Sexual-lineage-specific DNA methylation regulates meiosis in Arabidopsis

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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.

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Fig. 1: Male meiocytes exhibit high CG/CHG and low CHH methylation.
Fig. 2: SLHs in Arabidopsis.
Fig. 3: SLHs are produced by RdDM.
Fig. 4: SLMs are novel RdDM targets specific to the sexual lineage.
Fig. 5: SLMs target genes and regulate gene expression in meiocytes.
Fig. 6: Pre-tRNA genes are hypermethylated in the male sexual lineage.
Fig. 7: RdDM is important for the splicing of MPS1 and normal meiosis.

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Acknowledgements

We thank D. Zilberman for intellectual contributions to this work; D. Zilberman, C. Dean, K. Bomblies, V. Kumar, S. Brady and S. Kamoun for comments on the manuscript; H. Dickinson and J. Hellberg for developing the meiocyte isolation method; G. Oldroyd (Sainsbury Laboratory, Cambridge University) for the pGWB13-Bar vector; E. Fiume (Institut Jean-Pierre Bourgin, Paris) for the pMDC107-NTF vector; M. Hartley, M. Couchman and T. S. G. Olsson at the John Innes Centre Computing Infrastructure for Science Facility for bioinformatics support; and the Norwich Bioscience Institute Partnership Computing Infrastructure for Science group for High Performance Computing resources and the John Innes Centre Bioimaging Facility for assistance with microscopy. This work was funded by a Biotechnology and Biological Sciences Research Council (BBSRC) David Phillips Fellowship (BB/L025043/1) to X.F., a BBSRC grant (BB/M01973X/1) to J.D.H. and a Sainsbury PhD Studentship to J.W.

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Contributions

X.F. designed the study; J.W., H.G., J.Z., B.A., J.D.H. and X.F. performed the experiments; J.W., H.G., M.V. and X.F. analyzed the data; and X.F. wrote the manuscript.

Corresponding author

Correspondence to Xiaoqi Feng.

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The authors declare no competing financial interests.

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Integrated Supplementary Information

Supplementary Figure 1 DNA methylation profiles at genes and transposons in Arabidopsis male-sexual-lineage cells

A. thaliana genes (a, c, e) or transposable elements (TEs; b, d, f) were aligned at the 5’ end (left panel) or the 3’ end (right panel), and average methylation levels in the CG (a-b), CHG (c-d) or CHH (e-f) context for each 100-bp interval are plotted. drm, drm1drm2. The dashed line at zero represents the point of alignment.

Supplementary Figure 2 Examples of sexual-lineage-hypermethylated loci (SLHs)

Examples of typical SLHs located at the transcriptional start and termination sites and body of genes (a-c), and SLHs (that are also SLMs) with remnant CG methylation in drm1drm2 (drm) mutant sex cells and wild-type somatic tissues (d, e). SLHs are underlined in red (refer to Supplementary Table 2 for a full list).

Supplementary Figure 3 SLMs have little CHH/G methylation in the columella and embryo

Box plots showing the absolute methylation at SLMs (533 loci before filtering out columella overlaps; see Methods) 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).

Supplementary Figure 4 CG methylation in seedlings of wild type (WT) and RdDM mutants is strongly correlated

Scatter plots showing linear correlation between CG methylation at SLMs in seedlings of WT and drm2 (Pearson’s R = 0.58), and WT and rdr2 (Pearson’s R = 0.70).

Supplementary Figure 5 Examples of genes suppressed by sexual-lineage-specific methylation in meiocytes

Similar to Fig. 5b, snapshots of cytosine methylation in wild-type male sex cells, drm1drm2 (drm) meiocyte, and wild-type rosette leaves, and transcriptional expression (in log2RPKM) in wild-type and drm meiocyte are shown. SLMs are underlined in red.

Supplementary Figure 6 Examples of sexual-lineage-specific methylation at pre-tRNA genes

Snapshots of cytosine methylation, similar to Supplementary Fig. 2, in wild-type (WT) male sex cells (black), drm1drm2 (drm) mutant sex cells (red), and four somatic tissues (green). SLMs are underlined in red. a, Examples of SLMs at pre-tRNA genes encoding phenylalanine, methionine, glycine or valine anticodons. b, SLM at the methionine pre-tRNA gene (magenta box) located in the last intron of MPS1.

Supplementary Figure 7 Pre-tRNA genes are hypermethylated in male sex cells

Similar to Fig. 6b, these box plots show the absolute CHG (a) and CG (b) methylation at 3 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 (drm1drm2) mutant sex cells (dM, drm meiocyte; dS, drm sperm; dV, drm vegetative cell). Refer to Fig. 6 legend for the 3 groups of pre-tRNA genes.

Supplementary Figure 8 Meiotic defects in RdDM mutants and MPS1-interference lines

a-e, Male meiosis II in wild type (WT; a), drm1drm2 (drm; b, d) and rdr2 (c, e) mutants, and the MPS1 interference lines (g). All instances of WT male meiosis we observed (301 observations) were normal and lead to tetrads at the end of meiosis II (a). However, in 7.1% (380 total observations) and 7.8% (502 total observations) instances of drm (b) and rdr2 (c) male meiosis, respectively, chromosomes fail to separate, so that triads are observed at telophase II. Occasionally we also observed pentads in drm (d) and rdr2 (e) mutants. f, RT-PCR showing the expression of MPS1 transcript retaining last intron (149 bp) in drm mutant and 9 T1 plants of the interference lines, but not in WT. ACT8 as control shows 156 bp bands. g, Interference lines exhibit even higher percentages of triads (I1, 13.4%, 463 total observations; I2, 15.1%, 292 total observations). n, the number of chromosomes in the haploid genome. Scale bars, 10 μm.

Supplementary Figure 9 Separation of sperm- and vegetative-cell nuclei via fluorescence-activated cell sorting (FACS)

A representative flow cytometry plot showing two clear populations which indicate SYBR Green-stained sperm nuclei (SN) and vegetative cell nuclei (VN), respectively. AM and AN indicate the percentages of sorted SN and VN in total events, respectively.

Supplementary information

Supplementary Figures and Tables

Supplementary Figures 1–9 and Supplementary Tables 1, 3, 6 and 7

Life Sciences Reporting Summary.

Supplementary Table 2

List of loci that are significantly differentially methylated between the sexual lineage and somatic tissues.

Supplementary Table 4

Expression of 83 meiotic genes in our meiocyte RNA-seq data in comparison to those from published meiocyte transcriptomes.

Supplementary Table 5

List of differentially expressed genes and their association with SLMs.

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Walker, J., Gao, H., Zhang, J. et al. Sexual-lineage-specific DNA methylation regulates meiosis in Arabidopsis . Nat Genet 50, 130–137 (2018). https://doi.org/10.1038/s41588-017-0008-5

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