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An RNA polymerase II- and AGO4-associated protein acts in RNA-directed DNA methylation

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Abstract

DNA methylation is an important epigenetic mark in many eukaryotes1,2,3,4,5. In plants, 24-nucleotide small interfering RNAs (siRNAs) bound to the effector protein, Argonaute 4 (AGO4), can direct de novo DNA methylation by the methyltransferase DRM2 (refs 2, 4–6). Here we report a new regulator of RNA-directed DNA methylation (RdDM) in Arabidopsis: RDM1. Loss-of-function mutations in the RDM1 gene impair the accumulation of 24-nucleotide siRNAs, reduce DNA methylation, and release transcriptional gene silencing at RdDM target loci. RDM1 encodes a small protein that seems to bind single-stranded methyl DNA, and associates and co-localizes with RNA polymerase II (Pol II, also known as NRPB), AGO4 and DRM2 in the nucleus. Our results indicate that RDM1 is a component of the RdDM effector complex and may have a role in linking siRNA production with pre-existing or de novo cytosine methylation. Our results also indicate that, although RDM1 and Pol V (also known as NRPE) may function together at some RdDM target sites in the peri-nucleolar siRNA processing centre, Pol II rather than Pol V is associated with the RdDM effector complex at target sites in the nucleoplasm.

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Figure 1: Effects of rdm1 on RD29A-LUC and 35S-NPTII silencing, DNA methylation and small RNAs.
Figure 2: Effects of rdm1-1 on siRNA and transcript levels and DNA methylation at endogenous RdDM target loci.
Figure 3: Immunoblot analysis of RDM1 and its interaction with AGO4, DRM2 and NRPB1.
Figure 4: Sub-nuclear localization of RDM1 and its co-localization with other components of the RdDM pathway.

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Change history

  • 06 May 2010

    Reference 23 was updated to reflect a change in the title.

References

  1. Martienssen, R. A. & Richards, E. J. DNA methylation in eukaryotes. Curr. Opin. Genet. Dev. 5, 234–242 (1995)

    Article  CAS  Google Scholar 

  2. Baulcombe, D. RNA silencing in plants. Nature 431, 356–363 (2004)

    Article  ADS  CAS  Google Scholar 

  3. Tariq, M. & Paszkowski, J. DNA and histone methylation in plants. Trends Genet. 20, 244–251 (2004)

    Article  CAS  Google Scholar 

  4. Chan, S. W., Henderson, I. R. & Jacobsen, S. E. Gardening the genome: DNA methylation in Arabidopsis thaliana. Nature Rev. Genet. 6, 351–360 (2005)

    Article  CAS  Google Scholar 

  5. Matzke, M. A. & Birchler, J. A. RNAi-mediated pathways in the nucleus. Nature Rev. Genet. 6, 24–35 (2005)

    Article  CAS  Google Scholar 

  6. Matzke, M., Kanno, T., Daxinger, L., Huettel, B. & Matzke, A. J. M. RNA-mediated chromatin-based silencing in plants. Curr. Opin. Cell Biol. 21, 367–376 (2009)

    Article  CAS  Google Scholar 

  7. Gong, Z. et al. ROS1, a repressor of transcriptional gene silencing in Arabidopsis, encodes a DNA glycosylase/lyase. Cell 111, 803–814 (2002)

    Article  CAS  Google Scholar 

  8. Zhu, J., Kapoor, A., Sridhar, V. V., Agius, F. & Zhu, J. K. The DNA glycosylase/lyase ROS1 functions in pruning DNA methylation patterns in Arabidopsis. Curr. Biol. 17, 54–59 (2007)

    Article  CAS  Google Scholar 

  9. Penterman, J. et al. DNA demethylation in the Arabidopsis genome. Proc. Natl Acad. Sci. USA 104, 6752–6757 (2007)

    Article  ADS  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  11. He, X. J. et al. NRPD4, a protein similar to the RPB4 subunit of RNA polymerase II, is a component of RNA polymerases IV and V and is required for siRNA production, RNA-directed DNA methylation, and transcriptional gene silencing. Genes Dev. 23, 318–330 (2009)

    Article  CAS  Google Scholar 

  12. Huettel, B. et al. Endogenous targets of RNA-directed DNA methylation and Pol IV in Arabidopsis. EMBO J. 25, 2828–2836 (2006)

    Article  CAS  Google Scholar 

  13. Pontes, O. et al. The Arabidopsis chromatin-modifying nuclear siRNA pathway involves a nucleolar RNA processing center. Cell 126, 79–92 (2006)

    Article  CAS  Google Scholar 

  14. Li, C. F. et al. An ARGONAUTE4-containing nuclear processing center colocalized with Cajal bodies in Arabidopsis thaliana. Cell 126, 93–106 (2006)

