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• W2113643663 abstract "Article1 December 2002free access DNA methylation controls histone H3 lysine 9 methylation and heterochromatin assembly in Arabidopsis Wim J.J. Soppe Wim J.J. Soppe Present address: Max-Planck-Institut für Züchtungsforschung, Carl-von-Linné-Weg 10, D-50829 Köln, Germany Search for more papers by this author Zuzana Jasencakova Zuzana Jasencakova Department of Cytogenetics, Institute of Plant Genetics and Crop Plant Research (IPK), Corrensstrasse 3, D-06466 Gatersleben, Germany Search for more papers by this author Andreas Houben Andreas Houben Department of Cytogenetics, Institute of Plant Genetics and Crop Plant Research (IPK), Corrensstrasse 3, D-06466 Gatersleben, Germany Search for more papers by this author Tetsuji Kakutani Tetsuji Kakutani National Institute of Genetics, Mishima, Shizuoka, 411-8540 Japan Search for more papers by this author Armin Meister Armin Meister Department of Cytogenetics, Institute of Plant Genetics and Crop Plant Research (IPK), Corrensstrasse 3, D-06466 Gatersleben, Germany Search for more papers by this author Michael S. Huang Michael S. Huang Department of MCD Biology, Los Angeles, CA, 90095-1606 USA Search for more papers by this author Steven E. Jacobsen Steven E. Jacobsen Department of MCD Biology, Los Angeles, CA, 90095-1606 USA Molecular Biology Institute, UCLA, Los Angeles, CA, 90095-1606 USA Search for more papers by this author Ingo Schubert Corresponding Author Ingo Schubert Department of Cytogenetics, Institute of Plant Genetics and Crop Plant Research (IPK), Corrensstrasse 3, D-06466 Gatersleben, Germany Search for more papers by this author Paul F. Fransz Paul F. Fransz Swammerdam Institute for Life Sciences, University of Amsterdam, 1090 GB, Amsterdam, The Netherlands Search for more papers by this author Wim J.J. Soppe Wim J.J. Soppe Present address: Max-Planck-Institut für Züchtungsforschung, Carl-von-Linné-Weg 10, D-50829 Köln, Germany Search for more papers by this author Zuzana Jasencakova Zuzana Jasencakova Department of Cytogenetics, Institute of Plant Genetics and Crop Plant Research (IPK), Corrensstrasse 3, D-06466 Gatersleben, Germany Search for more papers by this author Andreas Houben Andreas Houben Department of Cytogenetics, Institute of Plant Genetics and Crop Plant Research (IPK), Corrensstrasse 3, D-06466 Gatersleben, Germany Search for more papers by this author Tetsuji Kakutani Tetsuji Kakutani National Institute of Genetics, Mishima, Shizuoka, 411-8540 Japan Search for more papers by this author Armin Meister Armin Meister Department of Cytogenetics, Institute of Plant Genetics and Crop Plant Research (IPK), Corrensstrasse 3, D-06466 Gatersleben, Germany Search for more papers by this author Michael S. Huang Michael S. Huang Department of MCD Biology, Los Angeles, CA, 90095-1606 USA Search for more papers by this author Steven E. Jacobsen Steven E. Jacobsen Department of MCD Biology, Los Angeles, CA, 90095-1606 USA Molecular Biology Institute, UCLA, Los Angeles, CA, 90095-1606 USA Search for more papers by this author Ingo Schubert Corresponding Author Ingo Schubert Department of Cytogenetics, Institute of Plant Genetics and Crop Plant Research (IPK), Corrensstrasse 3, D-06466 Gatersleben, Germany Search for more papers by this author Paul F. Fransz Paul F. Fransz Swammerdam Institute for Life Sciences, University of Amsterdam, 1090 GB, Amsterdam, The Netherlands Search for more papers by this author Author Information Wim J.J. Soppe2, Zuzana Jasencakova1, Andreas Houben1, Tetsuji Kakutani3, Armin Meister1, Michael S. Huang4, Steven E. Jacobsen4,5, Ingo Schubert 1 and Paul F. Fransz6 1Department of Cytogenetics, Institute of Plant Genetics and Crop Plant Research (IPK), Corrensstrasse 3, D-06466 Gatersleben, Germany 2Present address: Max-Planck-Institut für Züchtungsforschung, Carl-von-Linné-Weg 10, D-50829 Köln, Germany 3National Institute of Genetics, Mishima, Shizuoka, 411-8540 Japan 4Department of MCD Biology, Los Angeles, CA, 90095-1606 USA 5Molecular Biology Institute, UCLA, Los Angeles, CA, 90095-1606 USA 6Swammerdam Institute for Life Sciences, University of Amsterdam, 1090 GB, Amsterdam, The Netherlands ‡W.J.J.Soppe and