Article | Published:

Locus-specific control of the de novo DNA methylation pathway in Arabidopsis by the CLASSY family

Nature Geneticsvolume 50pages865873 (2018) | Download Citation


DNA methylation is essential for gene regulation, transposon silencing and imprinting. Although the generation of specific DNA methylation patterns is critical for these processes, how methylation is regulated at individual loci remains unclear. Here we show that a family of four putative chromatin remodeling factors, CLASSY (CLSY) 1–4, are required for both locus-specific and global regulation of DNA methylation in Arabidopsis thaliana. Mechanistically, these factors act in connection with RNA polymerase-IV (Pol-IV) to control the production of 24-nucleotide small interfering RNAs (24nt-siRNAs), which guide DNA methylation. Individually, the CLSYs regulate Pol-IV–chromatin association and 24nt-siRNA production at thousands of distinct loci, and together, they regulate essentially all 24nt-siRNAs. Depending on the CLSYs involved, this regulation relies on different repressive chromatin modifications to facilitate locus-specific control of DNA methylation. Given the conservation between methylation systems in plants and mammals, analogous pathways may operate in a broad range of organisms.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Additional information

Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


  1. 1.

    Castel, S. E. & Martienssen, R. A. RNA interference in the nucleus: roles for small RNAs in transcription, epigenetics and beyond. Nat. Rev. Genet. 14, 100–112 (2013).

  2. 2.

    Holoch, D. & Moazed, D. RNA-mediated epigenetic regulation of gene expression. Nat. Rev. Genet. 16, 71–84 (2015).

  3. 3.

    Haag, J. R. & Pikaard, C. S. Multisubunit RNA polymerases IV and V: purveyors of non-coding RNA for plant gene silencing. Nat. Rev. Mol. Cell Biol. 12, 483–492 (2011).

  4. 4.

    Zhou, M. & Law, J. A. RNA Pol IV and V in gene silencing: rebel polymerases evolving away from Pol II’s rules. Curr. Opin. Plant Biol. 27, 154–164 (2015).

  5. 5.

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

  6. 6.

    Matzke, M. A. & Mosher, R. A. RNA-directed DNA methylation: an epigenetic pathway of increasing complexity. Nat. Rev. Genet. 15, 394–408 (2014).

  7. 7.

    Zhai, J. et al. A one precursor one siRNA model for Pol IV-dependent siRNA biogenesis. Cell 163, 445–455 (2015).

  8. 8.

    Blevins, T. et al. Identification of Pol IV and RDR2-dependent precursors of 24 nt siRNAs guiding de novo DNA methylation in Arabidopsis. eLife 4, e09591 (2015).

  9. 9.

    Xie, Z. et al. Genetic and functional diversification of small RNA pathways in plants. PLoS Biol. 2, E104 (2004).

  10. 10.

    Mallory, A. & Vaucheret, H. Form, function, and regulation of ARGONAUTE proteins. Plant Cell 22, 3879–3889 (2010).

  11. 11.

    Wierzbicki, A. T., Haag, J. R. & Pikaard, C. S. Noncoding transcription by RNA polymerase Pol IVb/Pol V mediates transcriptional silencing of overlapping and adjacent genes. Cell 135, 635–648 (2008).

  12. 12.

    El-Shami, M. et al. Reiterated WG/GW motifs form functionally and evolutionarily conserved ARGONAUTE-binding platforms in RNAi-related components. Genes Dev. 21, 2539–2544 (2007).

  13. 13.

    Li, C. F. et al. An ARGONAUTE4-containing nuclear processing center colocalized with Cajal bodies in Arabidopsis thaliana. Cell 126, 93–106 (2006).

  14. 14.

    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).

  15. 15.

    Zhong, X. et al. Molecular mechanism of action of plant DRM de novo DNA methyltransferases. Cell 157, 1050–1060 (2014).

  16. 16.

