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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Mailand, N. et al. RNF8 ubiquitylates histones at DNA double-strand breaks and promotes assembly of repair proteins. Cell 131, 887–900 (2007)
Huen, M. S. et al. RNF8 transduces the DNA-damage signal via histone ubiquitylation and checkpoint protein assembly. Cell 131, 901–914 (2007)
Kolas, N. K. et al. Orchestration of the DNA-damage response by the RNF8 ubiquitin ligase. Science 318, 1637–1640 (2007)
Doil, C. et al. RNF168 binds and amplifies ubiquitin conjugates on damaged chromosomes to allow accumulation of repair proteins. Cell 136, 435–446 (2009)
Stewart, G. S. et al. The RIDDLE syndrome protein mediates a ubiquitin-dependent signaling cascade at sites of DNA damage. Cell 136, 420–434 (2009)
Jackson, S. P. & Durocher, D. Regulation of DNA damage responses by ubiquitin and SUMO. Mol. Cell 49, 795–807 (2013)
Hofmann, R. M. & Pickart, C. M. Noncanonical MMS2-encoded ubiquitin-conjugating enzyme functions in assembly of novel polyubiquitin chains for DNA repair. Cell 96, 645–653 (1999)
Mattiroli, F. et al. RNF168 ubiquitinates K13-15 on H2A/H2AX to drive DNA damage signaling. Cell 150, 1182–1195 (2012)
Fradet-Turcotte, A. et al. 53BP1 is a reader of the DNA-damage-induced H2A Lys 15 ubiquitin mark. Nature 499, 50–54 (2013)
Gatti, M. et al. A novel ubiquitin mark at the N-terminal tail of histone H2As targeted by RNF168 ubiquitin ligase. Cell Cycle 11, 2538–2544 (2012)
Panier, S. et al. Tandem protein interaction modules organize the ubiquitin-dependent response to DNA double-strand breaks. Mol. Cell 47, 383–395 (2012)
Kouzarides, T. Chromatin modifications and their function. Cell 128, 693–705 (2007)
Jenuwein, T. & Allis, C. D. Translating the histone code. Science 293, 1074–1080 (2001)
Cong, L. et al. Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819–823 (2013)
Mali, P. et al. RNA-guided human genome engineering via Cas9. Science 339, 823–826 (2013)
Bekker-Jensen, S. et al. HERC2 coordinates ubiquitin-dependent assembly of DNA repair factors on damaged chromosomes. Nature Cell Biol. 12, 80–86,–1–12 (2010)
Huen, M. S. et al. Noncanonical E2 variant-independent function of UBC13 in promoting checkpoint protein assembly. Mol. Cell. Biol. 28, 6104–6112 (2008)
Sims, J. J. et al. Polyubiquitin-sensor proteins reveal localization and linkage-type dependence of cellular ubiquitin signaling. Nature Methods 9, 303–309 (2012)
van Wijk, S. J. et al. Fluorescence-based sensors to monitor localization and functions of linear and K63-linked ubiquitin chains in cells. Mol. Cell 47, 797–809 (2012)
Wagner, S. A. et al. A proteome-wide, quantitative survey of in vivo ubiquitylation sites reveals widespread regulatory roles. Mol. Cell. Proteomics 10, M111.013284 (2011)
Kim, W. et al. Systematic and quantitative assessment of the ubiquitin-modified proteome. Mol. Cell 44, 325–340 (2011)
Petroski, M. D. et al. Substrate modification with lysine 63-linked ubiquitin chains through the UBC13-UEV1A ubiquitin-conjugating enzyme. J. Biol. Chem. 282, 29936–29945 (2007)
Christensen, D. E., Brzovic, P. S. & Klevit, R. E. E2-BRCA1 RING interactions dictate synthesis of mono- or specific polyubiquitin chain linkages. Nature Struct. Mol. Biol. 14, 941–948 (2007)
Windheim, M., Peggie, M. & Cohen, P. Two different classes of E2 ubiquitin-conjugating enzymes are required for the mono-ubiquitination of proteins and elongation by polyubiquitin chains with a specific topology. Biochem. J. 409, 723–729 (2008)
Catez, F., Ueda, T. & Bustin, M. Determinants of histone H1 mobility and chromatin binding in living cells. Nature Struct. Mol. Biol. 13, 305–310 (2006)
Murga, M. et al. Global chromatin compaction limits the strength of the DNA damage response. J. Cell Biol. 178, 1101–1108 (2007)
Pinato, S., Gatti, M., Scandiuzzi, C., Confalonieri, S. & Penengo, L. UMI, a novel RNF168 ubiquitin binding domain involved in the DNA damage signaling pathway. Mol. Cell. Biol. 31, 118–126 (2011)
