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. Author manuscript; available in PMC: 2014 Mar 7.
Published in final edited form as: Mol Cell. 2013 Feb 7;49(5):897–907. doi: 10.1016/j.molcel.2013.01.006

RNF111-dependent neddylation activates DNA damage-induced ubiquitination

Teng Ma 1, Yibin Chen 1, Feng Zhang 1, Chao-Yie Yang 2, Shaomeng Wang 2, Xiaochun Yu 1*
PMCID: PMC3595365  NIHMSID: NIHMS434987  PMID: 23394999

Summary

Ubiquitin-like proteins have been shown to be covalently conjugated to targets. However, the functions of these ubiquitin-like proteins are largely unknown. Here, we have screened most known ubiquitin-like proteins after DNA damage and found that NEDD8 is involved in the DNA damage response. Following various DNA damage stimuli, NEDD8 accumulated at DNA damage sites, and this accumulation was dependent on an E2 enzyme UBE2M and an E3 ubiquitin ligase RNF111. We further found that histone H4 was polyneddylated in response to DNA damage, and NEDD8 was conjugated to the N-terminal lysine residues of H4. Interestingly, the DNA damage-induced polyneddylation chain could be recognized by the MIU (Motif Interacting with Ubiquitin) domain of RNF168. Loss of DNA damage-induced neddylation negatively regulated DNA damage-induced foci formation of RNF168 and its downstream functional partners, such as 53BP1 and BRCA1, thus affecting the normal DNA damage repair process.

Introduction

Ubiquitination is one of the most important covalent protein modifications, shown to regulate a wide variety of biological processes(Baneres et al., 1997; Chen and Sun, 2009; Johnson, 2002; Pickart, 2001b; Sun and Chen, 2004). In the presence of E1, E2 and E3 enzymes, ubiquitin (Ub) is covalently conjugated to a substrate by the formation of an isopeptide bond between the carboxy-terminal glycine of Ub and lysine resides on the substrate(Pickart, 2001a). The first identified ubiquitination substrate was histone H2A(Goldknopf and Busch, 1977; Goldknopf et al., 1975). Subsequently, histone H2B was found to be ubiquitinated as well(West and Bonner, 1980). Both H2A and H2B are core histones in the nucleosome, in which a 146 bp of DNA is wrapped around a core histone octamer. The histone octamer consists of two copies each of H2A, H2B, H3 and H4(Kornberg, 1974; Kornberg and Lorch, 1999). In mammals, the ubiquitination sites have been mapped to lysines 119 and 120 on the tails of H2A and H2B, respectively. Most cellular H2A and H2B are mono-ubiquitinated(Jason et al., 2002; Weake and Workman, 2008). H2A and H2B contain only 131 and 125 residues respectively, while Ub is a 76 amino acid polypeptide. The large size of Ub relative to histone makes histone ubiquitination as a unique modification. Although structural analysis indicates that the histone-conjugated Ub protrudes to the outside of the nucleosome, addition of this bulky modification to the nucleosome potentially changes the chromatin structure(Fierz et al., 2011; Jason et al., 2001). Thus, it is not surprising that both H2A and H2B ubiquitination regulate chromatin remodeling during gene transcription(Shilatifard, 2006; Weake and Workman, 2008; Zhang, 2003). Interestingly, H2A and its variant H2AX can also be polyubiquitinated, especially during DNA damage response(Huen et al., 2007; Kolas et al., 2007; Mailand et al., 2007; Mattiroli et al., 2012). Following DNA double strand breaks (DSBs), RNF8 and RNF168, two E3 ligases, associated with Ubc13 and other E2 enzymes polyubiquitinate H2A and H2AX adjacent to chromatin lesions(Ikura et al., 2007; Mattiroli et al., 2012; Zhao et al., 2007). This polyubiquitination signal then further recruits downstream DNA damage response factors such as RAP80, BRCA1 and 53BP1 for DNA damage repair(Bennett and Harper, 2008; Panier and Durocher, 2009; Stewart, 2009; van Attikum and Gasser, 2009).

Besides Ub, there are a number of Ub-like polypeptides that share similar secondary and tertiary structures with Ub(Kerscher et al., 2006). They utilize similar chemical reactions for conjugation to substrate proteins, specifically an isopeptide bond between the carboxyl group of their terminal Gly residue and the ε-group of a lysine residue on the substrate(Schulman and Harper, 2009). One example is NEDD8 that is~ 60 % identical and ~ 80 % homologous to Ub, the highest among the Ub-like proteins. It has its own specific E1 and E2 enzymes for protein neddylation(Huang et al., 2009; Huang et al., 2007; Huang et al., 2004; Huang et al., 2005; Huang et al., 2008). Although neddylation substrates have been screened, the previous research on neddylation has mainly focused on Cullin, an essential subunit in the SCF E3 Ub ligase complex. Conjugated NEDD8 significantly changes the conformation of Cullin and potentially brings the E2 Ub conjugase to the substrate to promote protein ubiquitination(Duda et al., 2008).

Both Ub and members of the SUMO family have been shown to participate in DNA damage response (Dou et al., 2010; Galanty et al., 2009; Morris et al., 2009b). However, the function of NEDD8 in DNA damage remains unclear. Here, we have identified NEDD8 relocates to the damage sites and RNF111 acts as a neddylation E3 with E2 UBE2M. RNF111 mediated histone neddylation facilitates downstream RNF168 binding to the chromatin and regulates downstream repair.

