DNA methylation in an intron of the IBM1 histone demethylase gene stabilizes chromatin modification patterns
2012; Springer Nature; Volume: 31; Issue: 13 Linguagem: Inglês
10.1038/emboj.2012.141
ISSN1460-2075
AutoresMélanie Rigal, Zoltán Kevei, Thierry Pélissier, Olivier Mathieu,
Tópico(s)Genomics and Chromatin Dynamics
ResumoArticle11 May 2012free access DNA methylation in an intron of the IBM1 histone demethylase gene stabilizes chromatin modification patterns Mélanie Rigal Mélanie Rigal Centre National de la Recherche Scientifique (CNRS), UMR6293, GReD, INSERM U 1103, Clermont Université, Aubière, France Search for more papers by this author Zoltán Kevei Zoltán Kevei Centre National de la Recherche Scientifique (CNRS), UMR6293, GReD, INSERM U 1103, Clermont Université, Aubière, France Search for more papers by this author Thierry Pélissier Thierry Pélissier Centre National de la Recherche Scientifique (CNRS), UMR6293, GReD, INSERM U 1103, Clermont Université, Aubière, France Search for more papers by this author Olivier Mathieu Corresponding Author Olivier Mathieu Centre National de la Recherche Scientifique (CNRS), UMR6293, GReD, INSERM U 1103, Clermont Université, Aubière, France Search for more papers by this author Mélanie Rigal Mélanie Rigal Centre National de la Recherche Scientifique (CNRS), UMR6293, GReD, INSERM U 1103, Clermont Université, Aubière, France Search for more papers by this author Zoltán Kevei Zoltán Kevei Centre National de la Recherche Scientifique (CNRS), UMR6293, GReD, INSERM U 1103, Clermont Université, Aubière, France Search for more papers by this author Thierry Pélissier Thierry Pélissier Centre National de la Recherche Scientifique (CNRS), UMR6293, GReD, INSERM U 1103, Clermont Université, Aubière, France Search for more papers by this author Olivier Mathieu Corresponding Author Olivier Mathieu Centre National de la Recherche Scientifique (CNRS), UMR6293, GReD, INSERM U 1103, Clermont Université, Aubière, France Search for more papers by this author Author Information Mélanie Rigal1, Zoltán Kevei1, Thierry Pélissier1 and Olivier Mathieu 1 1Centre National de la Recherche Scientifique (CNRS), UMR6293, GReD, INSERM U 1103, Clermont Université, Aubière, France *Corresponding author. Centre National de la Recherche Scientifique (CNRS), UMR6293—INSERM U1103—GReD, Clermont Universite, 24 Avenue des Landais, BP 80026 63171 Aubière Cedex, France. Tel.:+33 473 407 405; Fax:+33 473 407 777; E-mail: [email protected] The EMBO Journal (2012)31:2981-2993https://doi.org/10.1038/emboj.2012.141 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The stability of epigenetic patterns is critical for genome integrity and gene expression. This highly coordinated process involves interrelated positive and negative regulators that impact distinct epigenetic marks, including DNA methylation and dimethylation at histone H3 lysine 9 (H3K9me2). In Arabidopsis, mutations in the DNA methyltransferase MET1, which maintains CG methylation, result in aberrant patterns of other epigenetic marks, including ectopic non-CG methylation and the relocation of H3K9me2 from heterochromatin into gene-rich chromosome regions. Here, we show that the expression of the H3K9 demethylase IBM1 (increase in BONSAI methylation 1) requires DNA methylation. Surprisingly, the regulatory methylated region is contained in an unusually large intron that is conserved in IBM1 orthologues. The re-establishment of IBM1 expression in met1 mutants restored the wild-type H3K9me2 nuclear patterns, non-CG DNA methylation and transcriptional patterns at selected loci, which included DNA demethylase genes. These results provide a mechanistic explanation for long-standing puzzling observations in met1 mutants and reveal yet another layer of control in the interplay between DNA methylation and histone modification, which stabilizes DNA methylation patterns at genes. Introduction Silent chromatin is typically associated with specific patterns of epigenetic modifications, which in plants include high levels of DNA methylation in all three cytosine contexts (CG, CHG and CHH, where H=A, T or C) and dense dimethylation at lysine 9 of histone H3 (H3K9me2) (Bender, 2004; Chan et al, 2005; Grewal and Jia, 2007). H3K9 methylation