Harshman, S. W., Young, N. L., Parthun, M. R. & Freitas, M. A. H1 histones: current perspectives and challenges. Nucleic Acids Res. 41, 9593–9609 (2013)
Gatti, M. et al. RNF168 promotes noncanonical K27 ubiquitination to signal DNA damage. Cell Reports 10, 226–238 (2015)
Povlsen, L. K. et al. Systems-wide analysis of ubiquitylation dynamics reveals a key role for PAF15 ubiquitylation in DNA-damage bypass. Nature Cell Biol. 14, 1089–1098 (2012)
Danielsen, J. M. et al. Mass spectrometric analysis of lysine ubiquitylation reveals promiscuity at site level. Mol. Cell. Proteomics 10, M110.003590 (2011)
Poulsen, M., Lukas, C., Lukas, J., Bekker-Jensen, S. & Mailand, N. Human RNF169 is a negative regulator of the ubiquitin-dependent response to DNA double-strand breaks. J. Cell Biol. 197, 189–199 (2012)
Bekker-Jensen, S., Lukas, C., Melander, F., Bartek, J. & Lukas, J. Dynamic assembly and sustained retention of 53BP1 at the sites of DNA damage are controlled by Mdc1/NFBD1. J. Cell Biol. 170, 201–211 (2005)
Hernández-Muñoz, I. et al. Stable X chromosome inactivation involves the PRC1 Polycomb complex and requires histone MACROH2A1 and the CULLIN3/SPOP ubiquitin E3 ligase. Proc. Natl Acad. Sci. USA 102, 7635–7640 (2005)
Ekkebus, R. et al. On terminal alkynes that can react with active-site cysteine nucleophiles in proteases. J. Am. Chem. Soc. 135, 2867–2870 (2013)
Ong, S. E. et al. Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics. Mol. Cell. Proteomics 1, 376–386 (2002)
Jensen, O. N., Wilm, M., Shevchenko, A. & Mann, M. Sample preparation methods for mass spectrometric peptide mapping directly from 2-DE gels. Methods Mol. Biol. 112, 513–530 (1999)
Cox, J. & Mann, M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nature Biotechnol. 26, 1367–1372 (2008)
Elias, J. E. & Gygi, S. P. Target-decoy search strategy for increased confidence in large-scale protein identifications by mass spectrometry. Nature Methods 4, 207–214 (2007)
Schaefer, M. H. et al. HIPPIE: Integrating protein interaction networks with experiment based quality scores. PLoS ONE 7, e31826 (2012)
Cline, M. S. et al. Integration of biological networks and gene expression data using Cytoscape. Nature Protocols 2, 2366–2382 (2007)
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.
The authors declare no competing financial interests.
Extended data figures and tables
a, b, Representative images of HCT116 wild-type (WT) or UBC13-knockout (KO) cells exposed to IR. Where indicated, cells were transfected with HA–RNF8 plasmid before IR (n = 2 experiments). c, Constructs used in Fig. 1d and their ability to restore IR-induced 53BP1 foci in UBC13-knockout cells. d, Representative images of HCT116 UBC13-knockout cells transfected with non-targeting control (CTRL) or RNF8 siRNAs and subsequently with plasmids encoding UBC13 or siRNA-resistant UBC13–RNF8 fusion constructs (c) (n = 2). e, Representative images of UBC13-knockout cells transfected with plasmids encoding wild-type or catalytically inactive (CI) HA–RNF168 (n = 2). Expression of HA–RNF168 wild type restores IR-induced 53BP1 foci formation (arrows). Scale bars, 10 μm.
a–c, Representative images of U2OS cells stably expressing GFP–K63-Super-UIM, transfected with siRNAs where indicated, and exposed to laser micro-irradiation or IR (n = 3). d, Representative images of U2OS cells stably expressing GFP- and nuclear localization signal (NLS)-tagged Vps27-UIM (with high affinity for binding to K63-linked ubiquitin18) transfected with the indicated siRNAs and exposed to laser micro-irradiation (n = 2). e, Loss of RNF8 or UBC13 has no impact on 53BP1 abundance. Immunoblot analysis of whole-cell extracts (WCEs) of U2OS cells transfected with indicated siRNAs. f, RNF8, but not RNF168, is modified by K63-linked ubiquitin chains. K63-Super-UIM pull-downs from U2OS cells transfected with empty vector (−), HA-tagged RNF8 (wild type (WT) or catalytically inactive (CI)) or HA–RNF168 plasmids were immunoblotted (IB) with indicated antibodies. Scale bars, 10 μm. e, f, Uncropped blots are shown in Supplementary Fig. 1.