    Article  CAS  Google Scholar 

  15. Li, C. F. et al. Dynamic regulation of ARGONAUTE4 within multiple nuclear bodies in Arabidopsis thaliana. PLoS Genet. 4, e27 (2008)

    Article  Google Scholar 

  16. Allard, S. T. et al. Structure at 1.6 Å resolution of the protein from gene locus At3g22680 from Arabidopsis thaliana. Acta Crystallogr. F 61, 647–650 (2005)

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  18. Zilberman, D. et al. Role of Arabidopsis ARGONAUTE4 in RNA-directed DNA methylation triggered by inverted repeats. Curr. Biol. 14, 1214–1220 (2004)

    Article  CAS  Google Scholar 

  19. Qi, Y. et al. Distinct catalytic and non-catalytic roles of ARGONAUTE4 in RNA-directed DNA methylation. Nature 443, 1008–1012 (2006)

    Article  ADS  Google Scholar 

  20. 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  Google Scholar 

  21. Zheng, B. et al. Intergenic transcription by RNA Polymerase II coordinates Pol IV and Pol V in siRNA-directed transcriptional gene silencing in Arabidopsis. Genes Dev. 23, 2850– (2009)

    Article  CAS  Google Scholar 

  22. 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  Google Scholar 

  23. Law, J. A. et al. A protein complex required for polymerase V dependent transcripts and RNA-directed DNA methylation in plants. Curr. Biol. (in the press)

  24. He, X. J. et al. An effector of RNA-directed DNA methylation in Arabidopsis is an ARGONAUTE 4-and RNA-binding protein. Cell 137, 498–508 (2009)

    Article  CAS  Google Scholar 

  25. Woo, H. R., Pontes, O., Pikaard, C. S. & Richards, E. J. VIM1, a methylcytosine-binding protein required for centromeric heterochromatinization. Genes Dev. 21, 267–277 (2007)

    Article  CAS  Google Scholar 

  26. Ishitani, M., Xiong, L., Stevenson, B. & Zhu, J. K. Genetic analysis of osmotic and cold stress signal transduction in Arabidopsis: interactions and convergence of abscisic acid-dependent and abscisic acid-independent pathways. Plant Cell 9, 1935–1949 (1997)

    Article  CAS  Google Scholar 

  27. Kapoor, A. et al. Mutations in a conserved replication protein suppress transcriptional gene silencing in a DNA-methylation-independent manner in Arabidopsis. Curr. Biol. 15, 1912–1918 (2005)

    Article  CAS  Google Scholar 

  28. Dorweiler, J. E. et al. Mediator of paramutation1 is required for establishment and maintenance of paramutation at multiple maize loci. Plant Cell 12, 2101–2118 (2000)

    Article  CAS  Google Scholar 

  29. Jasencakova, Z., Meister, A., Walter, J., Turner, B. M. & Schubert, I. Histone H4 acetylation of euchromatin and heterochromatin is cell cycle dependent and correlated with replication rather than with transcription. Plant Cell 12, 2087–2100 (2000)

    Article  CAS  Google Scholar 

  30. Gendrel, A. V., Lippman, Z., Yordan, C., Colot, V. & Martienssen, R. A. Dependence of heterochromatic histone H3 methylation patterns on the Arabidopsis gene DDM1. Science 297, 1871–1873 (2002)

    Article  ADS  CAS  Google Scholar 

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Acknowledgements

This work was supported by National Institutes of Health grants (J.-K.Z.), Austrian Fonds zur Förderung der wissenschaftlichen Forschung (M.M. and Z.J.L.), National Science Foundation Career Award (H.J.), Edward Mallinckrodt Foundation Award (O.P.), and Agence Nationale de la Recherche (T.L.). We thank E. Richards for his gift of methylated DNA oligonucleotides, B. Stevenson for technical assistance and T. Kanno for discussions.

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Z.G., H.-L.L, X.H., W.Q., H.L., M.X., S.Z., D.M., and X.Z. contributed Figs 1, 2, 3b–d, Supplementary Figs 1–6 and 9–12, and Supplementary Table 2. L.D, Z.J.L., A.J.M. and M.M. contributed the rdm1-4 allele (Supplementary Fig. 5) and data on its characterization (Supplementary Fig. 7). O.P. and C.S.P contributed Fig. 4, Supplementary Fig. 8 and Supplementary Table 1. D.P and T.L. contributed Fig. 3a. J.-K.Z designed the experiments and wrote the paper together with Z.G., H.J., O.P., C.S.P. and M.M.

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Correspondence to Jian-Kang Zhu.

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The authors declare no competing financial interests.

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Gao, Z., Liu, HL., Daxinger, L. et al. An RNA polymerase II- and AGO4-associated protein acts in RNA-directed DNA methylation. Nature 465, 106–109 (2010). https://doi.org/10.1038/nature09025

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