Z.Jasencakova contributed equally to this work *Corresponding author. E-mail: [email protected] The EMBO Journal (2002)21:6549-6559https://doi.org/10.1093/emboj/cdf657 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info We propose a model for heterochromatin assembly that links DNA methylation with histone methylation and DNA replication. The hypomethylated Arabidopsis mutants ddm1 and met1 were used to investigate the relationship between DNA methylation and chromatin organization. Both mutants show a reduction of heterochromatin due to dispersion of pericentromeric low-copy sequences away from heterochromatic chromocenters. DDM1 and MET1 control heterochromatin assembly at chromocenters by their influence on DNA maintenance (CpG) methylation and subsequent methylation of histone H3 lysine 9. In addition, DDM1 is required for deacetylation of histone H4 lysine 16. Analysis of F1 hybrids between wild-type and hypomethylated mutants revealed that DNA methylation is epigenetically inherited and represents the genomic imprint that is required to maintain pericentromeric heterochromatin. Introduction DNA methylation is essential for normal development of most higher eukaryotes and is involved in genomic imprinting, regulation of gene expression and defense against foreign DNA (Jost and Saluz, 1993; Finnegan et al., 1998). In concert with histone modifications, it contributes to chromatin remodeling (reviewed by Richards and Elgin, 2002). From fission yeast to mammals, methylation of histone H3 at lysine 9 (H3K9) is considered to be crucial for heterochromatin assembly, whereas methylation of H3 at lysine 4 (H3K4) occurs preferentially within transcriptionally competent chromatin (except for yeast rDNA, Briggs et al., 2001) (recently reviewed in Rice and Allis, 2001; Lachner and Jenuwein, 2002; Richards and Elgin, 2002). The interactions between DNA methylation, histone modifications and chromatin structure have mainly been studied at the molecular level for specific DNA sequences. Integrated genetic, molecular and cytological approaches can provide new insights into chromatin remodeling. For example, genome-wide H4 acetylation appeared to be tightly linked to DNA replication and possibly with post-replicative processes rather than with transcriptional activity (Jasencakova et al., 2000, 2001). Studies on DNA methylation and histone modifications at the nuclear level using DNA methylation mutants may elucidate the process of heterochromatin formation. Several mutants with reduced DNA methylation levels have been isolated in Arabidopsis. The strongest effects on DNA methylation were found in the recessive mutants decrease in DNA methylation1 (ddm1; Vongs et al., 1993) and methyltransferase1 (met1; Finnegan et al., 1996; Ronemus et al., 1996). DDM1 encodes a SWI/SNF-like protein, presumably a chromatin remodeling factor (Jeddeloh et al., 1999), while MET1 encodes a maintenance methyltransferase (Finnegan and Kovac, 2000). They are the plant homologs of the mammalian Lsh and Dnmt1 genes, respectively (Finnegan et al., 1996; Dennis et al., 2001). In both ddm1 and met1, repetitive and single-copy sequences become hypomethylated, causing a reduction in methylation level by ∼70% (Vongs et al., 1993; Ronemus et al., 1996). Remethylation of hypomethylated sequences is extremely slow or absent when ddm1 is backcrossed to the wild type (Kakutani et al., 1999). The mutants are further characterized by release of transcriptional gene silencing (TGS) and post-transcriptional gene silencing (PTGS) (Mittelsten Scheid et al., 1998; Morel et al., 2000) and by reactivation of some transposons (Hirochika et al., 2000; Singer et al., 2001). Morphological phenotypes of ddm1 and met1 include altered flower morphology and leaf shape, sterility and late flowering, and appear in the first homozygous mutant generation in met1, but only after several generations of inbreeding in ddm1 (Finnegan et al., 1996; Kakutani et al., 1996; Ronemus et al., 1996). The ddm1 and met1 mutants have been analyzed at the molecular and morphological level, but not in relation to histone modifications and heterochromatin formation. Heterochromatin in Arabidopsis nuclei is concentrated at DAPI-bright chromocenters that contain