Extended Data Figure 3 Experimental replicates of SILAC-based quantification of di-glycine-containing peptides and K63-Super-UIM pull-downs from wild-type and UBC13-knockout cells.
a, Schematic outline of SILAC-based mass spectrometry approach to quantify di-glycine-containing peptides in HCT116 wild-type (WT) and UBC13-knockout (KO) cells. b, c, Proportional Venn diagrams showing overlap between all identified di-glycine-containing peptides (b) and those with a SILAC ratio (UBC13-knockout/wild-type cells) <0.5 (c) in two independent experiments (Exp.) performed as shown in a (Supplementary Table 1). d, Scatter plot showing correlation between SILAC ratios of di-glycine-containing peptides. The Pearson’s correlation coefficient (R) is indicated. e, Schematic outline of SILAC-based mass spectrometry approach to identify UBC13-dependent K63-ubiquitylation targets in unperturbed HCT116 wild-type and UBC13-knockout cells. f, g, Proportional Venn diagrams showing overlap between all proteins identified in K63-Super-UIM pull-downs (f) and those with a SILAC ratio (UBC13-knockout/wild-type cells) <0.5 (g) in two independent experiments performed as shown in e (Supplementary Table 2). h, Scatter plot showing correlation between SILAC ratios of proteins identified in two experiments. The Pearson’s correlation coefficient (R) is indicated.
Extended Data Figure 4 Analysis of UBC13-dependent K63-ubiquitylated proteins in unperturbed cells and in response to DNA damage.
a, K63 linkages from extracts of HCT116 wild-type (WT) and UBC13-knockout (KO) cells were enriched by K63-Super-UIM pull-down (Supplementary Table 2). Interaction network shows proteins enriched at least twofold in wild-type cells. Proteins involved in UBC13-dependent activation of NF-κB signalling are highlighted in red. b, Functional annotation of potential UBC13-dependent K63-ubiquitylated proteins (a), showing enriched Gene Ontology (GO) biological process terms. c, Schematic outline of SILAC-based mass spectrometry approach to identify targets of K63 ubiquitylation in response to IR-induced DSBs. d, SILAC ratios of selected proteins from U2OS cells treated as in c. Data from a representative experiment are shown (n = 3).
a, Analysis of K63-linked ubiquitylation of histone H1.2 in RPE1 cells growing exponentially or kept quiescent by serum starvation. b, c, K63-Super-UIM pull-downs from U2OS cells exposed or not to IR were immunoblotted (IB) with the indicated antibodies. d, U2OS cells or U2OS cells stably expressing Strep–HA–ubiquitin (U2OS/Strep–Ub) were transfected with the indicated siRNAs and exposed or not to IR. Whole-cell extracts (WCEs) and Strep–ubiquitin-conjugated proteins immobilized on Strep-Tactin beads under denaturing conditions were analysed by immunoblotting. e, Proteins interacting with endogenous RNF8. U2OS cells stably expressing RNF8 shRNA in a doxycycline (DOX)-inducible manner1 was grown in light (L) or heavy (H) SILAC medium. Cells growing in light medium were induced to express RNF8 shRNA by treatment with DOX. Both cultures were then exposed to IR and processed for immunoprecipitation with RNF8 antibody. Bound proteins were analysed by mass spectrometry. Proteins displaying the highest H/L SILAC ratios are listed. a–d, The migration of molecular weight markers (kDa) is indicated on the left. Uncropped blots are shown in Supplementary Fig. 1.