Results

NEDD8 is accumulated at DNA damage sites

Since both ubiquitin and SUMO are conjugated at DNA damage sites and participate in DNA damage response, we wonder whether other Ub-like (Ubl) proteins are also involved in the DNA damage response. Thus, we screened the other 10 known Ub-like proteins. These proteins were expressed in U2OS cells and their localizations were examined by immunostaining following ionizing radiation (IR) treatment. Interestingly, we found that only NEDD8 formed nuclear foci and colocalized with γH2AX, the marker of DSBs(Fernandez-Capetillo et al., 2004) (Figure 1A). Although we did not detect other Ubl’s conjugation at DNA damage sites, we cannot exclude the possibility that these Ubl may participate in certain type of DNA damage repair. To confirm the specific NEDD8 conjugation at DNA damage sites, we deleted the carboxyl Gly residue of NEDD8 (NEDD8 ΔG mutant) to abolish its conjugation on target proteins(Kamitani et al., 1997). The ΔG mutant failed to accumulate at DNA damage sites (Figure S1A). Because IR mainly generates DSBs, we used other DNA damaging agents including MMC, UV and MMS to induce various types of DNA damage. Similarly, endogenous NEDD8 formed DNA damage-induced nuclear foci (Figure 1B and Figure S1B), and colocalized with Ub (Figure S1C), suggesting a close relationship between protein neddylation and protein ubiquitination during DNA damage response. To further examine the dynamic recruitment of NEDD8 to DNA damage sites, we performed time course experiments using laser micro-irradiation. As shown in Figure 1C, following laser treatment, NEDD8 is quickly recruited to DNA damage sites within 1 minute. The accumulation of NEDD8 at DNA damage sites reached the peak within 10 minutes and gradually diminished after around 16 hours. Thus, the NEDD8 conjugation rate at DNA damage sites is similar to that previously reported for Ub(Doil et al., 2009; Morris et al., 2006).

Figure 1. NEDD8 is accumulated at DNA damage sites.

Figure 1

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(A) NEDD8 relocates to DNA damage sites. U2OS cells transfected with HA-NEDD8, ISG15, FAU, UBL3, UBL4a, UBL4b, FAT10, ATG12, UBL5 and URM1 were irradiated with 10 Gyof IR. Cells were stained with anti-HA or γH2AX. Scale bar = 20 μm. Data are represented as mean +/− SEM. (B) U2OS cells were treated with or without IR (10 Gy), MMC (50 ng/ml), UV (40 J/m2) and MMS (0.5 mM). Foci formation of endogenous NEDD8 and γH2AX was examined by immunostaining. Scale bar = 20 μm. (C) U2OS cells were treated with laser micro-irradiation and NEDD8, 53BP1 or γH2AX at laser strip was examined at indicated time points (scale bar = 10 μm). For quantitative and comparative imaging, signal intensities of accumulated NEDD8, 53BP1 or γH2AX fluorescence at the laser line were converted into a numerical value using Axiovision software (version 4.5). Normalized fluorescent curves from 20 cells were averaged. The error bars represent the standard deviation. See also Figure S1.

RNF111 is a partner of UBE2M at DNA damage sites

To date, one E1 and two E2 enzymes have been identified that act in the neddylation system. Selection of the substrate is first determined by UBE2M and UBE2F (Huang et al., 2009), two E2 enzymes. Thus, we examined which E2 enzyme is involved in DNA damage response. Following IR treatment, UBE2M, but not UBE2F, was recruited to DSBs, and colocalized with NEDD8 and another DSB marker 53BP1(Stewart, 2009) (Figure S2A). And the UBE2M’s accumulation at the laser line showed the similar kinetics with that of NEDD8 (Figure S2B). During protein neddylation, a specific E3 ligase could facilitate the transfer of NEDD8 from an E2 enzyme to the substrate (Calabrese et al., 2011; Scott et al., 2010). Thus, we used yeast two hybrid screening to explore the possible E3s that function together with UBE2M in response to DNA damage. In the yeast two hybrid screening, we got ten candidates to interact with UBE2M including two known UBE2M partners Rbx1 and the IAPs (Figure S2C). To further validate which E3 ligase is involved in DNA damage, we examined the localization of these ten candidates in response to laser micro-irradiation. Among all the candidates, only RNF111 was recruited to DNA damage sites (Figure 2A). The kinetics of the accumulation of RNF111 at DNA damage sites is very similar to that of NEDD8 (Figure S2E). The interaction between UBE2M and RNF111 was validated by a protein pulldown assay (Figure 2B). RNF111 is a nucleus protein, which has been reported to function in the TGFβ signal pathway (Koinuma et al., 2011). To dissect which region of RNF111 is responsible for its localization at DNA damage sites, we generated a series of truncated RNF111 mutants based on the protein folding and monitored their localization in response to laser micro-irradiation (Figure S2F, G). Only the intact N-terminus of RNF111 (a.a. 1–543) but no other mutants relocated to the laser strip (Figure S2G). Interestingly, the N-terminus of RNF111 enriched with positively charged residues was tightly associated with chromatin (Figure 2C). We wonder whether the N-terminus of RNF111 recognizes DNA. To test the hypothesis, we used the DNA mobility shift assay and found that the N-terminal truncation mutant directly bound DNA (Figure 2D). Thus, the relocation of RNF111 to DNA damage sites is likely to be mediated by the naked DNA exposed at DNA damage sites. The mechanism of the relocation of RNF111 to DNA damage sites could be very similar to many other DNA damage response factors, such as the E3 ligases of SUMO (Galanty et al., 2009; Morris et al., 2009b). The computational modeling of the C-terminal Ring domain of RNF111 indicates that its tertiary structure is very similar to that of the Ring domain of Rbx1, a known E3 partner of UBE2M (Figure S3A). I44 and W87 in the Ring domain of Rbx1form a hydrophobic pocket that mediates the interaction with UBE2M (Calabrese et al., 2011). These two residues are conserved in the Ring domain of RNF111 and may form a similar hydrophobic pocket (Figure S3B).