major tandem repeats (the centromeric 180 bp repeat and rDNA genes) and dispersed pericentromeric repeats (Maluszynska and Heslop-Harrison, 1991; Heslop-Harrison et al., 1999; Fransz et al., 2002). The latter consist mainly of transposable elements and low-copy sequences (Fransz et al., 2000; The Arabidopsis Genome Initiative, 2000). All repeats are strongly methylated in wild-type plants but weakly in ddm1 and met1 single mutants (Vongs et al., 1993; Ronemus et al., 1996; Kakutani et al., 1999). To investigate the relationship between DNA methyl ation and genome-wide chromatin organization, and to elucidate the hierarchy of processes that control heterochromatin formation, we compared the location of (peri-) centromeric sequences, the nuclear patterns of DNA methylation and histone modifications, and the heterochromatin structure of leaf interphase nuclei of wild-type plants with those of ddm1 and met1 mutant plants. Results Hypomethylated mutants contain reduced amounts of heterochromatin After DAPI staining of wild-type nuclei from the Landsberg erecta (Ler) accession, conspicuous heterochromatic chromocenters can be distinguished. In nuclei of the hypomethylated mutants ddm1 and met1, the chromocenters are smaller, indicating a reduction of hetero chromatin (Figure 1A). This nuclear phenotype occurs in different organs, developmental stages and genetic backgrounds. We quantified this reduction of heterochromatin content by measuring the area and staining intensity of the chromocenters in relation to that of the entire nucleus (chromocenter fraction). The chromocenter fractions of ddm1 and met1 are reduced by ∼25–30% in comparison to the wild type (Figure 1B). The double mutant ddm1 met1 shows a further reduction of 20–25% compared with the single mutants, indicating an additive effect of both mutations for this feature. F1 hybrids between wild type and either ddm1 or met1 mutants contain nuclei with intermediate chromocenter fractions (Figure 1B), consistent with their intermediate methylation levels (Kakutani et al., 1999). Since the plant phenotypes in ddm1 appear in later generations (Kakutani et al., 1995), we determined (in Columbia background) whether these generations also show a further reduction in the chromocenter fraction. Although nuclei from plants of the eighth generation with a strong phenotype had smaller chromocenters than nuclei from plants of the second generation without phenotype, the difference was not significant (P = 0.122). Figure 1.Reduction of chromocenter size in hypomethylated mutants. (A) Phenotypes of representative DAPI-stained leaf interphase nuclei in a Ler background. Chromocenters are smaller and weaker stained in ddm1 and met1 nuclei than in wild-type nuclei, chromocenters in the ddm1 met1 double mutant show the weakest staining. Heterozygous DDM1 × ddm1 and MET1 × met1 F1 plants show an intermediate nuclear phenotype between wild type and mutants. Bar = 5 μm. (B) Chromocenter fractions are shown as the percentage of area and staining intensity of chromocenters in relation to the entire nucleus. This histogram quantifies the observations shown in (A). Furthermore, it is shown that chromocenters in ddm1 (in Col background) do not significantly reduce in size after two and eight selfing generations since the induction of the mutation. Percentages are derived from measurements of 50 nuclei each and the standard error of the mean is indicated on each bar. Download figure Download PowerPoint DNA hypomethylation causes dispersion of pericentromeric sequences away from chromocenters The reduced size of DAPI-bright chromocenters in ddm1 and met1 indicates that they contain less DNA than wild-type chromocenters, and the question arises which sequences are no longer within the chromocenters in ddm1 and met1. We examined this by fluorescent in situ hybridization (FISH) using tandem and dispersed repeats, which all localize in the wild-type chromocenters. The major tandem repeats (pAL1; see Figure 2A, 45S rDNA and 5S rDNA) co-localized with chromocenters in wild-type and mutants, and thus remained within the heterochromatin of ddm1 and met1 nuclei. Figure 2.Location of repetitive and single-copy sequences in leaf interphase nuclei. (A) Sequences