histone h1 couples initiation and amplification of
TRANSCRIPT
u n i ve r s i t y o f co pe n h ag e n
Histone H1 couples initiation and amplification of ubiquitin signalling after DNAdamage
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 versionPeer 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 ofubiquitin signalling after DNA damage. Nature, 527(7578), 389-93. https://doi.org/10.1038/nature15401
Download date: 10. apr.. 2022
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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:
2
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
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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
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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
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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
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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-
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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
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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,
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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.
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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.
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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
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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).
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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.
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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
17
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
18
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
19
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,
20
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
21
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
22
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.
23
Nuclesomes were incubated with 0.5 µM hUba1, 5 µM UbcH5c, 1 µM Ubc13-Mms2
complex, 5 µM RNF8351-485 fragment (purified as described in 8) 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
24
(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
25
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
26
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).
27
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
28
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
29
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.
30
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
31
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.
32
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.
a c d Thorslund et al, Figure 1
b
e
−MCM6 −Ubc13
Ubc
13 K
O
WT
WCE
HCT116
−Ub2
K6
K11
K27
K29
K33
K48
K63
M1
Ub2 input (100%)
K63-UIM MUT
Pull-down: K63-Super-UIM
K63-UIM WT + + + +
+ + + + + +
+ + + + + + K6 K11 K27 K29 K33 K48 K63 M1 Ub2
14–
28–
38–
17–
γ-H2AX 53BP1 merge
WT
+IR
−IR
Ubc
13 K
O
+IR
HA/Myc 53BP1 merge Ubc13 KO + IR
HA-
RN
F8ΔR
- U
bc13
H
A-
Ubc
13
Myc
- RN
F8ΔR
- U
bcH
2 H
A-
RNF8
Myc
- RN
F8ΔR
- U
bcH
7
WT
MDC1 Ubiquitin (FK2)
Ubc
13 K
O
f
UIM1 UIM2 UIM3 Bioease/GFP K63-Super-UIM
UIM1 UIM2 UIM3 MUT
* * * * * * WT
Bioease/GFP 0
20
40
60
80
100 K63-Super-UIM53BP1
DN
A d
amag
e st
ripes
(%
of c
ells
)
Ubc13 CTRL RNF8 RNF168 siRNA:
g
−IR
+laser microirradiation
Thorslund et al, Figure 2 d
b
+ + + + + + + +
−H1.2
*
39–
64–
97–
191–
51–
191–
H1.2-Ubn
Ubn
U2OS
IR
USP2cc
WT WT MUT MUT K63-UIM
Pull
dow
n: K
63-S
uper
-UIM
c
IR DOX
+ + + + + + + +
−H1.2
−RNF8
−MCM6
−RNF168
Pull
dow
n: K
63-S
uper
-UIM
W
CE
U2OS/shRNF8 U2OS/shRNF168
97−
191−
64−
97−
191−
H1.2-Ubn
Ubn
IR +
+ +
−H1.2
−MCM6
−Ubc13
Pull
dow
n: K
63-S
uper
-UIM
W
CE
siUbc13
97−
191−
64−
97−
191−
64−
H1.2-Ubn
Ubn
siCTRL + +
+ +
U2OS
H1.2-Ubn
−H1.2
Time (min) 60 120 90 30 RNF8351-485 + + +
In vitro ubiquitylation (Nucleosomes+E1+UbcH5c
+Ubc13-Mms2)
e a
0.0
0.5
1.0
1.5
2.0
K48 K63 K11 K6
Ubiquitin linkage type
SIL
AC
ratio (
Ubc
13 K
O/W
T)
Experiment 1
Experiment 2
N.S.
**
**
f
Thorslund et al, Figure 3
0
20
40
60
80
100siCTRLsiH1
DN
A d
amag
e st
ripe
inte
nsity
(r
elat
ive
to s
iCTR
L)
0
20
40
60
80
100
GFP
-K63
-UIM
stri
pe(%
cel
ls re
lativ
e to
siC
TRL)
c γ-H2AX+
DAPI
siC
TRL
siH
1
53BP1 GFP-K63-Super-UIM
U2OS/GFP-K63-Super-UIM
+laser microirradiation
d
GFP
53BP
1 G
FP
U2OS + IR
HMGB1-GFP GFP-H1.2 HMGB1-GFP+
GFP-H1.2
γ-H
2AX
b C
hrom
atin
−MCM6
−γ-H2AX (low exposure)
siH1 IR + +
U2OS
−γ-H2AX −γ-H2AX-Ub1 (K119 or K13/K15) γ-H2AX-Ub2 (K119 and K13/K15)
siCTRL +
+ + +
−H2AX
siH
1 si
CTR
L
GFP-K63-Super-UIM
U2OS/GFP-K63-Super-UIM WT + IR
γ-H2AX 53BP1
a
0
50
100
53B
P1
foci
(% c
ells
rel.