Figure 2. RNF111 is a partner of UBE2M at DNA damage sites.

Figure 2

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(A) RNF111 is accumulated at DNA damage sites. U2OS cells were treated with laser micro-irradiation and the cells were co-stained with anti-RNF111 and anti-53BP1 antibodies. (B) The Ring domain of RNF111 interacts with UBE2M. GST-RNF111 (Ring domain) protein was incubated with cell lysates from 293T cells expressing SFB-UBE2M. The protein pull-down assay was examined by western blot as indicated. GST and GST-Rbx1 (Ring domain) were used as the negative and positive controls respectively. (C) The N-terminus of RNF111 (a.a. 1–543) mainly exists in the chromatin fraction. The soluble and chromatin fraction of U2OS cells were collected as explained in Material and methods. Both fractions were subjected to SDS-PAGE and blotted with indicated antibody. (D) The N-terminus of RNF111 binds double-stranded DNA. GST or GST-RNF111 (a.a. 1–543) proteins were expressed in E. coli BL21 and purified. The purified proteins were incubated with 32P labeled DNA and then subjected to native page analysis. The gel was dried and exposed to X-ray film. The free DNA and RNF111-bound DNA were indicated. See also Figure S2 and S3.

RNF111 regulates neddylation at DNA damage sites

Since RNF111 interacts with UBE2M and relocates to DNA damage sites, we wonder whether RNF111 controls protein neddylation at DNA damage sites. We designed two different RNF111 siRNAs and both of them could successfully down-regulate the expression of RNF111 (Figure 3A). As shown in Figure 3B and C, depletion of RNF111 by siRNA treatment significantly suppressed the accumulation of NEDD8 at DNA damage sites. Moreover, the reconstitution of RNF111 siRNA treated cells with wild type RNF111 but neither the Ring domain deletion mutant nor the N-terminal deletion mutant of RNF111 restored the protein neddylation at DNA damage sites (Figure 3B and C, Figure S3C). Taken together, these results suggest that RNF111 regulates protein neddylation at DNA damage sites in vivo.

Figure 3. RNF111 regulates DNA damage-induced neddylation.

Figure 3

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(A) Control siRNA or RNF111 siRNAs were introduced into U2OS cells and the knockdown efficiency was determined by RT-PCR and Western blot. (B) RNF111 regulates DNA damage-induced neddylation. U2OS cells were transfected RNF111 siRNAs alone or together with siRNA-resistant wild type, the Ring domain deletion mutant or the N-terminal deletion mutant of RNF111. Cells were micro-irradiated and co-stained with anti-NEDD8 and anti-γH2AX antibodies. (C) Quantitative analysis of NEDD8 accumulation at the laser line. Data are represented as mean +/− SEM. See also Figure S3.

RNF111 regulates histone H4 neddylation

We next searched for the substrate of DNA damage-induced neddylation. 293T cell lysates were fractionated by different salt concentration and pH. Interestingly, a neddylated protein was remarkably increased by IR treatment in the soluble S3 fraction that mainly contains histones. Moreover, the molecular weight of this neddylated protein is similar to the predicted molecular weight of neddylated histones (Figure 4A). Thus, we examined whether histones could be neddylated. As shown in Figure 4B, using overexpression of histones and NEDD8, we found that histone H4, but not H3, could be clearly neddylated in vivo. Moreover, with deneddylation inhibitor 1,10-Phenanthroline treatment (1mM for 6 hours), we observed multi-neddylatedH4 in the cell lysates (Figure S4A). In addition, both H2A and H2B could be neddylated, although at very low level compared with that of H4 (Figure S4A). To validate that NEDD8 is covalently conjugated to H4, we examined the ΔG mutant and this mutant NEDD8 failed to be conjugated to H4 (Figure 4C). These data suggest that histone H4 is one of the histone targets of NEDD8 on the chromatin.

Figure 4. Histone H4 is a major neddylation substrate on the chromatin.

Figure 4

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(A) IR induces protein neddylation on the chromatin. 293T cells stably expressing SFB-NEDD8 were lysed. The lysates were separated by salt concentration and pH. Each fraction was examined by Western blot. (B) Histone H4 is multi-neddylated. SFB-tagged histone H2A, H2B, H3, or H4 were co-expressed with HA-tagged NEDD8 in 293T cells. Neddylated histones were examined by pull down with strepavidin beads and Western blot with anti-HA. An input control was analyzed with anti-FLAG antibody. Multi-neddylated H4 was indicated. (C) ΔG mutant of NEDD8 abolishes H4 neddylation. SFB-H4 was co-expressed with HA-NEDD8 or HA-NEDD8ΔG mutant into 293T cells. H4 neddylation was analyzed with indicated antibodies. (D) In vitro needylation assay. Recombinant H4 was incubated with NEDD8 (or methylated NEDD8), E1, UBE2M, ATP and recombinant RNF111 protein. After reaction, proteins were subjected to blot with anti-H4. Neddylated H4 was indicated. (E, F) IR induces endogenous H4 neddylation. 293T cells stably expressing SFB-NEDD8 (E) or 293T cells (F) were treated with or without IR (10 Gy). After 1 hour recovery, chromatin fractions were analyzed with indicated antibody. (G) RNF111 regulates IR-induced H4 neddylation. U2OS cells were transfected with control or RNF111 siRNA. Chromatin fraction was used to analyze H4 and H4 neddylation. See also Figure S4 and S5.

