1 umi, a novel rnf168 ubiquitin binding domain involved in the

1

UMI, a novel RNF168 ubiquitin binding domain involved in the DNA damage signaling 1

pathway 2

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Sabrina Pinato1, Marco Gatti

1, Cristina Scandiuzzi

1, Stefano Confalonieri

2 & Lorenza 5

Penengo1*

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1Department of DISCAFF and DFB Center, University of Piemonte Orientale ‘‘A. 8

Avogadro’’, 28100 Novara, Italy. 2IFOM, the FIRC Institute for Molecular Oncology 9

Foundation, 20139 Milan, Italy. 10

11

*Corresponding author. Mailing Address: Department of DISCAFF and DFB Center, 12

University of Piemonte Orientale ‘‘A. Avogadro’’, 28100 Novara, Italy. 13

Phone.: +39 321 375814, Fax.: +39 321 375821, E-mail: [email protected]

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Character count of Materials and Methods: 5001 18

Character count of Introduction, Results and Discussion: 19999 19

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Running title: A novel UBD involved in DNA damage response 22

Copyright © 2010, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.Mol. Cell. Biol. doi:10.1128/MCB.00818-10 MCB Accepts, published online ahead of print on 1 November 2010

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ABSTRACT 1

2

Ubiquitination regulates important cellular processes, including the DNA damage response 3

(DDR) and DNA repair. The complexity of the ubiquitin-mediated signals is decoded by 4

ubiquitin receptors, which contain protein modules named ubiquitin binding domains 5

(UBDs). We previously identified a new ubiquitin ligase, RNF168, involved in DDR and 6

endowed with two UBDs named MIU (motif interacting with ubiquitin). Here we provide the 7

identification of a novel UBD, the UMI (UIM- and MIU-related UBD), present in RNF168, 8

and characterize the interaction surface with ubiquitin, centered on two Leu residues. We 9

demonstrate that integrity of the UMI, in addition to the MIUs, is necessary for the proper 10

localization of RNF168 and for ubiquitination of nuclear proteins, including histone H2A. 11

Finally, we show that simultaneous inactivation of UMI and MIUs prevents the recruitment to 12

DDR foci of the crucial downstream mediator 53BP1. 13

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Subject categories: Genome Stability & Dynamics; Chromatin and Transcription 16

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Keywords: ubiquitination, ubiquitin binding domains, DNA damage response, chromatin 18

remodeling 19


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INTRODUCTION 1

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The post-translational modifications are one of the most versatile and effective intracellular 3

signaling system adopted by cells to rapidly counteract environmental changes. Among them, 4

the ubiquitination has a pivotal role as a master regulator of the most important cellular 5

functions (4). 6

Ubiquitination is a multi-step process involving a ubiquitin (Ub) activating enzyme (E1) that 7

activate the C-terminus of free Ub, which in turn is passed to an E2 conjugating enzyme and 8

finally, with the help of E3 Ub ligase, targets a lysine residue (Lys) of the substrate (21). 9

Notably, since Ub comprises seven Lys (K) residues within its sequence, Ub itself can be a 10

substrate of ubiquitination, resulting in the formation of different types of polyUb chains 11

endowed with distinct signaling significance (28, 30). Indeed, while K48 polyubiquitination is 12

involved in targeting proteins to proteasomal degradation, K63 chains are known to regulate a 13

variety of cellular processes including NF-κB signaling (3), DNA repair (2, 17), endocytosis 14

and vesicle trafficking (1). The function of other types of Ub linkages have now being clarified 15

(15). 16

The reversible covalent attachment of the bulky Ub moiety physically remodels the target 17

protein, conferring new interaction interfaces that can be recognized by protein modules known 18

as Ub binding domains (UBDs; (12). UBDs have the crucial role to decode Ub-mediated 19

network, allowing ubiquitinated proteins and Ub receptors (i.e. proteins carrying UBD) to 20

communicate and to translate the Ub signals into specific cellular functions. Lately, the key role 21

of non-proteolytic ubiquitination and UBDs system is emerging in processes relevant to the 22

surveillance and maintenance of genome integrity (10). Indeed, upon the formation of DNA 23

lesions, a number of proteins were found to be ubiquitinated, including proliferating cell 24

nuclear antigen (PCNA), proteins belonging to the Fanconi pathway as well as histones H2A 25


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and H2AX (2, 17, 19). Of particular interest, ubiquitination is assuming a prominent role in the 1

signaling pathway triggered by formation of DNA double strand breaks (DSBs), known as 2

DNA damage response (DDR). It has been demonstrated that, upon formation of DSBs, two Ub 3

ligases, namely RNF8 and RNF168, ubiquitinate histones H2A and H2AX at the site of damage 4

(6, 11, 14, 16, 22, 26, 27). RNF8 and RNF168 act epistatically in the DNA damage signaling 5

cascade, allowing relaxation of the chromatin structure and recruitment of important 6

downstream effectors, such as 53BP1 and BRCA1. Noteworthy, the localization of RNF8 to 7

DDR foci is due to upstream phosphorylation events induced by the PIKKs (PI3K-related 8

kinases) like ATM, while subsequent recruitment of RNF168 follows the first ubiquitination 9

events induced by RNF8 (6, 26). Two UBDs named MIU1 and MIU2 are important for 10

RNF168 localization and hence for its activity (6, 20, 22, 26). However, the inactivation of 11

these domains dramatically reduced RNF168 recruitment without abolishing it, thus suggesting 12

the involvement of additional mechanisms. 13

UBDs are structurally heterogeneous motifs, and can recognize different types of Ub chains (5, 14

