SRA- and SET-domain-containing proteins link RNA polymerase V occupancy to DNA methylation (original) (raw)

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).

References

  1. Pélissier, T. & Wassenegger, M. A DNA target of 30 bp is sufficient for RNA-directed DNA methylation. RNA 6, 55–65 (2000)
    Article PubMed Central PubMed Google Scholar
  2. Aufsatz, W., Mette, M. F., van der Winden, J., Matzke, A. J. & Matzke, M. RNA-directed DNA methylation in Arabidopsis. Proc. Natl Acad. Sci. USA 99 (Suppl. 4). 16499–16506 (2002)
    Article ADS CAS PubMed PubMed Central Google Scholar
  3. Law, J. A. & Jacobsen, S. E. Establishing, maintaining and modifying DNA methylation patterns in plants and animals. Nature Rev. Genet. 11, 204–220 (2010)
    Article CAS PubMed Google Scholar
  4. Pontier, D. et al. Reinforcement of silencing at transposons and highly repeated sequences requires the concerted action of two distinct RNA polymerases IV in Arabidopsis. Genes Dev. 19, 2030–2040 (2005)
    Article CAS PubMed Central PubMed Google Scholar
  5. Pikaard, C. S., Haag, J. R., Ream, T. & Wierzbicki, A. T. Roles of RNA polymerase IV in gene silencing. Trends Plant Sci. 13, 390–397 (2008)
    Article CAS PubMed Central PubMed Google Scholar
  6. Law, J. A. et al. A protein complex required for polymerase V transcripts and RNA-directed DNA methylation in Arabidopsis. Curr. Biol. 20, 951–956 (2010)
    Article CAS PubMed Central PubMed Google Scholar
  7. Wierzbicki, A. T., Ream, T. S., Haag, J. R. & Pikaard, C. S. RNA polymerase V transcription guides ARGONAUTE4 to chromatin. Nature Genet. 41, 630–634 (2009)
    Article CAS PubMed Google Scholar
  8. Johnson, L. M., Law, J. A., Khattar, A., Henderson, I. R. & Jacobsen, S. E. SRA-domain proteins required for DRM2-mediated de novo DNA methylation. PLoS Genet. 4, e1000280 (2008)
    Article PubMed Central PubMed CAS Google Scholar
  9. Kuhlmann, M. & Mette, M. F. Developmentally non-redundant SET domain proteins SUVH2 and SUVH9 are required for transcriptional gene silencing in Arabidopsis thaliana. Plant Mol. Biol. 79, 623–633 (2012)
    Article CAS PubMed Central PubMed Google Scholar
  10. Hashimoto, H. et al. The SRA domain of UHRF1 flips 5-methylcytosine out of the DNA helix. Nature 455, 826–829 (2008)
    Article ADS CAS PubMed Central PubMed Google Scholar
  11. Avvakumov, G. V. et al. Structural basis for recognition of hemi-methylated DNA by the SRA domain of human UHRF1. Nature 455, 822–825 (2008)
    Article ADS CAS PubMed Google Scholar
  12. Arita, K., Ariyoshi, M., Tochio, H., Nakamura, Y. & Shirakawa, M. Recognition of hemi-methylated DNA by the SRA protein UHRF1 by a base-flipping mechanism. Nature 455, 818–821 (2008)
    Article ADS CAS PubMed Google Scholar
  13. Rajakumara, E. et al. A dual flip-out mechanism for 5mC recognition by the Arabidopsis SUVH5 SRA domain and its impact on DNA methylation and H3K9 dimethylation in vivo. Genes Dev. 25, 137–152 (2011)
    Article CAS PubMed Central PubMed Google Scholar
  14. Zhang, X. et al. Structure of the Neurospora SET domain protein DIM-5, a histone H3 lysine methyltransferase. Cell 111, 117–127 (2002)
    Article CAS PubMed Central PubMed Google Scholar
  15. Zhang, X. et al. Structural basis for the product specificity of histone lysine methyltransferases. Mol. Cell 12, 177–185 (2003)
    Article PubMed Central PubMed Google Scholar
  16. Wu, H. et al. Structural biology of human H3K9 methyltransferases. PLoS ONE 5, e8570 (2010)
    Article ADS PubMed Central PubMed CAS Google Scholar
  17. 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 ADS CAS PubMed PubMed Central Google Scholar
  18. Zhong, X. et al. DDR complex facilitates global association of RNA polymerase V to promoters and evolutionarily young transposons. Nature Struct. Mol. Biol. 19, 870–875 (2012)
    Article CAS Google Scholar
  19. 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 CAS PubMed Central PubMed Google Scholar
  20. Aufsatz, W., Mette, M. F., Matzke, A. J. & Matzke, M. The role of MET1 in RNA-directed de novo and maintenance methylation of CG dinucleotides. Plant Mol. Biol. 54, 793–804 (2004)
    Article CAS PubMed Google Scholar
  21. 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 CAS PubMed Central PubMed Google Scholar
  22. Lister, R. et al. Highly integrated single-base resolution maps of the epigenome in Arabidopsis. Cell 133, 523–536 (2008)
    Article CAS PubMed Central PubMed Google Scholar
  23. Soppe, W. J. et al. The late flowering phenotype of fwa mutants is caused by gain-of-function epigenetic alleles of a homeodomain gene. Mol. Cell 6, 791–802 (2000)
    Article CAS PubMed Google Scholar
  24. Kakutani, T. Genetic characterization of late-flowering traits induced by DNA hypomethylation mutation in Arabidopsis thaliana. Plant J. 12, 1447–1451 (1997)
    Article CAS PubMed Google Scholar
  25. Chan, S. W., Zhang, X., Bernatavichute, Y. V. & Jacobsen, S. E. Two-step recruitment of RNA-directed DNA methylation to tandem repeats. PLoS Biol. 4, e363 (2006)
    Article PubMed Central PubMed CAS Google Scholar
  26. Law, J. A. et al. Polymerase IV occupancy at RNA-directed DNA methylation sites requires SHH1. Nature 498, 385–389 (2013)
    Article ADS CAS PubMed Central PubMed Google Scholar
  27. 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 ADS CAS PubMed PubMed Central Google Scholar
  28. 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 CAS PubMed Central PubMed Google Scholar
  29. Tariq, M. et al. Erasure of CpG methylation in Arabidopsis alters patterns of histone H3 methylation in heterochromatin. Proc. Natl Acad. Sci. USA 100, 8823–8827 (2003)
    Article ADS CAS PubMed PubMed Central Google Scholar
  30. 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 CAS PubMed Central PubMed Google Scholar
  31. Bernatavichute, Y. V., Zhang, X., Cokus, S., Pellegrini, M. & Jacobsen, S. E. Genome-wide association of histone H3 lysine nine methylation with CHG DNA methylation in Arabidopsis thaliana. PLoS ONE 3, e3156 (2008)
    Article ADS PubMed Central PubMed CAS Google Scholar
  32. Greenberg, M. V. et al. Interplay between active chromatin marks and RNA-directed DNA methylation in Arabidopsis thaliana. PLoS Genet. 9, e1003946 (2013)
    Article PubMed Central PubMed CAS Google Scholar
  33. Kinoshita, Y. et al. Control of FWA gene silencing in Arabidopsis thaliana by SINE-related direct repeats. Plant J. 49, 38–45 (2007)
    Article CAS PubMed Google Scholar
  34. Kolb, A. F. et al. Site-directed genome modification: nucleic acid and protein modules for targeted integration and gene correction. Trends Biotechnol. 23, 399–406 (2005)
    Article CAS PubMed Google Scholar
  35. Segal, D. J. et al. Evaluation of a modular strategy for the construction of novel polydactyl zinc finger DNA-binding proteins. Biochemistry 42, 2137–2148 (2003)
    Article CAS PubMed Google Scholar
  36. Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997)
    Article CAS PubMed Google Scholar
  37. Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D 66, 213–221 (2010)
    Article CAS PubMed PubMed Central Google Scholar
  38. Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D 66, 486–501 (2010)
    Article CAS PubMed PubMed Central Google Scholar
  39. Laskowski, R. A., Macarthur, M. W., Moss, D. S. & Thornton, J. M. PROCHECK: a program to check the stereochemical quality of protein structures. J. Appl. Cryst. 26, 283–291 (1993)
    Article CAS Google Scholar
  40. Larkin, M. A. et al. Clustal W and Clustal X version 2.0. Bioinformatics 23, 2947–2948 (2007)
    Article CAS PubMed Google Scholar
  41. Gouet, P., Courcelle, E., Stuart, D. I. & Metoz, F. ESPript: analysis of multiple sequence alignments in PostScript. Bioinformatics 15, 305–308 (1999)
    Article CAS PubMed Google Scholar

