Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

ROS3 is an RNA-binding protein required for DNA demethylation in Arabidopsis


DNA methylation is an important epigenetic mark for transcriptional gene silencing (TGS) in diverse organisms1,2,3,4,5,6. Recent studies suggest that the methylation status of a number of genes is dynamically regulated by methylation and demethylation7,8,9,10. In Arabidopsis, active DNA demethylation is mediated by the ROS1 (repressor of silencing 1) subfamily of 5-methylcytosine DNA glycosylases through a base excision repair pathway8,10,11,12,13. These demethylases have critical roles in erasing DNA methylation and preventing TGS of target genes7,8,10. However, it is not known how the demethylases are targeted to specific sequences. Here we report the identification of ROS3, an essential regulator of DNA demethylation that contains an RNA recognition motif. Analysis of ros3 mutants and ros1 ros3 double mutants suggests that ROS3 acts in the same genetic pathway as ROS1 to prevent DNA hypermethylation and TGS. Gel mobility shift assays and analysis of ROS3 immunoprecipitate from plant extracts shows that ROS3 binds to small RNAs in vitro and in vivo. Immunostaining shows that ROS3 and ROS1 proteins co-localize in discrete foci dispersed throughout the nucleus. These results demonstrate a critical role for ROS3 in preventing DNA hypermethylation and suggest that DNA demethylation by ROS1 may be guided by RNAs bound to ROS3.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: The ros3 mutation causes transcriptional gene silencing.
Figure 2: DNA hypermethylation in ros3 and suppression of ros3 by nrpd1a.
Figure 3: ROS3 binds small RNAs.
Figure 4: Co-localization of ROS3 with ROS1 in the nucleus of Arabidopsis mesophyll cells and assay of ROS1 and ROS3 mRNA levels.


  1. 1

    Bird, A. DNA methylation patterns and epigenetic memory. Genes Dev. 16, 6–21 (2002)

    CAS  Article  Google Scholar 

  2. 2

    Martienssen, R. A. & Colot, V. DNA methylation and epigenetic inheritance in plants and filamentous fungi. Science 293, 1070–1074 (2001)

    CAS  Article  Google Scholar 

  3. 3

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

    CAS  Article  Google Scholar 

  4. 4

    Bender, J. DNA methylation and epigenetics. Annu. Rev. Plant Biol. 55, 41–68 (2004)

    CAS  Article  Google Scholar 

  5. 5

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

    CAS  Article  Google Scholar 

  6. 6

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

    CAS  Article  Google Scholar 

  7. 7

    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)

    CAS  Article  Google Scholar 

  8. 8

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

    ADS  CAS  Article  Google Scholar 

  9. 9

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

    CAS  Article  Google Scholar 

  10. 10

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

    CAS  Article  Google Scholar 

  11. 11

    Agius, F., Kapoor, A. & Zhu, J. K. Role of the Arabidopsis DNA glycosylase/lyase ROS1 in active DNA demethylation. Proc. Natl Acad. Sci. USA 103, 11796–11801 (2006)

    ADS  CAS  Article  Google Scholar 

  12. 12

    Morales-Ruiz, T. et al. DEMETER and REPRESSOR OF SILENCING 1 encode 5-methylcytosine DNA glycosylases. Proc. Natl Acad. Sci. USA 103, 6853–6858 (2006)

    ADS  CAS  Article  Google Scholar 

  13. 13

    Gehring, M. et al. DEMETER DNA glycosylase establishes MEDEA polycomb gene self-imprinting by allele-specific demethylation. Cell 124, 495–506 (2006)

    CAS  Article  Google Scholar 

  14. 14

    Kapoor, A., Agius, F. & Zhu, J. K. Preventing transcriptional gene silencing by active DNA demethylation. FEBS Lett. 579, 5889–5898 (2005)

    CAS  Article  Google Scholar 

  15. 15

    Xiong, L. et al. FIERY1 encoding an inositol polyphosphate 1-phosphatase is a negative regulator of abscisic acid and stress signaling in Arabidopsis . Genes Dev. 15, 1971–1984 (2001)

    CAS  Article  Google Scholar 

  16. 16

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

    CAS  Article  Google Scholar 

  17. 17

    Zheng, X., Zhu, J., Kapoor, A. & Zhu, J. K. Role of Arabidopsis AGO6 in siRNA accumulation, DNA methylation and transcriptional gene silencing. EMBO J. 26, 1691–1701 (2007)

