The Arabidopsis nucleosome remodeler DDM1 allows DNA methyltransferases to access H1-containing heterochromatin

Abstract

Nucleosome remodelers of the DDM1/Lsh family are required for DNA methylation of transposable elements, but the reason for this is unknown. How DDM1 interacts with other methylation pathways, such as small-RNA-directed DNA methylation (RdDM), which is thought to mediate plant asymmetric methylation through DRM enzymes, is also unclear. Here, we show that most asymmetric methylation is facilitated by DDM1 and mediated by the methyltransferase CMT2 separately from RdDM. We find that heterochromatic sequences preferentially require DDM1 for DNA methylation and that this preference depends on linker histone H1. RdDM is instead inhibited by heterochromatin and absolutely requires the nucleosome remodeler DRD1. Together, DDM1 and RdDM mediate nearly all transposon methylation and collaborate to repress transposition and regulate the methylation and expression of genes. Our results indicate that DDM1 provides DNA methyltransferases access to H1-containing heterochromatin to allow stable silencing of transposable elements in cooperation with the RdDM pathway.

Figure 1. DDM1 and CMT2 mediate RdDM-independent CHH methylation of long TEs

(A) Patterns of TE DNA methylation (CG, CHG and CHH) in wild-type and indicated mutants. Arabidopsis TEs were aligned at the 5′ end or the 3′ end, and average methylation for all cytosines within each 100-bp interval is plotted. The dashed lines represent the points of alignment. (B) Patterns of TE methylation in met1, cmt3, cmt2 and drm2 plants. (C) CHH methylation, sRNA, GC content, nucleosomes, H3K9me2 and RNA levels of a representative region. Genes and TEs oriented 5′ to 3′ and 3′ to 5′ are shown above and below the line, respectively. (D) Phylogenetic tree of angiosperm chromomethylases, with Selaginella moellendorffii (black) as an outgroup. Dicots are shown in green and monocots in red. (E) LOWESS fit of CG, CHG and CHH methylation levels in wild-type and indicated mutants calculated in 50-bp windows and plotted against TE size. (F) DNA methylation in wild-type and indicated mutants was averaged specifically in long TEs (> 4 kb) as described in (A). See also Figure S1 and Table S1.

Figure 2. Heterochromatin requires DDM1 for DNA methylation and inhibits RdDM

(A) Spearman correlation coefficients between DDM1-mediated methylation (drd1 methylation minus ddm1drd1 methylation) and DNA sequence and chromatin features of TEs in 50-bp windows. (B) Box plots showing GC content, nucleosome occupancy and H3K9me2 levels in 50-bp windows within TEs and genes of the indicated size. (C) sRNA, GC content, nucleosome occupancy and H3K9me2 levels were averaged in long TEs (> 4 kb) as described in Figure 1. (D) Spearman correlation coefficients between DDM1-mediated methylation in short TEs (<500 bp) and chromatin features. (E) Spearman correlation coefficients between DRD1-mediated CHH methylation (ddm1 methylation minus ddm1drd1 methylation) and chromatin features, calculated for 50-bp windows with three different levels of sRNA. See also Figure S2 and Table S2.

Figure 3. Lack of H1 ameliorates the loss of methylation in ddm1 plants

(A) Kernel density plots of methylation differences between h1 and wild-type (positive numbers indicate greater methylation in h1). TEs with H3K9me2 log2 scores lower than -1 and higher than 1 are considered euchromatic and heterochromatic, respectively. The colored arrows emphasize global differences (a shifted peak) or extensive local differences (a broad shoulder). (B) Average methylation of TEs in sibling wild-type (WT), h1, ddm1, and h1ddm1 (two biological replicates) seedlings is plotted as in Figure 1. (C–D) M-spline curve fits of log2 DNA methylation ratios in 50-bp windows plotted against H3K9me2 level. See also Figure S3.

Figure 4. Methylation of TE families depends on sRNA abundance and chromatin features

(A–D) Averaged sRNA abundance (A) and CHH methylation levels (B–D) are plotted as in Figure 1 for TEs belonging to four distinct families. The ddm1 trace in (C) represents siblings of the wild-type (WT), h1 and h1ddm1 seedlings analyzed in this panel, and is independent of the ddm1 roots analyzed in (B). See also Figure S4.

Figure 5. DDM1 and RdDM collaborate to repress TE expression and transposition

(A) Venn diagram of significantly upregulated TEs in drd1, ddm1 and ddm1drd1 mutants. (B–D) Box plots of the sizes and H3K9me2 levels (B), absolute fractional CHH demethylation of 50-bp windows (C), and expression compared to wild-type (D) of TEs that are at least 32-fold overexpressed either in drd1 or in ddm1. (E) Box plots of TE family expression in the indicated mutants with respect to wild-type. (F) DNA sequencing coverage (log2(reads in mutant/reads in wild-type)), DNA methylation and RNA levels near the LTR retrotransposon EVADE (AT5TE20395). The sequence coverage is indicative of EVADE copy number relative to wild type (Tsukahara et al., 2009). See also Figure S5.

Figure 6. DDM1 mediates genic DNA methylation

(A) CG methylation was averaged in TEs (left panel) and genes for the indicated genotypes (right panels), and plotted against TE or gene size. Note the group of relatively short genes above the red line that are highly methylated in wild-type and significantly hypomethylated in ddm1 and ddm1drd1 mutants, similarly to TEs. (B) Box plots of averaged CHG methylation in TEs, euchromatic genes (mCG < 0.6), and heterochromatic genes (mCG > 0.6). (C) Box plots of H3K9me2 in euchromatic and heterochromatic genes. (D) Genes with average CG methylation between 20% and 60% (euchromatic genes) were aligned as described in Figure 1A. The Y-axis of the CHG plot was broken at 0.02 to improve visualization. (E) Distribution of CG methylation in representative genes AT1G04700, AT1G04750 and AT1G67220 (emphasized by horizontal black bars) that lose methylation in ddm1 but not in h1ddm1. (F) Box plots of wild-type CG methylation (left), absolute fractional CG demethylation in ddm1 (middle), and H3K9me2 (right) of 50-bp windows within long TEs (> 4 kb), short TEs (< 500 bp) and euchromatic genes. (G) Heat maps of CG (red) and CHG (yellow) DNA methylation in genes aligned at the 5' end (left half of each panel) and the 3' end (right half of each panel). More intense color indicates greater methylation. Genes without wild-type CG methylation (shown in the top half of each panel) were stacked from the top of chromosome 1 to the bottom of chromosome 5; genes containing CG methylation islands (shown at the bottom of each panel) were sorted based on the starting position (for 5' panels) or ending position (for 3' panels) of the wild-type CG methylation island. See also Figure S6.

Figure 7. DDM1 and RdDM synergistically regulate gene expression

(A–B) Venn diagram of significantly (p < 0.05) upregulated genes in drd1, ddm1 and ddm1drd1 mutants. (C) DNA methylation and RNA levels near AT1G46696, and the linked genes AT1G59920 and AT1G59930. (D) sRNA, H3K9me2, CHH methylation and RNA levels near SDC (AT2G17690). (E) Phenotypes of wild-type (flat leaves) and ddm1drd1 (leaves curled downward) plants. See also Figure S7.