is conserved from plants to mammals and relies on the activities of histone lysine methyltransferases in the Su(var)3-9 family (Rea et al, 2000). The Arabidopsis genome encodes nine homologues of the Drosophila Su(var)3-9 protein, which are referred to as SUVH proteins (for Su(var)3-9 homologues) (Baumbusch et al, 2001). Although SUVH4 (also known as KRYPTONITE or KYP), SUVH5 and SUVH6 seem to act redundantly in the maintenance of H3K9me2 at transposable elements (TEs) and repeats, only KYP appears to function at genes (Jackson et al, 2004; Ebbs and Bender, 2006; Inagaki et al, 2010). Conversely, enzymes of the JHDM2 family contain a jumonji C (jmjC) domain and can remove H3K9 methylation (Klose et al, 2006; Tsukada et al, 2006; Yamane et al, 2006). In Arabidopsis, experimental evidence supports the hypothesis that the jmjC domain-containing protein increase in BONSAI methylation 1 (IBM1) is a histone demethylase that is specific for H3K9me2 and H3K9 monomethylation (Saze et al, 2008; Inagaki et al, 2010). Mutants for IBM1 display ectopic H3K9me2 accumulation in the transcribed regions of a large number of genes, whereas TEs are unaffected (Inagaki et al, 2010). In Arabidopsis, CG methylation is propagated during DNA replication by the maintenance DNA methyltransferase METHYLTRANSFERASE 1 (MET1), which robustly copies methylation patterns on newly synthesized DNA strands. The maintenance of asymmetrical CHH methylation is mostly ensured by DOMAINS REARRANGED METHYLTRANSFERASE 2 (DRM2) in a process known as RNA-directed DNA methylation (RdDM), which involves the polymerases IV and V (Law and Jacobsen, 2010). The perpetuation of CHG methylation patterns is largely ensured by the plant-specific chromomethylase CMT3, and genetic analyses suggest that targeting of CMT3 to chromatin relies on H3K9me2, which indicates that H3K9me2 acts upstream of CHG methylation (Lindroth et al, 2004; Feng and Jacobsen, 2011). These two repressive marks are intimately associated, and at the genome level, ∼90% of CHG methylation coincides with H3K9me2-enriched regions (Bernatavichute et al, 2008). Additionally, the loss-of-function kyp and cmt3 alleles show a similar loss of cytosine methylation at CHG sites and induce transcriptional reactivation of a common set of silent targets (Jackson et al, 2002; Lippman et al, 2003; Lindroth et al, 2004; Ebbs et al, 2005; Tran et al, 2005; Ebbs and Bender, 2006). The SET and RING-associated (SRA) domain of KYP and SUVH6 binds to DNA that is methylated at CHGs in vitro, which suggests that CHG methylation also feeds back onto H3K9me2 (Johnson et al, 2007). In heterochromatin, methylation at CG sites and H3K9me2 are also linked. In wild-type (WT) plant nuclei, H3K9me2 is largely confined to heterochromatic chromosomal regions that are densely CG methylated. In the nuclei of the loss-of-function met1-3 mutant, CG methylation is lost, and H3K9me2 is redistributed away from the chromocentres (Soppe et al, 2002; Tariq et al, 2003; Mathieu et al, 2007). Therefore, although the molecular mechanism remains elusive, CG methylation appears to direct H3K9me2 in heterochromatin. Importantly, CG methylation is not restricted to heterochromatin, and genome-wide methylation profiling studies have highlighted that approximately one-third of Arabidopsis genes are CG methylated (Tran et al, 2005; Zhang et al, 2006; Zilberman et al, 2007; Cokus et al, 2008; Lister et al, 2008). Noticeably, gene-body methylation is almost exclusively restricted to CG sites, and it is not associated with H3K9me2 (Bernatavichute et al, 2008). Therefore, CG methylation and H3K9me2 show distinct interactions at different chromosomal locations. In addition, met1 mutants exhibit ectopic non-CG methylation (Soppe et al, 2002; Tariq et al, 2003; Zhang et al, 2006; Mathieu et al, 2007; Cokus et al, 2008; Lister et al, 2008). At heterochromatic sequences, this has been shown to result primarily from the misdirection of the RdDM pathway, while aberrant methylation at a few hundred genes likely originates from the transcriptional downregulation of the DNA demethylases (which notably include REPRESSOR OF SILENCING 1 (ROS1)), in the absence of CG methylation (Huettel et al, 2006; Mathieu et al, 2007). Importantly, several