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SRA- and SET-domain-containing proteins link RNA polymerase V occupancy to DNA methylation

A Corrigendum to this article was published on 08 February 2017

Abstract

RNA-directed DNA methylation in Arabidopsis thaliana depends on the upstream synthesis of 24-nucleotide small interfering RNAs (siRNAs) by RNA POLYMERASE IV (Pol IV)1,2 and downstream synthesis of non-coding transcripts by Pol V. Pol V transcripts are thought to interact with siRNAs which then recruit DOMAINS REARRANGED METHYLTRANSFERASE 2 (DRM2) to methylate DNA3,4,5,6,7. The SU(VAR)3-9 homologues SUVH2 and SUVH9 act in this downstream step but the mechanism of their action is unknown8,9. Here we show that genome-wide Pol V association with chromatin redundantly requires SUVH2 and SUVH9. Although SUVH2 and SUVH9 resemble histone methyltransferases, a crystal structure reveals that SUVH9 lacks a peptide-substrate binding cleft and lacks a properly formed S-adenosyl methionine (SAM)-binding pocket necessary for normal catalysis, consistent with a lack of methyltransferase activity for these proteins8. SUVH2 and SUVH9 both contain SRA (SET- and RING-ASSOCIATED) domains capable of binding methylated DNA8, suggesting that they function to recruit Pol V through DNA methylation. Consistent with this model, mutation of DNA METHYLTRANSFERASE 1 (MET1) causes loss of DNA methylation, a nearly complete loss of Pol V at its normal locations, and redistribution of Pol V to sites that become hypermethylated. Furthermore, tethering SUVH2 with a zinc finger to an unmethylated site is sufficient to recruit Pol V and establish DNA methylation and gene silencing. These results indicate that Pol V is recruited to DNA methylation through the methyl-DNA binding SUVH2 and SUVH9 proteins, and our mechanistic findings suggest a means for selectively targeting regions of plant genomes for epigenetic silencing.

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Figure 1: Crystal structure of SUVH9.
Figure 2: SUVH2 and SUVH9 are required for Pol V-dependent siRNA production, chromatin binding and transcription.
Figure 3: Pol V binding is dependent on DNA methylation.
Figure 4: Tethered SUVH2 recruits Pol V through DRD1, resulting in DNA methylation and a late-flowering phenotype.

Accession codes

Accessions

Gene Expression Omnibus

Protein Data Bank

Data deposits

The coordinates and structure factors of SUVH9 have been deposited in the RCSB Protein Data Bank with the accession code 4NJ5. Sequencing data have been deposited at GEO (GSE52041).

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Acknowledgements

We are grateful to W. Shi at the National Synchrotron Light Source (NSLS) at Brookhaven National Laboratory (BNL) for support in diffraction data collection. We thank C. Pikaard for the NRPE1 antibodies and M. Akhavan for technical assistance. High-throughput sequencing was performed in the UCLA BSCRC BioSequencing Core Facility. This work was supported by the Abby Rockefeller Mauze Trust and the Maloris and STARR foundations to D.J.P., and NIH grant GM60398 to S.E.J. C.J.H. is supported by the Damon Runyon post-doctoral fellowship, S.B. is supported by a post-doctoral fellowship of the Swiss National Science Foundation, S.F. is a Special Fellow of the Leukemia & Lymphoma Society, and X.Z. is supported by Ruth L. Kirschstein National Research Service grant F32GM096483-01. S.E.J. is an Investigator of the Howard Hughes Medical Institute.

Author information

Authors and Affiliations

Authors

Contributions

Protein purification and crystallography were designed and performed by J.D., G.M. and D.J.P., siRNA libraries were made by S.B., whole-genome bisulphite sequencing was performed and analysed by S.F., L.M.J., C.H., R.K.C. and M.P., Pol V transcription assays were performed by X.Z., ChIP-seq was performed and analysed by L.M.J. and C.H., cloning and flowering time experiments were performed by L.M.J., the Zn finger was designed by D.J.S. and S.E.J., pull-down experiments were performed by L.M.J. and S.B., the manuscript was written by L.M.J., J.D., D.J.P. and S.E.J., and S.E.J. participated in all experimental design.

Corresponding authors

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

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Interdomain interactions of SUVH9.

a, Colour-coded schematic representation of full length SUVH9 and the N-terminally truncated construct used for crystallization. b, The hydrophobic interactions and charged interactions within the two-helix bundle shown in two alternate views rotated by 180 degree. Residues involved in inter-helix hydrophobic interactions are highlighted in yellow. c, The N-terminal part of the first α-helix forms charged and hydrogen bonding interactions with the SRA domain and the SET domain. The interacting residues are shown in stick representation and the hydrogen-bonding interactions are shown with dashed red lines. d, The C-terminal part of the first α-helix exhibits extensive hydrophobic interactions with the SRA domain and the pre-SET/SET domains. The tip of a long loop from the SET domain covers over the first helix and forms hydrophobic interactions with it. e, The second α-helix forms some interactions with the SRA domain. f, The SRA domain forms a hydrophobic core that interacts with the pre-SET/SET domains. g, A long insertion loop of SUVH9 SET domain (highlighted in magenta) is enriched with hydrophobic residues and forms extensive hydrophobic interactions with the two-helix bundle, the pre-SET and SET domains.