    CAS  Article  Google Scholar 

  18. 18

    Wassenegger, M. RNA-directed DNA methylation. Plant Mol. Biol. 43, 203–220 (2000)

    CAS  Article  Google Scholar 

  19. 19

    Vaucheret, H. & Fagard, M. Transcriptional gene silencing in plants: targets, inducers and regulators. Trends Genet. 17, 29–35 (2001)

    CAS  Article  Google Scholar 

  20. 20

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

    ADS  CAS  Article  Google Scholar 

  21. 21

    Brodersen, P. & Voinnet, O. The diversity of RNA silencing pathways in plants. Trends Genet. 22, 268–280 (2006)

    CAS  Article  Google Scholar 

  22. 22

    Aravin, A. A. & Bourc’his, D. Small RNA guides for de novo DNA methylation in mammalian germ cells. Genes Dev. 22, 970–975 (2008)

    CAS  Article  Google Scholar 

  23. 23

    Kim, D. H., Villeneuve, L. M., Morris, K. V. & Rossi, J. J. Argonaute-1 directs siRNA-mediated transcriptional gene silencing in human cells. Nature Struct. Mol. Biol. 13, 793–797 (2006)

    CAS  Article  Google Scholar 

  24. 24

    Janowski, B. A. et al. Involvement of AGO1 and AGO2 in mammalian transcriptional silencing. Nature Struct. Mol. Biol. 13, 787–792 (2006)

    CAS  Article  Google Scholar 

  25. 25

    Li, L. C. et al. Small dsRNAs induce transcriptional activation in human cells. Proc. Natl Acad. Sci. USA 103, 17337–17342 (2006)

    ADS  CAS  Article  Google Scholar 

  26. 26

    Janowski, B. A. et al. Activating gene expression in mammalian cells with promoter-targeted duplex RNAs. Nature Chem. Biol. 3, 166–173 (2007)

    CAS  Article  Google Scholar 

  27. 27

    Carrington, J. C. & Ambros, V. Role of microRNAs in plant and animal development. Science 301, 336–338 (2003)

    ADS  CAS  Article  Google Scholar 

  28. 28

    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)

    CAS  Article  Google Scholar 

  29. 29

    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)

    CAS  Article  Google Scholar 

  30. 30

    Frommer, M. et al. A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands. Proc. Natl Acad. Sci. USA 89, 1827–1831 (1992)

    ADS  CAS  Article  Google Scholar 

  31. 31

    Lu, R. et al. Animal virus replication and RNAi-mediated antiviral silencing in Caenorhabditis elegans . Nature 436, 1040–1043 (2005)

    ADS  CAS  Article  Google Scholar 

  32. 32

    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)

    CAS  Article  Google Scholar 

  33. 33

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

    CAS  Article  Google Scholar 

  34. 34

    Sunkar, R. & Zhu, J. K. Novel and stress-regulated microRNAs and other small RNAs from Arabidopsis . Plant Cell 16, 2001–2019 (2004)

    CAS  Article  Google Scholar 

Download references


This work was supported by National Institutes of Health grants R01GM070795 and R01GM059138 (J.-K.Z.), R01GM077590 and 1R01GM060380 (C.S.P.), Edward Mallinckrodt Foundation (O.P.) and China Scholarship Council scholarship 2007104542 (F.Z.).

Author Contributions X.Z. did the cloning, mutant analysis, RNA binding and other experiments. J.Z. and A.K. contributed to mutant analysis. D.M., F.Z. and K.I. contributed to DNA methylation analysis. W.-X.L. contributed to small RNA results. O.P. and C.S.P. contributed immunostaining data. J.-K.Z. designed the project and wrote the paper.

Author information



Corresponding author

Correspondence to Jian-Kang Zhu.

Supplementary information

Supplementary Information

This file contains Supplementary Figures 1-12 with Legends and Supplementary Tables 1-7. (PDF 758 kb)

PowerPoint slides

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Zheng, X., Pontes, O., Zhu, J. et al. ROS3 is an RNA-binding protein required for DNA demethylation in Arabidopsis. Nature 455, 1259–1262 (2008).

Download citation

Further reading


By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing