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Polymerase IV occupancy at RNA-directed DNA methylation sites requires SHH1

Nature volume 498, pages 385–389 (2013)Cite this article

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Abstract

DNA methylation is an epigenetic modification that has critical roles in gene silencing, development and genome integrity. In Arabidopsis, DNA methylation is established by DOMAINS REARRANGED METHYLTRANSFERASE 2 (DRM2) and targeted by 24-nucleotide small interfering RNAs (siRNAs) through a pathway termed RNA-directed DNA methylation (RdDM)1. This pathway requires two plant-specific RNA polymerases: Pol-IV, which functions to initiate siRNA biogenesis, and Pol-V, which functions to generate scaffold transcripts that recruit downstream RdDM factors1,2. To understand the mechanisms controlling Pol-IV targeting we investigated the function of SAWADEE HOMEODOMAIN HOMOLOG 1 (SHH1)3,4, a Pol-IV-interacting protein3. Here we show that SHH1 acts upstream in the RdDM pathway to enable siRNA production from a large subset of the most active RdDM targets, and that SHH1 is required for Pol-IV occupancy at these same loci. We also show that the SHH1 SAWADEE domain is a novel chromatin-binding module that adopts a unique tandem Tudor-like fold and functions as a dual lysine reader, probing for both unmethylated K4 and methylated K9 modifications on the histone 3 (H3) tail. Finally, we show that key residues within both lysine-binding pockets of SHH1 are required in vivo to maintain siRNA and DNA methylation levels as well as Pol-IV occupancy at RdDM targets, demonstrating a central role for methylated H3K9 binding in SHH1 function and providing the first insights into the mechanism of Pol-IV targeting. Given the parallels between methylation systems in plants and mammals1,5, a further understanding of this early targeting step may aid our ability to control the expression of endogenous and newly introduced genes, which has broad implications for agriculture and gene therapy.

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Figure 1: Epigenetic profile of siRNA clusters affected in RdDM mutants.
Figure 2: Pol-IV levels at defined siRNA clusters.
Figure 3: The SHH1 SAWADEE domain recognizes H3K9 methylation and adopts a unique tandem Tudor domain-like fold.
Figure 4: Structural basis for recognition of H3(1–15)K9me2 peptide by the SHH1 SAWADEE domain and the functional impact of mutations of residues lining the K4 and K9me2 pockets.

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Accession codes

Primary accessions

Gene Expression Omnibus

Protein Data Bank

Data deposits

Coordinates and structure factors have been deposited in the RCSB Protein Data Bank with the accession codes: 4IUP for the Se–SAWADEE (L200M, L218M) complex and 4IUQ for the wild-type SAWADEE domain in the free state, 4IUR for the H3(1–15)K9me3–SAWADEE complex, 4IUT for the H3(1–15)K9me2– SAWADEE complex, 4IUU for the H3(1–15)K9me1–SAWADEE complex, and 4IUV for the H3(1–15)K4me1K9me1–SAWADEE complex. The genomics data are submitted to GEO under the accession number GSE45368.

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Acknowledgements

We are grateful to the staff of beamlines 24ID-C/E at the Argonne National Laboratory and X29A at the Brookhaven National Laboratory for support in diffraction data collection, to M. Akhavan and S. Fuchs for technical assistance, and to the UCLA BSCRC BioSequencing Core Facility for DNA sequencing. This work was supported by funds from the Abby Rockefeller Mauze Trust and Maloris and STARR Foundations to D.J.P. Work in the Jacobsen laboratory was supported by NIH grant GM60398. C.J.H. is supported by the Damon Runyon postdoctoral fellowship. S.F. is a Special Fellow of the Leukemia & Lymphoma Society. B.D.S. is supported by NIH grant GM85394. S.E.J. is an Investigator of the Howard Hughes Medical Institute.

Author information

Author notes

  1. Julie A. Law

    Present address: Present address: Plant Molecular and Cellular Biology, Salk Institute, La Jolla, California 92037, USA.,

  2. Julie A. Law, Jiamu Du and Christopher J. Hale: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Molecular, Cell and Developmental Biology, University of California at Los Angeles, Los Angeles, 90095, California, USA

    Julie A. Law, Christopher J. Hale, Suhua Feng & Steven E. Jacobsen

  2. Structural Biology Program, Memorial Sloan-Kettering Cancer Center, New York, 10065, New York, USA

    Jiamu Du & Dinshaw J. Patel

  3. Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, Los Angeles, California 90095, USA,

    Suhua Feng & Steven E. Jacobsen

  4. Howard Hughes Medical Institute, University of California at Los Angeles, Los Angeles, 90095, California, USA

    Suhua Feng & Steven E. Jacobsen

  5. Department of Biochemistry & Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, 27599, North Carolina, USA

    Krzysztof Krajewski & Brian D. Strahl

  6. Plant Molecular and Cellular Biology, Salk Institute, La Jolla, 92037, California, USA

    Ana Marie S. Palanca

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Contributions

J.A.L., J.D., C.J.H., S.F. and A.M.S.P. conducted the experiments, K.K. synthesized modified peptides, B.D.S., D.J.P. and S.E.J. directed the research, and J.A.L., J.D., C.J. H., D.J.P. and S.E.J. wrote the manuscript.

Corresponding authors

Correspondence to Dinshaw J. Patel or Steven E. Jacobsen.

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Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains Supplementary Figures 1-11 and Supplementary Tables 1-3. (PDF 1473 kb)

Supplementary Table 4

This file contains Genomic location (TAIR8) of sIRNA clusters and their genotypic dependencies. (XLSX 469 kb)

Supplementary Table 5

This file contains Genomic location (TAIR8) of PolIV peaks SHH1 and their dependency. (XLSX 42 kb)

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Law, J., Du, J., Hale, C. et al. Polymerase IV occupancy at RNA-directed DNA methylation sites requires SHH1. Nature 498, 385–389 (2013). https://doi.org/10.1038/nature12178

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