Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Non-CG methylation patterns shape the epigenetic landscape in Arabidopsis

Abstract

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.

This is a preview of subscription content

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

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.

Accession codes

Primary accessions

Gene Expression Omnibus

References

  1. Law, J.A. & Jacobsen, S.E. Establishing, maintaining and modifying DNA methylation patterns in plants and animals. Nat. Rev. Genet. 11, 204–220 (2010).

    CAS  Article  Google Scholar 

  2. Lister, R. et al. Human DNA methylomes at base resolution show widespread epigenomic differences. Nature 462, 315–322 (2009).

    CAS  Article  Google Scholar 

  3. Xie, W. et al. Base-resolution analyses of sequence and parent-of-origin dependent DNA methylation in the mouse genome. Cell 148, 816–831 (2012).

    CAS  Article  Google Scholar 

  4. Laurent, L. et al. Dynamic changes in the human methylome during differentiation. Genome Res. 20, 320–331 (2010).

    CAS  Article  Google Scholar 

  5. Meissner, A. et al. Reduced representation bisulfite sequencing for comparative high-resolution DNA methylation analysis. Nucleic Acids Res. 33, 5868–5877 (2005).

    CAS  Article  Google Scholar 

  6. Ramsahoye, B.H. et al. Non-CpG methylation is prevalent in embryonic stem cells and may be mediated by DNA methyltransferase 3a. Proc. Natl. Acad. Sci. USA 97, 5237–5242 (2000).

    CAS  Article  Google Scholar 

  7. Varley, K.E. et al. Dynamic DNA methylation across diverse human cell lines and tissues. Genome Res. 23, 555–567 (2013).

    CAS  Article  Google Scholar 

  8. Stroud, H., Greenberg, M.V., Feng, S., Bernatavichute, Y.V. & Jacobsen, S.E. Comprehensive analysis of silencing mutants reveals complex regulation of the Arabidopsis methylome. Cell 152, 352–364 (2013).

    CAS  Article  Google Scholar 

  9. Zemach, A. et al. The Arabidopsis nucleosome remodeler DDM1 allows DNA methyltransferases to access H1-containing heterochromatin. Cell 153, 193–205 (2013).

    CAS  Article  Google Scholar 

  10. Du, J. et al. Dual binding of chromomethylase domains to H3K9me2-containing nucleosomes directs DNA methylation in plants. Cell 151, 167–180 (2012).

    CAS  Article  Google Scholar 

  11. Ebbs, M.L. & Bender, J. Locus-specific control of DNA methylation by the Arabidopsis SUVH5 histone methyltransferase. Plant Cell 18, 1166–1176 (2006).

    CAS  Article  Google Scholar 

  12. Jackson, J.P., Lindroth, A.M., Cao, X. & Jacobsen, S.E. Control of CpNpG DNA methylation by the KRYPTONITE histone H3 methyltransferase. Nature 416, 556–560 (2002).

    CAS  Article  Google Scholar 

  13. Henikoff, S. & Comai, L. A DNA methyltransferase homolog with a chromodomain exists in multiple polymorphic forms in Arabidopsis. Genetics 149, 307–318 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Law, J.A. et al. Polymerase IV occupancy at RNA-directed DNA methylation sites requires SHH1. Nature 498, 385–389 (2013).

    CAS  Article  Google Scholar 

  15. Zhang, H. et al. DTF1 is a core component of RNA-directed DNA methylation and may assist in the recruitment of Pol IV. Proc. Natl. Acad. Sci. USA 110, 8290–8295 (2013).

    CAS  Article  Google Scholar 

  16. Johnson, L. et al. Mass spectrometry analysis of Arabidopsis histone H3 reveals distinct combinations of post-translational modifications. Nucleic Acids Res. 32, 6511–6518 (2004).

    CAS  Article  Google Scholar 

  17. Roudier, F. et al. Integrative epigenomic mapping defines four main chromatin states in Arabidopsis. EMBO J. 30, 1928–1938 (2011).

    CAS  Article  Google Scholar 

  18. Jullien, P.E., Susaki, D., Yelagandula, R., Higashiyama, T. & Berger, F. DNA methylation dynamics during sexual reproduction in Arabidopsis thaliana. Curr. Biol. 22, 1825–1830 (2012).

    CAS  Article  Google Scholar 

  19. Henderson, I.R. & Jacobsen, S.E. Tandem repeats upstream of the Arabidopsis endogene SDC recruit non-CG DNA methylation and initiate siRNA spreading. Genes Dev. 22, 1597–1606 (2008).

    CAS  Article  Google Scholar 

  20. Herr, A.J., Jensen, M.B., Dalmay, T. & Baulcombe, D.C. RNA polymerase IV directs silencing of endogenous DNA. Science 308, 118–120 (2005).

    CAS  Article  Google Scholar 

  21. Pontier, D. et al. Reinforcement of silencing at transposons and highly repeated sequences requires the concerted action of two distinct RNA polymerases IV in Arabidopsis. Genes Dev. 19, 2030–2040 (2005).

