DNA methylation controls histone H3 lysine 9 methylation and heterochromatin assembly in Arabidopsis

Abstract

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 F(1) 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.

Fig. 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.

Fig. 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.

Fig. 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.

Fig. 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 of the chromocenters but more dispersed around the other half. (C) The chromocenters with strong immunosignals for DNA methylation also show strong signals of H3K9 methylation. All genotypes have a Ler background. Images in black and white show DAPI-stained 3:1 in ethanol:acetic acid (A and B) or formaldehyde-fixed (C) nuclei. Immunosignals are in green for DNA methylation (A and C) and in red for H3K9 methylation (C). Bar = 5 µm.

Fig. 5. A model for heterochromatin assembly. (A) In wild-type Arabidopsis nuclei, strong methylation of DNA and H3K9, followed by histone deacetylation, leads after replication to re-establishment of heterochromatin by DDM1, MET1, a histone H3K9-specific methylase (KYP) and a histone deacetylase. DNA maintenance methylation mediated by MET1 and supported by DDM1 directs H3K9 methylation. H4K16 deacetylation at newly replicated nucleosomes is mediated by DDM1. (B) In the met1 mutant, maintenance DNA methylation is severely reduced, leading to decreased H3K9 methylation. Below a certain threshold, low-copy sequences disperse from heterochromatin and acquire euchromatin features. In the absence of DDM1, H4K16 de acetylation is additionally impaired.