Sexual-lineage-specific DNA methylation regulates meiosis in Arabidopsis

Abstract

DNA methylation regulates eukaryotic gene expression and is extensively reprogrammed during animal development. However, whether developmental methylation reprogramming during the sporophytic life cycle of flowering plants regulates genes is presently unknown. Here we report a distinctive gene-targeted RNA-directed DNA methylation (RdDM) activity in the Arabidopsis thaliana male sexual lineage that regulates gene expression in meiocytes. Loss of sexual-lineage-specific RdDM causes mis-splicing of the MPS1 gene (also known as PRD2), thereby disrupting meiosis. Our results establish a regulatory paradigm in which de novo methylation creates a cell-lineage-specific epigenetic signature that controls gene expression and contributes to cellular function in flowering plants.

Figure 1. Male meiocytes exhibit high CG/CHG and low CHH methylation.

a, Model of male sexual lineage development in Arabidopsis thaliana. n, the number of chromosomes in the haploid genome. b, Heat maps showing CG, CHG and CHH methylation of the male sexual lineage comprising the meiocyte (Me), microspore (Mi), sperm (Sp) and vegetative cell (Vg), in comparison to rosette leaf (Rs). Methylation is calculated and presented in 10 kb windows, with the maximum set at the highest value among the five tissues for each context. The region enriched with mitochondria sequences on Chromosome 2 (3.23 to 3.51 Mb) is removed. c, Box plots demonstrating CG methylation for individual CG sites located within annotated transposons, and with methylation greater than 50% and at least 10 informative sequenced cytosines. Each box encloses the middle 50% of the distribution, with the horizontal line making the median, and vertical lines marking the minimum and maximum values that fall within 1.5 times the height of the box.

Figure 2. Sexual-lineage hypermethylated loci (SLHs) in Arabidopsis.

a, Snapshots of cytosine methylation in wild-type male sex cells (black), rosette leaf (green) and drm1drm2 (drm) mutant meiocyte (red) at two SLH examples. SLHs (refer to Supplementary Data 1 for a full list) are underlined in red. Methylation patterns in other somatic tissues and drm sex cells are shown in Supplementary Fig. 2. b, Box plots showing absolute methylation difference between specific cells/tissues and rosette leaf for 50 bp windows that are CHH hypermethylated in meiocytes in comparison to rosette leaves.

Figure 3. Sexual-lineage hypermethylated loci (SLHs) are produced by RdDM.

a, Box plots showing the absolute methylation at SLHs in the drm1drm2 (drm) and rdr2 mutants in comparison to wild type. b, Box plots demonstrating the abundance of 24 nucleotide (nt) small RNA in pollen or shoot at SLHs and non-SLH RdDM target loci. *P < 0.001; Kolmogorov-Smirnov test. c, Box plots showing the absolute methylation at SLH and non-SLH 50 bp windows in ros1dml2dml3 (rdd) mutant rosette leaf, wild-type rosette leaf, and wild-type male sex cells (meiocyte, microspore and sperm).

Figure 4. SLMs are novel RdDM targets specific to the sexual lineage.

a,b, Box plots showing the absolute methylation at canonical SLHs (a) and SLMs (b) in somatic tissues (Sd, seedling; Rs, rosette leaf; Ca, cauline leaf; Ro, root), sex cells (Me, meiocyte; Mi, microspore; Sp, sperm), columella root cap (Co) and embryo (Em). c, Box plots illustrating CG methylation at SLMs in wild-type (WT) seedling (refer to b for other somatic tissues), and seedlings and sex cells from RdDM mutants: drm1drm2 (drm) and rdr2. d, Scatter plot showing the linear correlation between CG methylation in WT (y axis) and drd1 mutant (x axis) roots at SLMs (Pearson’s R = 0.80). e, Scatter plot showing the linear correlation between average CG methylation in WT somatic tissues (cauline leaf, rosette leaf, root and seedling; y axis) and that in drm mutant sex cells (meiocytes, sperm and vegetative cell; x axis) at SLMs (Pearson’s R = 0.76). f, Box plots demonstrating the absolute CG methylation at SLMs in WT seedling (same data as used in b and c), and published data^† including WT control seedlings (Sd^†), metl mutant, and MET1 reintroduction lines (T-METla T2 and T-MET1b T5).

Figure 5. SLMs target genes and regulate gene expression in meiocytes.

a, Pie charts illustrating percentages of SLMs, canonical SLHs and other RdDM target loci overlapping (green), within 500 bp (yellow), and more than 500 bp from (blue) genes or transposons (numbers shown in Supplementary Table 2). b, Snapshots of transcription (in log2RPKM) and DNA methylation (similar to Fig. 2a), at the RPS16B gene drm, drm1drm2; SLM is underlined in red. c, Quantitative RT-PCR showing the expression of three SLM-regulated genes. *P < 0.02 (t-test).

Figure 6. Pre-tRNA genes are hypermethylated in the male sexual lineage.

a, Snapshots of transcription and DNA methylation (similar to Fig. 5b) at the methionine pre-tRNA locus (magenta box) in the last intron of the MPS1 gene. drm, drm1drm2; SLM is underlined in red. Methylation patterns in other somatic tissues and drm sex cells are shown in Supplementary Fig. 6b. b, Box plots showing the absolute CHH methylation at three groups of pre-tRNA genes in sex cells (Me, meiocyte; Mi, microspore; Sp, sperm; Ve, vegetative cell), somatic tissues (Sd, seedling; Rs, rosette leaf; Ca, cauline leaf; Ro, root), and drm mutant sex cells (dM, drm meiocyte; dS, drm sperm; dV, drm vegetative cell). Group 1, the 24 pre-tRNA genes that overlap SLMs; Group 2, the additional 60 genes hypermethylated in the sexual lineage by RdDM; Group 3, the remaining 605 nuclear pre-tRNA genes. c, Box plots demonstrating the abundance of 24 nucleotide (nt) small RNA in pollen (Po) or shoot (Sh) at the abovementioned three groups of pre-tRNA genes.

Figure 7. RdDM is important for the splicing of MPS1 and normal meiosis.

a, Gene model illustrating that the methionine pre-tRNA SLM (magenta bar) located in the last intron of MPS1 affects the splicing of this intron. E, exon; black lollipops, DNA methylation. b, Quantitative RT-PCR showing the percentage of unspliced MPS1 transcript in wild type (WT) and drm1drm2 (drm) mutant meiocytes. *P < 0.02 (t-test). c, Percentage of meiotic triads in WT, drm and rdr2 mutants, two complementation lines (C1 and C2) and two interference lines (I1 and I2) (***P < 1x 10^-7, **P < 0.02, *P < 0.05, ****P < 1x 10^-14; Fisher’s exact test; C1, 514 observations; C2, 167 observations; for observation numbers of other genotypes refer to Supplementary Fig. 8 legend. d, Spindles (green) and nuclei (blue) of WT (tetrad) and drm (triad) meiotic products at tetrad stage. Scale bars, 10 μm.