    Böhmdorfer, G. et al. RNA-directed DNA methylation requires stepwise binding of silencing factors to long non-coding RNA. Plant J. 79, 181–191 (2014).

  17. 17.

    Calarco, J. P. et al. Reprogramming of DNA methylation in pollen guides epigenetic inheritance via small RNA. Cell 151, 194–205 (2012).

  18. 18.

    Ibarra, C. A. et al. Active DNA demethylation in plant companion cells reinforces transposon methylation in gametes. Science 337, 1360–1364 (2012).

  19. 19.

    Kawakatsu, T. et al. Unique cell-type-specific patterns of DNA methylation in the root meristem. Nat. Plants 2, 16058 (2016).

  20. 20.

    Slotkin, R. K. et al. Epigenetic reprogramming and small RNA silencing of transposable elements in pollen. Cell 136, 461–472 (2009).

  21. 21.

    Tang, W. W., Kobayashi, T., Irie, N., Dietmann, S. & Surani, M. A. Specification and epigenetic programming of the human germ line. Nat. Rev. Genet. 17, 585–600 (2016).

  22. 22.

    Seisenberger, S. et al. Reprogramming DNA methylation in the mammalian life cycle: building and breaking epigenetic barriers. Phil. Trans. R. Soc. Lond. B 368, 20110330 (2013).

  23. 23.

    Hsieh, T. F. et al. Genome-wide demethylation of Arabidopsis endosperm. Science 324, 1451–1454 (2009).

  24. 24.

    Widman, N., Feng, S., Jacobsen, S. E. & Pellegrini, M. Epigenetic differences between shoots and roots in Arabidopsis reveals tissue-specific regulation. Epigenetics 9, 236–242 (2014).

  25. 25.

    Schultz, M. D. et al. Human body epigenome maps reveal noncanonical DNA methylation variation. Nature 523, 212–216 (2015).

  26. 26.

    Stricker, S. H., Köferle, A. & Beck, S. From profiles to function in epigenomics. Nat. Rev. Genet. 18, 51–66 (2017).

  27. 27.

    Heard, E. & Martienssen, R. A. Transgenerational epigenetic inheritance: myths and mechanisms. Cell 157, 95–109 (2014).

  28. 28.

    Pikaard, C. S. & Mittelsten Scheid, O. Epigenetic regulation in plants. Cold Spring Harb. Perspect. Biol. 6, a019315 (2014).

  29. 29.

    Springer, N. M. & Schmitz, R. J. Exploiting induced and natural epigenetic variation for crop improvement. Nat. Rev. Genet. 18, 563–575 (2017).

  30. 30.

    Smith, Z. D. & Meissner, A. DNA methylation: roles in mammalian development. Nat. Rev. Genet. 14, 204–220 (2013).

  31. 31.

    Klutstein, M., Nejman, D., Greenfield, R. & Cedar, H. DNA methylation in cancer and aging. Cancer Res. 76, 3446–3450 (2016).

  32. 32.

    Liu, J. et al. An atypical component of RNA-directed DNA methylation machinery has both DNA methylation-dependent and -independent roles in locus-specific transcriptional gene silencing. Cell Res. 21, 1691–1700 (2011).

  33. 33.

    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).

  34. 34.

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

  35. 35.

    Law, J. A., Vashisht, A. A., Wohlschlegel, J. A. & Jacobsen, S. E. SHH1, a homeodomain protein required for DNA methylation, as well as RDR2, RDM4, and chromatin remodeling factors, associate with RNA polymerase IV. PLoS Genet. 7, e1002195 (2011).

  36. 36.

    Smith, L. M. et al. An SNF2 protein associated with nuclear RNA silencing and the spread of a silencing signal between cells in Arabidopsis. Plant Cell 19, 1507–1521 (2007).

  37. 37.

    Greenberg, M. V. et al. Identification of genes required for de novo DNA methylation in Arabidopsis. Epigenetics 6, 344–354 (2011).

  38. 38.

    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).

  39. 39.