12). So far, six classes of UBDs have been involved in, although not restricted to, the regulation 15

of DDR: UIM (Ub interacting motif), MIU (motif interacting with Ub), UBA (Ub associated) 16

and UBM (Ub binding motif) present an α-helical structure, UEV (Ub conjugating enzyme 17

variant) displays the Ub conjugating (UBC) domain while UBZ (Ub binding zinc finger) is a 18

zinc finger (10). Here we have identified and characterized in RNF168 a novel UBD that shows 19

similarities with the previously described UIM (23) and MIU (20) domains, and thereby we 20

called it UMI (UIM- and MIU-related UBD). We found that isolated UMI domain binds 21

polyUb chains, with a preference for the K63 linkage, and we determined the amino acid 22

residues within the UMI sequence crucial for Ub binding. We found that simultaneous 23

inactivation of UMI, MIU1 and MIU2 almost completely abolishes RNF168-dependent 24

formation of K63-specific polyubiquitinated proteins within the nucleus and the ubiquitination 25


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of histone H2A. Functionally, we demonstrated that this impairment has dramatic 1

consequences in the signaling cascade activated by DSBs, resulting in the inadequate 2

recruitment of 53BP1 at the DDR foci, which is a critical step for the proper activation of the 3

downstream effectors of DDR. 4

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6

RESULTS 7

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Identification of a novel Ub binding region in RNF168 9

We previously found that RNF168 is endowed with two UBDs named MIU (motif interacting 10

with Ub) and we demonstrated that isolated MIU1 (amino acids 168-191) and MIU2 (amino 11

acids 439-462) are able to interact with both K48 and K63 Ub chains (20). The integrity of the 12

MIU domains is required for the binding of full-length RNF168 to K48 Ub chains, for its 13

appropriate localization to the site of genomic lesions and for the correct assembly of the DDR 14

foci(6, 22, 26). Hence, due to the importance of the MIU domains in RNF168 function, we 15

further investigated its Ub binding ability. By in vitro pull-down experiments, we found that the 16

MIU1 plays a prominent role in RNF168 binding to K48 Ub chains. In fact, MIU1 inactivation 17

by point mutation (A179G, MIU1*) strongly affected K48 Ub chain binding, while inactivation 18

of MIU2 (A450G, MIU2*) resulted only in a slight reduction (Fig. 1A, left panel). This result is 19

in accordance with our previous finding revealing that MIU1 is more efficient in binding K48 20

Ub chains compared to MIU2 (20). Consistently, the double mutant affecting integrity of the 21

MIU domains (A179G, A450G, MIU1-2**) almost completely abolished Ub binding. 22

K48 ubiquitination is generally considered a signal for proteasomal degradation, while other 23

types of polyUb chains target proteins to different fates. In particular, since K63 ubiquitination 24

is a signaling device largely used in DNA damage response and repair (9, 13, 25), we asked 25


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whether RNF168 shows the same specificity for binding to K48- and K63-linked Ub chains. 1

Surprisingly, we found that the mutant MIU1-2** still interacts with K63 Ub chains, unveiling 2

the existence of additional Ub binding region within the protein, which shows preferential 3

binding to the K63 linkage compared to the K48 (Fig. 1A, right panel). 4

Therefore, to map the region involved in binding to K63 polyUb chains, we used a panel of 5

deletion mutants encompassing the whole protein, in combination with point mutants 6

addressing MIU1 and MIU2, summarized in Fig. 1B. We found that the sequence within amino 7

acids 56 and 166, which does not contain any obvious UBD, interacts with K63 polyUb chains. 8

Secondary structure prediction of this sequence highlighted the presence of a coiled-coil motif 9

(amino acids 115-166). Within this sequence, a putative amphipathic α-helix is present (amino 10

acids 134-166), highly conserved throughout evolution (Fig. 1C). Thus, we asked whether this 11

short sequence was responsible for the binding to Ub chains. Indeed, we found that the region 12

including amino acids 134-166 retained Ub binding ability, while the adjacent region (amino 13

acids 56-134) did not (Fig. 1D). Interestingly we observed that this isolated region binds also to 14

K48 polyUb chains, although to a lesser extent (Fig. S1), suggesting that Ub-chain specificity 15

might be influenced by the surrounding sequence. 16

Our bioinformatic analysis suggested an amino acid spatial distribution that is reminiscent of 17

the amphipathic α-helix in the UIM/MIU motifs (Fig. 2A), with Y145, I146 L149 and L150 18

providing the core of the hydrophobic side, while E143, E144, Q147 and R148 make up the 19

hydrophilic side (Fig. 2A). Consistently, a single substitution of each of the two leucine 20

residues at position 149 and 150, almost abrogated the Ub binding of the domain (Fig. 2B). In 21

contrast, mutation of alanine 151 (marked with an asterisk in Fig. 2A), crucial for the function 22

of the MIU domain (20), did not exert a major effect on the interaction with Ub chains, as 23

predicted by its positioning in the hydrophilic side of the helix in our model (Fig. 2B). Due to 24

the similarity with the UIM and MIU domains, we named it UMI (UIM- and MIU-related Ub 25


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binding domain). 1

To better evaluate the Ub-linkage specificity, we analyzed the effect of the point mutations 2

addressing UMI and MIU domains on the binding to K63 and K48 polyUb chains. Since we 3

observed that the N-terminus of the protein displayed a residual Ub binding that is currently 4

under investigation, we introduced the point mutations in a sequence deleted of this region 5