    CAS  Article  Google Scholar 

  22. Zilberman, D., Cao, X. & Jacobsen, S.E. ARGONAUTE4 control of locus-specific siRNA accumulation and DNA and histone methylation. Science 299, 716–719 (2003).

    CAS  Article  Google Scholar 

  23. Kasschau, K.D. et al. Genome-wide profiling and analysis of Arabidopsis siRNAs. PLoS Biol. 5, e57 (2007).

    Article  Google Scholar 

  24. Mosher, R.A., Schwach, F., Studholme, D. & Baulcombe, D.C. PolIVb influences RNA-directed DNA methylation independently of its role in siRNA biogenesis. Proc. Natl. Acad. Sci. USA 105, 3145–3150 (2008).

    CAS  Article  Google Scholar 

  25. Zhang, X., Henderson, I.R., Lu, C., Green, P.J. & Jacobsen, S.E. Role of RNA polymerase IV in plant small RNA metabolism. Proc. Natl. Acad. Sci. USA 104, 4536–4541 (2007).

    CAS  Article  Google Scholar 

  26. Wierzbicki, A.T. et al. Spatial and functional relationships among Pol V-associated loci, Pol IV-dependent siRNAs, and cytosine methylation in the Arabidopsis epigenome. Genes Dev. 26, 1825–1836 (2012).

    CAS  Article  Google Scholar 

  27. McCue, A.D., Nuthikattu, S. & Slotkin, R.K. Genome-wide identification of genes regulated in trans by transposable element small interfering RNAs. RNA Biol. 10, 1379–1395 (2013).

    CAS  Article  Google Scholar 

  28. Zhong, X. et al. DDR complex facilitates global association of RNA polymerase V to promoters and evolutionarily young transposons. Nat. Struct. Mol. Biol. 19, 870–875 (2012).

    CAS  Article  Google Scholar 

  29. Johnson, L.M. et al. The SRA methyl-cytosine-binding domain links DNA and histone methylation. Curr. Biol. 17, 379–384 (2007).

    CAS  Article  Google Scholar 

  30. Inagaki, S. et al. Autocatalytic differentiation of epigenetic modifications within the Arabidopsis genome. EMBO J. 29, 3496–3506 (2010).

    CAS  Article  Google Scholar 

  31. Mathieu, O., Probst, A.V. & Paszkowski, J. Distinct regulation of histone H3 methylation at lysines 27 and 9 by CpG methylation in Arabidopsis. EMBO J. 24, 2783–2791 (2005).

    CAS  Article  Google Scholar 

  32. Soppe, W.J. et al. DNA methylation controls histone H3 lysine 9 methylation and heterochromatin assembly in Arabidopsis. EMBO J. 21, 6549–6559 (2002).

    CAS  Article  Google Scholar 

  33. Johnson, L., Cao, X. & Jacobsen, S. Interplay between two epigenetic marks. DNA methylation and histone H3 lysine 9 methylation. Curr. Biol. 12, 1360–1367 (2002).

    CAS  Article  Google Scholar 

  34. Deleris, A. et al. Loss of the DNA methyltransferase MET1 induces H3K9 hypermethylation at PcG target genes and redistribution of H3K27 trimethylation to transposons in Arabidopsis thaliana. PLoS Genet. 8, e1003062 (2012).

    CAS  Article  Google Scholar 

  35. Wierzbicki, A.T., Ream, T.S., Haag, J.R. & Pikaard, C.S. RNA polymerase V transcription guides ARGONAUTE4 to chromatin. Nat. Genet. 41, 630–634 (2009).

    CAS  Article  Google Scholar 

  36. Chan, S.W. et al. RNAi, DRD1, and histone methylation actively target developmentally important non-CG DNA methylation in Arabidopsis. PLoS Genet. 2, e83 (2006).

    Article  Google Scholar 

  37. Feng, S., Rubbi, L., Jacobsen, S.E. & Pellegrini, M. Determining DNA methylation profiles using sequencing. Methods Mol. Biol. 733, 223–238 (2011).

    CAS  Article  Google Scholar 

  38. Stroud, H. et al. DNA methyltransferases are required to induce heterochromatic re-replication in Arabidopsis. PLoS Genet. 8, e1002808 (2012).

    CAS  Article  Google Scholar 

  39. Langmead, B., Trapnell, C., Pop, M. & Salzberg, S.L. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 10, R25 (2009).

    Article  Google Scholar 

  40. Lu, C., Meyers, B.C. & Green, P.J. Construction of small RNA cDNA libraries for deep sequencing. Methods 43, 110–117 (2007).

    Article  Google Scholar 

  41. Moissiard, G. et al. MORC family ATPases required for heterochromatin condensation and gene silencing. Science 336, 1448–1451 (2012).

    CAS  Article  Google Scholar 

Download references

Acknowledgements

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.

Author information

Authors and Affiliations

Authors

Contributions

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.

Corresponding author

Correspondence to Steven E Jacobsen.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

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 (http://www.genome.jp/tools/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 (http://david.abcc.ncifcrf.gov/). (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.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–6 and Supplementary Tables 1–3 (PDF 8888 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

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). https://doi.org/10.1038/nsmb.2735

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsmb.2735

Further reading

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing