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The AAA-ATPase VCP/p97 promotes 53BP1 recruitment by removing L3MBTL1 from DNA double-strand breaks

Nature Structural & Molecular Biology volume 18, pages 1345–1350 (2011)Cite this article

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Abstract

The accumulation of the human tumor suppressor 53BP1 at DNA damage sites requires the ubiquitin ligases RNF8 and RNF168. As 53BP1 recognizes dimethylated Lys20 in histone H4 (H4K20me2), the requirement for RNF8- and RNF168-mediated ubiquitylation has been unclear. Here we show that RNF8-mediated ubiquitylation facilitates the recruitment of the AAA-ATPase valosin-containing protein (VCP, also known as p97) and its cofactor NPL4 to sites of double-strand breaks. RIDDLE cells, which lack functional RNF168, also show impaired recruitment of VCP to DNA damage. The ATPase activity of VCP promotes the release of the Polycomb protein L3MBTL1 from chromatin, which also binds the H4K20me2 histone mark, thereby facilitating 53BP1 recruitment. Consistent with this, nematodes lacking the VCP orthologs CDC-48.1 or CDC-48.2, or cofactors UFD-1 or NPL-4, are highly sensitive to ionizing radiation. Our data suggest that human RNF8 and RNF168 promote VCP-mediated displacement of L3MBTL1 to unmask 53BP1 chromatin binding sites.

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Figure 1: VCP is involved in the DNA damage response.
Figure 2: Chromatin tethering of RNF8 causes VCP recruitment.
Figure 3: RNF8 and RNF168 are involved in recruitment of VCP to DSBs.
Figure 4: VCP is required for efficient 53BP1 accumulation at DSBs.
Figure 5: VCP-dependent extraction of L3MBTL1 protein from DSBs.
Figure 6: DNA damage–induced L3MBTL1 ubiquitylation and VCP binding.

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Acknowledgements

We thank J. Lukas (Danish Cancer Society), N. Mailand (University of Copenhagen), T. Misteli (US National Institutes of Health Bethesda) and G. Stewart (University of Birmingham) for cell lines, O. Gozani (Stanford University) for the Flag-L3MBTL1 expression vector, K. Lindsten for technical advice, P. Young for stimulating discussions and the Caenorhabditis Genetics Center (funded by the US National Institutes of Health, National Center for Research Resources) for strains. This work was supported by the Swedish Cancer Society (N.P.D.), the Swedish Research Council (N.P.D.), the European Community Network of Excellence RUBICON (project no. LSHC-CT-2005-018683; N.P.D. and T.H.), the Netherlands Organization for Scientific Research (NWO Rubicon grant 2007/09198/ALW/825.07.042; M.S.L.), the European Molecular Biology Organization (EMBO long-term fellowship; M.S.L.), the Federation of European Biochemical Societies (FEBS long-term fellowship to M.S.L.) and the Deutsche Forschungsgemeinschaft (especially the Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases, Research Unit FOR885, and DIP grant HO 2541/4-1; T.H.). T.H. is an EMBO Young Investigator.

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  1. Martijn S Luijsterburg

    Present address: Present address: Department of Toxicogenetics, Leiden University Medical Center, Leiden, The Netherlands.,

  2. Klara Acs and Martijn S Luijsterburg: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Cell and Molecular Biology, Karolinska Institutet, Stockholm, Sweden

    Klara Acs, Martijn S Luijsterburg, Florian A Salomons & Nico P Dantuma

  2. Institute for Genetics and Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases, University of Cologne, Cologne, Germany

    Leena Ackermann & Thorsten Hoppe

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Contributions

K.A., M.S.L. and N.P.D. initiated the study; K.A., M.S.L. and N.P.D. designed the experiments in mammalian cells; L.A. and T.H. designed the experiments in C. elegans; K.A. and M.S.L. conducted the experiments with mammalian cells; L.A. conducted the experiments in C. elegans; F.A.S. assisted with the micro-irradiation experiments; K.A., M.S.L. and N.P.D. wrote the manuscript. All authors analyzed the data, discussed the results and commented on the manuscript.

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Correspondence to Nico P Dantuma.

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Acs, K., Luijsterburg, M., Ackermann, L. et al. The AAA-ATPase VCP/p97 promotes 53BP1 recruitment by removing L3MBTL1 from DNA double-strand breaks. Nat Struct Mol Biol 18, 1345–1350 (2011). https://doi.org/10.1038/nsmb.2188

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