    Johnson, N. R., Yeoh, J. M., Coruh, C. & Axtell, M. J. Improved placement of multi-mapping small RNAs. G3 (Bethesda) 6, 2103–2111 (2016).

  40. 40.

    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).

  41. 41.

    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).

  42. 42.

    Love, M. I., Huber, W. & Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15, 550 (2014).

  43. 43.

    Cokus, S. J. et al. Shotgun bisulphite sequencing of the Arabidopsis genome reveals DNA methylation patterning. Nature 452, 215–219 (2008).

  44. 44.

    Lister, R. et al. Highly integrated single-base resolution maps of the epigenome in Arabidopsis. Cell 133, 523–536 (2008).

  45. 45.

    Bewick, A. J. & Schmitz, R. J. Gene body DNA methylation in plants. Curr. Opin. Plant Biol. 36, 103–110 (2017).

  46. 46.

    Chan, S. W. et al. RNA silencing genes control de novo DNA methylation. Science 303, 1336 (2004).

  47. 47.

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

  48. 48.

    Inagaki, S. et al. Gene-body chromatin modification dynamics mediate epigenome differentiation in Arabidopsis. EMBO J. 36, 970–980 (2017).

  49. 49.

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

  50. 50.

    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).

  51. 51.

    Blevins, T. et al. A two-step process for epigenetic inheritance in Arabidopsis. Mol. Cell 54, 30–42 (2014).

  52. 52.

    Kim, J. M., To, T. K. & Seki, M. An epigenetic integrator: new insights into genome regulation, environmental stress responses and developmental controls by histone deacetylase 6. Plant Cell Physiol. 53, 794–800 (2012).

  53. 53.

    Andersen, P. R., Tirian, L., Vunjak, M. & Brennecke, J. A heterochromatin-dependent transcription machinery drives piRNA expression. Nature 549, 54–59 (2017). advance online publication.

  54. 54.

    Hale, C. J., Stonaker, J. L., Gross, S. M. & Hollick, J. B. A novel Snf2 protein maintains trans-generational regulatory states established by paramutation in maize. PLoS Biol. 5, e275 (2007).

  55. 55.

    Hu, Y. et al. Analysis of rice Snf2 family proteins and their potential roles in epigenetic regulation. Plant Physiol. Biochem. 70, 33–42 (2013).

  56. 56.

    Yelina, N. E. et al. Epigenetic remodeling of meiotic crossover frequency in Arabidopsis thaliana DNA methyltransferase mutants. PLoS Genet. 8, e1002844 (2012).

  57. 57.

    Alonso, J. M. et al. Genome-wide insertional mutagenesis of Arabidopsis thaliana. Science 301, 653–657 (2003).

  58. 58.

    Woody, S. T., Austin-Phillips, S., Amasino, R. M. & Krysan, P. J. The WiscDsLox T-DNA collection: an Arabidopsis community resource generated by using an improved high-throughput T-DNA sequencing pipeline. J. Plant Res. 120, 157–165 (2007).

  59. 59.

    Kleinboelting, N., Huep, G., Kloetgen, A., Viehoever, P. & Weisshaar, B. GABI-Kat SimpleSearch: new features of the Arabidopsis thaliana T-DNA mutant database. Nucleic Acids Res. 40, D1211–D1215 (2012).

  60. 60.

    Sessions, A. et al. A high-throughput Arabidopsis reverse genetics system. Plant Cell 14, 2985–2994 (2002).

  61. 61.

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

  62. 62.

    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).

  63. 63.

    Saze, H., Mittelsten Scheid, O. & Paszkowski, J. Maintenance of CpG methylation is essential for epigenetic inheritance during plant gametogenesis. Nat. Genet. 34, 65–69 (2003).

  64. 64.

    Vongs, A., Kakutani, T., Martienssen, R. A. & Richards, E. J. Arabidopsis thaliana DNA methylation mutants. Science 260, 1926–1928 (1993).

  65. 65.