(amino acids 56-571). As expected, the mutant that is simultaneously defective of UMI, MIU1 6

and MIU2 (UMI*MIU1-2**) is unable to interact with K48 and poorly with K63 Ub chains, 7

indicating that there are no additional UBDs in this region (Fig. 2C). Oddly, we found that the 8

mutant 56-571(MIU1-2**) still interacts with K48 Ub chains, in contrast to what observed in 9

the case of full-length protein (Fig. 1A). This result, in agreement with other data obtained in 10

the lab, suggests that the region encompassing the RING finger of RNF168 might influence the 11

Ub binding specificity, by limiting the type of Ub chain that can access the UMI domain. 12

13

The proper localization of RNF168 relies on the integrity of the UMI domain 14

Localization of DDR proteins at the site of DNA damage is mandatory for the full activation of 15

the cellular response aimed at counteracting the formation of harmful DNA DSBs (7). Proper 16

subcellular localization of RNF168 largely depends on the integrity of the MIU domains, with a 17

pivotal role of MIU2 (22, 26). In fact, inactivation of MIU2 by point mutation (A450G) highly 18

impairs the accumulation of RNF168 at the DSBs upon etoposide treatment. Nevertheless, we 19

previously showed that such impairment is not complete, since a small population of MIU1-2** 20

still localizes at DDR foci (22), suggesting a possible role of other domains in RNF168 21

localization. To investigate the role of the UMI domain in RNF168 localization, we adopted 22

experimental conditions where the expression of endogenous protein is abrogated, by using 23

U2OS cells conditionally expressing RNF168-targeting shRNA (6) (see in Fig. S2A in the 24

supplemental material); here, we introduced the shRNA-resistant version of the different 25


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RNF168 constructs (see in Fig. S2B in the supplemental material). We observed that 1

inactivation of the UMI by Leu to Ala substitution at position 149 and 150 (UMI*) partially 2

altered the localization of RNF168, resulting in a more diffuse localization compared to the 3

wild type protein (Fig. 3A). A more dramatic effect is obtained by replacing the endogenous 4

protein with the construct carrying simultaneously the UMI, MIU1 and MIU2 inactivating 5

mutations (UMI*MIU1-2**). In this case, the protein displayed a diffuse nuclear pattern and it 6

failed to localize at the DSB foci even in the presence of DNA damage (Fig. 3A). We 7

previously showed that a significant population of RNF168 still localized to DDR foci even 8

when MIUs are inactivated (22), suggesting the existence of a MIU-independent mechanism 9

for RNF168 recruitment to DDR foci. Thus, we adopted the same approach to clarify if the 10

simultaneous inactivation of the three UBDs further affects localization of the protein at the 11

sites of DNA lesion. Indeed, we found that the triple mutant (UMI*MIU1-2**) is unable to re-12

locate to the DDR foci, although it still resides in detergent-resistant chromatin structures (Fig. 13

3B, right panels). This result is quite surprisingly, since we expected the mutant UMI*MIU1-14

2** to be soluble upon detergent treatment. To investigate this point, we performed a GST pull 15

down using chromatin extracts to evaluate the ability of the mutants to interact with chromatin 16

(Fig. 3C). Consistently with the IF data, we found that all of RNF168 mutants, including the 17

UMI*MIU1-2**, retain the ability to bind histones at comparable levels, either in damaged or 18

undamaged cells. These results indicate that, in addition to the UBD-dependent binding of 19

RNF168 to ubiquitinated histone H2A upon DNA lesions (26), there are additional binding 20

sites on RNF168 to chromatin. 21

22

RNF168-induced ubiquitination is impaired in cells expressing the UMI-defective mutant 23

We and others previously demonstrated that RNF168 is a nuclear E3 Ub ligase, which induces 24

in vitro ubiquitination of histones H2A and H2AX in a RING finger-dependent manner (6, 22, 25


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26). In addition, it has also been ascribed to the two MIU domains an important role in the in 1

vivo ubiquitination activity. Indeed, inactivation of MIU1 and MIU2 caused a 70% reduction of 2

the amount of ubiquitinated proteins at the DDR foci as compared to the wild type protein (6). 3

Thus we asked whether inactivation of the UMI domain could also impair the activity of 4

RNF168, as a consequence of the altered localization. By using the U2OS RNF168-targeting 5

shRNA cells complemented with the shRNA-resistant constructs of either the wild type 6

RNF168 or the different mutants, we found that the sole inactivation of the UMI domain 7

reduces the number of Ub-positive foci in the nucleus (UMI*), as revealed by the use of anti-8

Ub antibody (FK2) that recognized Ub only when conjugated in chains (Fig. 4A, 4B). This 9

resembles what observed with the mutant in the MIU domains (MIU1-2**). Noteworthy, 10

inactivation of the UMI domain in the context of a MIU-defective protein completely abolished 11

the formation of Ub conjugates at the DSBs (UMI*MIU1-2**; Fig. 4A, 4B). The same results 12

were recapitulated by the use of the K63-linkage specific anti-Ub antibody (Apu3.A8(18)), as 13

shown in Fig. S3 in the supplemental material. Furthermore, we addressed the ubiquitination 14

status of chromatin-bound proteins, by performing acidic extraction of nuclear components 15

derived from cells ectopically expressing wild type and different RNF168 mutants (Fig. 4C). In 16

the presence of wild type protein, anti-Ub immunoblot revealed the formation of a ladder of 17

about five bands, which differ in molecular weight, reminiscent of the multiple ubiquitinated 18

forms of histone H2A. Notably, inactivation of the three UBDs (UMI*MIU1-2**) prevented 19

the formation of these ubiquitinated proteins, similarly to what observed in cells transfected 20

with to the empty vector. The Apu3.A8 anti-Ub immunoblotting showed that expression of 21

wild type RNF168 induces formation of K63-ubiquitinated proteins with molecular weight of 22

about 30 and 38 kDa (the latter with lower intensity), compatible with the di- and tri-23

ubiquitinated forms of histone H2A. Inactivation of any single UBD (UMI*, MIU1*, MIU2*) 24

and of the double MIU mutant (MIU1-2**) still induced the formation of the 30 kDa band, but 25