to c
ontro
l)
GFP HMGB1-GFP GFP-H1.2
g
e
+ + +
+ +
a
Thorslund et al, Figure 4
c
1714
P4D1 (S.C)
K6 K11 K27 K29 K33 K48 K63 M1
Pull-
dow
n:
Stre
p In
put
−Ub2
−UDM1
−Ub2
Ub2 K6
K11
K27
K29
K33
K48
K63
M1
Strep-RNF168(UDM1) + Ub2
MDC1
RNF8 P P
Ubc13
∫∫∫∫H1
DSB
P
γ-H2AX
MDC1
RNF8 P P
Ubc13
P MDC1
RNF8 P P
Ubc13
P
53BP1 53BP1
UbMe
H2A ∫∫ ∫∫ ∫∫∫∫ ∫∫∫∫ ∫∫H1 ∫∫∫∫ ∫∫
H1
UbUb
Ub
RAD18 Ub
Ub
RAP80 BRCA1-
A
UbUb
RNF168 Ub
UbK63 RNF168
E2
Ub
UbK63
E2
1
HsRNF168
MIU2 (443-459)
571
RING (15-58)
MIU1 (171-188)
UMI (141-156)
LRM1 (110-128)
Binds RNF8-generated ubiquitylation products
(H1-K63-Ub)
Binds RNF168-generated ubiquitylation products
(H2A-Ub)
LRM2 (466-478)
UDM1 UDM2
Inpu
t
−UDM1
−H1.0
−UDM2 −UDM1
−H2A
−UDM2
UDM1 UDM2
Pull-down
+ +
Inpu
t UDM1 UDM2
Pull-down
+ +
b
e
d
IR + + +
−Strep-UDM1
H1x-Ubn
Pull
dow
n: S
trep
UDM2 UDM1 − U2OS + Strep-RNF168 UDM
−MCM6
WC
E
−Strep-UDM2
− H1x −
98
62
49
WT
ΔUDM
2 ΔU
DM1
γ-H2AX GFP-RNF168 53BP1 U2OS/shRNF168 + GFP-RNF168 + IR
GFP
-RN
F168
f
Thorslund et al, Extended Data Figure 1 +I
R W
T
HA-RNF8 53BP1 merge +I
R −I
R a
c
Ubc
13 K
O
d
HA-
RNF8
(*FH
A)
ΔR-U
bc13
HCT116 Ubc13 KO + IR
HA 53BP1 merge
siC
TRL
siRN
F8
HA-
RNF8
ΔR
-Ubc
13
HA-
Ubc
13
− RNF168 γ-H2AX DAPI
+IR
WT
+IR
Ubc
13 K
O
b
HA-RNF8 ΔR-Ubc13
HA-RNF168 R RNF168
Ubc13 HA
Ubc13 RNF8 HA
R RNF8 HA
UbcH7 RNF8 Myc HA-RNF8 ΔR-UbcH2
HA-RNF8
HA-Ubc13
UbcH2 RNF8 Myc HA-RNF8 ΔR-UbcH7
+
Rescues 53BP1 foci
+
+
−
−
−
HA
e
CI
53BP1 merge HA-RNF168
WT
HA-
RNF1
68
HCT116 Ubc13 KO + IR
Thorslund et al, Extended Data Figure 2 a
b
c WT
U2OS/GFP-K63-Super-UIM MUT
GFP
-K63
-Su
per-U
IM
+laser microirradiation
γH2A
X
53BP1 GFP-K63-Super-UIM
U2OS/GFP-K63-Super-UIM WT
siC
TRL
siU
bc13
si
RNF1
68
siRN
F8
γ-H2AX + DAPI
+ laser microirradiation
53BP1 GFP-NLS-Vps27-UIM
U2OS/GFP-NLS-Vps27-UIM
siC
TRL
siU
bc13
si
RNF1
68
siRN
F8
γ-H2AX
+ laser microirradiation
d
e
WC
E
−RNF8
−Ubc13
si53BP1
U2OS
−53BP1
siUbc13 siRNF8
+ +
+
−Actin
siC
TRL
siRN
F8
siRN
F168
si
Ubc
13
γ-H2AX GFP-K63-Super-UIM 53BP1
U2OS/GFP-K63-Super-UIM WT + IR
WC
E
−Ubc13 −53BP1
−Actin
Ubc
13 K
O
WT
HCT116
IB: HA
IB: H1.2
IB: HA
Pull
dow
n:
K63-
Supe
r-UIM
W
CE
IR + + + +
HA- RNF8
CI
HA- RNF8 WT −
HA- RNF168
WT
f
Thorslund et al, Extended Data Figure 3
diGly sites identified
b c
diGly sites showing ≥2-fold enrichment in WT cells
d
f g
K63-Super-UIM bound proteins identified
