Histone H1 Couples Initiation and Amplification of Ubiquitin Signalling After DNA Damage

  

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

Thorslund, Tina; Ripplinger, Anita; Hoffmann, Saskia; Wild, Thomas; Uckelmann, Michael; Villumsen, Bine; Narita, Takeo; Sixma, Titia K; Choudhary, Chuna Ram; Bekker-Jensen, Simon; Mailand, Niels

Published in: Nature

DOI: 10.1038/nature15401

Publication date: 2015

Document version Peer reviewed version

Citation for published version (APA): Thorslund, T., Ripplinger, A., Hoffmann, S., Wild, T., Uckelmann, M., Villumsen, B., Narita, T., Sixma, T. K., Choudhary, C. R., Bekker-Jensen, S., & Mailand, N. (2015). Histone H1 couples initiation and amplification of ubiquitin signalling after DNA damage. Nature, 527(7578), 389-93. https://doi.org/10.1038/nature15401

Download date: 01. Oct. 2021 Histone H1 couples initiation and amplification of ubiquitin signaling after DNA damage

Tina Thorslund1,*, Anita Ripplinger1,*, Saskia Hoffmann1,*, Thomas Wild2,*, Michael

Uckelmann3, Bine Villumsen1, Takeo Narita2, Titia K. Sixma3, Chunaram Choudhary2, Simon

Bekker-Jensen1, Niels Mailand1,#

1Ubiquitin Signaling Group, Protein Signaling Program; and 2Proteomics Program, The

Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical

Sciences, University of Copenhagen, Blegdamsvej 3B, DK-2200 Copenhagen, Denmark;

3Division of Biochemistry, Cancer Genomics Center, Netherlands Cancer Institute,

Amsterdam, Netherlands

Running title: RNF8 ubiquitylates histone H1 after DNA damage

*These authors contributed equally to this study

#Correspondence and requests for materials to N.M.; phone: +45 35 32 50 23; e-mail: [email protected]

1 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 1-6, and Ubc13, an E2 ubiquitin-conjugating enzyme that specifically generates K63-linked ubiquitin chains 7. While RNF168 catalyzes ubiquitylation of H2A- type histones leading to recruitment of repair factors such as 53BP1 8-10, 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. 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 ubiquitylations 11 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 signaling and expand the concept of the histone code 12,13 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.

To mechanistically explain the critical role of Ubc13 in the RNF8/RNF168 pathway, we generated human Ubc13 knockout cells using CRISPR/Cas9 technology 14,15 (Fig 1a). As expected, loss of Ubc13 abrogated the accumulation of RNF168, RNF168-dependent ubiquitin conjugates, and 53BP1 and other readers of these marks at DSB sites, while RNF8

2 recruitment was normal (Fig. 1b,c; Extended Data Fig. 1a,b). Reintroducing Ubc13 into these cells restored 53BP1 focus formation, as did a fusion protein mimicking an RNF8-Ubc13 E3-

E2 complex 16,17, independently of endogenous RNF8 (Fig. 1d; Extended Data Fig. 1c,d). In contrast, expression of RNF8 alone or other RNF8-E2 chimeras failed to support 53BP1 accumulation at DSBs in these cells (Fig. 1d; Extended Data Fig. 1c). Unlike RNF8, overexpression of RNF168 was sufficient to restore 53BP1 recruitment to damaged DNA in

Ubc13 knockout cells (Extended Data Fig. 1e). These data suggest that Ubc13 mainly cooperates with RNF8 but not RNF168 to catalyze K63 ubiquitylation of DSB-flanking chromatin to promote recruitment of repair factors.

To understand the molecular basis of this process, we employed a tandem ubiquitin-binding entity (‘K63-Super-UIM’) 18 that showed remarkable specificity for binding to K63-linkages but not other ubiquitin chain types (Fig. 1e,f). Indeed, GFP-tagged K63-Super-UIM, but not a ubiquitin binding-deficient mutant (Fig. 1e,f), was efficiently recruited to microlaser- and ionizing radiation (IR)-generated DSBs (Extended Data Fig. 2a-c), thus providing an efficient sensor of DSB-associated K63 ubiquitylation 19. Depletion of Ubc13 or RNF8, but not

RNF168, abrogated recruitment of the K63-Super-UIM, as well as an independently derived high-affinity binder of K63-linked ubiquitin chains, to DSB sites, and autoubiquitylation- generated K63 ubiquitin chains were abundantly present on RNF8 but not RNF168 (Fig. 1g;

Extended Data Fig. 2b-f). This suggests that the roles of RNF8 and RNF168 in promoting

DSB-associated chromatin ubiquitylation can be functionally uncoupled and that RNF8, but not RNF168, is a primary mediator of Ubc13-dependent K63 ubiquitylation at DSB sites.

To identify RNF8-Ubc13-dependent ubiquitylation processes underlying RNF168 recruitment to DSBs, we first characterized the impact of Ubc13 loss on global K63-linked ubiquitylation

3 under steady state conditions. While K63-linked ubiquitylation is thought to be mainly catalyzed by Ubc13 7, the extent to which this E2 contributes to the total pool of K63 ubiquitin linkages in human cells is not known. To address this, we quantified the relative abundance of different ubiquitin chains in HCT116 wild type (WT) and Ubc13 knockout cells using SILAC-based mass spectrometry. Ablation of Ubc13 decreased the global level of K63 ubiquitin linkages by ~50%, while the abundance of other chain types was not markedly affected (Fig. 2a). Using the di-Gly approach 20,21, we quantified over 3,000 ubiquitylation sites, of which less than 1% showed a >2-fold decrease in Ubc13 knockout cells (Extended

Data Fig. 3a-d; Supplementary Table 1). Thus, Ubc13 has little impact on the conjugation of the initial ubiquitin moiety to substrates, consistent with previous reports that Ubc13 primarily acts at the ubiquitin chain elongation step 22-24. To identify Ubc13-dependent K63- ubiquitylated proteins, we analyzed K63-Super-UIM pull-downs from WT and Ubc13 knockout cells under stringent buffer conditions by mass spectrometry. We identified 371 proteins that showed >2-fold enrichment in WT cells, several of which are known targets of

K63 ubiquitylation (Extended Data Fig. 3e-h and 4a,b; Supplementary Table 2).

We extended this proteomic strategy to search for the chromatin-bound substrate(s), whose

K63 ubiquitylation by RNF8-Ubc13 promotes recruitment of RNF168 and downstream factors to DSB sites (Extended Data Fig. 4c). Surprisingly, only few cellular proteins reproducibly displayed elevated K63-linked ubiquitylation upon IR-induced DSBs, and H1 linker histones but not core histones were major chromatin-associated factors showing such behaviour (Extended Data Fig. 4d). Using the K63-Super-UIM we confirmed biochemically for two endogenous histone H1 isoforms (H1.2 and H1x) that they were modified with K63- linked ubiquitin chains and, more importantly, that these ubiquitylations were markedly upregulated after DSBs (Fig. 2b; Extended Data Fig. 5a,b). Basal levels of H1 K63

4 polyubiquitylation in non-irradiated cells dropped substantially upon serum starvation

(Extended Data Fig. 5a), suggesting that they result mostly from endogenous DNA damage generated by cell cycle-dependent processes. Little if any K63-linked ubiquitylation of core histones was detectable, regardless of whether DSBs had been inflicted or not (Extended Data

Fig. 5c), thus H1 appears unique among histones in undergoing robust DSB-stimulated K63 ubiquitylation. Importantly, the DNA damage-induced increase in K63-linked polyubiquitylation of H1 was dependent on RNF8 and Ubc13 but not RNF168 (Fig. 2c,d), whereas none of these enzymes appreciably affected H1 monoubiquitylation (Extended Data

Fig. 5d). Moreover, mass spectrometry analysis showed that H1 was a major factor co- purifying with endogenous RNF8 (Extended Data Fig. 5e). Finally, recombinant RNF8 catalyzed robust ubiquitylation of nucleosome-associated H1 with Ubc13 in vitro (Fig. 2e).

These data suggest that histone H1 is a major chromatin-associated substrate of RNF8-Ubc13.