There are seven Lys residues on either N-terminal or C-terminal tail of H4. To examine which Lys residue(s) could be neddylated, we mutated each Lys into Arg. However, none of these solo mutations could abolish H4 neddylation (Figure S4B). Since mutation of the only Lys residue (K91) within the C-terminal tail of H4 did not abolish H4 neddylation, we mutated multiple lysines at the N-terminal tail of H4 (Figure S4C). However, only when all six Lys residues at the N-terminus of H4 were mutated, could neddylation on H4 be abolished. Moreover, with five mutated N-terminal Lys, H4 could still be multi-neddylated. To further validate that N-terminal Lys residues are neddylated, we mutated all the 11 lysine residues into arginine (the H4 NOK mutant). The H4NOK mutant was not able to covalently conjugate on H4 (Figure S4D). Moreover, we kept each lysine residue at the N-terminus of H4 and mutated remaining other lysine residues into arginine (H4K5only, H4K8only, H4K12only, H4K16only, H4K20only and H4K31only mutant). All these mutants could be neddylated, while H4K44only, H4K59only, H4K78/80only and H4K91only mutants could not be neddylated. These results suggest that only N-terminal lysine residues of H4 could be neddylated and neddylation of these lysine residues is likely interchangeable. Similar to wild type H4, both the 6R and H4 NOK mutants existed in the histone fraction, but not in the NP-40 soluble fraction (Figure S4E), suggesting that the 6R and H4 NOK mutants are incorporated into the chromatin. Moreover, we used another approach to confirm that these mutants of H4 are deposited into the genome. Using a ChIP assay, we found that all these H4 mutants, like wild type H4, existed in the p21 gene. Upon DNA damage, these mutants did not significantly affect the transcription of p21 (Figure S4F). Taken together, these H4 mutants are incorporated into the genome and function in the p21 gene transcription as wild type H4 does. Interestingly, with only one lysine residue at the N-terminus of H4, H4 still could be multi-neddylated (Figure S4D), suggesting that NEDD8 polymer could be conjugated on H4. Moreover, we mutated all the 9 lysine residues in Nedd8 into arginine (the NEDD8 K null mutant) to abolish the potential poly-NEDD8 chain formation. This NEDD8 mutant was conjugated onto H4 as the mono-mer (Figure S4D). We also established an in vitro neddylation assay and found that RNF111 facilitated the H4 multi-neddylation in vitro (Figure 4D). However, when we used the lysine methylated Nedd8 to block the poly-chain formation, the H4 multi-neddylation was suppressed (Figure 4D). Finally, to study whether NEDD8 could form poly-chain, we performed mass spectrometry analysis and found that NEDD8 could be covalently linked into polymer at K22 and K48 (Figure S5A and B).

Next, we examined DNA damage-induction of H4 neddylation. Since anti-NEDD8 antibody is not efficient for immunoprecipitation in the histone fraction in which proteins are denatured with 0.2 M HCl treatment, we established 293T cells stably expressing SFB(S -FLAG-SBP)-tagged NEDD8. Without DNA damage stimulation we detected very little neddylation on endogenous H4. However, upon IR treatment, polyneddylated H4 could be clearly detected (Figure 4E). To examine the endogenous H4 neddylation, H4 was precipitated with anti-H4 antibodies. Again, DNA damage clearly induced H4 neddylation (Figure 4F). Moreover, depletion of RNF111 abrogated DNA damage-induced H4 neddylation (Figure 4G), confirming that this specific NEDD8 E3 regulates DNA damage-induced H4 neddylation.