thousand genes specifically display CHG hypermethylation in their body sequence in the met1 background, and the majority of these genes contain CG methylation in the WT background (Lister et al, 2008; Reinders et al, 2008). This suggests that CG methylation (and/or MET1 itself) may exclude CHG methylation from genes; however, the molecular mechanism that links CG and CHG methylation at genes remains to be elucidated. The recent analyses of the IBM1 loss-of-function mutant have revealed widespread ectopic CHG DNA methylation and H3K9me2 at genes (Saze et al, 2008; Miura et al, 2009; Inagaki et al, 2010). These new epigenetic patterns are dependent on the function of CMT3 and KYP and interestingly, genes that contain CHG hypermethylation in the met1 and ibm1 mutant backgrounds largely overlap (Miura et al, 2009). These similarities between the met1 and ibm1 mutants have led us to hypothesize that CG methylation and/or MET1 may protect genes from ectopic CHG methylation and H3K9me2 because they are required for proper IBM1 expression. IBM1 encodes two mRNA variants; the longer variant (IBM1-L) specifically encodes the functional IBM1 protein that contains the jmjC domain. We found that the proper accumulation of IBM1-L mRNA is controlled by DNA methylation and depends on the simultaneous presence of CG and CHG methylation in an unusually large intron of the IBM1 gene. The re-establishment of IBM1-L expression in met1 mutants largely suppressed the abnormal H3K9me2, DNA methylation and transcriptional patterns that were induced by the mutation at selected target genes. Interestingly, the expression of the ROS1 DNA demethylase was recovered when IBM1-L accumulation was restored in the met1 background, which thereby also contributed to the removal of aberrant DNA methylation patterns that occur in this mutant background. Therefore, by controlling the proper expression of H3K9 and DNA demethylases, CG methylation insures the maintenance of proper genic DNA methylation and histone modification patterns through a self-regulatory loop. These results highlight the importance of CG methylation as a central coordinator of epigenetic stability at genes and provide mechanistic explanations for long-standing enigmatic observations in met1 mutants. Results DNA methylation is required for proper IBM1 expression To understand how CHG gene-body methylation occurs in met1 mutant plants, we examined the transcription of the IBM1 gene with respect to DNA methylation. IBM1 is predicted to produce two different transcripts; only the longer transcript (hereafter referred to as IBM1-L) is predicted to encode the jmjC domain and thus the functional IBM1 protein (Figure 1A). Northern blotting and reverse transcription (RT)–PCR analyses confirmed the presence of the two predicted IBM1 transcripts (Figure 1B and C). In WT plants that were treated with the DNA methylation inhibitor 5-aza-2′-deoxycytidine (5-aza-dC) and in plants that were homozygous for the met1-3 null MET1 allele (Saze et al, 2003), the accumulation of IBM1-L mRNA was specifically downregulated, whereas the accumulation of the short IBM1 RNA transcript (IBM1-S) was not significantly affected (Figure 1B and C). To further investigate the impact of DNA methylation on IBM1 expression, we assayed transcript accumulation in mutants of additional regulators of genomic DNA methylation. Triple mutants for the VARIATION IN METHYLATION (VIM) 1, 2 and 3 genes, which encode co-factors that are required for CG methylation maintenance (Woo et al, 2007, 2008; Kraft et al, 2008), also showed specific downregulation of the IBM1-L mRNA transcript (Figure 1D). Together with the results from the met1 mutants, this suggests that CG methylation is required for the proper accumulation of IBM1 transcripts that contain the putative H3K9 demethylase domain jmjC. Figure 1.DNA methylation is required for IBM1-L transcript accumulation. (A) Schematic representation of the two IBM1 mRNA variants, IBM1-S and IBM1-L. The boxes represent exons; coding regions are indicated in black and untranslated regions are indicated in white. The region encoding the jmjC domain is shown in green (Saze et al, 2008). The intronic DNA-methylated zone (horizontal red bar), the positions of the probe