To address whether linker histones represent critical RNF8 substrates in DSB signaling, we downregulated overall H1 levels using an siRNA cocktail targeting different isoforms

(Extended Data Fig. 6a,b). Under these conditions, accumulation of K63-linked ubiquitin chains, RNF168, 53BP1 and BRCA1, but not MDC1, at DSB sites was clearly diminished

(Fig. 3a-d; Extended Data Fig. 6c-g), suggesting that loss of H1 uncouples ubiquitin- dependent DSB signaling at the level of RNF168 accrual. Consistently, like RNF8 or RNF168 knockdown, H1 downregulation suppressed DSB-induced H2A ubiquitylation catalyzed by

RNF168 8,10 (Fig. 3e; Extended Data Fig. 6h). As an alternative strategy to interfere with H1 functionality, we overexpressed the high mobility group protein HMGB1 that competes with

H1 for chromatin binding in a non-site-specific manner 25. Elevated HMGB1 levels impaired

K63 ubiquitylation and 53BP1 accumulation at DSB sites despite enhancing γ-H2AX formation, and this could be rescued by co-overexpression of H1 or by WT but not

5 catalytically inactive RNF168 (Fig. 3f,g; Extended Data Fig. 7a-c). A similar strong increase in γ-H2AX formation accompanied by only mildly elevated 53BP1 accumulation at DSBs, indicating a partial uncoupling of these events, was reported for murine cells lacking 3 of 6

H1 isoforms 26. Together, these data suggest that H1-type histones are functionally critical targets of RNF8- and Ubc13-dependent K63 ubiquitylation at DSB-flanking chromatin areas.

We asked whether K63-ubiquitylated H1 provides a platform for initial, RNF8-Ubc13- dependent RNF168 recruitment to DSBs. RNF168 contains two DSB-targeting modules, both of which contain ubiquitin-binding domains 4,5,27 (Fig. 4a). While the C-terminal LRM2-

MIU2 region, which we termed Ubiquitin-dependent DSB recruitment Module 2 (UDM2), recognizes RNF168-catalyzed forms of ubiquitylated H2A, thereby effectively enabling

RNF168 to autonomously propagate H2A ubiquitylation at DSB sites once recruited, its

LRM1-UMI-MIU1 region (UDM1) has been suggested to bind as-yet undefined RNF8- generated ubiquitylation products required for initial relocalization of RNF168 to break sites

(Fig. 4a) 11. Therefore, if K63-ubiquitylated H1 functions as a recruitment platform for

RNF168 at DSB-modified chromatin, the UDM1 module should be capable of recognizing this modified form of H1. Consistent with this idea, we found that UDM1 bound to K63- linked ubiquitin but no other chain types (Fig. 4b). RNF168 UDM2 also interacted with K63 linkages, albeit with much lower affinity, and unlike UDM1 it also recognized other ubiquitin chains (Extended Data Fig. 8a,b), This likely reflects that UDM1, but not UDM2, has two adjacent ubiquitin-binding domains (UMI and MIU1), which together may confer specific binding to K63-linked chains. We also found that UDM1 interacted with histone H1 but not

H2A in vitro, whereas UDM2 displayed the inverse preference, binding only to H2A, as previously shown 11 (Fig. 4c). The LRM1 part of UDM1 has been hypothesized to impart target specificity to the ubiquitin-binding affinity of this module 11. Consistently, the H1-

6 binding ability of UDM1 was at least partially mediated by the LRM1 motif (Fig. 4a), which interacted strongly with H1 in vitro, unlike LRM2 (Extended Data Fig. 8c). However, additional sequence elements within UDM1 were also able to bind H1 (data not shown), possibly due to the very acidic nature of the UDM1 region (Extended Data Fig. 8d), which could facilitate robust interaction with the highly basic H1 proteins 28. Unlike UDM1, UDM2 interacted with neither modified nor unmodified forms of H1 (Fig. 4c,d). When expressed in cells, the UDM1 domain efficiently bound to high molecular weight forms of endogenous H1 isoforms in an IR-stimulated manner, suggesting that they correspond to K63-ubiquitylated

H1 (Fig. 4d; Extended Data Fig. 8e). Supporting this, point mutations in the UMI or MIU1 domains that impair their ubiquitin-binding activity and RNF168 recruitment to DSBs 11,27 markedly reduced the binding of UDM1 to modified H1 (Extended Data Fig. 8f). Moreover,

UDM1 did not interact with ubiquitylated forms of core histones, in sharp contrast to its binding to ubiquitylated H1 (Extended Data Fig. 9a). Finally, in-frame deletion of UDM1 but not UDM2 impaired the ability of ectopically expressed RNF168 to promote IR-induced

53BP1 foci in cells lacking endogenous RNF168 (Fig. 4e). We conclude that the RNF168

UDM1 is a high-affinity recognition module for K63-ubiquitylated histone H1. While the isolated UDM1 domain was distributed pan-cellularly and interacted with a range of proteins when overexpressed in cells, the only such protein showing DSB-stimulated K63 polyubiquitylation was an H1 isoform (Extended Data Fig. 9b,c), further suggesting that ubiquitylated H1 is a major receptor for RNF168 at DSB-modified chromatin.

Collectively, our findings suggest an integrated model for how RNF8 and Ubc13 promote ubiquitin-dependent protein recruitment to DSB sites. We propose that H1-type linker histones represent key chromatin-associated RNF8 substrates whose Ubc13-dependent K63- linked ubiquitylation at DSB-containing chromatin provide an initial binding platform for

7 RNF168 via the UDM1 (Fig. 4f). RNF168 then ubiquitylates H2A at K13/K15 and possibly other proteins to trigger recruitment of DSB repair factors. The notable absence of K63-linked ubiquitin chains on H2A suggests that RNF168 does not efficiently modify H2A in conjunction with Ubc13, but may instead catalyze formation of other ubiquitin chains or monoubiquitylation of the H2A K13/K15 mark. Indeed, RNF168 was recently shown to catalyze the formation of K27-linked ubiquitin chains, at least when overexpressed 29. It is possible, however, that RNF168 cooperates with Ubc13 in K63-linked ubiquitylation of other factors at damaged chromatin. While we observed no significant DSB-induced change in overall nuclear H1 mobility and no overt enrichment or depletion of H1 isoforms at DSB sites, the K63-ubiquitylated forms of H1 proteins were more loosely associated with chromatin than their unmodified counterparts (Extended Data Fig. 10a,b; data not shown).

This suggests that in addition to triggering RNF168 recruitment, the DSB-associated K63 ubiquitylation of H1 may play a role in facilitating chromatin remodeling to allow efficient repair.

H1-type histones consist of almost 30% lysine residues 28, and proteomic studies have shown that many of these can be ubiquitylated 20,21,30. Because Ubc13 only generates few de novo ubiquitylation marks, it is conceivable that RNF8-Ubc13 mainly extends pre-existing H1 ubiquitylations in response to DNA damage, rendering identification of the key H1 sites targeted by RNF8-Ubc13-dependent K63 ubiquitylation challenging. Although numerous

PTMs have been mapped on different H1 isoforms 28, these marks have not so far been connected directly to the 'histone code' specifying protein recruitment to different chromatin states 12,13. In addition to providing the first insights into the functional role of H1 ubiquitylation, our findings also show that linker histones can indeed form part of a dynamic histone code for DNA repair, wherein RNF8 and RNF168 function as writer and reader,

8 respectively, of K63-ubiquitylated H1. The multitude of site-specific H1 PTMs raises the possibility that some of these marks may play an integral role in the histone code, an area of study warranting further investigation.

9 Acknowledgements

We thank Drs. Daniel Durocher and Marco Bianchi for providing reagents and Jiri Lukas for helpful discussions. This work was supported by grants from the Novo Nordisk Foundation

(Grants no. NNF14CC0001 and NNF12OC0002114), European Research Council (ERC),

NWO-CW, The Danish Cancer Society, and The Danish Council for Independent Research.

Author contributions

T.T. initiated the project, designed and performed cell biological and biochemical experiments, and analyzed data; A.R. and S.H. performed cell biological and biochemical experiments and analyzed data; T.W. generated Ubc13 knockout cells, designed and performed mass spectrometry experiments and analyzed the data; M.U. performed in vitro ubiquitylation assays with purified nucleosomes; B.V. helped T.T. with biochemical experiments; T.N. analyzed mass spectrometry data; T.K.S. supervised M.U.; C.C. supervised

T.W. and T.N., designed mass spectrometry experiments and analyzed the data; S.B-J. performed and designed experiments and analyzed data; N.M. conceived and supervised the project, designed experiments, analyzed data and wrote the manuscript with input from the other authors.

Conflict of Interest

The authors declare that they have no conflict of interest.

10 References

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12 Figure legends

Figure 1.