K63-Super-UIM bound proteins showing ≥2-fold enrichment in WT cells
Light (L) SILAC (Arg0/Lys0)
Combine lysates
Pull-down: di-Gly antibody
In-solution digestion
Medium (M) SILAC (Arg6/Lys4)
LC-MS/MS analysis
Ubc13 KO WT
Light (L) SILAC (Arg0/Lys0)
Combine lysates
In-solution digestion
Pull-down: K63-Super-UIM
Heavy (H) SILAC (Arg8/Lys10)
LC-MS/MS analysis
Ubc13 KO WT
a
e
h
Exp. 1 Exp. 2 Exp. 1 Exp. 2
Exp. 1 Exp. 2 Exp. 1 Exp. 2
Thorslund et al, Extended Data Figure 4 a
b
Positive regulation of I-κB kinase/NF-κB signaling
Protein K63-linked ubiquitylation
Protein K48-linked ubiquitylation Vesicle docking
GO biological process ATP hydrolysis coupled protein transport
Pull-down: K63-Super-UIM
Light (L) SILAC (Arg0/Lys0)
Combine lysates
In-gel digestion
LC-MS/MS analysis
+IR Mock
Heavy (H) SILAC (Arg10/Lys8) Protein SILAC ratio
(H/L) TRAF6 3.41 TRAF2 2.91 HP1BP3 2.62 Histone H1.2 1.85 Histone H1x 1.66 Histone H1.0 1.40 CENPF 1.36 SUMO1 1.36 Histone H2A 1.10 Histone H3 1.09 Histone H2B 0.96 Histone H4 0.88
c d
Thorslund et al, Extended Data Figure 5 a
c
e
SILAC ratio (H/L)
RNF8 * 2.29 Histone H1 1.60 HERC2 1.50 PRPF19 1.42 TPR 1.38 RUVBL2 1.28 ERCC1 1.24 XPF 1.16
Protein
d
−Ubc13
−H1x-Strep-Ub1
−RNF168 −RNF8
−MCM6
H1x − −
−H1x-Strep-Ub2 −H1x-Strep-Ub3
64
50
35
WC
E
Pull-
dow
n:
Stre
p
siC
TRL
IR + + + + + +
siC
TRL
siH
1x
siC
TRL
siC
TRL
siRN
F8
siRN
F168
si
Ubc
13
U2OS/Strep-Ub U2OS
+ + + + +
+ + +
−H1x-Strep-Ub4
IR Serum
+ + + +
−H1.2
−γ-H2AX −MCM6
−Cyclin A
Pull
dow
n: K
63-S
uper
-UIM
WC
E RPE1
97−
191−
97−
191−
H1.2-Ubn
Ubn
IB: H1.2 IB: H2A IB: H2B IB: H3 IB: H4
Pull-down: K63-UIM MUT
IB: Ub
+ +
98
62 49
38
28
17 Pull-
dow
n: K
63-S
uper
-UIM
W
CE
+ +
IR Pull-down: K63-UIM WT
+ + +
+
+ + +
+
+ + +
+
+ + +
+
+ + +
+
H1.2-Ubn
+
+ + +
+
+ + +
98
62
49
38
28
IB: H1x IB: Ub
WC
E
PD: K63-UIM MUT
IR PD: K63-UIM WT
Pull-
dow
n: K
63-U
IM
b
IB: H1x
+
+ + +
Thorslund et al, Extended Data Figure 6 a b
99kD
anti-MCM6
siMM
si5914
siMM
si5914
anti-H1
anti-H1.0
anti-H1.1
anti-H1.2
- 39 kD
- 39 kD
- 39 kD
- 39 kD
P3.3 w42 Western Blot_checking H1 KD efficiency with different H1 antibodies
(double KD 72h and 48h before lysis)
IB: pan-H1
IB: MCM6 WC
E
siCTRL siH1(#1,4,5,9) +
+ U2OS
99kD
anti-MCM6
siMM
si5914
siMM
si5914
anti-H1
anti-H1.0
anti-H1.1
anti-H1.2
- 39 kD
- 39 kD
- 39 kD