Download references

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

Author notes

  1. Xuehua Zhong
    Present address: Present address: Wisconsin Institute for Discovery, Laboratory of Genetics, University of Wisconsin, Madison, Wisconsin 53706, USA.,
  2. Lianna M. Johnson and Jiamu Du: 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
    Lianna M. Johnson, Christopher J. Hale, Sylvain Bischof, Suhua Feng, Ramakrishna K. Chodavarapu, Xuehua Zhong, Matteo Pellegrini & Steven E. Jacobsen
  2. Structural Biology Program, Memorial Sloan-Kettering Cancer Center, New York, 10065, New York, USA
    Jiamu Du, Giuseppe Marson & Dinshaw J. Patel
  3. Howard Hughes Medical Institute, University of California at Los Angeles, Los Angeles, 90095, California, USA
    Suhua Feng & Steven E. Jacobsen
  4. Genome Center and Department of Biochemistry and Molecular Medicine, University of California at Davis, Davis, 95616, California, USA
    David J. Segal

Authors

  1. Lianna M. Johnson
  2. Jiamu Du
  3. Christopher J. Hale
  4. Sylvain Bischof
  5. Suhua Feng
  6. Ramakrishna K. Chodavarapu
  7. Xuehua Zhong
  8. Giuseppe Marson
  9. Matteo Pellegrini
  10. David J. Segal
  11. Dinshaw J. Patel
  12. Steven E. Jacobsen

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 toDinshaw J. Patel or Steven E. Jacobsen.

Ethics declarations

Competing interests

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 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.

Supplementary information

PowerPoint slides

Rights and permissions

About this article

Cite this article

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

Download citation