RNF8 but not RNF168 is a primary mediator of Ubc13-dependent protein recruitment to DSB-flanking chromatin a. Immunoblot analysis of HCT116 wild type (WT) and Ubc13 knockout (KO) cells. MCM6 was a loading control. b-d. Representative images of HCT116 WT and Ubc13 KO cells exposed to IR or laser microirradiation (n=3 experiments). Cells in (d) were transfected with indicated expression constructs (Extended Data Fig. 1c). e,f. Binding of recombinant wild- type (WT) and mutant (MUT) K63-Super-UIM to di-ubiquitin (Ub2) linkages. The migration of molecular weight markers (kDa) is indicated on the left. g. Quantification of GFP-K63-

Super-UIM and 53BP1 accumulation at DSBs in U2OS/GFP-K63-Super-UIM cells transfected with indicated siRNAs. Data are mean ± s.d. from three independent experiments.

Representative images are shown in Extended Data Fig. 2b. Scale bars, 10 µm. WCE, Whole cell extract. a,g. Uncropped blots are shown in Supplementary Fig. 1.

Figure 2.

H1-type linker histones are major chromatin-bound targets of RNF8-Ubc13-dependent

K63 ubiquitylation a. Tukey boxplot showing levels of ubiquitin linkages in HCT116 Ubc13 knockout cells relative to WT cells, quantified by SILAC-based proteomics. Data from two independent experiments are shown. **, p < 0.01; N.S., not significant (Welch’s t-test). b-d. Pull-down analysis of IR-induced K63-linked ubiquitylation of histone H1.2 in U2OS cells (b,d) or derivative cell lines expressing RNF8 or RNF168 shRNAs in a Doxycycline (DOX)-inducible manner (c) using recombinant WT or mutant (MUT) K63-Super-UIM. Where indicated, ubiquitin conjugates were digested with recombinant USP2 catalytic domain (USP2cc) prior

13 to SDS-PAGE. e. In vitro ubiquitylation of purified H1-containing oligonucleosomes by

RNF8351-485 and indicated factors. WCE, Whole cell extract. The migration of molecular weight markers (kDa) is indicated on the left. b-e: Uncropped blots are shown in

Supplementary Fig. 1.

Figure 3.

Histone H1 is required for K63-linked ubiquitylation and RNF168-dependent protein retention at DSB sites a,b. Representative images of GFP-K63-Super-UIM-expressing cells transfected with siRNAs. c. Proportion of cells with visible accumulation of GFP-K63-Super-UIM at laser- induced DSBs (a). d. Intensity of GFP-K63-Super-UIM or 53BP1 accumulation at laser- induced DSBs normalized to γ-H2AX signal (a). e. Analysis of IR-induced γ-H2AX ubiquitylation (Ub) by RNF168 (marked by arrow) in chromatin fractions of U2OS cells transfected with indicated siRNAs. f. Representative images of U2OS cells expressing indicated constructs and exposed to IR. HMGB1 overexpression impairs 53BP1 recruitment to DSBs (marked by arrows). g. Quantification of data in (f). Data in c and d are mean ± s.d. from three independent experiments. Data in g are mean ± s.e.m. from two independent experiments. Scale bars, 10 µm. e: Uncropped blots are shown in Supplementary Fig. 1.

Figure 4.

The RNF168 UDM1 domain is a high-affinity reader of K63-ubiquitylated H1 a. Composition and reported functions of ubiquitin-dependent DSB recruitment modules

(UDMs) in human RNF168. b. Binding of recombinant UDM1 to di-ubiquitin (Ub2) linkages. c. Binding of recombinant UDM1 and UDM2 to purified histones (H1.0 and H2A).

14 d. Pull-down assays of Strep-tagged UDM1 and UDM2 modules expressed in U2OS cells.

The migration of molecular weight markers (kDa) is indicated on the left. e. Representative images of shRNF168-expressing cells transfected with GFP-RNF168 constructs and exposed to IR (n=2 experiments). Deletion of UDM1 (ΔUDM1) impairs restoration of 53BP1 foci by

GFP-RNF168 (indicated by arrows). Scale bar, 10 µm. f. Model of RNF8-Ubc13 function in ubiquitin-dependent signaling after DSBs. WCE, Whole cell extract. b-d: Uncropped blots are shown in Supplementary Fig. 1.

15 Methods

Plasmids cDNAs encoding K63-Super-UIM (WT and mutant) and the Vps27-based K63 binder 18 containing C-terminal His6 tags were produced as synthetic genes (Eurofins) and inserted into pDONRTM221 by BP reactions (Invitrogen). By means of LR reactions (Invitrogen) the inserts were then transferred to the Champion™ pET104 BioEase™ Gateway® Biotinylation

System (Invitrogen) for recombinant protein production or pcDNA-DEST53 (Invitrogen) for

GFP-tagged constitutive mammalian expression. For inducible expression of GFP-tagged

K63-Super-UIM, the GFP-K63-Super-UIM cDNA was inserted into pcDNA4/TO

(Invitrogen). Plasmids encoding HA-tagged WT and catalytically inactive (CI) RNF8

(C403S), WT and CI (C16S/C19S) forms of RNF168, and Ubc13, as well as chimeras between RNF8 and different E2 enzymes were described previously 1,4,16. The *FHA mutation (R42A) in HA-RNF8ΔR-Ubc13 was generated by site-directed mutagenesis. RNF8 constructs were made resistant to RNF8-siRNA by introducing 3 silent mutations (underlined) in the siRNA targeting sequence (5’-TGCGGAGTATGAGTACGAG-3’) in the plasmids by site-directed mutagenesis. The RNF168 UDM1 (amino acids 110-201) and UDM2 (amino acids 419-487) fragments were amplified by PCR and inserted into either pTriExTM-5

(Novagen) for Strep- and His-tagged expression in E. coli and mammalian cells, or pEGFP-

C1 (Clontech) for expression of GFP-tagged versions. The Strep-RNF168 UDM1 mutants used in this study (*UMI (L149A) and *MIU1 (A179G)) were generated using the

QuikChange site-directed mutagenesis kit (Stratagene). Constructs encoding GFP-H1 isoforms were cloned by inserting the respective cDNAs into the BglII and BamHI sites of pEGFP-C1 (Clontech). A plasmid encoding HMGB1-GFP was kindly provided by Dr. Marco

Bianchi (San Raffaele University, Milan, Italy). A FLAG-HMGB1 expression construct was generated by inserting the HMGB1 ORF into pFLAG-CMV2 (Sigma). All constructs were

16 verified by sequencing. Plasmid transfections were done with FuGene 6 (Promega) or

Genejuice (Novagene), siRNA transfections were done with Lipofectamine RNAiMAX

(Invitrogen), according to the manufacturers' instructions. siRNA siRNA sequences used in this study were:

Non-targeting control (CTRL) (5’-GGGAUACCUAGACGUUCUATT-3’); Ubc13 (5’-

GAGCAUGGACUAGGCUAUATT-3’); RNF8 (5’-UGCGGAGUAUGAAUAUGAATT-3’);

RNF168 (5’-GUGGAACUGUGGACGAUAATT-3’) or (5’-

GGCGAAGAGCGAUGGAAGATT-3’); Histone H1(#1) (5’-

GCUACGACGUGGAGAAGAATT-3’); H1(#2) (5’-GCUCCUUUAAACUCAACAATT-

3’); H1(#3) (5’-GAAGCCAAGCCCAAGGUUATT-3’);

H1(#4) (5’-CCUUUAAACUCAACAAGAATT-3’); H1(#5) (5’-

CCUUCAAACUCAACAAGAATT-3’); H1(#6) (5’-UCAAGAGCCUGGUGAGCAATT-

3’); H1(#7) (5’-GGACCAAGAAAGUGGCCAATT-3’); H1(#8) (5’-

GCAUCAAGCUGGGUCUCAATT-3’); H1(#9) (5’-CAGUGAAACCCAAAGCAAATT-

3’); H1(#10) (specific for H1x) (5’-CCUUCAAGCUCAACCGCAATT-3’); 53BP1 (5’-

GAACGAGGAGACGGUAAUATT-3’); USP7 (5’-GGCGAAGUUUUAAAUGUAUTT-3’); and USP9x (5’-GCAGUGAGUGGCUGGAAGUTT-3’).

Cell culture

Human U2OS, HCT116 and RPE1 cells were obtained from ATCC. U2OS and HCT116 were cultured in DMEM containing 10% FBS and 1xPen-Strep, while RPE1 cells were grown in a

1:1 mixture of Ham’s F12 and DMEM supplemented with 10% FBS and 1xPen-Strep.

