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

Abstract

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.

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Fig. 1: The CLSY family controls 24nt-siRNA levels in a locus-specific manner.
Fig. 2: Specific CLSY pairs regulate 24nt-siRNAs at non-overlapping and spatially distinct genomic loci.
Fig. 3: 24nt-siRNA losses in clsy mutants result in reduced DNA methylation.
Fig. 4: The CLSY family controls the expression of RdDM targets.
Fig. 5: The CLSY proteins are required for Pol-IV chromatin association at 24nt-siRNA producing loci.
Fig. 6: The CLSY1/2 and CLSY3/4 proteins regulate Pol-IV in connection with repressive chromatin marks.

References

  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).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  2. 2.

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

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  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).

    Article  PubMed  CAS  Google Scholar 

  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).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  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).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  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).

    Article  PubMed  CAS  Google Scholar 

  7. 7.

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

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  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).

    Article  PubMed  PubMed Central  Google Scholar 

  9. 9.

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

    Article  PubMed  PubMed Central  Google Scholar 

  10. 10.

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

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  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).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  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).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  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).

    Article  PubMed  CAS  Google Scholar 

  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).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  15. 15.

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

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  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).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  17. 17.

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

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  18. 18.

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

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  19. 19.

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

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  20. 20.

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

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  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).

    Article  PubMed  CAS  Google Scholar 

  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).

    Article  CAS  Google Scholar 

  23. 23.

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

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  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).

    Article  PubMed  CAS  Google Scholar 

  25. 25.

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

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  26. 26.

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

    Article  PubMed  CAS  Google Scholar 

  27. 27.

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

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  28. 28.

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

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  29. 29.

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

    Article  PubMed  CAS  Google Scholar 

  30. 30.

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

    Article  PubMed  CAS  Google Scholar 

  31. 31.

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

    Article  PubMed  CAS  Google Scholar 

  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).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  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).

    Article  PubMed  PubMed Central  Google Scholar 

  34. 34.

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

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  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).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  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).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  37. 37.

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

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  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).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  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).

    Article  CAS  Google Scholar 

  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).

    Article  PubMed  PubMed Central  Google Scholar 

  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).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  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).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  43. 43.

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

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  44. 44.

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

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  45. 45.

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

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  46. 46.

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

    Article  PubMed  CAS  Google Scholar 

  47. 47.

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

    Article  PubMed  CAS  Google Scholar 

  48. 48.

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

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  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).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  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).

    Article  PubMed  CAS  Google Scholar 

  51. 51.

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

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  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).

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  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).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  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).

    Article  PubMed  CAS  Google Scholar 

  56. 56.

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

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  57. 57.

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

    Article  PubMed  Google Scholar 

  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).

    Article  PubMed  CAS  Google Scholar 

  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).

    Article  PubMed  CAS  Google Scholar 

  60. 60.

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

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  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).

    Article  PubMed  CAS  Google Scholar 

  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).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  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).

    Article  PubMed  CAS  Google Scholar 

  64. 64.

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

    Article  PubMed  CAS  Google Scholar 

  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).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  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).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  67. 67.

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

    Article  PubMed  CAS  Google Scholar 

  68. 68.

    Martin, M. Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet.journal http://doi.org/10.14806/ej.17.1.200 (2011).

    Article  Google Scholar 

  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).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  70. 70.

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

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  71. 71.

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

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  72. 72.

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

    PubMed  PubMed Central  Article  Google Scholar 

  73. 73.

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

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  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).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  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).

    Article  CAS  Google Scholar 

  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).

    Article  PubMed  CAS  Google Scholar 

  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).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

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Acknowledgements

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.

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Contributions

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.

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Correspondence to Julie A. Law.

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Supplementary information

Supplementary Figures

Supplementary Figures 1–9

Reporting Summary

Supplementary Table 1

Summary of the mRNA-seq data

Supplementary Table 2

Summary of the smRNA-seq data

Supplementary Table 3

The numbers of 21-24nt-siRNA clusters

Supplementary Table 4

Reduced 24nt-siRNA clusters

Supplementary Table 5

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

Supplementary Table 6

Summary of the MethylC-seq data

Supplementary Table 7

Hypo DMRs lists

Supplementary Table 8

Upregulated loci in clsy and pol-iv mutants

Supplementary Table 9

Gene expression matrix for Fig. 4b

Supplementary Table 10

Summary of the ChIP-seq data

Supplementary Table 11

Primer oligos for qPCR in this study

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Zhou, M., Palanca, A.M.S. & Law, J.A. Locus-specific control of the de novo DNA methylation pathway in Arabidopsis by the CLASSY family. Nat Genet 50, 865–873 (2018). https://doi.org/10.1038/s41588-018-0115-y

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