corresponding to the 180 bp centromeric pAL repeat (red) are always located at chromocenters. Sequences corresponding to the pericentromeric BAC F28D6 (green) are located at chromocenters in wild type, but yield additional dispersed signals in the single and double mutants. Similar results were obtained with other pericentromeric BACs (F17A20, F10A2 and F21I2, DDBJ/EMBL/GenBank accession Nos AF147262, AF147259 and AF147261). (B) Schematic representation of BAC F28D6 (top). The different sequence elements are shown in accordance with the GenBank annotation; green boxes above (A-H) indicate the position and size of different PCR fragments used as probes in FISH experiments. Red signals on the nuclei, corresponding to the location of Athila elements, are always located at chromocenters. Green signals, corresponding to the location of PCR fragment C, are located at chromocenters in the wild type but yield dispersed signals in ddm1. (C) All tested repetitive elements (Tat1 from the Ty3-gypsy group of LTR retrotransposons, Ta1 from the Ty1-copia group of LTR retrotransposons, the MITE Emi12, the repetitive DNA element AthE1.4 and the chromomeric repeat ATR63) are located at chromocenters in wild-type and mutant nuclei. (D) The CAC1 sequence was most frequently detected at chromocenters in the wild-type and outside chromocenters in the ddm1 mutant nuclei (arrow). The position of CAC1 on BAC T10J7 is indicated by a yellow box in the scheme. FISH with this BAC yielded multiple signals (red), due to the presence of repetitive elements. Green signal is from four PCR fragments (green in the scheme), amplified from a sequence adjacent to CAC1, and indicates its original position. This signal is masked by the strong DAPI staining of chromocenters in the left image of the same wild-type nucleus. (E) The FWA sequence was usually located outside chromocenters, as detected by FISH with two BACs (T30C3 and F14M19), adjacent to the gene, in red and a probe of 10.5 kb, covering the gene, in green. (F) The SUP sequence was usually located outside chromocenters, as detected with a BAC (K14B15) that contains SUP, in red and a probe of 6.7 kb, covering the gene, in green. (A-C, E and F) Nuclei from plants with Ler background; (D) nuclei from plants with Col background. Images in black and white show DAPI-stained nuclei; color images show FISH signals on the same nuclei. Bar = 5 μm. Download figure Download PowerPoint However, BAC DNA clones that represent sequences from the pericentromeric regions hybridized exclusively with chromocenters in the wild type, but showed a dispersed pattern at and around the chromocenters in the hypomethylated mutants (Figure 2A). This suggests that some pericentromeric sequences are located away from the chromocenter in the mutants. Most pericentromeric BAC clones contain many different transposable elements as well as non-transposable sequences (The Arabidopsis Genome Initiative, 2000). To determine which of these sequences are released from heterochromatin in the mutants, we probed wild-type and mutant nuclei with four highly repetitive elements mapped in pericentromeric regions and two (Emi12 and AthE1.4) mapped in various regions along the chromosome arms. Each of these elements belongs to different families: Athila (Pélissier et al., 1995) and Tat1 (Wright and Voytas, 1998) from the Ty3-gypsy group of LTR retrotransposons, Ta1 (Voytas et al., 1990; Konieczny et al., 1991) from the Ty1-copia group of LTR retrotransposons, the miniature inverted-repeat transposable element (MITE) Emi12 (Casacuberta et al., 1998), the repetitive element AthE1.4 (Surzycki and Belknap, 1999) and the chromomeric repeat ATR63, which is derived from the heterochromatic knob hk4S (Fransz et al., 2000). All transposable elements hybridized to chromocenters in wild-type and mutant nuclei (Figure 2B and C). This implies that pericentromeric sequences other than transposable elements are relocated away from heterochromatin in the mutant nuclei. We tested this by FISH with different PCR fragments (A, B, C, D, E, F, G and H in Figure 2B) of BAC F28D6 (DDBJ/EMBL/GenBank accession No. AF147262) that represent putative genes and unannotated sequences. The fragments A, B, D, E, F, G and H contained