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Uhrf1-dependent H3K23 ubiquitylation couples maintenance DNA methylation and replication



Faithful propagation of DNA methylation patterns during DNA replication is critical for maintaining cellular phenotypes of individual differentiated cells1,2,3,4,5. Although it is well established that Uhrf1 (ubiquitin-like with PHD and ring finger domains 1; also known as Np95 and ICBP90) specifically binds to hemi-methylated DNA through its SRA (SET and RING finger associated) domain and has an essential role in maintenance of DNA methylation by recruiting Dnmt1 to hemi-methylated DNA sites6,7,8,9,10, the mechanism by which Uhrf1 coordinates the maintenance of DNA methylation and DNA replication is largely unknown. Here we show that Uhrf1-dependent histone H3 ubiquitylation has a prerequisite role in the maintenance DNA methylation. Using Xenopus egg extracts, we successfully reproduce maintenance DNA methylation in vitro. Dnmt1 depletion results in a marked accumulation of Uhrf1-dependent ubiquitylation of histone H3 at lysine 23. Dnmt1 preferentially associates with ubiquitylated H3 in vitro though a region previously identified as a replication foci targeting sequence11. The RING finger mutant of Uhrf1 fails to recruit Dnmt1 to DNA replication sites and maintain DNA methylation in mammalian cultured cells. Our findings represent the first evidence, to our knowledge, of the mechanistic link between DNA methylation and DNA replication through histone H3 ubiquitylation.

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Figure 1: xUhrf1- and DNA-replication-dependent DNA methylation and ubiquitylation of H3 at lysine 23 in Xenopus egg extracts.
Figure 2: Dnmt1 preferentially binds to UbH3.
Figure 3: hUhrf1- and S-phase-dependent ubiquitylation of hH3 at lysine 23 in HeLa cells.
Figure 4: The RING finger domain of Uhrf1 is crucial for recruitment of Dnmt1 to DNA replication sites and for maintenance of DNA methylation in mammalian cells.


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

    Article  CAS  Google Scholar 

  2. Jones, P. A. & Liang, G. Rethinking how DNA methylation patterns are maintained. Nature Rev. Genet. 10, 805–811 (2009)

    Article  CAS  Google Scholar 

  3. Alabert, C. & Groth, A. Chromatin replication and epigenome maintenance. Nature Rev. Mol. Cell Biol. 13, 153–167 (2012)

    Article  CAS  Google Scholar 

  4. Margueron, R. & Reinberg, D. Chromatin structure and the inheritance of epigenetic information. Nature Rev. Genet. 11, 285–296 (2010)

    Article  CAS  Google Scholar 

  5. Probst, A. V., Dunleavy, E. & Almouzni, G. Epigenetic inheritance during the cell cycle. Nature Rev. Mol. Cell Biol. 10, 192–206 (2009)

    Article  CAS  Google Scholar 

  6. Bostick, M. et al. UHRF1 plays a role in maintaining DNA methylation in mammalian cells. Science 317, 1760–1764 (2007)

    Article  ADS  CAS  Google Scholar 

  7. Sharif, J. et al. The SRA protein Np95 mediates epigenetic inheritance by recruiting Dnmt1 to methylated DNA. Nature 450, 908–912 (2007)

    Article  ADS  CAS  Google Scholar 

  8. 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  Google Scholar 

  9. 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  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  Google Scholar 

  11. Leonhardt, H., Page, A. W., Weier, H. U. & Bestor, T. H. A targeting sequence directs DNA methyltransferase to sites of DNA replication in mammalian nuclei. Cell 71, 865–873 (1992)

    Article  CAS  Google Scholar 

  12. Arita, K. et al. Recognition of modification status on a histone H3 tail by linked histone reader modules of the epigenetic regulator UHRF1. Proc. Natl Acad. Sci. USA 109, 12950–12955 (2012)

    Article  ADS  CAS  Google Scholar 

  13. Hu, L., Li, Z., Wang, P., Lin, Y. & Xu, Y. Crystal structure of PHD domain of UHRF1 and insights into recognition of unmodified histone H3 arginine residue 2. Cell Res. 21, 1374–1378 (2011)

    Article  CAS  Google Scholar 

  14. Rajakumara, E. et al. PHD finger recognition of unmodified histone H3R2 links UHRF1 to regulation of euchromatic gene expression. Mol. Cell 43, 275–284 (2011)

    Article  CAS  Google Scholar 

  15. Rothbart, S. B. et al. Association of UHRF1 with methylated H3K9 directs the maintenance of DNA methylation. Nature Struct. Mol. Biol. 19, 1155–1160 (2012)

    Article  CAS  Google Scholar 

  16. Nady, N. et al. Recognition of multivalent histone states associated with heterochromatin by UHRF1 protein. J. Biol. Chem. 286, 24300–24311 (2011)

    Article  CAS  Google Scholar 

  17. Song, J., Rechkoblit, O., Bestor, T. H. & Patel, D. J. Structure of DNMT1-DNA complex reveals a role for autoinhibition in maintenance DNA methylation. Science 331, 1036–1040 (2011)

    Article  ADS  CAS  Google Scholar 

  18. Felle, M. et al. The USP7/Dnmt1 complex stimulates the DNA methylation activity of Dnmt1 and regulates the stability of UHRF1. Nucleic Acids Res. 39, 8355–8365 (2011)

    Article  CAS  Google Scholar 

  19. Achour, M. et al. The interaction of the SRA domain of ICBP90 with a novel domain of DNMT1 is involved in the regulation of VEGF gene expression. Oncogene 27, 2187–2197 (2008)

    Article  CAS  Google Scholar 

  20. Lutzmann, M. & Mechali, M. MCM9 binds Cdt1 and is required for the assembly of prereplication complexes. Mol. Cell 31, 190–200 (2008)

