Letter | Published:

Histone H1 couples initiation and amplification of ubiquitin signalling after DNA damage

Nature volume 527, pages 389393 (19 November 2015) | Download Citation


DNA double-strand breaks (DSBs) are highly cytotoxic DNA lesions that trigger non-proteolytic ubiquitylation of adjacent chromatin areas to generate binding sites for DNA repair factors. This depends on the sequential actions of the E3 ubiquitin ligases RNF8 and RNF168 (refs 1, 2, 3, 4, 5, 6), and UBC13 (also known as UBE2N), an E2 ubiquitin-conjugating enzyme that specifically generates K63-linked ubiquitin chains7. Whereas RNF168 is known to catalyse ubiquitylation of H2A-type histones, leading to the recruitment of repair factors such as 53BP1 (refs 8, 9, 10), the critical substrates of RNF8 and K63-linked ubiquitylation remain elusive. Here we elucidate how RNF8 and UBC13 promote recruitment of RNF168 and downstream factors to DSB sites in human cells. We establish that UBC13-dependent K63-linked ubiquitylation at DSB sites is predominantly mediated by RNF8 but not RNF168, and that H1-type linker histones, but not core histones, represent major chromatin-associated targets of this modification. The RNF168 module (UDM1) recognizing RNF8-generated ubiquitylations11 is a high-affinity reader of K63-ubiquitylated H1, mechanistically explaining the essential roles of RNF8 and UBC13 in recruiting RNF168 to DSBs. Consistently, reduced expression or chromatin association of linker histones impair accumulation of K63-linked ubiquitin conjugates and repair factors at DSB-flanking chromatin. These results identify histone H1 as a key target of RNF8–UBC13 in DSB signalling and expand the concept of the histone code12,13 by showing that posttranslational modifications of linker histones can serve as important marks for recognition by factors involved in genome stability maintenance, and possibly beyond.

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We thank D. Durocher and M. Bianchi for providing reagents and J. Lukas for helpful discussions. This work was supported by grants from the Novo Nordisk Foundation (grants NNF14CC0001 and NNF12OC0002114), European Research Council, Nederlandse Organisatie voor Wetenschappelijk Onderzoek- Chemische Wetenschappen (NWO-CW), The Danish Cancer Society, and The Danish Council for Independent Research.

Author information

Author notes

    • Tina Thorslund
    • , Anita Ripplinger
    • , Saskia Hoffmann
    •  & Thomas Wild

    These authors contributed equally to this work.


  1. Ubiquitin Signaling Group, Protein Signaling Program, The Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3B, DK-2200 Copenhagen, Denmark

    • Tina Thorslund
    • , Anita Ripplinger
    • , Saskia Hoffmann
    • , Bine Villumsen
    • , Simon Bekker-Jensen
    •  & Niels Mailand
  2. Proteomics Program, The Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3B, DK-2200 Copenhagen, Denmark

    • Thomas Wild
    • , Takeo Narita
    •  & Chunaram Choudhary
  3. Division of Biochemistry, Cancer Genomics Center, Netherlands Cancer Institute, 1066 CX Amsterdam, the Netherlands

    • Michael Uckelmann
    •  & Titia K. Sixma


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T.T. initiated the project, designed and performed cell biological and biochemical experiments, and analysed data; A.R. and S.H. performed cell biological and biochemical experiments and analysed data; T.W. generated UBC13-knockout cells, designed and performed mass spectrometry experiments and analysed the data; M.U. performed in vitro ubiquitylation assays with purified nucleosomes; B.V. helped T.T. with biochemical experiments; T.N. analysed mass spectrometry data; T.K.S. supervised M.U.; C.C. supervised T.W. and T.N., designed mass spectrometry experiments and analysed the data; S.B.-J. performed and designed experiments and analysed data; N.M. conceived and supervised the project, designed experiments, analysed data and wrote the manuscript with input from the other authors.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Niels Mailand.

Extended data

Supplementary information

PDF files

  1. 1.

    Supplementary Figure 1

    This file contains the uncropped scans of immunoblot data with size marker indications.

Excel files

  1. 1.

    Supplementary Table 1

    This table contains the Mass spectrometry data for SILAC-based quantification of di-glycine containing peptides isolated from HCT116 WT and Ubc13 KO cells. See Extended Data Fig. 3a for experimental set-up.

  2. 2.

    Supplementary Table 2

    This table contains the Quantitative mass spectrometry data for proteins identified in K63-Super-UIM pull-downs from HCT116 WT and Ubc13 KO cells. See Extended Data Fig. 3e for experimental set-up.

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