low-copy sequences and yielded poor FISH signals outside the mutant chromo centers. However, fragment C, which has ∼75 highly homologous sites in pericentromeric regions, is present at chromocenters in wild type but occupies more dispersed positions in ddm1 and met1 nuclei (Figure 2B). Thus, the dispersed signals from pericentromeric BACs in the hypomethylated mutants seem to be due to sequences separating the transposable elements. The transposable elements tested above are inactive in wild type and largely inactive in the hypomethylated mutants. To find out whether the spatial position relative to heterochromatin might be related to transposon activity, we examined the location of the single-copy CAC1 transposon located in the pericentromeric region of chromosome arm 2L, which is silent and methylated in Columbia (Col) wild-type plants but active and hypomethylated in the ddm1 mutant (Miura et al., 2001). FISH with a combination of the BAC that contains CAC1 (T10J7; DDBJ/EMBL/GenBank accession No. AC005897) and four PCR fragments, located on either side of the transposon (Figure 2D), revealed that activation and hypomethylation of the CAC1 transposon in ddm1 are correlated with its relocation away from the heterochromatin (Table I). Table 1. Number of FISH signals, present within and outside chromocenters, for different single-copy sequences and genotypes Gene Accession Genotype No. of scored nuclei No. of signals in chromocenters No. of signals out of chromocenters CAC1 Col Wild type 55 65 13 CAC1 Col ddm1 58 30 65 FWA Ler Wild type 104 24 154 FWA Ler fwa-1 100 14 158 FWA Col Wild type 52 2 89 FWA Col ddm1 selfed 2× 51 6 86 FWA Col ddm1 selfed 8× 50 5 87 SUP Ler Wild type 56 6 88 SUP Ler clk-3 51 5 89 SUP Ler ddm1 52 3 93 Not all silenced genes reside in chromocenters The correlation between silencing and the nuclear position of the CAC1 transposon prompted us to investigate whether such a correlation also exists for other genes like FWA (mapped at the long arm of chromosome 4) and SUPERMAN (SUP; mapped at the short arm of chromosome 3), which differ in their DNA methylation and expression levels between wild type and hypomethylation mutants (Jacobsen and Meyerowitz, 1997; Soppe et al., 2000). The FWA sequence was localized with a combination of three probes. Two BACs, positioned on either side of the gene (T30C3, DDBJ/EMBL/GenBank accession No. AL079350; and F14M19, accession No. AL049480) were detected in red and a small probe of 10.5 kb, containing FWA, in green (Figure 2E). In adult wild-type plants, the FWA gene is not expressed and the 5′-region of the gene is strongly methylated, in contrast to its hypomethylation and constitutive expression in the fwa-1 mutant (Soppe et al., 2000). For both wild type and the fwa-1 mutant, the FWA sequence was detected outside chromocenters in the majority of nuclei (Table I). We also compared the location of FWA between Col wild type (methylated and not expressed), a ddm1 line of the second generation (methylated and not expressed) and a ddm1 line of the eighth generation (hypomethylated and expressed). In all these genotypes, FWA was located mainly out side chromocenters (Table I). Therefore, silencing and methylation of the FWA gene do not mediate a shift of its nuclear position toward the chromocenters. Similar results were obtained for the SUP gene. In wild-type plants, SUP is hypomethylated and expressed in developing flowers (Sakai et al., 1995). Several hypermethylated alleles of SUP have been found [clark kent (clk) alleles], which show decreased expression (Jacobsen and Meyerowitz, 1997). The SUP gene is also methylated and silenced in ddm1 and met1 mutant plants (Jacobsen and Meyerowitz, 1997; Jacobsen et al., 2000). Although SUP is not expressed in leaves, the pattern and extent of methylation are the same in leaves and flowers (Kishimoto et al., 2001). The SUP gene was localized in leaf nuclei by FISH with two probes: the entire BAC K14B15 (DDBJ/EMBL/GenBank accession No. AB025608) containing the SUP gene was detected in red and a small probe of 6.7 kb, comprising the gene, in green color (Figure 2F). In all genetic backgrounds, SUP was preferentially located outside chromocenters (Table I). Decreased DNA and H3K9 