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The histone H4 Lys 20 methyltransferase PR-Set7 regulates replication origins in mammalian cells

Nature Cell Biology volume 12, pages 1086–1093 (2010)Cite this article

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Abstract

The initiation of DNA synthesis is governed by the licensing of replication origins, which consists of assembling a pre-replication complex (pre-RC) on origins during late M- and G1-phases1,2. In metazoans, functional replication origins do not show defined DNA consensus sequences, thus evoking the involvement of chromatin determinants in the selection of these origins3. Here, we show that the onset of licensing in mammalian cells coincides with an increase in histone H4 Lys 20 monomethylation (H4K20me1) at replication origins by the methyltransferase PR-Set7 (also known as Set8 or KMT5A). Indeed, tethering PR-Set7 methylase activity to a specific genomic locus promotes the loading of pre-RC proteins on chromatin. In addition, we demonstrate that PR-Set7 undergoes a PCNA- and Cul4–Ddb1-driven degradation during S phase that contributes to the disappearance of H4K20me1 at origins and the inhibition of replication licensing. Strikingly, expression of a PR-Set7 mutant insensitive to this degradation causes the maintenance of H4K20me1 and repeated DNA replication at origins. These results elucidate a critical role for PR-Set7 and H4K20me1 in the chromatin events that regulate replication origins.

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Figure 1: Cell-cycle changes in PR-Set7 levels are regulated by the ubiquitin–proteasome pathway.
Figure 2: PCNA binding is required for S-phase degradation of PR-Set7 by Cul4–Ddb1-mediated ubiquitylation.
Figure 3: Neutralization of PCNA-mediated PR-Set7 degradation causes a G2 arrest with re-replicated DNA.
Figure 4: PR-Set7-induced H4K20me1 at replication origins is associated with the onset of replication licensing.
Figure 5: PR-Set7 contributes to the assembly of pre-RC complexes on chromatin.

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Acknowledgements

We thank K. Helin, C. S. Sørensen, S. Jørgensen, K. Hansen, M. DePamphilis, D. Tempé and G. Bossis for providing reagents, L. Linares for technical help with ubiquitylation assay, P. Coulombe for technical help with the CsCl gradient centrifugation experiments, and G. Pawlak, E. Fabbrizio, R. Hipskind, and E. Schwob for critical reading of the manuscript. M.T. was supported by a fellowship from Ministère de la Recherche and from Association de la Recherche contre le Cancer (ARC). J.B. was supported by a fellowship from Fondation pour la Recherche Médicale (FRM). O.K. was supported by a fellowship from Agence Nationale de la Recherche (ANR). This work was supported by the institutional support of the CNRS and a grant from the Institut National du Cancer Cancéropôle Grand Sud-Ouest to C.S. and E.J., by an ANR grant (ANR-09-JCJC-0014-01) to E.J, and by institutional grants from INSERM, the University Joseph Fourier, and the Fondation de France to C.L. and M.C.

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Author notes

  1. Mathieu Tardat and Julien Brustel: These authors contributed equally to this work.

Authors and Affiliations

  1. Institut de Génétique Moléculaire de Montpellier (IGMM), UMR 5535 CNRS, Montpellier, France

    Mathieu Tardat, Julien Brustel, Olivier Kirsh, Claude Sardet & Eric Julien

  2. Université Montpellier 2, Montpellier, France

    Mathieu Tardat, Julien Brustel, Olivier Kirsh, Claude Sardet & Eric Julien

  3. Université Montpellier 1, Montpellier, France

    Mathieu Tardat, Julien Brustel, Olivier Kirsh, Claude Sardet & Eric Julien

  4. INSERM U823, Institut Albert Bonniot, Grenoble, France

    Christine Lefevbre & Mary Callanan

  5. Université Joseph-Fourier, Grenoble, France

    Christine Lefevbre & Mary Callanan

  6. Department of Hematology, Onco-Hematology Unit, Onco-genetics and Immunology, Pôle de Biologie, CHU-Grenoble, Grenoble, France

    Christine Lefevbre & Mary Callanan

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  1. Mathieu Tardat
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Contributions

E.J. conceived the project and designed and supervised the study. M.T., J.B., O.K. and E.J. performed and analysed experiments. C.L. and M.C. performed and analysed FISH experiments. C.S. provided conceptual advice on study design and the interpretation of the results. The manuscript was written by E.J. and edited by C.S.

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Correspondence to Claude Sardet or Eric Julien.

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Tardat, M., Brustel, J., Kirsh, O. et al. The histone H4 Lys 20 methyltransferase PR-Set7 regulates replication origins in mammalian cells. Nat Cell Biol 12, 1086–1093 (2010). https://doi.org/10.1038/ncb2113

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