that was used for the northern blot analysis (horizontal grey bar) and the primers that were used for the RT–PCR analysis of the IBM1-L and IBM1-S variants (arrows) are shown. The position of the T-DNA insertion in ibm1-4 is indicated with a triangle. (B) Northern blot analysis of the IBM1 transcripts using poly(A)+ RNAs for the indicated mutant genotypes. Hybridization with a probe corresponding to ACTIN2 (ACT2) is shown as a loading control. (C) RT–PCR analysis of IBM1-S and IBM1-L transcripts in WT and met1-3 plants that were grown on medium containing (+) or lacking (−) 5-aza-dC. (D) RT–PCR analysis of the IBM1-S and IBM1-L transcripts in the indicated DNA methylation-deficient mutant backgrounds. (E) RT–PCR analysis of IBM1-L expression in met1 cmt3 compared with the WT and single mutants. The amplification of ACT2 or 18S rRNA (18S) was used to normalize the RNA template levels. The negative controls (no RT) lacked reverse transcriptase. Download figure Download PowerPoint Noticeably, the downregulation of IBM1-L that was observed in the met1 plants was further enhanced when the met1 plants were grown on 5-aza-dC, which suggests that non-CG methylation may also participate in the control of IBM1 expression (Figure 1C). In the nrpd1 mutant of polymerase IV, as well as in the nrpe2 (nrpd2a) mutant of the common subunit of polymerases IV and V, IBM1 transcription was similar to the WT, and the drm1 drm2 double mutant also exhibited no detectable IBM1 transcriptional variation, which indicates that the RdDM pathway/CHH methylation does not control IBM1 expression (Figure 1B and D). H3K9me2 and DNA methylation at CHG sites are intimately coupled, and a self-reinforcing feedback loop between KYP and CMT3 maintains these two marks in heterochromatin. We analysed the cmt3 and kyp mutants to determine whether CHG methylation influences IBM1 expression. RNA-gel blot and RT–PCR analyses showed that accumulation of the IBM1-L transcript was drastically downregulated in both cmt3 and kyp (Figure 1B and D). Triple mutants of the histone H3K9 methyltransferases KYP(SUVH4)/SUVH5/SUVH6 mimic the kyp single mutant, indicating that SUVH5 and SUVH6 do not significantly contribute IBM1 transcriptional control (Figure 1D). Together, these observations indicate that CG and CHG methylation are required for the proper expression of IBM1. This reveals another layer of interdependence and control between DNA methylation and H3K9 methylation, in which, somehow paradoxically with their role in the maintenance of DNA methylation, MET1, CMT3 and KYP are also involved in the exclusion of CHG methylation/H3K9me2 from genes. The mutation of DECREASE IN DNA METHYLATION 1 (DDM1) did not alter IBM1 transcript accumulation (Figure 1B). Intronic DNA methylation controls IBM1 expression To understand how DNA methylation controls IBM1 expression, we examined DNA methylation profiles of the IBM1 gene by bisulphite sequencing. It has been reported that gene-body methylation is preferentially targeted to nucleosomes on exons (Chodavarapu et al, 2010), and accordingly, IBM1 carries body methylation at CG sites in several exons in WT plants (http://neomorph.salk.edu/epigenome/epigenome.html). In Arabidopsis, the average intron size is ∼180 bp (The Arabidopsis Genome Initiative, 2000). The IBM1 gene contains an unusually large intron that is >2 kb; this intron must be spliced out to generate the IBM1-L mRNA that encodes the jmjC histone demethylase domain. Interestingly, the large intron contains a zone that is densely methylated at both CG and CHG positions in the WT (http://neomorph.salk.edu/epigenome/epigenome.html; Figure 2). In the met1-3 null mutant, exonic CG methylation was lost in the IBM1 gene (http://neomorph.salk.edu/epigenome/epigenome.html). In the large intron, bisulphite sequencing confirmed that CG methylation was also erased; however, non-CG methylation, which was mostly represented by CHG methylation, was essentially maintained (Figure 2). Figure 2.Bisulphite sequencing analysis of the DNA methylation pattern in the large intron of IBM1. The position of the methylated zone is indicated in Figure 1. The proportions of methylated cytosines at the CG (red), CHG (blue) and CHH (green) sites are given as percentages. Download