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not that of the higher molecular weight (Fig. 4C). Strikingly, the UMI*MIU1-2** mutant failed 1

to form any K63-linked ubiquitinated proteins, strongly indicating that inactivation of the sole 2

MIU domains is not enough to abolish RNF168 Ub ligase activity, but that the concurrent 3

inactivation of the UMI is strictly required. Interestingly, the decreased ubiquitinating activity 4

of the mutants is not ascribable to a reduced intrinsic Ub ligase activity, since we performed an 5

in vitro ubiquitination assay showing that the activity of the RNF168 mutants is comparable to 6

that of the wild type protein (Fig. S4). 7

8

Inactivation of the UMI affects in vivo histone ubiquitination 9

Histones H2A and H2AX are the sole RNF168 substrate identified at the moment. With the 10

purpose of verifying the role of the new UBD on histone ubiquitination, we performed a 11

biochemical analysis of chromatin histones using an antibody that specifically recognizes the 12

ubiquitinated forms of histones H2A (uH2A; Fig. 4D). Anti-uH2A immunoblot revealed that 13

inactivation of the UMI, as well as of the MIU1, reduced the level of histone H2A 14

ubiquitination induced by ectopical expression of RNF168, failing to form the signal 15

corresponding to tri-ubiquitinated histone H2A present in wild type protein and markedly 16

reducing the di-ubiquitinated form. Simultaneous inactivation of the three UBDs (UMI*MIU1-17

2**) completely abolished both di- and tri-ubiquitination of H2A, with a level of ubiquitination 18

comparable to that of cells transfected with the empty vector alone (Fig. 4D). Interestingly, 19

inactivation of the sole MIU2 did not have significant effect, despite its major role in the 20

localization of RNF168 at DDR foci. We confirmed these results by immunofluorescence 21

analysis, where we used anti-uH2A antibody to detect the amount of ubiquitinated histone H2A 22

in U2OS (shRNF168) cells complemented with RNF168 mutants (Fig. 4E, 4F). 23

Overall these results clearly indicated that UMI domain has a pivotal role to ensure proper 24

localization of RNF168, which is instrumental for the execution of its full Ub ligase activity on 25


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physiological substrates, which are histones. 1

2

Integrity of the UMI domain is required for the proper recruitment of downstream 3

signaling proteins 4

The ubiquitination events driven by RNF8/RNF168 are strictly required for the proper 5

execution of the DDR program. It has been demonstrated that the depletion of RNF168, either 6

by naturally occurring mutations (26) or by using the U2OS RNF168-targeting shRNA (6), 7

prevents the recruitment of 53BP1 to DDR foci, thereby affecting the downstream signaling 8

events. Relevantly, such effect may be reverted by reintroducing the wild type form of 9

RNF168. 10

The results obtained supporting the functional relevance of the UMI domain in the localization 11

and activity of RNF168 prompted us to test whether this domain is also required for the 12

recruitment of 53BP1 to DDR foci. To this purpose, we adopted the U2OS cell-system, 13

previously used in other experiments, to analyze the ability of the different RNF168 mutants 14

complementing the lack of endogenous protein. Accordingly to what already described in the 15

literature (26), we found that, upon DNA damage, the reintroduction of a construct carrying 16

both MIU1 and MIU2 inactivating mutations (MIU1-2**) restores 53BP1 localization only 17

partially (Fig. 5A and quantitation in Fig. 5B). Similar results were obtained upon expression of 18

the UMI* mutant. Strikingly, the simultaneous inactivation of UMI and MIUs in RNF168 19

(UMI*MIU1-2**) generates a protein that is almost completely unable to complement the 20

phenotype (Fig. 5A and Fig. 5B), with an effect comparable to that obtained in the absence of 21

the protein. 22

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DISCUSSION 25


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1

UBDs have a crucial role in Ub-mediated events by determining localization, activity and 2

interaction partners of proteins. We previously identified and characterized a new family of 3

UBDs, named MIU, present in a number of proteins with unrelated function, such as the 4

guanine nucleotide-exchange factor Rabex-5, the EGF-induced phosphoprotein Ymer, Myosin-5

6 and the RING finger protein RNF168 (20). Further we demonstrated that the two isolated 6

MIUs of RNF168 are able to interact with Ub chains, and that their inactivation by point 7

mutation impedes interaction with K48-linked Ub chains. 8

Here we report the identification of a novel UBD in RNF168, called UMI, which manifests 9

preference for binding to K63- as compared to K48-linked polyUb chains. At a sequence level, 10

the UMI shows similarity with the previously characterized UIM and MIU, being a short 11

amphipathic α-helix with adjacent glutamate-rich regions both at N- and C-terminus. 12

Interestingly, the interaction surface of the UMI with Ub is centered on two Leu residues 13

(L149, L150), which are expected, for the predicted positioning of amino acid residues on the 14

helix, to be exposed to the hydrophobic part of the helix. On the contrary the Ala residue, 15

crucial for UIM and MIU activity (20), is embedded in the hydrophilic side of the helix and 16

does not participate in Ub binding ability of the UMI. 17

The MIU domains of RNF168 bind ubiquitinated histone H2A, and this event accounts for the 18

proper localization of RNF168 at DDR foci and for the formation of RNF168-dependent 19

ubiquitinated proteins within the nucleus, including histones. In line with this, the MIU-20

defective mutant impairs 53BP1 recruitment at the site of lesions (26), without abolishing it. 21

