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



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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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:

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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

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