    Article  CAS  Google Scholar 

  21. Citterio, E. et al. Np95 is a histone-binding protein endowed with ubiquitin ligase activity. Mol. Cell. Biol. 24, 2526–2535 (2004)

    Article  CAS  Google Scholar 

  22. Jenkins, Y. et al. Critical role of the ubiquitin ligase activity of UHRF1, a nuclear RING finger protein, in tumor cell growth. Mol. Biol. Cell 16, 5621–5629 (2005)

    Article  CAS  Google Scholar 

  23. Karagianni, P., Amazit, L., Qin, J. & Wong, J. ICBP90, a novel methyl K9 H3 binding protein linking protein ubiquitination with heterochromatin formation. Mol. Cell. Biol. 28, 705–717 (2008)

    Article  CAS  Google Scholar 

  24. Peng, J. et al. A proteomics approach to understanding protein ubiquitination. Nature Biotechnol. 21, 921–926 (2003)

    Article  CAS  Google Scholar 

  25. Kim, W. et al. Systematic and quantitative assessment of the ubiquitin-modified proteome. Mol. Cell 44, 325–340 (2011)

    Article  CAS  Google Scholar 

  26. Shimamura, S. & Ishikawa, F. Interaction between DNMT1 and DNA replication reactions in the SV40 in vitro replication system. Cancer Sci. 99, 1960–1966 (2008)

    CAS  PubMed  Google Scholar 

  27. Takebayashi, S., Tamura, T., Matsuoka, C. & Okano, M. Major and essential role for the DNA methylation mark in mouse embryogenesis and stable association of DNMT1 with newly replicated regions. Mol. Cell. Biol. 27, 8243–8258 (2007)

    Article  CAS  Google Scholar 

  28. Plechanovová, A., Jaffray, E. G., Tatham, M. H., Naismith, J. H. & Hay, R. T. Structure of a RING E3 ligase and ubiquitin-loaded E2 primed for catalysis. Nature 489, 115–120 (2012)

    Article  ADS  Google Scholar 

  29. Muraki, K., Nabetani, A., Nishiyama, A. & Ishikawa, F. Essential roles of Xenopus TRF2 in telomere end protection and replication. Genes Cells 16, 728–739 (2011)

    Article  CAS  Google Scholar 

  30. Nishiyama, A., Frappier, L. & Mechali, M. MCM-BP regulates unloading of the MCM2–7 helicase in late S phase. Genes Dev. 25, 165–175 (2011)

    Article  CAS  Google Scholar 

  31. Fujinoki, M. et al. Identification of 36-kDa flagellar phosphoproteins associated with hamster sperm motility. J. Biochem. 133, 361–369 (2003)

    Article  CAS  Google Scholar 

  32. Daigo, K. et al. The proteomic profile of circulating pentraxin 3 (PTX3) complex in sepsis demonstrates the interaction with azurocidin 1 and other components of neutrophil extracellular traps. Mol. Cell. Proteomics 11, M111.015073 (2012)

    Article  Google Scholar 

  33. Keller, A., Nesvizhskii, A. I., Kolker, E. & Aebersold, R. Empirical statistical model to estimate the accuracy of peptide identifications made by MS/MS and database search. Anal. Chem. 74, 5383–5392 (2002)

    Article  CAS  Google Scholar 

  34. Nesvizhskii, A. I., Keller, A., Kolker, E. & Aebersold, R. A statistical model for identifying proteins by tandem mass spectrometry. Anal. Chem. 75, 4646–4658 (2003)

    Article  CAS  Google Scholar 

  35. Chen, C. & Okayama, H. High-efficiency transformation of mammalian cells by plasmid DNA. Mol. Cell. Biol. 7, 2745–2752 (1987)

    Article  CAS  Google Scholar 

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We are grateful to T. S. Takahashi, K. Shintomi, K. Muraki, H. Nakaoka, M. Iwabuchi and K. Ohsumi for reagents and technical advice, J. Maller for Xenopus Orc2 antibody, H. Miyoshi for lentiviral vectors, K. Helin for reading of the manuscript and M. Orii for designing the schematic diagram shown in Supplementary Fig. 1. We thank A. Hosoi and C. Yamada-Namikawa for technical assistance. M.N. was supported by a Grant-in-Aid for Scientific Research on Innovative Areas ‘Cell fate control’, Scientific Research (A), and Challenging Exploratory Research from MEXT Japan. A.N. was supported by a Research Activity Start-up and Grant-in-Aid for Young Scientists (B) from the Japan Society for the Promotion of Science.

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Authors and Affiliations



M.N. and A.N. planned studies and interpreted the data. A.N. and L.Y. performed most of the Xenopus studies. J.S. and H.K validated the mUhrf1 knockout lines and performed bisulphite DNA sequencing analysis. Y.J. and K.N. performed most of the mammalian studies. T.Kaw. and T.Kod. performed LC–MS/MS analysis. S.S. and F.I. generated purified recombinant mDnmt1 proteins. K.A. generated the purified recombinant hUhrf1 protein. M.N. and A.N wrote the paper.

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Correspondence to Atsuya Nishiyama or Makoto Nakanishi.

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The authors declare no competing financial interests.

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Supplementary Information

This file contains a Supplementary Discussion, Supplementary references and Supplementary Figures 1-15. Please note that Supplementary Figure 15 contains the un-cropped images, which show the molecular weight markers for all the immunoblotting for all the figures, both in the main paper and in the supplementary information. (PDF 3578 kb)

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Nishiyama, A., Yamaguchi, L., Sharif, J. et al. Uhrf1-dependent H3K23 ubiquitylation couples maintenance DNA methylation and replication. Nature 502, 249–253 (2013).

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