Histone neddylation tethers RNF168 to DNA damaged sites

Besides NEDD8, both Ub and SUMO modifications are accumulated at DNA damage sites(Morris, 2010). To study the functional correlation between these posttranslational modifications in response to DNA damage, cells were depleted RNF111 by siRNA to abolish the ionizing radiation-induced foci (IRIF) of NEDD8. Interestingly, we found that depletion of RNF111 significantly down-regulated DNA damage-induced Ub foci formation (Figure 5A), suggesting that protein neddylation regulates ubiquitination in response to DNA damage. Consistently, ubH2A foci formation was also reduced by siRNA knock-down of RNF111 (Figure 5A). Knockdown of UBE2M, but not UBE2F in cells resulted in similar effect as the RNF111 knockdown (Figure S6A and B). It further demonstrates that UBE2M function together with RNF111 in response to DNA damage. The DNA damage-induced ubiquitination cascade is controlled by two E3 Ub ligases, RNF8 and RNF168 (Bennett and Harper, 2008; Panier and Durocher, 2009; Stewart, 2009; van Attikum and Gasser, 2009). Previous studies have shown that DNA damage-induced MDC1 phosphorylation recruits RNF8 to DNA damage sites (Huen et al., 2007; Kolas et al., 2007; Mailand et al., 2007). Upon reaching DNA lesions, RNF8 promotes initial histone ubiquitination. The MIU domain of RNF168 then recognizes ubiquitination signals at DNA damage sites and further catalyzes protein polyubiquitination, which facilitates DNA damage repair by recruiting important mediators, such as BRCA1 and 53BP1, to the DNA damage site(Doil et al., 2009; Stewart et al., 2009). Surprisingly, depletion of RNF111 only affected the recruitment of RNF168 to DNA damage sites, but not that of RNF8 and MDC1 (Figure 5B and Figure S7), suggesting that partial loss of RNF168 at DNA damage sites could be the reason for the affected IRIF of Ub and ubH2A. Since the MIU domain targets RNF168 to DNA damage sites, we hypothesize that the RNF168 MIU domain might bind to NEDD8 at DNA damage sites. Of note, NEDD8 has almost identical secondary and tertiary structure with Ub and also shares 80 % primary sequence homology with Ub. Particularly, a hydrophobic binding pocket composed of Ile44 in Ub, recognized by the MIU domain, is well conserved in NEDD8(Whitby et al., 1998). Thus, we asked whether one of the two RNF168 MIU domains could directly interact with NEDD8. To test our hypothesis, we generated recombinant RNF168 MIU1 and MIU2 proteins. Both MIU motifs could directly interact with Ub as well as NEDD8. Mutation of conserved residues that are critical for Ub interaction also abolished the interaction with NEDD8 (Figure 5C). These results suggest that the MIU motifs of RNF168 could not distinguish between Ub and NEDD8 in vitro. To examine whether RNF168 MIU motifs recognize polyneddylated H4, we performed pull down experiments and found that RNF168 MIU2 has a stronger affinity for polyneddylated H4 than that of RNF168 MIU1 (Figure 5D). This is consistent with previous studies indicating that MIU2 has a stronger affinity for K63 based poly-Ub and is essential to target RNF168 to DNA damage sites(Doil et al., 2009; Stewart et al., 2009). Again, mutation of key residues in MIU2 abolished the binding to the poly-NEDD8 chain (Figure 5D). To quantitatively measure the affinity between MIU2 and NEDD8, we used the proprietary BioLayer Interferometry. The KD between MIU2 and NEDD8 is 14±3 μM, which is similar to that between MIU2 and Ub (17±3 μM) (Figure S8). Taken together, these results suggest that the MIU domain of RNF168 recognizes not only protein ubiquitination but also protein neddylation.

Figure 5. RNF111-dependent neddylation regulates RNF168-dependent ubiquitination during DNA damage response.

Figure 5

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(A) Protein neddylation regulates foci formation of Ub and ubH2A. U2OS cells were treated with 10 Gy IR. Foci formation of Ub was examined by FK2 monoclonal antibody. Data are represented as mean +/− SEM. (B) Protein neddylation regulates foci formation of RNF168 but not that of RNF8. IR-induced RNF8 and RNF168 foci were examined using indicated antibodies. Data are represented as mean +/− SEM. (C) MIU motifs of RNF168 direct bind NEDD8 in vitro. GST-MIU1, GST-MIU1A179G, GST-MIU2, or GST-MIU2A450G was incubated with His-NEDD8 or His-Ubiquitin respectively, and binding was analyzed by Western blot with anti-His antibodies. (D) MIU motifs of RNF168 interact with neddylated H4.SFB - H4 and HA-NEDD8 were co-expressed in 293T cells. The chromatin fraction was extracted and incubated with GST-MIU1, GST-MIU1A179G, GST-MIU2, or GST-MIU2A450G. Interactions were analyzed by GST pull down and Western blot with anti-Flag antibody. (E) RNF111 depletion reduces chromatin-bound RNF168. U2OS cells were treated with control or RNF111 siRNA. The status of RNF168 in both NETN100 soluble fraction and chromatin fraction was analyzed by IP and western blot with indicated antibodies. Chromatin-bound RNF168 was compared with histone H4 and evaluated by Glyko Bandscan software. (F) RNF111 depletion abolishes the IRIF of RNF168 in RNF8 −/− cells. RNF111 was depleted by siRNA, and RNF168 foci were observed after 10 Gy of IR treatment. The percentage of RNF111 foci positive cells were summarized in the right graph. Data are represented as mean +/− SEM. See also Figure S6, S7, S8 and S9.

To examine whether polyneddylation signals also target RNF168 to DNA damage sites for Ub cascade, we used siRNA to down-regulate RNF111 and its-associated DNA damage-induced protein neddylation, and found that chromatin-bound RNF168 was reduced by this treatment (Figure 5E). Although RNF8 is upstream of RNF168 during the DNA damage-induced Ub cascade, we still found that RNF168 formed foci in a fraction of RNF8 null cells (Figure 5F). With RNF111 siRNA treatment, these RNF168 foci were diminished whereas the MDC1 foci remained intact (Figure 5F, Figure S7, and S9). Thus, these results suggest that DNA damage-induced protein neddylation are recognized by RNF168, which accounts for targeting or retaining a fraction of RNF168 at DNA damage sites.