Serum-starvation of RPE1 cells was done by incubating cells for 24 h in medium supplemented with 0.25% FBS. A HCT116 Ubc13 knockout cell line was generated using the

CRISPR/Cas9 technology 14,15. A donor plasmid bearing a splice acceptor site and a

17 puromycin resistance marker, flanked by homology arms, was co-transfected with pX300 14 targeting the GGCGCGCGGGAATCGCGGCG sequence within the first intron of the Ubc13 gene. To generate cell lines capable of Doxycycline-induced expression of GFP-tagged K63-

Super-UIM, U2OS cells were transfected with GFP-K63-Super-UIM plasmid and pcDNA6/TR and positive clones were selected with Zeocin (Invitrogen) and Blasticidin S

(Invitrogen). Stable U2OS cell lines expressing RNF8 or RNF168 shRNA in a doxycycline- inducible manner or Strep-HA-ubiquitin were described previously 1,4,31. All cell lines were regularly tested for mycoplasma infection. Unless otherwise indicated, cells were exposed to

DSBs using IR (4 Gy for microscopy experiments and 10 Gy for biochemical analyses) or laser microirradiation (as described in 32), and collected 1 h later.

Recombinant protein production

Purified biotinylated K63-Super-UIM WT and mutant proteins containing an N-terminal, biotinylated BioEase tag and a C-terminal His6-tag were obtained by expressing the proteins in an E.coli strain expressing the BirA biotin ligase. Bacteria were grown in LB medium containing 0.5 mM biotin, induced with 0.25 mM IPTG for 3 h at 30 °C, and then lysed by

French press. The K63-Super-UIM constructs were purified using IMAC followed by SEC.

Purity and complete biotinylation of the proteins was verified by MS. Recombinant Strep-

His6-RNF168 UDM-1/2 was produced in Rosetta2(DE3)pLacI (Novagen) bacteria induced with 0.5 mM IPTG for 3 h at 30 °C, lysed using Bugbuster (Novagen) supplemented with

Protease Inhibitor Cocktail without EDTA (Roche). The proteins were purified on Ni2+-NTA- agarose (Qiagen). Recombinant hUba1, UbcH5c, RNF8 and ubiquitin used for in vitro ubiquitylation assays were purified as described 8.

Antibodies

Antibodies used in this study included: Ubc13 (#4919, Cell Signaling), MCM6 (sc-9843,

Santa Cruz), 53BP1 (sc-22760, Santa Cruz), γ-H2A.X (05-636, Millipore) or (2577, Cell

18 Signaling), H2A.X (2595, Cell Signaling), MDC1 (ab11171, Abcam), Conjugated ubiquitin

(FK2) (BML-PW8810-0500, Enzo Life Sciences), HA (11867423991, Roche; and sc-7392,

Santa Cruz), Myc (sc-40, Santa Cruz), His6 (631212, Clontech), GFP (sc-9996, Santa Cruz;

11814460001, Roche), Ubiquitin (sc-8017, Santa Cruz), Histone H1.2 (ab17677, Abcam),

Histone 1x (A304-604A, Bethyl Labs), Histone H1 (pan, #AE-4 clone) (ab7789, Abcam),

Histone H2A (07-146, Millipore), Histone H2B (ab1790, Abcam), Histone H3 (ab1791,

Abcam), Histone H4 (ab7311, Abcam), USP7 (A300-033A, Bethyl Labs), USP9x (A301-

351A, Bethyl Labs), Cyclin A (sc-751, Santa Cruz), Actin (MAB1501, Millipore), BRCA1

(sc-6954, Santa Cruz), RNF168 for IF (06-1130, Millipore) and antibody to RNF168 5 used for WB was a kind gift from Dr. Daniel Durocher (University of Toronto, Canada). Antibody to RNF8 has been described previously 1.

Immunochemical methods

For pull-down of K63-ubiquitylated proteins, cells were lysed in high-stringency buffer (50 mM Tris, pH 7.5; 500 mM NaCl; 5 mM EDTA; 1% NP40; 1 mM DTT, 0.1% SDS) containing 1.25 mg/mL N-Ethylmaleimide, 50 µM DUB inhibitor PR619 (LifeSensors), and protease inhibitor cocktail (Roche). Recombinant biotionylated K63-Super-UIM (25 µg/ml) was added immediately upon lysis, followed by sonication and centrifugation. Streptavidin

M-280 Dynabeads (Invitrogen) was added to immobilize the K63-Super-UIM, and bound material was washed extensively in high-stringency buffer. A Benzonase (Sigma) and MNase

(NEB) treatment step was included to remove any contaminating nucleotides. Proteins were resolved by SDS-PAGE and analyzed by immunoblotting. Where indicated, bound complexes were subjected to deubiquitylation by incubation with USP2cc (1 µM, Boston Biochem) in

DUB buffer (50 mM HEPES, pH 7.5; 100 mM NaCl; 1 mM MnCl2; 0.01% Brij-35; 2 mM

DTT) overnight at 30 ºC before boiling in Laemmli Sample Buffer. Immunoblotting, Strep-

Tactin pull-downs, and chromatin enrichment were done essentially as described 32. Briefly,

19 Strep-RNF168 UDM pull down experiments from cells were performed after lysing cells in

EBC buffer (50 mM Tris, pH 7.4; 150 mM NaCl; 0.5% NP-40; 1 mM EDTA) containing 1.25 mg/mL NEM, 50 µM PR619 (LifeSensors) and protease inhibitor cocktail (Roche). The soluble fraction was subsequently used for immunoprecipitation using Strep-Tactin sepharose

(IBA). After washing in EBC buffer, proteins were eluted and analyzed by immunoblotting.

To isolate Strep-HA-ubiquitin conjugated proteins, cells were lysed in denaturing buffer (20 mM Tris, pH 7.5; 50 mM NaCl; 1 mM EDTA; 1 mM DTT; 0.5% NP-40; 0.5% sodium deoxycholate; 0.5% SDS) containing 1.25 mg/mL NEM, 50 µM PR619 (LifeSensors) and protease inhibitor cocktail (Roche). After sonication and centrifugation, Strep-HA-ubiquitin- conjugated proteins were immobilized on Strep-Tactin sepharose (IBA). After extensive washing in denaturing buffer, proteins were eluted and analyzed by immunoblotting. For chromation fractionation, cells were first lysed in Buffer 1 (100 mM NaCl; 300 mM sucrose;

3 mM MgCl2; 10 mM PIPES, pH 6.8; 1 mM EGTA; 0.2% Triton X-100) containing protease, phosphatase and DUB inhibitors and incubated on ice for 5 min. After centrifugation, the soluble proteins were removed and the pellet was resuspended in Buffer 2 (50 mM Tris-HCl, pH 7.5; 150 mM NaCl; 5 mM EDTA; 1% Triton X-100; 0.1% SDS) containing protease, phosphatase and DUB inhibitors. Lysates were then incubated 10 min on ice, sonicated, and solubilized chromatin-enriched fractions were collected after centrifugation.

Immunofluorescence staining and microscopy

For immunofluorescence staining, cells were fixed in 4% paraformaldehyde for 15 min, permeabilized with PBS containing 0.2% Triton X-100 for 5 min, and incubated with primary antibodies diluted in DMEM for 1 h at room temperature. Following staining with secondary antibodies (Alexa Fluor; Life Technologies) for 1 h, coverslips were mounted in Vectashield mounting medium (Vector Laboratories) containing nuclear stain DAPI. Images of GFP-K63-

Super-UIM were all obtained from our stable inducible cell line where GFP-K63-Super-UIM

20 was induced by incubating with 1 µg/mL Doxycycline for approximately 24 h unless otherwise stated. Images were acquired with an LSM 780 confocal microscope (Carl Zeiss

Microimaging Inc.) mounted on Zeiss-Axiovert 100M equipped with Plan-Apochromat

40x/1.3 oil immersion objective, using standard settings. Image acquisition and analysis was carried out with ZEN2010 software. For ImageJ-based image analysis, images were acquired with an AF6000 widefield microscope (Leica Microsystems) equipped with a Plan-

Apochromat 40x/0.85 CORR objective, using the same microscopic settings. Fluorescence intensities of the micro-irradiated region (demarcated by γ-H2AX positivity) and the nucleus were first corrected for the general image background. Using these values, relative recruitment to DNA damage sites (relative fluorescence units, RFU) was calculated by normalizing the nuclear background-corrected signal at the micro-irradiated region to that of the nuclear background. Finally, the RFU of the protein of interest was normalized to the

RFU of the γH2AX signal and plotted as the average of biological triplicates. Fluorescense

Recovery After Photobleaching (FRAP) was performed essentially as described 33. Briefly,

U2OS cells stably expressing GFP-H1 were grown in glass-bottom dishes (LabTek) in the presence of CO2-independent medium. A 2-µm wide rectangular strip spanning the entire width of the cell was bleached by excitation with the maximal intensity of a 488 nm laser line, after which 95 frames of the bleached region were acquired at 4 s intervals. Mean fluorescence intensites were processed, normalized and analyzed as described 33.