Extended Data Figure 2 SUVH9 SRA and pre-SET/SET domains.

a, A model positioning the mCHH DNA to the active site of SUVH9 SRA domain following superposition of the structures of the SUVH5 SRA–mCHH complex (PDB code 3Q0F) and SUVH9 in the free state. SUVH9 domains are depicted with the same colour-coding as in Fig. 1a and the modelled DNA is coloured in yellow. The DNA fits well into the SRA domain without significant steric clashes. Some surrounding residues on the second α-helix of the two-helix bundle, which can potentially be involved in the binding to the DNA, are highlighted in a stick representation. b, A stereo view of the superposition of the structure of SUVH9 in the free state and the structure of human GLP catalytic fragment complexed with SAH (PDB code 2IGQ). The GLP pre-SET and SET domains are coloured in silver and its post-SET domain is coloured in cyan. The zinc-binding motif of GLP post-SET domain and SET domain, the bound SAH molecule, and the corresponding Thr 597 of SUVH9 are highlighted in a stick representation.

Extended Data Figure 3 Structure-based sequence alignment of SUVH family proteins from Arabidopsis.

The secondary structural elements of SUVH9 are labelled on the top of the sequence alignment. The domain boundaries are marked on the top and depicted with colour-coding as in Fig. 1a. Conserved residues involved in the interaction with flipped 5mC base and the DNA backbone available from the published SUVH5–DNA complex structures are highlighted with cyan circles and blue hexagons, respectively. The insertions in the SET domains are highlighted with a purple box. The zinc-coordinating Cys residues are highlighted with black stars in the SET domain and grey stars in the post-SET domain. Two-tyrosine residues that are conserved and normally important for enzymatic activity are highlighted with red dots.

Extended Data Figure 4 SUVH2 and SUVH9 act redundantly genome-wide.

a, Metaplots of CHH methylation over DMRs identified in the various SUVH mutants. b, Metaplots of CHH methylation over Pol V binding sites. c, Venn diagram detailing the overlaps between CHH hypo-methylated regions in SUVH mutants.

Extended Data Figure 5 Pol V occupancy in WT versus met1.

Chromosome 1 showing Pol V ChIP in WT versus met1 as mapped over TAIR10 (green genes, red transposable elements (TEs)).

Extended Data Figure 6 Screen shot of Pol V binding in WT versus met1.

An example of reduced Pol V binding in met1 at sites that become hypomethylated.

Extended Data Figure 7 Screen shot of Pol V binding in WT versus met1.

Reduction in Pol V binding in a met1 hypomethylated site.

Extended Data Figure 8 Screen shot of Pol V binding at a hyper-CHH methylated site in WT versus met1.

An example of Pol V being redistributed to regions that gain methylation in met1.

Extended Data Figure 9 Pol V binding at hyper-CHH methylated site that is also transcribed.

Strong Pol V binding was detected at regions in the genome that not only retained high levels of non-CG methylation, but also were transcriptionally activated in met1.

Extended Data Figure 10 ZF–SUVH2 construct stably recruits Pol V to FWA through a direct interaction with DRD1.

a, Top, diagram of SUVH2 with Zn finger (ZF) inserted immediately before the HA tag. Bottom, schematic of FWA gene showing the two small and two large repeats (blue arrows), the regions amplified by PCR (promoter and transcript, green lines), the start and direction of transcription (red arrow), and the sites bound by the ZF (indicated by two orange arrows). b, Flag-ChIP in WT versus ZF–KYP (Flag-tagged) showing enrichment at FWA in both the promoter and transcript region (see above). c, Per cent methylation at each C in the FWA repeat region from three individual T1 plants. Per cent methylation was determined from 20–25 clones of bisulphite-treated DNA. d, BS-seq of FWA from a Basta-resistant line containing the ZF–SUVH2 transgene and two Basta-sensitive siblings which had lost the ZF–SUVH2 transgene. e, Pull-down of DRD1–Flag with ZF–SUVH2. Input, DRD1–Flag extract from Arabidopsis; Beads-mock, elution from DRD1–Flag pull-down using HA-magnetic beads pre-bound with Nicotiana benthamiana extract; Beads–ZF–SUVH2, elution from DRD1–Flag pull-down using HA-magnetic beads pre-bound with Nicotiana benthamiana ZF–SUVH2 extract. Top, Flag blot; bottom, HA blot.

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Johnson, L., Du, J., Hale, C. et al. SRA- and SET-domain-containing proteins link RNA polymerase V occupancy to DNA methylation. Nature 507, 124–128 (2014). https://doi.org/10.1038/nature12931

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