Histone neddylation is involved in DSB repair

The RNF168-dependent Ub cascade is required for the recruitment of other DNA damage repair factors, such as BRCA1 and 53BP1(Doil et al., 2009; Stewart et al., 2009). Consistently, down-regulation of DNA damage-induced neddylation also impaired the IRIF of BRCA1 and 53BP1 (Figure 6A and Figure S7). Again, the reconstitution of RNF111 siRNA treated cells with wild type RNF111 but neither the Ring domain deletion mutant nor the N-terminal deletion mutant of RNF111 restored the IRIF of BRCA1, 53BP1 and RNF168 (Figure S7). Likewise, the IRIF of Rad51(Huen et al., 2010), a downstream partner of BRCA1 during DNA damage repair, was abrogated (Figure 6B and Figure S9D). Of note, since we observe IRIF of RAD51 6 hours after IR treatments, most of the cells are stacked in S and G2 phase due to the activation of intra-S checkpoint. Thus, we observed the IRIF of RAD51 in more than 80 % control cells. Both BRCA1 and Rad51 are critical players during homologous recombination (HR) repair. Thus, we examined HR repair and found that the efficiency of HR repair was reduced, along with the down-regulation of the IRIF of BRCA1 and Rad51 in the absence of RNF111 (Figure 6C). Moreover, depleting RNF111 also induced slightly prolonged retention of γH2AX at DNA damage sites in G0/G1 cells, indicating that the NHEJ (non-homologous end joining) repair might also regulated by RNF111-dependent protein neddylation (Quennet et al., 2011) (Figure S10). Finally, with rendered DNA damage repair, cells depleted of RNF111 were hypersensitive to IR (Figure 6D). Although protein neddylation facilitates the recruitment of many DNA damage repair proteins to DNA damage sites, suppression of DNA damage-induced neddylation by knock-down RNF111 did not significantly affect the G2/M checkpoint (Figure S11A and B). It is possible that loss of NEDD8 pathway only partially affects foci formation of RNF168, 53BP1 and BRCA1, which is insufficient to attenuate ATM activation and its-dependent G2/M checkpoint. Thus, RNF111 was depleted in RNF8-deficient cells. Again, we found that lacking of RNF111 did not significantly impair the G2/M checkpoint activation in response to DNA damage (Figure S11C). Taken together, these results demonstrate that protein neddylation plays an important role for DNA damage repair.

Figure 6. Protein neddylation is important for DNA damage repair.

Figure 6

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(A, B) RNF111-dependent neddylation is important for IRIF of downstream DNA damage response factors. IRIF of BRCA1 (A) and Rad51 (B) was examined after cells were treated with RNF111 siRNA. The percentage of foci-positive cells is summarized. Data are represented as mean +/− SEM. (C) RNF111-dependent neddylation is important for HR repair. U2OS-DR-GFP cells were treated with indicated siRNAs. The siRNA-treated cells were then infected with adeno-I-SceI. HR efficiency was analyzed by FACS. The mean values are obtained from three independent experiments. Data are represented as mean +/− SEM. (D) Cells with impaired RNF111-dependent neddylation are hypersensitive to IR. U2OS cells transfected with control or RNF111 siRNA were treated with indicated dose of IR. Survival cell colonies were counted. The error bars represent the standard deviation. (E) A model of IR-induced H4 neddylation. See also Figure S10 and S11.

Discussion

In this study, we have demonstrated that protein neddylation is important for DNA damage response. In response to DNA damage, H2AX is phosphorylated by a group of PI3K-like kinases, including ATM, ATR and DNAPKc(Durocher and Jackson, 2001; Harper and Elledge, 2007). Phosphorylated H2AX stabilizes most DNA damage response factors at DNA lesions by recruiting MDC1 to DNA damage sites(Stucki and Jackson, 2006). MDC1 at DNA damage sites is also phosphorylated and recruits RNF8 and Ubc13 to initiate the Ub cascade(Huen et al., 2007; Ikura et al., 2007; Kolas et al., 2007; Mailand et al., 2007; Zhao et al., 2007). Other E3 ligases, such as RNF168, recognize the initial Ub signals set up by RNF8 and amplify the Ub cascade(Doil et al., 2009; Mattiroli et al., 2012; Stewart et al., 2009). Here, we found that neddylation signals at DNA damage sites also recruited RNF168 and facilitated the amplification of the Ub cascade in response to DNA damage. The amplified Ub cascade is important for stabilization of other DNA damage repair proteins at DNA damage sites in order to repair DNA lesions (Figure 6e).

It is intriguing that the MIU motifs of RNF168 directly bind to NEDD8. MIU belongs to a family of ubiquitin binding domains (UBDs) which contains at least 15 other members including UBA, UIM, DUIM, CUE, GAT, NZF, A20 ZnF, UBP ZnF, UBZ, Ubc, UEV, UBM, GLUE, Jab1/MPN and PFU(Dikic et al., 2009; Harper and Schulman, 2006). Most of these domains bind Ub with low affinity by interacting with a key residue, the Ile44 of Ub. Interestingly, this Ile reside is also present in NEDD8(Whitby et al., 1998). Thus, it is likely other UBDs may also bind NEDD8, and neddylation may cooperate with ubiquitination in other biological events besides DNA damage response.

