi, 2003) or GST alone (as a negative control) essentially as described (Grafi et al., 1996). GST-MBD2 was reconstructed using a cDNA library (kindly provided by the ABRC) as a template and 5'-GAGAGGATCCATGAGTATGTCGCAGTCTCGAGC-3' as sense primer and 5'-GAGAGAATTCTTATCTATCAGCAAGTTCGTCG-3' as antisense primer. The PCR product was digested with BamHI and EcoRI and subcloned into the same sites of pGEX-2T.
Image Analysis
Image analysis was performed using the NIH Image program version 1.61 (http://rsb.info.nih.gov/nih-image). Briefly, to obtain the coefficient of variation for fluorescence intensity, the SD of the pixel values for each nucleus was divided by the mean pixel value. The mean of the coefficient of variation for a given population and the SD were determined for each set of nuclei transformed with AtMBD6-GFP (shown in Supplemental Figure 3A online). Statistical significance was determined using TTEST.
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Acknowledgments |
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Footnotes |
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Online version contains Web-only data. 
Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.105.031567.
Received February 6, 2005; accepted March 14, 2005.
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REFERENCES |
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