These data indicate the existence of other mechanisms responsible for RNF168 recruitment to 22

DDR foci, in addition to the one mediated by the MIU domains. 23

Now we show that integrity of the new UMI domain is largely required for RNF168 function, 24

since the simultaneous inactivation of the three UBDs generates a protein unable to localize at 25


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DDR foci (Fig. 3A, 3B), to induce K63-specific polyubiquitination of proteins (see in Fig. 4C 1

and in Fig. S3 in the supplemental material), including ubiquitination of histone H2A (Fig. 4D, 2

4E, 4F), and to allow the recruitment of 53BP1 (Fig. 5). 3

The need of three different UBDs for the proper activity of RNF168 can be explained in 4

different, not mutually exclusive, ways. The first invokes the “avidity-effect” (12), with the 5

three UBDs collaborating to ensure stable binding of RNF168 to chromatin at the site of DNA 6

damage, thereby allowing sustained ubiquitination of histones. The second regards the 7

functions of the three UBDs in regulating RNF168 activity that have to be considered as 8

separate. In this respect, we can envision the possibility that the UMI binds to a different, yet 9

unidentified substrate, and that the contemporary binding of RNF168 to histones H2A and to 10

this unknown substrate may be mandatory for recruitment/stability of the protein to DNA 11

damage sites. 12

The third model relies on two different data: i) inactivation of UMI and MIU1 affects in vivo 13

ubiquitination of histone H2A (Fig. 4D), while the MIU2 is mainly involved in the proper 14

localization of RNF168 at DDR foci (22, 26); ii) in cells, RNF168 self-associates to form 15

dimers/oligomers (our unpublished data). In addition, it is well established that, upon formation 16

of DSBs, a vast area surrounding the damaged chromosome undergoes massive 17

phosphorylation and ubiquitination (24). How this event occurs is still under investigation. 18

Here, we hypothesize that RNF168 MIU domains, and MIU2 in particular, might help in 19

keeping the dynamic status of RNF168-uH2A association, thereby allowing the protein “to 20

walk” along the ubiquitinated histones. In such way, RNF168 would be able to propagate the 21

damage signal by stepping from a nucleosome to the other. In this scenario, due to the 22

contiguity of UMI and MIU1 and their proximity with the RING finger domain, we 23

hypothesize that UMI and MIU1 might facilitate the local polyubiquitination of histones by 24

transiently sustaining the binding of RNF168 to the growing Ub chain appended to uH2A. 25


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Finally, a speculative explanation takes into consideration the ubiquitination status of the 1

protein. We observed that in cells RNF168 is ubiquitinated (our unpublished data), although the 2

meaning of such modification is still elusive. It has been described that a number of UBDs, 3

including UIM and MIU, sustain the so-called “coupled mono-ubiquitination” (8, 29), a process 4

in which ubiquitination of a target protein is driven by its own UBD, through an unclear 5

mechanism. It will be of interest to investigate the role of UMI, MIU1 and MIU2 in RNF168 6

ubiquitination to verify whether the UBDs are important not only for their intrinsic Ub binding 7

ability, but also because they affect RNF168 function by regulating its own ubiquitination 8

status. 9

In conclusion, our results give new insights into the complex Ub-mediated regulation of 10

RNF168 activation. Nevertheless, much more need to be done to characterize in more detail 11

how a single UBD regulates RNF168 localization and function. This will necessary pass 12

through the identification of additional interacting partners, substrates and regulators of 13

RNF168. 14

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MATERIALS AND METHODS 1

2

Cell culture and DNA transfection 3

293T cells were grown in Dulbecco's modified Eagle's medium (SIGMA) supplemented with 4

10% fetal bovine serum (GIBCO) and 2 mM L-Glutamine (SIGMA). U2OS cell lines 5

expressing the RNF168-targeting shRNA in a Doxycycline-inducible manner, kindly 6

provided by J. Lukas, were cultured in Dulbecco’s modified Eagle’s medium (SIGMA) 7

supplemented with 10% fetal bovine serum Tetracycline-free (EuroClone), 1 µg/ml 8

Puromycin (SIGMA), 5 µg/ml Blasticidin S (SIGMA). Etoposide (SIGMA) was used at a 9

concentration of 5 µM for one hour to induce DSBs. Depletion of endogenous RNF168 was 10

obtained treating U2OS derivative cell line with 0.1 µg/ml Doxycycline (SIGMA) for 72 11

hours (see in Fig. S2 in the supplemental material). Cells were transfected either with Calcium 12

Phosphate method for biochemical analysis (293T cells) or with FuGENE (Roche) for 13

immunofluorescence (U2OS cells). 14

15

Antibodies and construct design 16

Antibodies used in this study included mouse monoclonal anti-Ub P4D1 (Santa Cruz) and 17

FK2 (Stressgen Bioreagents), rabbit polyclonal anti-GST was home-made, mouse monoclonal 18

anti-FLAG (M2, SIGMA), rabbit polyclonal anti-FLAG (SIGMA), mouse monoclonal anti-19

ubiquityl-Histone H2A (Upstate), anti-phospho-Histone H2AX (Ser139; Upstate). The 20

linkage specific antibody directed to K63 (Apu3.A8) was from Genentec. Mouse anti-53BP1 21

was a gift from Dr T. Halazonetis. Anti-RNF168 polyclonal antibodies were raised against 22

two different GST-RNF168 constructs, comprising the following regions: amino acid 1-191 23