Using unbiased approach, we have identified the E3 ligase, RNF111, which functions together with UBE2M to promote IR-induced histone H4 neddylation. Following DNA damage, RNF111 relocates to DNA damage sites together with UBE2M. Similar to the PIAS family, the SUMO E3 ligases (Galanty et al., 2009; Morris et al., 2009a), the relocation of RNF111 to DNA damage sites is dependent on its DNA-binding domain. The DNA-binding domain of RNF111 recognizes dsDNA. We did not observe that this DNA-binding domain could specifically recognize any DNA intermediate during DNA damage repair. Thus, it is likely that γH2AX-dependent chromatin remolding exposes the dsDNA at the vicinity of DNA lesions, which recruits RNF111 to neddylate histone H4 at the vicinity of DNA damage sites for the next step DNA damage repair.

DNA damage-induced H4 neddylation occurs on the H4 N-terminus containing six lysine residues. Neddylation can occur among any of these lysines, which is similar to other modifications at these sites. For example, acetylation of these lysines is also interchangeable(Dion et al., 2005). Structural analyses of nucleosomes indicate that the N-terminal tail of H4 interacts with the H2A/H2B dimer in the adjacent nucleosome(Luger et al., 1997). Compared to the size of H4, mono-NEDD8 is already a bulky modification in the nucleosome, while poly-NEDD8 chain is significantly larger that H4 itself. Thus, H4 neddylation may also drastically change chromatin orientation and break inter-nucleosome interactions, which could facilitate access of DNA damage response factors to DNA lesions.

In summary, we have identified a neddylation pathway during DNA damage response, which is another layer of regulation for DNA damage repair.

Experimental Procedures

Cell culture and treatment

293T, U2OS, and HCC1937 cells were cultured in RPMI 1640 medium with 10 % fetal bovine serum. Mouse embryo broblasts (MEFs) were cultured in Dulbecco Modified Eagle medium with 10 % fetal bovine serum. For IR treatment, cells were irradiated with the indicated doses using a JL Shepherd 137Cs radiation source. Cells were then recovered in normal culture condition for 4 hours or the indicated time. For UV treatment, cells were irradiated with the indicated dose using UV Stratalinker 1800. Cells were recovered for 4 hours and examined by immunofluorescence staining. For MMS (from Sigma) and Mitomycin C (MMC, from Sigma) treatments, drugs were administrated for 16 hours with indicated doses.

Laser micro-irradiation and imaging of cells

U2OS cells with or without transfection of indicated plasmids were plated on glass-bottomed culture dishes (Mat Tek Corporation). Laser micro-irradiation was performed using an IX 71 microscope (Olympus) coupled with the MicoPoint Laser Illumination and Ablation System (Photonic Instruments, Inc.). A 337.1 nm laser diode (3.4 mW) transmits through a specific Dye Cell and then yields 365 nm wavelength laser beam that is focused through ×60 UPlanSApo/1.35 oil objective to yield a spot size of 0.5–1 μm. The time of cell exposure to the laser beam was around 3.5 ns. The pulse energy is 170 μJ at 10 Hz. Images were taken by the same microscope with the CellSens software (Olympus).

Antibodies and other materials

Anti-RNF8, phospho-H2AX, BRCA1, Rad51 and 53BP1 were previously described(Huen et al., 2007; Ward et al., 2003; Yu et al., 2003). Rabbit anti-RNF168 antibodies were raised against N-terminus (aa: 1–300) and C-terminus (aa: 301–571) of RNF168. Anti-FK2 antibody was purchased from Millipore. Rabbit anti-NEDD8 antibody was purchased from Genetex. Rabbit anti-UBE2M antibody was purchased from BostonBiochem. Goat anti-UBE2F antibody was purchased from Abcam. Anti-ubH2A antibody and anti-histone H4 antibody (07–108) were purchased from Upstate. Anti-β-actin and FLAG (M2) antibodies were purchased from Sigma. Anti-HA antibody was purchased from Covance. Anti-RNF111 antibody was purchased from Abnova Taiwan Corporation. The siRNA duplexes were purchased from Dharmacon Research. All the siRNA sequences against UBE2M, UBE2F and RNF111 are listed in Table S1. siRNAs were transfected into cells using lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions, or transfected into MEF cells using electroporation (Genepulser Xcell™; BioRad).

Vector constructs

All primers for gene cloning and mutagenesis are listed in Supplementary Table S2 and S3. NEDD8, ISG15, FAU, UBL3, UBL4a, UBL4b, FAT10, ATG12, UBL5, URM1 and SUMO1 were PCR amplified and cloned into pCMV-HA (Clontech). UBE2F, UBE2M, RNF168, RNF111 and H4 were PCR amplified and cloned into pS-FLAG-SBP (SFB) vector. SFB-NEDD8 was sub-cloned from HA-NEDD8. HA-NEDD8ΔG mutant was generated by deletion of the Gly 76 in NEDD8 based on previous report(Kamitani et al., 1997). SFB-H4 K5R, K8R, K12R, K16R, K20R, K31R, K91R; and K5,8,12R (3R); K5,8,12,16R (4R); K5,8,12,16,20R (5R); K5,8,12,16,20,31R (6R) mutants were generated using QuikChange Site-Directed Mutagenesis Kit (Stratagene). SFB-H4 K5,8,12,16,20,31,44,59,78,80,91 only mutants were generated using QuikChange Site-Directed Mutagenesis Kit (Stratagene) based on SFB-H4 NOK mutant. GST-RNF168-MIU1 (168–191) and GST-RNF168-MIU2 (439–462) were PCR amplified and cloned into pGEX4T-1. Subsequent point mutations (alanine to glycine) to obtain MIU1A179G and MIU2A450G were introduced using QuikChange Site-Directed Mutagenesis Kit (Stratagene). The RNF111 siRNA-resistant mutant and the RING mutant (C942S/C945S) were generated using QuickChange Site-directed Mutagenesis Kit (Stratagene). The N-terminus deletion mutant of RNF111 (a.a. 544 – 994) was generated by PCR amplification.