figure Download PowerPoint In the kyp mutant, CG methylation remained largely intact. However, methylation at CHG positions drastically decreased from ∼40 to 10% (Figure 2). This indicates that CHG methylation in the large IBM1 intron strongly depends on KYP activity. The remaining low level of CHG methylation in the mutant probably results from the redundant activity of other SUVH proteins, such as SUVH5 and/or SUVH6, which was previously shown for other loci (Ebbs et al, 2005; Ebbs and Bender, 2006); although the contribution of SUVH5 and/or SUVH6 was not visualized at the transcriptional level (Figure 1D). DRM2 and CMT3 have been reported to be redundantly required for the maintenance of CHG methylation at particular genomic loci (Cao and Jacobsen, 2002; Cao et al, 2003). At the large intron of IBM1, CHG methylation was completely lost in cmt3, which indicates that CMT3 is solely responsible for CHG methylation at this locus; this is in agreement with the absence of detectable difference in IBM1-L accumulation in the drm1 drm2 background (Figures 1D and 2). Based on the impact of the met1, cmt3 and kyp mutations on IBM1-L transcript accumulation, these data suggest that the simultaneous presence of CG and CHG methylation in the large IBM1 intron is required for the proper production of IBM1-L transcripts. Accordingly, the met1 cmt3 double mutant showed a stronger reduction in IBM1-L accumulation relative to each single mutant (Figure 1E). As mentioned above, the accumulation of IBM1 transcripts was not altered by the ddm1-2 mutation (Figure 1B), and consistent with our previous conclusion, the large IBM1 intron was not hypomethylated but rather hypermethylated at all of the cytosine sequence contexts in this mutant (Figure 2). Regardless of the mechanism responsible for IBM1 hypermethylation in the ddm1 mutant background, this result supports the conclusion that DNA methylation in the large IBM1 intron positively correlates with IBM1-L accumulation. To further confirm that DNA methylation in the large IBM1 intron controls the accumulation of RNA that encodes the protein that contains the H3K9me2 demethylase jmjC domain, we outcrossed the met1, cmt3 and kyp mutants (all in the Col-0 background) with WT Ler-0 plants and assayed for DNA methylation and the transcription of the mutant-derived IBM1 allele in the resultant F1 plants. Once they are altered in met1 mutants, the CG methylation patterns cannot be re-established upon the reintroduction of MET1 activity (Soppe et al, 2000; Kankel et al, 2003; Saze et al, 2003; Mathieu et al, 2007). Consistently, PCR from bisulphite-treated DNA followed by digestion with a restriction enzyme showed that the met1-derived IBM1 allele was still unmethylated at CG sites in the Ler × met1 F1 hybrids (Figure 3A). This absence of remethylation correlated with a low accumulation of IBM1-L mRNA that originated from the met1-derived IBM1 allele, which was similar to the IBM1 transcription in the mutant parent (Figure 3B). In contrast to MET1, the reintroduction of CMT3 largely restores the developmental phenotypes induced by the loss of silencing that is associated with non-CG methylation in the drm1/2 cmt3 mutants (Chan et al, 2006). Methylation at CHG sites, which was completely lost in the cmt3 mutants, reappeared to a certain level in the large intron of the mutant-derived IBM1 allele in the Ler × cmt3 F1 individuals (Figure 3C). RT–PCR analyses revealed that the accumulation of the IBM1-L transcript from the cmt3-derived allele was restored to WT levels, such that the IBM1 transcriptional patterns in the Ler × cmt3 F1s and the Ler × Col control F1s were indistinguishable (Figure 3D). Likewise, the accumulation of IBM1-L mRNA from the kyp-derived IBM1 allele was re-established to WT levels in the F1 progeny of the Ler × kyp cross (Figure 3D). Restoration of CHG methylation at the mutant-derived IBM1 allele appeared to be more efficient in the Ler × kyp F1s when compared with the Ler × cmt3 F1 plants (Figure 3C), and this was confirmed with bisulphite sequencing (Supplementary Figure S1). Because CHG methylation was completely lost in cmt3 but not in kyp, CHG remethylation is likely more efficient or occurs faster in the presence of residual CHG