methylation accompany the size reduction of chromocenters in ddm1 and met1 We compared the distribution patterns of methylated DNA in wild type and hypomethylated mutants using antibodies against 5-methylcytosine. Wild-type nuclei showed strong signals, especially at chromocenters (Figure 3A). In contrast, in nuclei of the ddm1 and met1 mutants, the immunosignals were dispersed and no longer clustered at chromocenters. This phenotype appeared to be stronger in the double mutant ddm1 met1, consistent with its further reduced chromocenter fraction (Figure 3A). Figure 3.Chromatin modifications in wild-type and hypomethylated mutant nuclei. (A) Immunosignals for DNA methylation (green) are strongly clustered at chromocenters in wild-type nuclei; ddm1 and met1 nuclei have more weakly labeled chromocenters. This effect is even stronger in the double mutant ddm1 met1. (B) Histone H3 K9 methyl ation. In wild-type nuclei, immunosignals for H3dimethylK9 (red) localize preferentially to chromocenters, whereas ddm1 and met1 nuclei showed a significantly lower intensity of labeling. (C) Histone H3 K4 methylation (red) occurs at euchromatin, while chromocenters and nucleoli remained unlabeled in wild-type as well as in ddm1 and met1 nuclei. (D-G) H4Ac16 labeling patterns (green) in wild-type nuclei. Three distinct patterns can be distinguished. (D) Type 1: euchromatin intensely labeled, nucleoli and chromocenters unlabeled. (E) Type 2: chromatin more or less uniformly labeled, nucleoli unlabeled. (F and G) Type 3: chromocenters with signal clusters, nucleoli unlabeled (inactive rDNA components of chromocenters remained unlabeled in type 2 and 3 nuclei). FISH with centromeric (pAL) or 45S rDNA repeats (red, on the right). (H) DNA hypomethylated mutants show similar labeling patterns as wild-type nuclei. For both mutants, labeling patterns of chromocenters correlate with the reduced size of chromocenters. DAPI staining (left), immunosignals of H4Ac16 (green, middle) and the merge of both (right). All genotypes have a Ler background. Images in black and white show DAPI-stained 3:1 in ethanol:acetic acid (A) or formaldehyde-fixed (B-H) nuclei. Adjacent color images show immunosignals on the same nuclei. Bar = 5 μm. Download figure Download PowerPoint Since DNA methylation has recently been reported to be tightly correlated with histone H3 methylation (for reviews, see Rice and Allis, 2001; Lachner and Jenuwein, 2002; Richards and Elgin, 2002), we analyzed the nuclear distribution of methylated histone H3 isoforms. In wild-type nuclei, immunosignals for H3methylK9 were clustered at the chromocenters. The area and intensity of H3methylK9 signals were reduced in ddm1 and met1 chromocenters (Figure 3B). This indicates that methyl ation of H3K9 is controlled by DNA methylation. Immunolabeling of H3methylK4 gave an opposite pattern that was similar for wild-type and mutant nuclei. Euchromatin was strongly labeled, while nucleoli and chromocenters (size reduced in the mutants) were unlabeled (Figure 3C). The data suggest that the decrease in DNA methylation leads to a reduction in methylated H3K9 at chromocenters. H4K16 acetylation in ddm1 deviates from that of wild type and met1 Apart from the effects of DNA methylation and histone methylation, chromatin structure is also modified by histone acetylation. Antibodies recognizing isoforms of histone H4 acetylated at lysine 5, 8 and 12, and histone H3 acetylated at lysine 9, all yielded similar patterns of immunosignals in wild-type nuclei. Euchromatin was intensely labeled, while nucleoli and heterochromatic chromocenters were unlabeled. A comparable pattern was observed in ddm1 and met1 nuclei (Table II), although the unlabeled domains were smaller than in wild type, correlating with the smaller chromocenters. Table 2. Compilation of chromatin modification data in leaf nuclei of Arabidopsis wild type and DNA methylation mutants Wild type ddm1 met1 H4Ac5 eu+ nu− cc− eu+ nu− cc− eu+ nu− cc− H4Ac8 eu+ nu− cc− eu+ nu− cc− eu+ nu− cc− H4Ac12 eu+ nu− cc− eu+ nu− cc− eu+ nu− cc− H4Ac16 (see Table III) tri-/tetra-AcH4 eu+ nu− cc− eu+ nu− cc− eu+ nu− cc− H3Ac9 eu+ nu− cc− eu+ nu− cc− eu+ nu− cc− H3 methyl K4 eu+ nu− cc− eu+ nu− cc− eu+ nu− cc− H3 methyl K9 