- 39 kD
P3.3 w42 Western Blot_checking H1 KD efficiency with different H1 antibodies
(double KD 72h and 48h before lysis)
c
siC
TRL
MDC1 DAPI+γ-H2AX U2OS
siH
1
+laser micro-irradiation
f
d
g si
CTR
L BRCA1 DAPI+γ-H2AX
U2OS si
H1
+laser micro-irradiation
siC
TRL
GFP-K63-UIM U2OS/GFP-K63-UIM WT + IR
γ-H2AX 53BP1
siH
1 si
CTR
L
RNF168 γ-H2AX U2OS + laser micro-irradiation
siH
1
DAPI
WC
E
−GFP-H1.0 −GFP-H1.1 −GFP-H1.2
−GFP-H1.3
−GFP-H1.4
−GFP-H1.5
siC
TRL
siH
1(#1
) si
H1(
#2)
siH
1(#3
) si
H1(
#4)
siH
1(#5
)
siH
1(#6
) si
H1(
#7)
siH
1(#8
) si
H1(
#9)
siH
1(#1
0) U2OS
e
siC
TRL
RNF168 γ-H2AX U2OS + IR
siH
1
DAPI
h
Chr
omat
in
−MCM6
−H2AX
siRNF168 IR + + +
U2OS
−γ-H2AX −γ-H2AX-Ub1 (K119 or K13/K15) γ-H2AX-Ub2 (K119 and K13/K15)
siCTRL siRNF8
+ +
+ +
Thorslund et al, Extended Data Figure 7
a
b
HA-RNF168 53BP1 HMGB1-GFP
HA-
RNF1
68 W
T H
A-RN
F168
WT
HA-RNF168 γ-H2AX HMGB1-GFP
HA-
RNF1
68 C
I H
A-RN
F168
CI
U2OS + HMGB1-GFP + HA-RNF168 + IR
U2OS + HMGB1-GFP + HA-RNF168 + IR
c
FLAG-HMGB1
γ-H2AX + DAPI
GFP-K63-Super-UIM
U2OS/GFP-K63-Super-UIM + FLAG-HMGB1
+laser micro-irradiation
Thorslund et al, Extended Data Figure 8
a
b
−Strep-UDM1
H1.2-Ubn
Pull
dow
n:
Stre
p W
CE
Strep-RNF168 UDM1
− WT
*UM
I
*MIU
1
f
d
−K63 Ub2
K63
Ub 2
inpu
t
Strep- UDM1
[KCl]
Strep- UDM2
Pull-down: Strep
−Strep-UDM1 −Strep-UDM2
LSKPGELRREYEEEISKVAAERRASEEEENKASEEYIQRLLAEEEEEEKRQAEKRRRAMEEQLKSDEELARKLSIDINN LRM1 UMI MIU1
IR + + +
−Strep-UDM1
−H1.2
H1.2-Ubn
Pull
dow
n: S
trep
UDM2 UDM1 −
Strep-RNF168 UDM
−H1.2
WC
E
−Strep-UDM2
Ub 2
inpu
t (20
%)
Stre
p
Stre
p-U
DM2
−Ub2(K6)
−Ub2(K11)
−Ub2(K27)
−Ub2(K29) −Ub2(K33)
−Ub2(K48) −Ub2(K63)
−Ub2(M1)
c
Bio-LRM2 peptide Bio-LRM1 peptide
+ +
14− 17−
28−
38−
+
Pull-down: Streptavidin Input (50%)
MW
mar
ker
−H1.0
−H2A −Streptavidin
H1.
0
H2A
+
+H2A +H1.0
e
Colloidal Blue
110
LRM1 peptide *UMI (L149A)
188
*MIU1 (A179G)
Thorslund et al, Extended Data Figure 9
IB: H1.2 IB: H2A
−Strep-
+ + +
+ + +
IR
Strep- UDM1
Pull
dow
n W
CE
IB: H2B IB: H3 IB: H4
−MCM6
+ + +
+ + +
+ + +
+ + +
UDM1
WCE Pull-down: Strep-RNF168-UDM1
b GFP-UDM1 γ-H2AX 53BP1 merge
Increased K63 ubiquitylation
after IR
Pulled down by GFP-UDM1
172 34 1
(H1x)
SILAC ratio > 1.5 c
U2OS + GFP-UDM1
+laser micro-irradiation
a
Thorslund et al, Extended Data Figure 10
0
0.2
0.4
0.6
0.8
1
1.2
0 1 2 3 4 5 6 7
Time (min) Bleach
GFP
-H1.
