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Non-CG methylation patterns shape the epigenetic landscape in Arabidopsis


DNA methylation occurs in CG and non-CG sequence contexts. Non-CG methylation is abundant in plants and is mediated by CHROMOMETHYLASE (CMT) and DOMAINS REARRANGED METHYLTRANSFERASE (DRM) proteins; however, its roles remain poorly understood. Here we characterize the roles of non-CG methylation in Arabidopsis thaliana. We show that a poorly characterized methyltransferase, CMT2, is a functional methyltransferase in vitro and in vivo. CMT2 preferentially binds histone H3 Lys9 (H3K9) dimethylation and methylates non-CG cytosines that are regulated by H3K9 methylation. We revealed the contributions and redundancies between each non-CG methyltransferase in DNA methylation patterning and in regulating transcription. We also demonstrate extensive dependencies of small-RNA accumulation and H3K9 methylation patterning on non-CG methylation, suggesting self-reinforcing mechanisms between these epigenetic factors. The results suggest that non-CG methylation patterns are critical in shaping the landscapes of histone modification and small noncoding RNA.

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Figure 1: In vitro activity of CMT2.
Figure 2: CMT2 is mediated by H3K9 dimethylation.
Figure 3: Dissecting contributions of non-CG methyltransferases in DNA methylation patterning.
Figure 4: Non-CG methyltransferases cooperatively silence TEs and genes.
Figure 5: Relationship between non-CG methylation and 24-nt siRNA accumulation.
Figure 6: Relationship between non-CG methylation and H3K9 methylation.
Figure 7: Non-CG methylation pathways.

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We thank M. Akhavan for Illumina sequencing. Sequencing was performed at the University of California, Los Angeles (UCLA) Broad Stem Cell Research Center BioSequencing Core Facility. We thank W. Yang and M. Pellegrini for help with the UCSC browser. We thank L. Tao and D. Chen for technical help with experiments. We thank F. Berger for helpful comments. This work was supported by the Abby Rockefeller Mauzé Trust, the Maloris and Starr foundations (D.J.P.) and US National Institutes of Health grant GM60398 and National Science Foundation grant 0701745 (S.E.J.). H.S. was supported by a UCLA Dissertation Year Fellowship. X.Z. is supported by a Ruth L. Kirschstein National Research Service Award (F32GM096483-01). S.F. is supported as a Special Fellow of the Leukemia & Lymphoma Society. S.E.J. is supported as an investigator of the Howard Hughes Medical Institute.

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Authors and Affiliations



H.S., T.D., J.D., X.Z., S.F. and L.J. performed the experiments. S.E.J. and D.J.P. oversaw the study. H.S. designed the study, analyzed data and wrote the manuscript.

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Correspondence to Steven E Jacobsen.

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Integrated supplementary information

Supplementary Figure 1 CMT2 does not methylate CG sites in vitro, and its activity is distinct from that of CMT3.

(a) RT-PCR on CMT2 and ACTIN transcripts in wild type and cmt2-7 mutants. A no RT control for the CMT2 amplification is also shown. Cartoon of T-DNA insertion site in the CMT2 gene (At4g19020) is also shown. Thick lines, exons; thin lines, introns. (b) CMT3 in vitro methyltransferase activity assay performed in parallel to the CMT2 assay. Error bars represent SD for two technical replicates. (c) CMT2 in vitro methyltransferase activity on CG sites. Error bars represent SD for two technical replicates

Supplementary Figure 2 CMT2 preferentially binds H3K9 di- and trimethylated peptides in vitro.

(a) Sequence comparison between CMT2, CMT3, and ZMET2 (the maize CMT3 homolog) by ClustalW ( BAH and CHROMO domains are shaded. (b) Single modification peptide reactivity from the peptide array generated by the manufacturer software (Active Motif).

Supplementary Figure 3 Contributions of non-CG methyltransferases in DNA methylation patterning.

(a) CG, CHG, and CHH methylation levels across chromosomes. (b) Genome browser views of CHG and CHH methylation in chromosome 1. Blue bars, TEs; Yellow bars, genes. (c) Overlap between drm1 drm2 cmt2 and drm1 drm2 cmt2 cmt3 CHH DMRs. (d) Overlap between drm1 drm2 and cmt2 CHH DMRs.

Supplementary Figure 4 Roles of non-CG methyltransferases in gene silencing.

(a) Genome browser views of RNA-seq data. Blue bars, TEs; Yellow bars, genes. (b) Types of TEs upregulated in drm1 drm2 cmt2 cmt3 mutants. (c) Wild-type CHG and CHH methylation levels over genes. (d) Gene ontology analysis of genes upregulated in drm1 drm2 cmt2 cmt3 mutants using DAVID ( (e) Expression levels of SDC. Error bars represent SD from two biological replicates. (f) Photo of plants of indicated genotypes.

Supplementary Figure 5 24-nt siRNAs produced at CMT2 sites do not guide DNA methylation in cis.

(a) Average distribution of Pol IV over CMT2 target sites. ChIP-seq data on epitope-tagged CMT31, Pol IV2, Pol V3 relative to the respective controls, as well as Pol II relative to input genomic DNA was plotted over CMT2 target sites. (b) Heatmaps of CHH methylation levels4 within cmt2 CHH DMRs.

Supplementary Figure 6 Non-CG methylation shapes the histone-modification landscape.

(a) Genome browser views of DNA methylation, expression levels, and H3K9me2 in wild type, drm1 drm2 cmt2 cmt3, and kyp suvh5 suvh6 mutants in chromosome 1. Yellow bars, genes. (b) Average distribution of H3K9me2 over previously defined met1 CHG hypomethylation DMRs (black) and CHH hypomethylation DMRs (blue). Wild-type data is plotted in solid lines, and met1 data is plotted in faded lines. (c) Distribution of H3K9me2 over chromosomes in wild type and met1 mutants. (d) Average distribution of wild-type H3K23ac levels over genes categorized by indicated wild-type expression levels. (e) Distribution of H3K23ac relative to H3 across chromosomes. (f) Genome browser views of DNA methylation, expression levels, H3K23ac, and H3K9me2 in wild type, drm1 drm2 cmt2 cmt3, and kyp suvh5 suvh6 mutants in chromosome 1. Yellow bars, genes. (g) Chromocenter decondensation assay in indicated genotypes. >100 nuclei were assayed per genotype.

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Stroud, H., Do, T., Du, J. et al. Non-CG methylation patterns shape the epigenetic landscape in Arabidopsis. Nat Struct Mol Biol 21, 64–72 (2014).

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