(RF-MIU1) and 439-571 (MIU2-C-term); rabbit immunization was performed by Yorkshire 24


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Bioscience (UK), and the purified antibodies were tested as described in Supplementary Fig.s 1

S2C, S2D. 2

The cDNA of human full length RNF168 wild type and MIU mutants were obtained as 3

described previously (22). The point mutations in UMI domain were introduced by site-4

directed mutagenesis using the following nucleotides: forward 5

GAAGAATACATACAGAGGGCAGCAGCAGAGGAGGAAGAAGAG and reverse 6

CTCTTCTTCCTCCTCTGCTGCTGCCCTCTGTATGTATTCTTC. The truncated forms of 7

RNF168 constructs were generated by PCR amplification followed by cloning into pGEX6P2 8

vector. The oligonucleotide sequences are available upon request. The constructs were made 9

resistant to shRNA by site-directed mutagenesis using the following oligonucleotides: 10

forward AGACAGGCAGAAAAAAGAAGGCGAGCGATGGAAGAACAAC and reverse 11

GTTGTTCTTCCATCGCTCGCCTTCTTTTTTCTGCCTGTCT. All the constructs were 12

sequence verified. 13

14

GST pull-down assays 15

Recombinant proteins were expressed in E. Coli strain BL21 pLys with a glutathione S-16

transferase (GST) tag and purified as previously described14

. Chromatin was extracted by 17

293T cells, treated or not with etoposide (20 µM), by resuspending cells in PBS buffer with 18

inhibitors (1 mM PMSF, protease inhibitor cocktail (SIGMA), 20 mM Sodium Piruvate, 50 19

mM NaF, 1 mM Na3PO4, 20 µM NEM, 80 U/ml Benzonase), and clarified by centrifugation 20

at 3500 rpm for 5 min at 4°C. Pellet was lysed with Lysis Buffer (50mM Tris HCl pH 8.1, 10 21

mM EDTA, 1% Triton supplemented with inhibitors), incubated on ice for 10 min and 22

sonicated to shear DNA at 80% amplitude, 2 x 10 sec impulses, 20 sec pauses, on ice. The 23

shared chromatin was centrifuged at 14000 rpm in a 4°C for 30 min. For Ub chain pull down, 24

1 µM of GST fusion proteins, immobilized onto GSH beads, were incubated with 0.5 µg of 25


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K48- or K63-linked polyUb2-7 chains for one hour at 4°C in a buffer containing 50 mM Hepes 1

pH 7.5, 150 mM NaCl, 10% glycerol, and 0.5% Triton X-100. Proteins were separated by 2

SDS-PAGE (15%) and transferred onto PVDF membranes (SIGMA). Membranes were 3

denaturated using a solution containing 6 M guanidine hydrochloride, 20 mM Tris-HCl pH 4

7.4, 1 mM PMSF, 5 µM β-mercaptoethanol for 30 min at 4°C. After extensive washing in 5

Tris-buffered saline (TBS) buffer, membranes were blocked in TBS buffer containing BSA 6

(5%) over night and then incubated with anti-Ub P4D1 antibody (Santa Cruz) for one hour. 7

For chromatin binding, 10 µg of GST-fusion proteins immobilized onto GSH beads were 8

incubated with 1 mg of the chromatin extracts for 2 hours a 4°C. Bound proteins were 9

resolved on SDS-PAGE (15%), transferred on nitrocellulose and analyzed by immunoblotting 10

as indicated. 11

12

In vivo ubiquitination assay 13

Transfected 293T cells were collected in Phosphate Saline Buffer (PBS) containing protease 14

inhibitor cocktail (SIGMA), 1 mM PMSF and 20 µM NEM. 1/10 of the samples were 15

separately processed for protein normalization, while the remaining cell pellets were 16

subjected to acid extraction of histones as previously described(22). Samples were resolved 17

on SDS-PAGE, transferred onto PVDF membranes (SIGMA) and detected with the indicated 18

antibodies. 19

20

In vitro ubiquitination assay 21

5 µg of purified GST-RNF168 constructs were incubated with 0.1 µg human recombinant E1 22

Ub-activating enzyme (Boston Biochem), 200 ng of purified Ubc13/Mms2 complex 23

(provided by Dr E. Maspero, IFOM, Milan), 2 µg of Ub (home made) in 25 mM Tris-HCl, 24

pH7.4, 5 mM MgCl2, 100 mM NaCl, 1 µM DTT, 2 mM ATP at 30°C for 1.5 hours. The 25


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reaction was stopped by boiling in Laemmli buffer. Ubiquitination was detected by anti-Ub 1

(P4D1) immunoblotting. 2

3

Immunofluorescence analysis 4

Twenty-four hours after transfection, U2OS cells were fixed in 4% paraformaldehyde, 5

permeabilized with a solution of 0.5% Triton X-100 in 0.2% BSA for 7 min at room 6

temperature and blocked with PBG (PBS, 0.5% BSA, 0.2% gelatin) for one hour. Coverslips 7

were incubated for one hour with a primary antibody and, after extensive washing, incubated 8

with the appropriate secondary antibody (Alexa fluor 488 goat anti-mouse or anti-rabbit IgG, 9

Alexa fluor 546 goat anti-mouse or anti-rabbit IgG; Invitrogen) for 30 min at RT. Images 10

were acquired by confocal scanning laser microscope (Leica TCS2; Leica Lasertechnik, 11