eu− nu− cc+ eu− nu− cc+/− eu− nu− cc+/− DNA methylation eu− nu− cc+ eu− nu− cc+/− eu− nu− cc+/− eu, euchromatin; nu, nucleoli; cc, chromocenter. The intensity of labeling is indicated by + or −. In contrast, antibodies against H4Ac16 yielded three classes of labeling patterns (Figure 3D-H; Table III). Type 1, showing strongly labeled euchromatin and unlabeled chromocenters and nucleoli, comprises 66.7% of the nuclei (Figure 3D). Type 2 displayed a uniformly labeled chromatin with unlabeled nucleoli and represents 24.2% of nuclei (Figure 3E). Type 3 is characterized by chromocenters that are more intensely labeled than euchromatin, while nucleoli and chromocenters containing inactive rDNA genes remain unlabeled. This class comprises 9.1% of nuclei (Figure 3F). The H4Ac16 patterns resemble the cell cycle-dependent modulation of acetylation at this lysine position observed in root meristems of faba bean (Jasencakova et al., 2000). However, leaf nuclei are mitotically inactive but frequently endopolyploid (Galbraith et al., 1991). Therefore, the high intensity of acetylation of H4K16 might reflect a link with DNA (endo-)replication or post-replicative processes, particularly at chromocenters. Table 3. H4Ac16 labeling patterns of leaf nucleia of Arabidopsis wild type and DNA methylation mutants Labeling patternsb Wild type ddm1 met1 F1 (Ler × ddm1) n % n % n % n % 1 nu− cc− eu+ 132 66.7 35 19.8 50 53.2 79 71.2 2 nu− cc+ eu+(+) 48 24.2 95 53.7 43 45.7 23 20.7 3 nu− cc++ eu+ 18 9.1 47 26.5 1 1.1 9 8.1 2+3 66 33.3 142 80.2c 44 46.8d 32 28.8 Σ 198 100.0 177 100.0 94 100.0 111 100.0 a 4C nuclei as the major fraction of leaf nuclei, 2C and 8C nuclei showed similar results. b nu, nucleoli and rDNA component of cc. c P < 0.001. d P = 0.037. We observed comparable labeling patterns for leaf nuclei of ddm1 and met1 mutants (Figure 3H). However, the proportion of nuclei with labeled chromocenters (type 2 and 3) was increased somewhat in met1 (46.8%) and drastically in ddm1 (80.2%) compared with the wild type (33.3%; Table III). This indicates that deacetylation of H4K16 depends on DDM1 activity and implies a functional difference between DDM1 and MET1 with respect to histone acetylation. DNA methylation and histone H3K9 methylation, but not histone H4K16 acetylation patterns, are inherited epigenetically Inheritance of ddm1-induced DNA hypomethylation is stable, even in wild-type/DDM1 hybrid background (Kakutani et al., 1999). We therefore examined chromatin structure in F1 plants, heterozygous for either ddm1 or met1. The nuclei of heterozygotes showed two groups of chromocenters that differed strikingly in methylation level. One group displayed the wild-type morphology, whereas chromocenters of the other were similar to those of the mutants (Figure 4A). The difference in parental origin of chromocenters was supported by FISH with 45S rDNA. This probe hybridized to four chromocenters, of which two were heavily methylated, whereas the other two were not (data not shown). This means that the DDM1 and MET1 activities in the F1 nuclei do not restore the wild-type level of DNA methylation in mutant-derived chromocenters. Considering the recessive nature of the ddm1 and met1 mutations (Vongs et al., 1993; E.Richards, personal communication), this indicates that DNA methyl ation is inherited epigenetically. When the pericentromeric BAC F28D6 was probed to DDM1ddm1 or MET1met1 F1 nuclei, one half of the chromocenters showed the wild-type pattern and the other half showed the mutant pattern with more dispersed signals (Figure 4B). This indicates that the formation of pericentromeric heterochromatin requires DNA methylation as an epigenetic imprint. Figure 4.The epigenetic inheritance of DNA methylation and H3K9 methylation is visible in nuclei of F1 plants (wild type × mutant). (A) Half of the chromocenters show strong immunosignals for DNA methylation and the other half weak signals. (B) FISH signals for BAC F28D6 (green) are strongly clustered at half" @default.
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• W2113643663 title "DNA methylation controls histone H3 lysine 9 methylation and heterochromatin assembly in Arabidopsis" @default.
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