methylation. Together, these observations indicate that DNA methylation in the large intron of IBM1 is required for the proper accumulation of the IBM1-L transcript that encodes the jmjC domain. Figure 3.Inheritance of IBM1 intronic methylation and IBM1-L transcript accumulation. (A, C) DNA methylation of the IBM1 large intron was assayed in the indicated genotypes by Col-0-specific PCR from bisulphite-treated DNA that was followed by digestion with TaqI (A) or HpyCH4V (C). CG and CHG methylation protect the TaqI and HpyCH4V sites, respectively, from bisulphite conversion, which facilitates restriction digestion after bisulphite treatment and PCR. Undigested (U) and digested (D) samples are shown. (B, D) Allele-specific RT–PCR analysis of IBM1-L transcript accumulation in Ler-0 × met1 (B) Ler-0 × cmt3 and Ler-0 × kyp (D) F1 individuals. The Ler × Col-0 individuals are shown as controls. The ACTIN 2 (ACT2) gene was used as a control. Download figure Download PowerPoint RT–PCR analyses with primer sets that were designed along the length of the large IBM1 intron revealed that the reduction of the IBM1-L transcript accumulation in the met1 background occurred inside the methylated region of the large intron (Supplementary Figure S2). When primer sets located upstream of the intron methylation region were used, no clear difference in IBM1 pre-mRNA level was detected between the WT and met1. Furthermore, northern blot analyses using poly(A)+ RNA samples revealed no stable alternative polyadenylated IBM1 transcript variants in met1, cmt3 or kyp relative to the WT, indicating no differential transcript polyadenylation in these mutants (Figure 1B). Collectively, these results favour the hypothesis that intronic DNA methylation at CG and CHG sites is required for proper IBM1-L transcript elongation. Enhanced IBM1-L mRNA accumulation complements the ibm1 mutation and largely restores H3K9me2 patterns in met1 The loss of CG methylation in met1 mutants results in aberrant patterns of other epigenetic marks, including non-CG methylation and H3K9me2. In met1-3 mutant nuclei, H3K9me2 is relocated away from heterochromatic chromocentres into euchromatic chromosomal regions (Tariq et al, 2003; Mathieu et al, 2007). To date, the underlying molecular mechanism that is responsible for this relocation has not been elucidated. Because IBM1 targets genes for H3K9 demethylation, we hypothesized that the lower accumulation of IBM1-L may account for the ectopic accumulation of H3K9me2 at genes in met1. To test this hypothesis, we cloned the IBM1-L cDNA under the endogenous IBM1 promoter and used the resulting construct (pIBM1:IBM1-L) to transform ibm1-4/IBM1 and met1-3/MET1 heterozygous plants. Unlike untransformed ibm1 mutant segregants, the ibm1 individuals expressing IBM1-L (ibm1::IBM1-L) exhibited WT-like leaf size and morphology, as well as restored fertility (Figure 4A and B). Additionally, ibm1::IBM1-L plants showed reduced ectopic DNA methylation at the BONSAI gene (Supplementary Figure S3). These observations indicate that the pIBM1:IBM1-L construct efficiently complements the ibm1-4 mutation. In WT nuclei, H3K9me2 is essentially clustered at heterochromatic chromocentres (Figure 4C and E). The H3K9me2 signal in euchromatic regions was notably enhanced in nuclei of the ibm1-4 mutant compared with the WT (Figure 4C), which is consistent with the fact that IBM1 targets a large number of genes for H3K9 demethylation (Inagaki et al, 2010). The nuclei of complemented ibm1::IBM1-L lines displayed H3K9me2 patterns that were indistinguishable from the WT (Figure 4C). In nuclei from first-generation met1-3 mutants, H3K9me2 was frequently associated with euchromatic nuclear regions (Figure 4D and E; Tariq et al, 2003; Mathieu et al, 2007). The restored accumulation of IBM1-L in the met1-3 background in met1::IBM1-L plants (see Figure 5C and Supplementary Figure S5) drastically increased the proportion of nuclei that exhibited WT-like H3K9me2 patterns; this mark was preferentially associated with heterochromatin and less-intense euchromatin staining (Figure 4D and E). These results indicate that the downregulation of IBM1-L largely accounts for the relocation of H3K9me2 at gene-rich euchromatic regions that occurs