Extended Data Figure 6 Knockdown of H1-type histones impairs accumulation of K63-linked ubiquitin conjugates, RNF168 and BRCA1 at DSB sites.
a, Immunoblot analysis of U2OS cells transfected sequentially with plasmids encoding GFP-tagged histone H1 isoforms (H1.0–H1.5) and the indicated siRNAs. Knockdown efficiency of the siRNAs (#1, #4, #5 and #9) used in the pan-H1 siRNA cocktail to reduce global H1 expression level is highlighted in red boxes. b, Immunoblot analysis of U2OS cells transfected with H1 siRNA cocktail (a). c–g, Representative images of siRNA-transfected U2OS/GFP–K63-Super UIM (c) or U2OS cells (d–g) exposed to IR or laser micro-irradiation (n = 2). h, Analysis of IR-induced γ-H2AX ubiquitylation (Ub) by RNF168 (marked by arrow) in chromatin fractions of U2OS cells transfected with indicated siRNAs. Scale bars, 10 μm. a, b, h, Uncropped blots are shown in Supplementary Fig. 1.
Extended Data Figure 7 HMGB1 overexpression impairs the RNF8/RNF168-dependent signalling response at DSB sites at the level of K63 ubiquitylation and RNF168 recruitment.
a, b, Representative images of U2OS cells co-transfected with constructs encoding HMGB1–GFP and wild-type (WT) or catalytically inactive (CI) HA–RNF168 and exposed to IR (n = 3). Arrows indicate cells expressing HA–RNF168 CI, in which 53BP1 foci formation is not restored. c, Representative images of U2OS/GFP–K63-Super-UIM cells transfected with Flag–HMGB1 construct and subjected to laser micro-irradiation (n = 3). Flag–HMGB1-expressing cells show reduced K63 ubiquitylation at DSB sites (indicated by arrows). Scale bars, 10 μm.
a, Immunoblot analysis of immobilized recombinant RNF168 UDM1 or UDM2 incubated with K63 linked di-ubiquitin (Ub2) in the presence of increasing salt concentrations (75 mM, 150 mM and 250 mM KCl, respectively). b, Binding of immobilized recombinant Strep–UDM2 or empty Strep-Tactin beads to indicated di-ubiquitin (Ub2) linkages was analysed by immunoblotting. c, Biotinylated peptides corresponding to the LRM1 and LRM2 motifs in human RNF168 were analysed for binding to recombinant H2A or H1.0 in vitro by Streptavidin pull-down followed by SDS–PAGE and Colloidal Blue staining. The migration of molecular weight markers (kDa) is indicated on the left. d, Sequence of the UDM1 region in human RNF168, showing the location of the LRM1, UMI and MIU1 motifs. Acidic amino acids are highlighted in red. The sequence corresponding to the LRM1 peptide (c) and mutations introduced to generate UDM1 *UMI and *MIU1 (f) are indicated. e, f, Pull-down assays of Strep-tagged UDM1 and UDM2 constructs expressed in U2OS cells. a–c, e, f, Uncropped blots are shown in Supplementary Fig. 1.
a, Pull-downs of Strep-tagged RNF168 UDM1 expressed in U2OS cells were immunoblotted (IB) with antibodies to indicated histones. b, Localization pattern of GFP-tagged UDM1 expressed in U2OS cells. Scale bar, 10 μm. c, Venn diagram showing overlap between proteins displaying increased K63-linked ubiquitylation after IR (SILAC ratio (IR/mock) >1.5) and proteins showing potential interaction with overexpressed GFP–UDM1 (SILAC ratio (GFP–UDM1/mock) >1.5). Only one protein, histone H1x, was common to both of these subsets of cellular proteins. a, Uncropped blots are shown in Supplementary Fig. 1.
a, FRAP analysis of U2OS cells stably expressing GFP–H1.2 and exposed or not to IR (10 Gy). Individual data points represent mean values from ten independent measurements and error bars represent twice the s.d. b, U2OS cells left untreated or exposed to IR were lysed in EBC buffer. Soluble and resolubilized, EBC-insoluble fractions were incubated with recombinant K63-Super-UIM and washed thoroughly. Bound material and input fractions were analysed by immunoblotting with indicated antibodies. b, Uncropped blots are shown in Supplementary Fig. 1.
This file contains the uncropped scans of immunoblot data with size marker indications. (PDF 5793 kb)
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. (XLSX 1569 kb)
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Thorslund, T., Ripplinger, A., Hoffmann, S. et al. Histone H1 couples initiation and amplification of ubiquitin signalling after DNA damage. Nature 527, 389–393 (2015). https://doi.org/10.1038/nature15401
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