In vitro binding and ubiquitylation assays

Binding of K63-Super-UIM to di-ubiquitin (Ub2) linkages (Boston Biochem) was done by incubating 100 ng Ub2 with 2.5 µg K63-Super-UIM immobilized on Streptavidin M-280

Dynabeads (Invitrogen) in Buffer A (50 mM Tris, pH 7.5; 10% glycerol; 400 mM NaCl;

0.5% NP40; 2 mM DTT; 0.1 mg/mL BSA). After extensive washing, bound complexes were resolved by SDS-PAGE and analyzed by immunoblotting. Binding of RNF168 UDM1/2 to

21 di-ubiquitin (Ub2) linkages was analyzed by incubating 100 ng Ub2 with 5 µg Strep-RNF168-

UDM1/2 immobilized on Strep-Tactin sepharose (IBA BioTAGnology) in Buffer B (50 mM

Tris, pH 8; 5% glycerol; 0.5% NP40; 2 mM DTT; 0.1 mg/mL BSA; 2 mM MgCl2, added 250 mM KCl for UDM1 binding and 100 mM KCl for UDM2 binding). After extensive washing, bound complexes were resolved by SDS-PAGE and analyzed by immunoblotting. Where indicated, UDM1/2 binding to K63-linked Ub2 was analyzed in the presence of increasing

KCl concentrations (75 mM, 150 mM, and 250 mM). To analyze binding of RNF168

UDM1/2 to recombinant histones, purified Strep-RNF168 UDM1/2 (10 µg) was pre-bound to

Strep-Tactin sepharose in buffer C (for binding to H1.0) (50 mM, Tris pH 8; 5% glycerol; 150 mM KCl; 0.5% NP40; 2 mM DTT; 0.1 mg/mL BSA) or D (for binding to H2A) (50 mM, Tris pH 8; 5% glycerol; 75 mM KCl; 0.05% NP40; 2 mM DTT; 0.1 mg/mL BSA), and incubated with 500 ng recombinant histone H1.0 or H2A (New England Biolabs). Bound complexes were washed and analyzed by immunoblotting. To analyze binding of LRM1 and LRM2 peptides to histone H1.0 or H2A, magnetic Streptavidin beads were incubated with buffer E

(25 mM, Tris pH 8.5; 5% glycerol; 50 mM KCl; 0.5% TX-100; 1 mM DTT; 0.1 mg/mL BSA) in the absence (control) or presence of 1.5 µg purified, biotinylated RNF168 LRM1 (amino acids 110-133) or LRM2 (amino acids 463-485) peptide. Samples were then incubated with

250 ng recombinant H2A or H1.0 for 2 h at 4 °C, and immobilized complexes were washed and analyzed by SDS-PAGE and Colloidal Blue staining (Invitrogen).

For in vitro ubiquitylation assays, histone H1-containing oligonucleosomes (10 µM) were purified in the presence of 55 mM Iodoacetamide, essentially as described previously 34, with the exception that micrococcal nuclease digestion was stopped with 20 mM EGTA and dialysis was started right after the second homogenization in buffer containing 50 mM Tris, pH 7.5; 150 mM NaCl; 1 mM TCEP; and 340 mM Sucrose. Dialyzed samples were then incubated with DUB inhibitor (Ubiquitin-PA 35, 20 µM) for 20 min at room temperature.

22 Nuclesomes were incubated with 0.5 µM hUba1, 5 µM UbcH5c, 1 µM Ubc13-Mms2

8 complex, 5 µM RNF8351-485 fragment (purified as described in ) and 75 µM ubiquitin in reaction buffer (50 mM Tris, pH 7.5; 100 mM NaCl; 3 mM ATP; 3 mM MgCl; 1 mM TCEP) at 31 ºC. Samples were analyzed by immunoblot analysis.

SILAC based quantification of di-glycine containing peptides

For SILAC experiments, U2OS or HCT116 cells were grown in medium containing unlabeled

L-arginine and L-lysine (Arg0/Lys0) as the light condition, or isotope labeled variants of L- arginine and L-lysine (Arg6/Lys4 or Arg10/Lys8) as the heavy condition 36. SILAC labeled

HCT116 wildtype and Ubc13 knockout cells were lysed in modified RIPA buffer (50 mM

Tris-HCl, pH 7.5; 150 mM NaCl; 1% Nonidet P-40; 0.1% sodium-deoxycholate; 1 mM

EDTA) supplemented with protease inhibitors (Complete protease inhibitor mixture tablets,

Roche Diagnostics) and N-ethylmaleimide (5 mM). Lysates were incubated for 10 min on ice and cleared by centrifugation at 16,000 × g. An equal amount of protein from the two SILAC states was mixed and precipitated by adding 5-fold acetone and incubating at -20 °C overnight. Precipitated proteins were dissolved in denaturing buffer (6 M urea; 2 M thiourea;

10 mM HEPES, pH 8.0), reduced with DTT (1 mM) and alkylated with chloroacetamide (5.5 mM). Proteins were digested with Lysyl endoproteinase C (Lys-C) for 6 h, diluted four-fold with water and digested overnight with trypsin. The digestion was stopped by addition of trifluoroacetic acid (0.5% final concentration), incubated at 4 °C for 2 h and centrifuged for

15 min at 4000×g. Peptides from the cleared solution were purified by reversed-phase Sep-

Pak C18 cartridges (Waters Corporation). Di-glycine-lysine modified peptides were enriched using the Ubiquitin Remnant Motif Kit (Cell Signaling Technology), according to the manufacturer´s intructions. Briefly, Peptides were eluted from the Sep-Pak C18 cartridges with 50% acetonitrile, which was subsequently removed by centrifugal evaporation. Peptides were incubated with 40 µl of anti-di-Gly-lysine antibody resin in immunoaffinity purification

23 (IAP) buffer for 4 h at 4 °C. Beads were washed three times with IAP buffer, two times with water and peptides eluted with 0.15% trifluoroacetic acid. Eluted peptides were fractionated by microcolumn-based strong cation exchange chromatography (SCX) and cleaned by reversed phase C18 stage-tips.

SILAC K63-Super-UIM pull-down and in-gel digestion

SILAC labeled cells were lysed in high-stringency RIPA buffer (50 mM Tris-HCl, pH 7.5;

500 mM NaCl; 1% Nonidet P-40; 0.1% sodium-deoxycholate; 1 mM EDTA) containing 1.25 mg/mL N-Ethylmaleimide, 50 µM DUB inhibitor PR619 (LifeSensors), and protease inhibitor cocktail (Roche). Lysates from different SILAC states were separately incubated for 10 min on ice and cleared by centrifugation at 16,000×g. Extract (5 mg) were incubated for 4 h at 4

°C with K63-Super-UIM immobilized to streptavidin beads (approximately 5 µg K63-Super-

UIM per experiment). Beads were washed three times with high-stringency RIPA, beads from the different SILAC conditions were mixed, and proteins were eluted with SDS sample buffer, incubated with DTT (10 mM) for 10 min at 70 °C and alkylated with chloroacetamide

(5.5 mM) for 60 min at 25 °C. Proteins were separated by SDS-PAGE using a 4%-12% gradient gel and visualized with colloidal blue stain. Gel lanes were sliced into six pieces, and proteins were digested in-gel using standard methods 37.

Mass spectrometry and data analysis

Peptides were analyzed on a quadrupole Orbitrap (Q Exactive, Thermo Scientific) mass spectrometer equipped with a nanoflow HPLC system (Thermo Scientific). Peptide samples were loaded onto C18 reversed-phase columns and eluted with a linear gradient (1-2 h for in- gel samples, and 3-4 h for di-glycine-lysine enriched samples) from 8 to 40% acetonitrile containing 0.5% acetic acid. The mass spectrometer was operated in a data-dependent mode automatically switching between MS and MS/MS. Survey full scan MS spectra (m/z 300–

1200) were acquired in the Orbitrap mass analyzer. The 10 most intense ions were

24 sequentially isolated and fragmented by higher-energy C-trap dissociation (HCD). Peptides with unassigned charge states, as well as peptides with charge state less than +2 for in-gel samples and +3 for di-glycine-lysine enriched samples were excluded from fragmentation.