in met1 mutants. Figure 4.IBM1-L expression complements the ibm1-4 mutation and restores H3K9me2 patterns in met1::IBM1-L plants. (A) Representative images of 3-week-old siblings segregating from self-pollination of an ibm1-4/IBM1::IBM1-L/+ parent are shown. (B) Inflorescences of ibm1 and ibm1::IBM1-L plants. H3K9me2 patterns in WT, ibm1-4, ibm1::IBM1-L (C), met1 and met1::IBM1-L plants (D) were analysed by immunocytology with a specific antibody against this mark. Representative images from one experiment are shown; two independent ibm1::IBM1-L and met1::IBM1-L (#22 and #23) lines were analysed, and they provided similar results. Two different H3K9me2 patterns were commonly observed in the met1-3 and met1::IBM1-L plants, and the signals were either dispersed from or clustered at the chromocentres. (E) Proportion of both types of nuclei was monitored and is represented as a histogram (±s.d.). Between 60 and 80 nuclei from three independent experiments were scored. Scale bar=5 μm. Download figure Download PowerPoint Figure 5.Restoration of IBM1-L transcript accumulation in met1 plants suppresses ectopic CHG hypermethylation and H3K9me2 enrichment at genes. (A) CHG methylation was determined in the WT, met1-3, met1::IBM1-L (#23) and ibm1-4 at the body of the indicated genes using bisulphite sequencing. Each region analysed contains 11–23 CHG sites. The full DNA methylation profiles are provided in Supplementary Figure S4. (B) Association of the indicated genes with H3K9me2 was determined by ChIP with a specific antibody against this mark and is represented as a relative enrichment over the WT. Quantifications are from three independent experiments (±standard error of the mean). (C) RT–PCR analysis of transcript accumulation at the indicated genes. 'end.' endogenous IBM1 transcript, 'trans.' transcript from the IBM1-L transgene. Amplification of ACTIN2 (ACT2) was used to normalize the RNA template levels. The negative controls (no RT) lacked reverse transcriptase. Download figure Download PowerPoint Downregulation of IBM1-L is responsible for ectopic CHG methylation at genes in met1 mutants Because the maintenance of H3K9me2 and CHG methylation are mechanistically linked, we sought to determine whether the relocation of H3K9me2 in heterochromatin in met1::IBM1-L was accompanied by the suppression of ectopic CHG methylation at genes. Using bisulphite sequencing, we analysed DNA methylation at 11 genes that are body methylated at CG positions in the WT (http://neomorph.salk.edu/epigenome/epigenome.html). Methylation at CHG sites was virtually absent in the WT at all these genes (Figure 5A). In agreement with the fact that IBM1 targets a large number of genes for H3K9me2 demethylation, all genes but one (AT1G58030) showed ectopic CHG methylation in the ibm1 mutant background compared with the WT. Among these, six also exhibited ectopic CHG methylation (∼10–40%) in the met1 background (Figure 5A; Supplementary Figure S4). In met1::IBM1-L, CHG methylation levels were strongly decreased in the body of these six genes, indicating that the enhanced expression of IBM1-L in met1 largely suppresses ectopic body methylation at CHG sites (Figure 5A; Supplementary Figure S4). Interestingly, four genes (AT5G39960, AT2G42600, AT3G23750, AT4G24740) were CHG hypermethylated in ibm1 but not in met1 (Figure 5A), suggesting that some genes are protected from ectopic CHG methylation in met1, although they are targeted for H3K9me2 demethylation by IBM1. Chromatin immunoprecipitation (ChIP) assays confirmed that the gene-body CHG hypermethylation in both met1 and ibm1 mutants was associated with enrichment in H3K9me2 at the same regions analysed for DNA methylation relative to the WT (Figure 5B). Consistent with the fact that IBM1-L encodes the functional form of the H3K9 demethylase, nearly WT H3K9me2 levels were restored in met1::IBM1-L at genes, showing ectopic H3K9me2 in the met1 mutant background (Figure 5B). Interestingly, the intronic DNA-methylated region of the IBM1 gene also showed H3K9me2 enrichment in ibm1, indicating that IBM1 targets its large intron for H3K9me2 demethylation (Figure 5B). However, the persistence of high CHG methylation levels at t
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