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Uhrf1-dependent H3K23 ubiquitylation couples maintenance DNA methylation and replication

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Abstract

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.

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Acknowledgements

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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Contributions

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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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). https://doi.org/10.1038/nature12488

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