Fragment spectra were acquired in the Orbitrap mass analyzer. Raw MS data were analyzed using MaxQuant software (version 1.3.9.21). Parent ion and tandem mass spectra were searched against protein sequences from the UniProt knowledge database using the

Andromeda search engine. Spectra were searched with a mass tolerance of 6 ppm in the MS mode, 20 ppm for MS/MS mode, strict trypsin specificity and allowing up to two missed cleavage sites. Cysteine carbamidomethylation was searched as a fixed modification, whereas amino-terminal protein acetylation, methionine oxidation and N-ethylmaleimide modification of cysteines, and di-glycine-lysine were searched as variable modifications. Di-glycine- lysines were required to be located internally in the peptide sequence. Site localization probabilities were determined using MaxQuant (PTM scoring algorithm) as described previously 38. A false discovery rate of less than one percent was achieved using the target- decoy search strategy 39 and a posterior error probability filter. Information about previously known protein-protein interactions among putative Ubc13-dependent K63-Super-UIM interacting proteins was extracted using the HIPPIE database 40 (version 1.6), and interactions were visualized in Cytoscape 41. The Gene Ontology (GO) term Biological Process analysis for Ubc13-dependent K63-Super-UIM interacting proteins was filtered for categories annotated with at least 20 and not more than 300 genes. Redundant GO terms (less than 30% unique positive scoring genes compared to more significant GO term) were removed and the five most significant (Fisher’s exact t-test) remaining GO term categories depicted. To determine the variation within the quantification of ubiquitin linkage types, an F-test was performed and the p-values were adjusted using the Bonferroni method. A significant difference in the variances between K48 and K11, and K48 and K6 ubiquitin linkages was

25 detected. To test the significance of the difference between the SILAC ratios measured for ubiquitin linkage types, the Welch Two Sample t-test was performed and the obtained p- values were adjusted using the Bonferroni method.

31 Danielsen, J. M. et al. Mass spectrometric analysis of lysine ubiquitylation reveals promiscuity at site level. Mol Cell Proteomics 10, M110 003590, doi:M110.003590 [pii] 10.1074/mcp.M110.003590 (2011). 32 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, doi:jcb.201109100 [pii] 10.1083/jcb.201109100 (2012). 33 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, doi:jcb.200503043 [pii] 10.1083/jcb.200503043 (2005). 34 Hernandez-Munoz, 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 U S A 102, 7635-7640, doi:10.1073/pnas.0408918102 (2005). 35 Ekkebus, R. et al. On terminal alkynes that can react with active-site cysteine nucleophiles in proteases. Journal of the American Chemical Society 135, 2867-2870, doi:10.1021/ja309802n (2013). 36 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). 37 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). 38 Cox, J. & Mann, M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat Biotechnol 26, 1367-1372, doi:nbt.1511 [pii] 10.1038/nbt.1511 (2008). 39 Elias, J. E. & Gygi, S. P. Target-decoy search strategy for increased confidence in large-scale protein identifications by mass spectrometry. Nat Methods 4, 207-214, doi:10.1038/nmeth1019 (2007). 40 Schaefer, M. H. et al. HIPPIE: Integrating protein interaction networks with experiment based quality scores. PLoS One 7, e31826, doi:10.1371/journal.pone.0031826 (2012). 41 Cline, M. S. et al. Integration of biological networks and gene expression data using Cytoscape. Nat Protoc 2, 2366-2382, doi:10.1038/nprot.2007.324 (2007).

26 Extended Data

Extended Data Figure 1.

Protein recruitment to DSB sites in Ubc13 knockout cells 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 prior to IR

(n=2 experiments). c. Constructs used in Fig. 1d and their ability to restore IR-induced 53BP1 foci in Ubc13 KO cells. d. Representative images of HCT116 Ubc13 KO 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 KO cells transfected with plasmids encoding WT or catalytically inactive (CI) HA-RNF168 (n=2). Expression of HA-RNF168 WT restores IR- induced 53BP1 foci formation (arrows). All scale bars, 10 µm.

Extended Data Figure 2.

RNF8- and Ubc13-dependent K63-linked ubiquitylation at DSB sites a-c. Representative images of U2OS cells stably expressing GFP-K63-Super-UIM, transfected with siRNAs where indicated, and exposed to laser microirradiation or IR (n=3). d. Representative images of U2OS cells stably expressing GFP- and NLS-tagged Vps27-UIM

(with high affinity for binding to K63-linked ubiquitin 18) transfected with indicated siRNAs and exposed to laser microirradiation (n=2). e. Loss of RNF8 or Ubc13 has no impact on

53BP1 abundance. Immunoblot analysis of whole cell extracts (WCE) 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 (WT or catalytically inactive (CI)) or HA-RNF168 plasmids were

27 immunoblotted (IB) with indicated antibodies. All 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 WT and Ubc13 KO cells a. Schematic outline of SILAC-based mass spectrometry approach to quantify di-glycine- containing peptides in HCT116 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 KO/WT cells) < 0.5 (c) in two independent experiments 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 WT and Ubc13 KO 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 KO/WT 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 WT and Ubc13 knockout cells were enriched by

K63-Super-UIM pull-down (Supplementary Table 2). Interaction network shows proteins

28 enriched at least 2-fold in WT cells. Proteins involved in Ubc13-dependent activation of NF-

κB signaling are highligted 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).

Extended Data Figure 5.

DSB-induced K63 ubiquitylation of H1-type linker histones 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 indicated antibodies. d. U2OS or U2OS stably expressing Strep-HA-Ubiquitin (U2OS/Strep-Ub) were transfected with indicated siRNAs and exposed or not to IR. Whole cell extracts (WCE) and Strep-ubiquitin-conjugated proteins immobilized on Strep-Tactin beads under denaturing conditions were analyzed by immunoblotting. e. Proteins interacting with endogenous RNF8. U2OS cells stably expressing

RNF8 shRNA in a Doxycycline (DOX)-inducible manner 1 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 analyzed 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.

29 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 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 microirradiation

(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. All 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 signaling 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 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). All scale bars, 10 µm.

Extended Data Figure 8.

Interaction of RNF168 UDM1 with K63-ubiquitylated H1

30 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 analyzed by immunoblotting. c. Biotinylated peptides corresponding to the LRM1 and LRM2 motifs in human RNF168 were analyzed 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.

Extended Data Figure 9.

RNF168 UDM1 recognizes ubiquitylated forms of H1 but not core histones 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.

Extended Data Figure 10.

31 Impact of DSBs on H1 chromatin association 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 10 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 analyzed by immunoblotting with indicated antibodies. b: Uncropped blots are shown in

Supplementary Fig. 1.

32 Thorslund et al, Figure 1 a c Ubiquitin (FK2) MDC1 d Ubc13 KO + IR HCT116 53BP1 HA/Myc merge WT HA- Ubc13 WT Ubc13 KO −Ubc13 −MCM6

WCE Ubc13 KO HA- RNF8 b +laser microirradiation 53BP1 γ-H2AX merge HA- Ubc13 − IR RNF8 Δ R- WT Myc- UbcH7 +IR RNF8 Δ R- Myc- UbcH2 − IR RNF8 Δ R- g 100 K63-Super-UIM

Ubc13 KO 53BP1 80 +IR 60 e K63-Super-UIM 40 (% of cells) of (% Bioease/GFP UIM1 UIM2 UIM3 20

WT stripes damage DNA * * * * * * 0 Bioease/GFP UIM1 UIM2 UIM3 siRNA: CTRL Ubc13 RNF8 RNF168 MUT f Pull-down: K63-Super-UIM

Ub2 input (100%) K6 K11 K27 K29 K33 K48 K63 M1 Ub2 + + + + + + + + K63-UIM WT M1 K48 K63 K27 K33 K29 K11 K6 + + + + + + + + K63-UIM MUT

38–

28–

−Ub2 17– 14– Thorslund et al, Figure 2 a d U2OS e In vitro Experiment 1 + + siCTRL ubiquitylation Experiment 2 + + siUbc13 (Nucleosomes 2.0 ** + + IR +E1+UbcH5c ** +Ubc13-Mms2) 191− + N.S. + + + RNF8351-485 1.5 97− 90 30 60 120 Time (min) H1.2-Ubn

64− 1.0 H1.2-Ubn 191−

Ubn 97− 0.5 −H1.2 Pull down: K63-Super-UIM SILAC ratio ( Ubc13 KO /WT ) 64− −H1.2 0.0 −Ubc13

K48 K63 K11 K6 WCE Ubiquitin linkage type −MCM6 b U2OS c U2OS/shRNF8 U2OS/shRNF168 MUT WT MUT WT K63-UIM + + + + DOX + + + + IR + + + + USP2cc + + + + IR 191− 191– H1.2-Ubn 97−