Heidelberg, Germany). 12

13


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FIGURE LEGENDS 1

2

FIG. 1. Identification of a new Ub binding region in RNF168. (A) We performed in vitro pull-3

down assay using the indicated GST-tagged RNF168 constructs. GST-fusion proteins were 4

incubated with synthetic K48- (left panel) or K63-linked (right panel) polyUb2-7 chains and 5

separated by SDS-PAGE. Immunoblot (IB) was performed with antibodies directed against 6

Ub and GST, as described in Methods. (B) Schematic representation of RNF168 deletion 7

constructs used in pull-down experiments (numbers refer to the amino acid position within the 8

sequence; RF, RING finger domain); their ability to bind K63 polyUb chains, resumed on the 9

left, is shown in the anti-Ub immunoblot of the in vitro pull-down assay (lower panels). 10

Normalization is visualized by anti-GST immunoblotting. (C) Multiple alignments of region 11

134-166 RNF168 homologues in vertebrates. Secondary structure prediction was obtained 12

using SAM-T08, an HMM-based Protein Structure Prediction software 13

(http://compbio.soe.ucsc.edu/SAM_T08/T08-query.html). (D) Mapping of the minimal 14

sequence responsible for Ub binding. In vitro pull-down assay was performed using the 15

indicated GST-tagged deletion mutants of RNF168 and incubated with K63 polyUb chains. 16

IB was performed with anti-Ub and anti-GST antibodies. 17

18

FIG. 2. Mapping of the amino acid residues required for Ub binding. (A) Comparison of the 19

UMI, UIM and MIU motifs, where residues involved in the binding to Ub are in bold; 20

underlined sequences were drawn as projected helices using the Helical Wheel Projections 21

tool (http://rzlab.ucr.edu/scripts/wheel/wheel.cgi). (B) The point mutants were generated by 22

introduction of single amino acid substitutions (A151G, L149A, L150A) or double (L149A, 23

L150A) addressing the sequence 134-166, and tested by in vitro pull-down assay as in FIG. 24

1D. (C) The indicated RNF168 point mutants were inserted in the sequence 56 amino acids 25


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and analyzed by in vitro pull-down assay using K48 and K63 poly-Ub chains. Anti-Ub and 1

anti-GST immunoblot is shown. 2

3

FIG. 3. Inactivation of the UMI domain affects localization of RNF168. (A) The U2OS cells 4

conditionally expressing RNF168-targeting shRNA were transfected with the indicated 5

FLAG-tagged shRNA-resistant constructs. After twenty-four hours, cells were treated with 6

etoposide for one hour or left untreated (Eto + and Eto - respectively). (B) The U2OS cells, 7

treated as in a, were either fixed (TX100 -) or pre-treated with Triton X-100 before fixing 8

(TX100 +). Immunostaining of both panels was performed using anti-FLAG and anti-9

phospho-H2AX (γH2AX) antibodies. (C) Pull-down assay was performed using GST-10

RNF168 constructs and chromatin extracted from 293T cells, treated or not with etoposide. 11

H2A and H2B antibodies (upper and lower panels, respectively) revealed the presence of 12

specific bands (of about 15 kDa) in all RNF168 constructs but not in the lane of GST alone. 13

14

FIG. 4. UMI, MIU1 and MIU2 are required for the ubiquitination events mediated by 15

RNF168. (A) U2OS shRNF168 cells transfected either with the indicated FLAG-tagged 16

shRNA-resistant constructs or with the vector alone were treated with etoposide for one hour 17

before fixing and immunostained using anti-FLAG and anti-Ub (FK2) antibodies. (B) 18

Quantitation of RNF168-positive cells with a number of foci labeled with anti-FK2 higher 19

than 30. (C) Acid extraction of histones from 293T cells transfected with the indicated FLAG-20

tagged RNF168 constructs. Ubiquitinated proteins were detected by immunoblot using P4D1 21

(Ub; upper panel) and Apu3.A8 (K63; medium panel) antibodies. Cell extracts were analyzed 22

for equal expression of the different constructs (FLAG; lower panel). (D) 293T cells were 23

processed as in C and immunodecorated with antibody directed to ubiquitinated forms of 24

histone H2A (uH2A). Cell loading was normalized by FLAG immunoblot. (E) 25


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Immunofluorescence (IF) analysis was performed with anti-FLAG and with anti-uH2A 1

antibodies. (F) Quantitation of RNF168-positive cells with a number of foci labeled with anti-2

Ub higher than 5. 3

4

FIG. 5. Inactivation of RNF168 UBDs abolishes 53BP1 recruitment to DDR foci. (A) The 5

U2OS cells conditionally expressing RNF168-targeting shRNA were transfected with the 6

indicated FLAG-tagged shRNA-resistant RNF168 constructs. Twenty-four hours after 7

transfection, cells were treated with etoposide for one hour, fixed and immunostained with 8

anti-FLAG and anti-53BP1 antibodies. (B) Quantitation of the two independent experiments 9

described in A, representing the percentage of transfected cells with 53BP1-positive foci. 10

11

12

13


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ACKNOWLEDGMENTS 1

We wish to thank Fabrizio Condorelli for reading the manuscript and Grant Stewart for 2

discussion. We are grateful to Jiri Lukas for kindly providing U2OS cells conditionally 3

expressing RNF168-targeting shRNA and to Genentec for providing antibodies specific for 4

K63 poly-Ub chains. L.P. designed research; S.P. and M.G. performed research; C.S. 5

contributed new reagents/analytical tools; S.C. performed sequence analysis; S.P. and L.P. 6

wrote the paper. This work was supported by grants from Associazione Italiana per la Ricerca 7

sul Cancro (AIRC) and Regione Piemonte RSF. M.G. is supported by Lagrange Fellowship. 8