    Jacob, Y. et al. ATXR5 and ATXR6 are H3K27 monomethyltransferases required for chromatin structure and gene silencing. Nat. Struct. Mol. Biol. 16, 763–768 (2009).

  66. 66.

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

  67. 67.

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

  68. 68.

    Martin, M. Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet.journal (2011).

  69. 69.

    Heinz, S. et al. Simple combinations of lineage-determining transcription factors prime cis-regulatory elements required for macrophage and B cell identities. Mol. Cell 38, 576–589 (2010).

  70. 70.

    Kestler, H. A. et al. VennMaster: area-proportional Euler diagrams for functional GO analysis of microarrays. BMC Bioinformatics 9, 67 (2008).

  71. 71.

    Neph, S. et al. BEDOPS: high-performance genomic feature operations. Bioinformatics 28, 1919–1920 (2012).

  72. 72.

    Li, D. et al. The MBD7 complex promotes expression of methylated transgenes without significantly altering their methylation status. Elife 6, e19893 (2017).

  73. 73.

    Dobin, A. et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29, 15–21 (2013).

  74. 74.

    Kim, D. et al. TopHat2: accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions. Genome Biol. 14, R36 (2013).

  75. 75.

    Ramírez, F. et al. deepTools2: a next generation web server for deep-sequencing data analysis. Nucleic Acids Res. 44 W1, W160–W165 (2016).

  76. 76.

    Clough, S. J. & Bent, A. F. Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 16, 735–743 (1998).

  77. 77.

    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).

Download references


We thank laboratory members and colleagues for comments and discussions, the Salk Institute NGS core for sequencing, the bioinformatics core for technical support, and L. and C. Greenfield for charitable contributions. This work was supported by the NIH (GM112966) and Hearst Foundation to J.L. M.Z. was funded by a Pioneer Fund Postdoctoral Award. M.Z. and A.M.S.P. were funded by postdoctoral fellowships from the Glenn Center for Aging Research at the Salk Institute. This work was also supported by the NGS Core Facility and the Integrative Genomics and Bioinformatics Core Facility at the Salk Institute with funding from NIH-NCI CCSG P30 014195, the Glenn Center for Aging Research at the Salk Institute, the Chapman Foundation and the Helmsley Charitable Trust.

Author information


  1. Plant Molecular and Cellular Biology Laboratory, Salk Institute for Biological Studies, La Jolla, CA, USA

    • Ming Zhou
    • , Ana Marie S. Palanca
    •  & Julie A. Law


  1. Search for Ming Zhou in:

  2. Search for Ana Marie S. Palanca in:

  3. Search for Julie A. Law in:


M.Z. and J.L. conducted the experiments, A.M.S.P. contributed to preparation of genetic materials as well as the MethylC-seq samples, J.L. directed the research, and M.Z., A.M.S.P. and J.L. wrote the manuscript.

Competing interests

The authors declare no competing interests.

Corresponding author

Correspondence to Julie A. Law.

Supplementary information

  1. Supplementary Figures

    Supplementary Figures 1–9

  2. Reporting Summary

  3. Supplementary Table 1

    Summary of the mRNA-seq data

  4. Supplementary Table 2

    Summary of the smRNA-seq data

  5. Supplementary Table 3

    The numbers of 21-24nt-siRNA clusters

  6. Supplementary Table 4

    Reduced 24nt-siRNA clusters

  7. Supplementary Table 5

    Down-regulated 21nt- and 22nt-siRNA clusters

  8. Supplementary Table 6

    Summary of the MethylC-seq data

  9. Supplementary Table 7

    Hypo DMRs lists

  10. Supplementary Table 8

    Upregulated loci in clsy and pol-iv mutants

  11. Supplementary Table 9

    Gene expression matrix for Fig. 4b

  12. Supplementary Table 10

    Summary of the ChIP-seq data

  13. Supplementary Table 11

    Primer oligos for qPCR in this study

  14. Source Data

    Custom code

About this article

Publication history