97– H1.2-Ubn 64−

64– * 191− Ubn Pull down: K63-Super-UIM 51– 97−

39– −H1.2 −H1.2

Pull down: K63-Super-UIM −RNF8

WCE −RNF168 Ubn 191– −MCM6 Thorslund et al, Figure 3 a U2OS/GFP-K63-Super-UIM c d 100 100 GFP-K63- γ-H2AX+ siCTRL Super-UIM 53BP1 DAPI 80 80 siH1

60 60

40 40 siCTRL 20 (relativesiCTRL) to 20 GFP-K63-UIM stripe (% cells(% relative siCTRL) to 0 intensity stripe damage DNA 0 siH1

+laser microirradiation e U2OS b + + siCTRL U2OS/GFP-K63-Super-UIM WT + IR + + siH1 GFP-K63- + + IR Super-UIM γ-H2AX 53BP1 γ-H2AX-Ub (K119 and K13/K15) 2 −γ-H2AX-Ub1 (K119 or K13/K15) −γ-H2AX siCTRL −γ-H2AX (low exposure)

Chromatin −H2AX

siH1 −MCM6 f U2OS + IR g HMGB1-GFP+ GFP HMGB1-GFP GFP-H1.2 GFP-H1.2 100 GFP

50 53BP1 53BP1 foci (% cells rel. to control) to rel. cells (% foci 53BP1 0 + GFP + + HMGB1-GFP

γ -H2AX + + GFP-H1.2 Thorslund et al, Figure 4

a LRM1 d U2OS + Strep-RNF168 UDM (110-128) − UDM1 UDM2 UMI MIU2 (141-156) (443-459) + + + IR MIU1 LRM2 RING (171-188) (466-478) 98 (15-58) H1x-Ubn HsRNF168 62 49 1 571 UDM1 UDM2 Pull down: Strep −Strep-UDM1 −Strep-UDM2 Binds RNF8-generated Binds RNF168-generated ubiquitylation products ubiquitylation products − H1x

(H1-K63-Ub) (H2A-Ub) WCE −

b Strep-RNF168(UDM1) + Ub2 −MCM6

Ub e M1 K48 K63 K27 K33 K29 K11 K6 2 U2OS/shRNF168 + GFP-RNF168 + IR GFP-RNF168 53BP1 γ-H2AX −Ub2

Strep −UDM1 Pull-down: WT K6 K11 K27 K29 K33 K48 K63 M1 17 P4D1 (S.C) 14 −Ub2 Input c Pull- Pull- down down Δ UDM1 + +

UDM1 UDM1 GFP-RNF168 Input Input + UDM2 + UDM2 −H1.0 −H2A −UDM1 −UDM1 −UDM2 −UDM2 Δ UDM2 f Ubc13 Ubc13 Ubc13 RNF8 RNF8 Ub RNF8 Ub P P P P K63 RNF168 P P K63 RNF168 MDC1 MDC1 Ub E2 MDC1 Ub E2 P H1 P H1 P H1 H2A DSB ∫∫ ∫∫ ∫∫ ∫∫ ∫∫ ∫∫ ∫∫ ∫∫ ∫∫ Ub ∫∫ ∫∫ ∫∫ 53BP1 γ-H2AX Ub Ub Me Ub 53BP1 RAP80 Ub Ub Ub BRCA1- Ub RAD18 A Thorslund et al, Extended Data Figure 1

a HA-RNF8 53BP1 merge d HCT116 Ubc13 KO + IR 53BP1 HA merge − siCTRL WT +IR − IR HA-Ubc13 +IR Ubc13 KO siRNF8 HA-RNF8 Δ R -Ubc13 b RNF168 γ-H2AX DAPI WT +IR Δ R -Ubc13 HA-RNF8(*FHA)

e HCT116 Ubc13 KO + IR 53BP1 HA-RNF168 merge +IR Ubc13 KO

c Rescues WT 53BP1 foci

HA-Ubc13 HA Ubc13 +

− HA-RNF8 HA RNF8 R HA-RNF168 CI HA-RNF8 + ΔR-Ubc13 HA RNF8 Ubc13 HA-RNF8 − ΔR-UbcH7 Myc RNF8 UbcH2 HA-RNF8 ΔR-UbcH2 Myc RNF8 UbcH7 −

HA-RNF168 HA R RNF168 + Thorslund et al, Extended Data Figure 2

a U2OS/GFP-K63-Super-UIM c U2OS/GFP-K63-Super-UIM WT + IR WT MUT GFP-K63- Super-UIM 53BP1 γ-H2AX GFP-K63- Super-UIM siCTRL γ H2AX

+laser microirradiation siUbc13

b U2OS/GFP-K63-Super-UIM WT GFP-K63- γ-H2AX + Super-UIM 53BP1 DAPI siRNF8 siCTRL siRNF168

d U2OS/GFP-NLS-Vps27-UIM GFP-NLS- siUbc13 Vps27-UIM 53BP1 γ-H2AX siRNF8 siCTRL siUbc13 siRNF168 + laser microirradiation f HA- HA- HA- RNF8 RNF8 RNF168 siRNF8 − WT CI WT + + + + IR

HCT116 siRNF168 IB: HA + laser microirradiation e U2OS Pull down: + siUbc13 K63-Super-UIM

WT Ubc13 KO + siRNF8 −53BP1 + si53BP1 IB: HA −Ubc13 −53BP1 WCE

−Actin WCE −Ubc13

IB: H1.2 WCE −RNF8 −Actin Thorslund et al, Extended Data Figure 3 a b Exp. 1 Exp. 2 c Light (L) SILAC Medium (M) SILAC Exp. 1 Exp. 2 (Arg0/Lys0) (Arg6/Lys4)

WT Ubc13 KO

Combine lysates diGly sites showing ≥2-fold diGly sites identified In-solution digestion enrichment in WT cells d Pull-down: di-Gly antibody

LC-MS/MS analysis

e f Exp. 1 Exp. 2 g Light (L) SILAC Heavy (H) SILAC Exp. 1 Exp. 2 (Arg0/Lys0) (Arg8/Lys10)

WT Ubc13 KO

Combine lysates

K63-Super-UIM bound K63-Super-UIM bound Pull-down: K63-Super-UIM proteins identified proteins showing ≥2-fold

h enrichment in WT cells In-solution digestion

LC-MS/MS analysis Thorslund et al, Extended Data Figure 4 a

b GO biological process

ATP hydrolysis coupled protein transport Protein K63-linked ubiquitylation Positive regulation of I-κB kinase/NF-κB signaling Protein K48-linked ubiquitylation Vesicle docking c d SILAC ratio Light (L) SILAC Heavy (H) SILAC Protein (Arg0/Lys0) (Arg10/Lys8) (H/L)

Mock +IR TRAF6 3.41 TRAF2 2.91 HP1BP3 2.62 Combine lysates Histone H1.2 1.85 Histone H1x 1.66 Histone H1.0 1.40 Pull-down: K63-Super-UIM CENPF 1.36 SUMO1 1.36 In-gel digestion Histone H2A 1.10 Histone H3 1.09 LC-MS/MS analysis Histone H2B 0.96 Histone H4 0.88 Thorslund et al, Extended Data Figure 5 a RPE1 d + U2OS + + Serum + + + + + + + U2OS/Strep-Ub + + IR

191− siH1x siCTRL siCTRL siCTRL siRNF8 siRNF168 siUbc13 H1.2-Ub siCTRL 97− n + + + + + + IR

64 −H1x-Strep-Ub4 −H1x-Strep-Ub3 191− 50 −H1x-Strep-Ub2

Strep −H1x-Strep-Ub Ub 1 n Pull-down: 35 97− Pull down: K63-Super-UIM − −H1.2 − H1x −RNF8 −Cyclin A −RNF168 WCE WCE −γ-H2AX −Ubc13 −MCM6 −MCM6 b PD: K63-UIM MUT + + + PD: K63-UIM WT + + + + + + e IR + + + Protein SILAC ratio (H/L) WCE * RNF8 2.29 98 IB: H1x Histone H1 1.60 HERC2 1.50 62 PRPF19 1.42 49 TPR 1.38 RUVBL2 1.28 38 ERCC1 1.24 Pull-down: K63-UIM 28 XPF 1.16 c IB: H1x IB: Ub Pull-down: K63-UIM MUT + + + + + + Pull-down: K63-UIM WT + + + + + + + + + + + + IR + + + + + +