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INPUT K

48

GST

RNF16

8

MIU

1*

MIU

2*

MIU

1-2*

*

INPUT K

63

GST

RNF16

8

MIU

1*

MIU

2*

MIU

1-2*

*

INPUT K

63

GST

56-1

66

56-1

34

134-

166

A

C

D

17

26

55

72

17

26

55

72

17

26

55

72

B

Ub

GST

IB

Ub

GST

IB

Ub

GST

IB

MIU1 MIU2RFK63

+

+

+

+

/+

+

-

1-571

1-166

56-166

168-462

168-462MIU1-2**

439-571

439-571MIU2*

Hs_RNF168

Pt_RNF168

Cj_RNF168

Bt_RNF168

Ec_RNF168

Cf_RNF168

Mm_RNF168

Rn_RNF168

Gg_RNF168

Xt_RNF168

Xl_RNF168

Dr_RNF168

134

134

134

134

134

134

136

134

134

136

136

143

166

166

166

166

166

166

168

166

165

168

168

172

SEEEENKASEEYIQRLLAEEEEEEKRQAEKRRR

SEEEENKASEEYIQRLLAEEEEEEKRQAEKRRR

TEEEENKASEEYIQRLLAEEEEEDRRQAEKRRR

CEEEENKASEEYIQKLLAEEEEEEKRQAEKRHR

NEEEENKASEEYIQRLLAEEEEEEKRQAEKRQR

SEEEENKASEECIQRLLAEEEEEEKRQAEKRRR

SKEEENKASEEYIQRLLAEEEEEEKRQREKRRS

SKEEENKASEEYIQRLLAEEEEEEKRRTERRRS

HEQEENKASEEYIQKLLAEEEEEH-RLAEERRK

QEEAERKASEDYIQKLLAEEEAEENLHAEASQR

QEEAERKASEDYIQKLLAEEAAQDRLHSEAVQR

LEEAERRASEEYIQRLLAEEEE---RLEEERRR

SAM-T08 pred.

Figure 1

-

-

192-439

1-57

1

1-16

6

56-1

66

168-

462

168-

462M

IU1-

2**

439-

571

439-

571M

IU2*

192-

439

GST

INPUT

K63

Ub

GST

IB

17

26

55

72


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INPUT K

63

GST

134-

166

134-

166 A15

1G

134-

166 L1

49A

134-

166 L1

50A

134-

166 L1

49A, L

150A

A B

17

26

5572

IB

Ub

GST

E

Q

A

E

R

L

I

L

Y E

Q

E

A

E

L

I

L

A

D L

W

E

R

E

R

Q

A

L

LE

UMI_RNF168

UIM_EPS15

MIU_RABEX

141

877

48

156

892

63

ASEEYIQRLLAEEEEE

QEQEDLELAIALSKSE

QIQEDWELAERLQREE

*

RNF168 UMI EPS15 UIM RABEX MIU

*

Figure 2

INPUT K

48

GST

RNF16

8

UM

I*

MIU

1-2*

*

UM

I*M

IU1-

2**

17

26

55

72

INPUT K

63

GST

RNF16

8

UM

I*

MIU

1-2*

*

UM

I*M

IU1-

2**

C

17

26

55

72

IB

Ub

GST

IB

Ub

GST

56-571 56-571


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RNF168

EtoTX100

- + ++--

FLAG

γH2AX

merge

MIU1-2** UMI*MIU1-2**

- + ++--

- + ++--

- + ++--

UMI*

EtoTX100

FLAG

γH2AX

merge

Figure 3

FLAG

γH2AX

merge

RNF168

- +

UMI*

- +

MIU1-2**

- +

UMI*MIU1-2**

- +

A

B

C

Eto

Eto - Eto +

IB

H2A

H2B

GST

TCL

GST

RNF16

8

UM

I*

MIU

1-2*

*

UM

I*M

IU1-

2**

TCL

GST

RNF16

8

UM

I*

MIU

1-2*

*

UM

I*M

IU1-

2**

IB

H2A

H2B

GST


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vector

RNF16

8

UM

I*

MIU

1*

MIU

2*

MIU

1-2*

*

UM

I*M

IU1-

2**

Ub

K63

FLAG

vector

RNF16

8

UM

I*

MIU

1*

MIU

2*

MIU

1-2*

*

UM

I*M

IU1-

2**

uH2A

FLAG

A

C

E

vecto

rR

NF

168

UM

I*M

IU1-2

**U

MI*

MIU

1-2

**

FLAG FK2 merge

vecto

rR

NF

168

UM

I*M

IU1-2

**U

MI*

MIU

1-2

**

FLAG uH2A merge

26

34

55

26

34

55

26

72

72

IB

IB

******

******

F

*****

% c

ells

with

uH

2A

foci (>

10)

vector

UM

I*

RNF16

8

MIU

1-2*

*

UM

I*M

IUs*

*

D

100

20

80

60

40

0

Figure 4

B

% c

ells

with F

K2 foci (>

30)

vector

UM

I*

RNF16

8

MIU

1-2*

*

UM

I*M

IUs*

*

100

20

80

60

40

0


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AB

vectorRNF168UMI*MIU1-2**UMI*MIU1-2**

FLA

G53B

P1

merg

e

% cells with 53BP1 foci (>10)

vector

RNF168

UM

I*

MIU

1-2**

UM

I*MIU

s**

10

0

20

80

60

400

Fig

ure

5


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1 umi, a novel rnf168 ubiquitin binding domain involved in the

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