Immunofluorescence staining

Cells grown on coverslips were fixed with 3 % paraformaldehyde for 20 minutes and permeabilized with 0.5 % Triton X-100 in PBS for 5 minutes at room temperature. Samples were blocked with 5 % goat serum in PBS and then incubated with primary antibody for 1 hour. Samples were washed three times and incubated with secondary antibody for 30 minutes. The coverslips were mounted onto glass slides and samples were visualized with a fluorescence microscope. For NEDD8, UBE2M, UBE2F and ubH2A staining, cells were pretreated in 0.5 % Triton X-100 for 5 minutes before fixation. To visualize IR-induced foci, cells were cultured on coverslips and treated with 10 Gy IR (1 Gy =100 rads), followed by recovery for 4 hours or indicated time. To visualize RAD51 foci, IR-treated cells were recovered for 6 hours. Presented data were collected from 1000 cells or 1000 positive transfectants. The mean values were obtained from three independent experiments.

Colony formation assay

500 cells were seeded in a 6-well plate immediately after irradiation. After incubation for 14 days in the regular cell culture medium, the surviving cell colonies were fixed, stained with crystal violet and counted. The number of colonies of the irradiated samples was compared with those of non-irradiated controls. The results were obtained from three independent experiments.

Homologous Recombination Assay

The assay was established previously(Weinstock et al., 2006). U2OS cells with a single copy of DR-GFP were transfected with control siRNA, UBE2M siRNA or UBE2F siRNA. siRNA-treated cells were infected with adenovirus-encoded I-SceI (adeno-I-SceI). Cells were harvested 3 days after infection and subjected to flow cytometry analysis. The GFP-positive cell population was measured. A set of raw data were shown. The mean values were obtained from three independent experiments. The experiment was performed under the control of equal adenovirus infection. Adenovirus infection efficiency was examined in U2OS cells prior to the HR assays. At MOI of 1000, the infection efficiency was close 100 % using control Adeno-GFP or Adeno-RFP. With the same MOI, we expect the same packaged Adeno-I-SceI expressed in almost all the cells. Each experiment has been performed at least three times. Little variation was observed from three independent experiments, suggesting the consistent adenovirus infection efficiency. Meanwhile, the cell viability was also examined under microscope and by trypan blue staining before adenovirus infection. All the groups showed more than >90% viability.

Immunoprecipitation, pull down assay and Western blot

293T or U2OS cells were lysed with NETN-100 buffer (20 mM Tris-HCl pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5 % Nonidet P-40) on ice. Soluble fractions were collected and the pellets were washed twice with PBS and once with H2O followed by treatment with 0.2 M HCl to release histones or chromatin-bound proteins, which were then neutralized with 1 M Tris-HCl pH 8.5. Both fractions were subjected to electrophoresis and Western blot, and probed with antibodies as indicated, or subjected to immunoprecipitation.

To search for DNA damage-induced needylation substrates, after 1 hour recovery from the IR treatment, 293T cells stably expressing SFB-NEDD8 were lysed with NETN-100. The soluble fraction is named as S1. The pellet was washed with PBS three times and incubated with NETN-300 buffer (20 mM Tris-HCl pH 8.0, 300 mM NaCl, 1 mM EDTA, 0.5 % Nonidet P-40). The eluted fraction was named as S2. The remained pellet was washed with H2O three times and incubated with 0.2 M HCl. The soluble fraction was neutralized with 1 M Tris-HCl pH 8.5 and named as S3.

GST fusion protein expression and pull-down assay

GST fusion proteins were expressed in Escherichia coli and purified under standard procedure. Purified GST-fusion proteins were immobilized on GST beads. 100 pmol purified His6-ubiquitin or His6-NEDD8 was incubated with GST fusion proteins for two hours at 4 °C. For pull down from cell extracts, 293T cells were lysed with NETN-100 buffer and the histone fraction was collected with 0.2 M HCl treatment. 100 μg histone extract with 30–40% nucleosomal histones were incubated with 10 pmol GST fusion protein at 4 °C for 2 hours. After washing with NETN-100 buffer four times, the samples were analyzed by Western blot.

Since SBP peptide has high affinity to strepavidin, strepavidin-conjugated beads were used for pull down SFB-tagged proteins.

Supplementary Material

01

Highlights.

  1. Histone neddylation is a DNA damage-induced epigenetic modification.

  2. Neddylation regulates DNA damage-induced ubiqutination cascade.

  3. Poly-neddylation occurs in response to DNA damage in vivo.

  4. Poly-neddylation can be recognized by an ubiquitin-binding motif.

Acknowledgments

We thank Jiaxue Wu, Lin-yu Lu, and Degui Wang and for technical support. We thank Henry Kuang and Chunjing Bian for proofreading the manuscript. This work was supported by the National Institute of Health (CA132755 and CA130899 to X.Y.). X.Y. is a recipient of the Era of Hope Scholar Award from the Department of Defense. T.M is a recipient of the Ann Schreiber Program Excellence Award from the Ovarian Cancer Research Fund.

Footnotes

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