98 H1.2-

Ubn 62 49 38

28

17 Pull-down: K63-Super-UIM IB: Ub WCE IB: H1.2 IB: H2A IB: H2B IB: H3 IB: H4 P3.3 w42 Western Blot_checking H1 KD efciency with diferent H1 antibodies Thorslund et al, Extended Data Figure 6 P3.3 w42 Western Blot_checking(double H1 KD e KDfciency 72h and with 48h dif beforeerent H1 lysis) antibodies a U2OS b U2OS + siCTRL (double KD 72h and 48h before lysis)

siMM si5914 siMM +si5914 siH1(#1,4,5,9) - 39 kD siCTRL siH1(#1) siH1(#2) siH1(#3) siH1(#4) siH1(#5) siH1(#6) siH1(#7) siH1(#8) siH1(#9) siH1(#10)

siMM si5914 IB: pan-H1siMM si5914 anti-H1 −GFP-H1.0

WCE - 39 kD IB:- 39 MCM6 kD anti-H1.0anti-H1 anti-MCM6−GFP-H1.1 - 39 kD 99kDanti-MCM6 - 39 kD anti-H1.0 −GFP-H1.2 f U2OS anti-H1.1 99kD - 39 kD −GFP-H1.3 MDC1- 39DAPI kD +γanti-H1.2-H2AX anti-H1.1 WCE - 39 kD −GFP-H1.4 anti-H1.2

−GFP-H1.5 siCTRL siCTRL c U2OS/GFP-K63-UIM WT + IR GFP-K63-UIM γ-H2AX 53BP1 siH1

siCTRL siCTRL +laser micro-irradiation

g U2OS BRCA1 DAPI+γ-H2AX siH1 d U2OS + laser micro-irradiation

RNF168 γ-H2AX DAPI siCTRL siCTRL siCTRL siH1

+laser micro-irradiation

siH1 h U2OS + + siCTRL e U2OS + IR + siRNF8 + siRNF168 RNF168 γ-H2AX DAPI + + + IR

γ-H2AX-Ub2 (K119 and K13/K15) −γ-H2AX-Ub1 (K119 or K13/K15)

siCTRL siCTRL −γ-H2AX −H2AX Chromatin −MCM6 siH1 Thorslund et al, Extended Data Figure 7

a U2OS + HMGB1-GFP + HA-RNF168 + IR HMGB1-GFP HA-RNF168 53BP1 HA-RNF168 WT HA-RNF168 CI b U2OS + HMGB1-GFP + HA-RNF168 + IR HMGB1-GFP HA-RNF168 γ-H2AX HA-RNF168 WT HA-RNF168 CI c U2OS/GFP-K63-Super-UIM + FLAG-HMGB1 GFP-K63- FLAG- γ-H2AX + Super-UIM HMGB1 DAPI

+laser micro-irradiation Thorslund et al, Extended Data Figure 8 a e Strep-RNF168 UDM Pull-down: Strep

input − UDM1 UDM2 2 Strep- Strep- UDM1 UDM2 + + + IR [KCl] K63 Ub H1.2-Ubn

−K63 Ub2

−Strep-UDM1 −Strep-UDM2 −H1.2 b Pull down: Strep −Strep-UDM1 −Strep-UDM2

−H1.2 WCE input (20%) 2 Strep Strep-UDM2 Ub f Strep-RNF168 −Ub2(K6) UDM1

−Ub2(K11)

−Ub2(K27) − WT *UMI *MIU1

−Ub2(K29) H1.2-Ubn Strep −Ub2(K33) Pull down:

−Ub2(K48) −Strep-UDM1 WCE −Ub2(K63)

−Ub2(M1) c Input Pull-down: Streptavidin (50%) +H2A +H1.0 + + Bio-LRM1 peptide H2A H1.0 MW marker + + Bio-LRM2 peptide

38− −H1.0 28−

17− −H2A 14− −Streptavidin Colloidal Blue d LRM1 UMI MIU1

110 LSKPGELRREYEEEISKVAAERRASEEEENKASEEYIQRLLAEEEEEEKRQAEKRRRAMEEQLKSDEELARKLSIDINN188 LRM1 peptide *UMI (L149A) *MIU1 (A179G) Thorslund et al, Extended Data Figure 9 a Pull-down: Strep-RNF168-UDM1 WCE Strep- + + + + + + + + + + + + UDM1 + + + + + + IR

−Strep- UDM1

−MCM6 Pull down WCE

IB: H1.2 IB: H2A IB: H2B IB: H3 IB: H4 b U2OS + GFP-UDM1 GFP-UDM1 γ-H2AX 53BP1 merge

+laser micro-irradiation c SILAC ratio > 1.5

1 34 (H1x) 172

Increased K63 Pulled down by ubiquitylation GFP-UDM1 after IR Thorslund et al, Extended Data Figure 10

a b U2OS + 1.2 EBC buffer

1

0.8 + + IR

0.6 191−

97− H1.2-Ubn 0.4 Mock IR 64− 0.2 191− H1x-Ub 97− n to pre-bleach) GFP-H1.2 intensity (rel. 0 0 1 2 3 4 5 6 7 Time (min) Bleach 191−

97− Ubn Pull down: K63-Super-UIM 64−

−MCM6 −53BP1

−RNF168

−H1.2

Input −H1x

−γH2AX-Ub1 −γH2AX

−H2AX

−H3 Supplementary Figure – Uncropped scans with size marker (kDa) indicaons

Figure 1a Figure 1f Ubc13 Ubiquin

191 MCM6 97 64 51 38 28 17 14 6 3

Figure 2b Figure 2c/shRNF8 Input/RNF8 H1.2 PD/H1.2 191 191 191 191 97 97 97 97 64 64 64 64 51 51 51 39 51 39 39 28 39 28 28 PD/Ubiquin 28 191 Input/MCM6 51 Input/H1.2 39 28 64 Ubiquin 51 Figure 2c/shRNF168 191 PD/H1.2 191 Input/H1.2 97 39 97 191 28 64 64 Input/MCM6 51 64 51 191 39 51 97 39 28 39 PD/Ubi 19 64 28 14 51 Input/RNF168

1 Figure 2d Figure 2e Figure 3e PD/H1.2 H1.2 y-H2AX 191 97

64

191 97 64 51 PD/Ubiquin y-H2AX Input/MCM6 191 97 64 51 Figure 4b 28 PD/Ubiquin Input/H1.2 Input/Ubiquin 17 39 14 MCM6 28 6 188 Ponceau 98 62 Input/Ubc13 49 38 H2AX 17 28 14 17 14 6 6

Figure 4c Figure 4d H2A Ext Data 2e PD/H1x Acn 188 98 62 53BP1 49 H1.0 49 Ubc13 38 38 28 28 17 17 RNF8 14 14 6 Input/H1x Ext Data 2f PD/HA His6 His6

188 188 98 62 62 Input/His 49 49 38 38 28 28 17 17 14 14 Input/MCM6 Input/HA

Input/H1.2

2 Ext Data 5c Ext Data 5a

39 Ubiquin H1.2/long H1.2/short 191 191 28 97 97 H1.2 51 Cyclin A 64 64 51 51 28 y-H2AX 14 39 39 97 28 14 MCM6 MCM6 14 PD/H1.2 PD/Ubiquin H2A/long Ubiquin H2A/short Ext Data 5b H1x/High Ubi

H2B/long H2B/short Ubiqun

H1x/low

MCM6

MCM6

H3/long Ubiquin H3/short

MCM6

H4/long H4/short Ubiquin

MCM6

3 Ext Data 5d Input/H1x

PD/H1x 250 148 Input/RNF8 98 Input/RNF168 64

50

35 Input/Ubc13 Input/H1x PD/H1x

Input/MCM6

Ext Data 6a

GFP-H1.1/GFP GFP-H1.4/GFP GFP-H1.2/GFP 191 191 191 97 97 97 64 64 51 51 64 51 39 39 28 28 39 19 19 28 19

GFP-H1.5/GFP GFP-H1.0/GFP 191 191 97 64 97 64 51 64 51 39 51 28 39 39 19 28 28 GFP-H1.3/GFP 19 19

4 Ext Data 6b Ext Data 6h Ext Data 8a y-H2AX Ubiquin

39 191

28 His6 97

Pan-H1 64 MCM6

H2AX Ext Data 8e

MCM6

Ext Data 8b PD/H1.2 Ubiquin

Input/H1.2

Input/His6

Ubiquin

Ext Data 8f

H1.2

His6 5 Ext Data 9a

H1.2/long H1.2/short

MCM6

His6

H2A/long H2A/short MCM6

His6

H2B/long

H2B/short MCM6

H1.2/medium

His6

H3/long MCM6 H3/short

His6

H4/long H4/short MCM6

His6

6 Ext Data 10b

PD/H1.2 53BP1

MCM6

yH2AX H1.2

PD/H1x

RNF168

H3

H1x

PD/Ubiquin

H2AX

7