2 in
tens
ity (r
el. t
o pr
e-bl
each
)
IR + +
−H1.2
−MCM6
Pull
dow
n: K
63-S
uper
-UIM
Inpu
t
97−
191−
64−
H1.2-Ubn
Ubn
97−
191− H1x-Ubn
97−
191−
64−
−γH2AX −γH2AX-Ub1
−53BP1
−H1x
−RNF168
−H2AX
a b
Mock IR
−H3
U2OS + EBC buffer
Supplementary Figure – Uncropped scans with size marker (kDa) indica=ons
Figure 2b
Ubiqui=n
H1.2
191
191
97
64
97
64
51
39
51
28
39
28
1
Figure 1a Ubc13
MCM6
Figure 1f
17 14
6
3
28 38 51 64
97 191
Ubiqui=n
Figure 2c/shRNF8 PD/H1.2
PD/Ubiqui=n
Input/RNF8
Input/MCM6 Input/H1.2
191 191 191
191
97 97 97
64 64 64
64
51 51 51
51
39 39
39
39
51
28 28 28
28
Figure 2c/shRNF168
191
191
191
PD/H1.2
PD/Ubi
Input/MCM6
Input/H1.2
Input/RNF168
97
97
64
64
64 51
51
39 28
28 39
51 39
19 14
Figure 2d
191
191
191
97
97
97
64
64
64 51
51
39 28
17 14
6
PD/H1.2
PD/Ubiqui=n
Input/MCM6
Input/H1.2
Input/Ubc13 H2AX
MCM6
Figure 3e y-‐H2AX
y-‐H2AX
Figure 4b Input/Ubiqui=n PD/Ubiqui=n
Ponceau 188 98 62 49 38 28
28 17
17 14
14 6
6
Figure 2e H1.2
Figure 4c
H1.0
H2A
His6 His6
188
188 188 98
98 62
62 62 49 49
49 49 38 38
38 38 28 28
28 28 17 17
17 17 14 14
14 14
6
Figure 4d PD/H1x
Input/H1x
Input/His
Input/MCM6
Ext Data 2e Ac=n
53BP1 Ubc13
RNF8
Ext Data 2f PD/HA
Input/HA
Input/H1.2
2
Ext Data 5a
PD/H1.2 PD/Ubiqui=n
H1.2
Cyclin A
y-‐H2AX
MCM6
191 191 97 97
97
64 64
51 51
51
39 39
39
28
28
28
14
14 14
Ext Data 5b H1x/High
H1x/low
Ubi
Ext Data 5c
H1.2/long H1.2/short
MCM6
Ubiqui=n
H2A/long H2A/short Ubiqui=n
Ubiqu=n H2B/short H2B/long
MCM6
MCM6
H3/long
H3/short
Ubiqui=n
MCM6
H4/long H4/short
MCM6
Ubiqui=n
3
Ext Data 5d
Input/H1x
PD/H1x
Input/H1x
Input/RNF8
Input/RNF168
Input/Ubc13
Input/MCM6
250 148
98
64
50
35
PD/H1x
Ext Data 6a
191
191
191
191
191
97 97 97
97 97 64
64
64 64 64
64
51 51 51
51
51 51
39
39 39 39
39
39
28
28 28 28
28
28
19
19 19 19
19
19
GFP-‐H1.1/GFP
GFP-‐H1.0/GFP
GFP-‐H1.4/GFP
GFP-‐H1.5/GFP
GFP-‐H1.2/GFP
GFP-‐H1.3/GFP
4
Ext Data 6b
39
28
Pan-‐H1 MCM6
97
64
191
Ext Data 6h y-‐H2AX
H2AX
MCM6
Ext Data 8a Ubiqui=n
His6
Ext Data 8b
Ubiqui=n
Ubiqui=n
Ext Data 8e
PD/H1.2
Input/H1.2
Input/His6
Ext Data 8f
H1.2
His6
5
Ext Data 9a H1.2/long
H1.2/short
H2A/short H2A/long
H2B/long
H2B/short
H3/short
H3/long
H4/long H4/short
MCM6
MCM6
MCM6
H1.2/medium
MCM6
MCM6
His6
His6
His6
His6
His6
6
Ext Data 10b
PD/H1.2
PD/H1x
PD/Ubiqui=n
53BP1
yH2AX
H3
H1x
H2AX
RNF168
H1.2
MCM6
7