A20/TNFAIP3 Regulates the DNA Damage Response and Mediates

Published OnlineFirst December 12, 2017; DOI: 10.1158/0008-5472.CAN-17-2143

Cancer Translational Science Research

A20/TNFAIP3 Regulates the DNA Damage Response and Mediates Tumor Cell Resistance to DNA-Damaging Therapy Chuanzhen Yang1,2, Weicheng Zang1,2, Zefang Tang3, Yapeng Ji1,2, Ruidan Xu1,2, Yongfeng Yang1,2, Aiping Luo4, Bin Hu1,2, Zemin Zhang3, Zhihua Liu4, and Xiaofeng Zheng1,2

Abstract

A competent DNA damage response (DDR) helps prevent cancer, after repair. Conversely, A20 deletion increased the efficiency of but once cancer has arisen, DDR can blunt the efficacy of chemo- error-prone nonhomologous DNA end-joining and decreased error- therapy and radiotherapy that cause lethal DNA breakage in cancer free DNA homologous recombination, destablizing the genome cells. Thus, blocking DDR may improve the efficacy of these modal- and increasing sensitivity to DNA damage. In clinical specimens of ities. Here, we report a new DDR mechanism that interfaces with invasive breast carcinoma, A20 was widely overexpressed, consistent inflammatory signaling and might be blocked to improve anticancer with its candidacy as a therapeutic target. Taken together, our outcomes. Specifically, we report that the ubiquitin-editing enzyme findings suggest that A20 is critical for proper functioning of the A20/TNFAIP3 binds and inhibits the E3 ubiquitin ligase RNF168, DDR in cancer cells and it establishes a new link between this NFkB- which is responsible for regulating histone H2A turnover critical for regulated ubiquitin-editing enzyme and the DDR pathway. proper DNA repair. A20 induced after DNA damage disrupted Significance: This study identifies the ubiquitin-editing enzyme RNF168–H2A interaction in a manner independent of its enzymatic A20 as a key factor in mediating cancer cell resistance to DNA- activity. Furthermore, it inhibited accumulation of RNF168 and damaging therapy, with implications for blocking its function to downstream repair protein 53BP1 during DNA repair. A20 was also leverage the efficacy of chemotherapy and radiotherapy. Cancer Res; required for disassembly of RNF168 and 53BP1 from damage sites 78(4); 1069–82. 2017 AACR.

Introduction to kinase signaling, ubiquitination also plays an important role in the DDR. E3 ligase RNF8 is recruited by phosphorylated Mammalian cells are exposed to various physical and chem- MDC1 and ubiquitinates histone H1 (8). Next, another E3 ical agents that induce DNA damage. A single cell is likely to ligase, RNF168, is recruited to catalyze monoubiquitination of encounter tens of thousands of DNA lesions per day (1, 2). H2A and H2AX at Lys13 and Lys15, which initiates the sub- DNA double strand breaks (DSB) are among the most danger- sequent formation of a lysine 63–linked polyubiquitin chain. ous types of DNA damage, and unrepaired or incorrectly The ubiquitin signaling catalyzed by the RNF8/RNF168 cascade repaired DSBs lead to genome instability, cancer, and aging promotes recruitment of downstream repair proteins such as (3, 4). To maintain genomic integrity, cells have evolved a set of 53BP1 (9, 10). Error-free homologous recombination (HR) and complex signaling cascades known as the DNA damage error-prone nonhomologous end-joining (NHEJ) are the major response (DDR; ref. 5). In response to DSBs, checkpoint kinase pathways to repair DSBs (1, 3). 53BP1 is a crucial effector that ATM (Ataxia Telangiectasia Mutated) phosphorylates H2AX promotes DSB repair through NHEJ (3, 11). NHEJ is important (also designated as gH2AX) near the damage sites, leading to for maintaining genome stability; however, overuse of NHEJ for recruitment and phosphorylation of MDC1 (6, 7). In addition repair leads to chromosomal translocation and genome instability (12, 13). Therefore, a proper cellular response to DNA 1State Key Laboratory of Protein and Plant Gene Research, School of Life damage is crucial for the maintenance of normal cell function. Sciences, Peking University, Beijing, China. 2Department of Biochemistry and For instance, defective DNA repair results in a human immu- Molecular Biology, School of Life Sciences, Peking University, Beijing, China. nodeficiency disorder called RIDDLE (radio sensitivity, immu- 3Biodynamic Optical Imaging Center, School of Life Sciences, Peking University, nodeficiency dysmorphic features, and learning difficulties) 4 Beijing, China. State Key Laboratory of Molecular Oncology, Cancer Institute syndrome and enhanced DNA repair capacity renders cancer and Hospital, Chinese Academy of Medical Sciences and Peking Union Medical cells resistant to radiotherapy and chemotherapy (14, 15). This College, Beijing, China. connection reveals the importanceofdelicateregulationofthe Note: Supplementary data for this article are available at Cancer Research DDR at DSBs. Online (http://cancerres.aacrjournals.org/). In addition to E3 ligases, deubiquitinating enzymes (DUB) Corresponding Author: Xiaofeng Zheng, Department of Biochemistry and Molec- also participate in the DDR pathway through regulating H2A ular Biology, School of Life Sciences, Peking University, Beijing 100871, China. ubiquitination. The human genome encodes approximately Phone: 8610-6275-5712; Fax: 86-10-62757924; E-mail: [email protected] 100 DUBs, which are divided into five families: UCH (ubiqui- doi: 10.1158/0008-5472.CAN-17-2143 tin C-terminal hydrolases), USP (ubiquitin specificproteases), 2017 American Association for Cancer Research. OTU (ovarian tumor proteases), Josephin, and JAMM (JAB1/

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MPN/Mov34 metalloenzyme). So far, most of the identified of Cell Line Resource in 2014. The identities of all cell DUBs that antagonize DSB-induced H2A ubiquitination lines were authenticated by short tandem repeat analysis in belong to the USP family. USP3 and USP44 abolish accumu- 2016. Cell lines were tested for mycoplasma contamination lation of RNF168 and 53BP1 at DNA damage sites (16, 17). by PCR. All cell lines were passaged for fewer than 2 months Recently, USP51 was demonstrated to specifically deubiquiti- after resuscitation and were used at the fourth through nate H2A at Lys13 and Lys15 and fine-tune the DDR (18). twelfth passage in culture for this study. The cell lines were USP16 deubiquitinates H2A at Lys 119 and represses gene cultured in DMEM (Gibco) supplemented with 10% FBS transcription (19). OTUB1 is the only reported deubiquitinat- (Gibco). HEK293T cells were transfected with PEI according ing enzyme in the OTU family that inhibits RNF168-mediated to the manufacturer's instructions (Polyscience). HeLa H2A ubiquitination. Independent of its DUB catalytic activity, cells were transfected with X-tremeGENE HP DNA Transfec- OTUB1 antagonizes H2A ubiquitination via direct binding tion Reagent according to the manufacturer's instructions to and inhibition of E2 UBC13 (20). Although recent studies (Sigma). have revealed the importance of deubiquitinating enzymes in tightly controlling histone ubiquitination during the DDR, it His-ubiquitin pull-down assay remains unclear whether other DUBs from the OTU family can HEK293T cells were transfected with his-ubiquitin and the regulate DSB-induced H2A ubiquitination and the DDR. indicated plasmids. Cells were harvested 48 hours after transfec- TNFAIP3/A20, a member of the OTU deubiquitinase family, tion. His-ubiquitin pull-down assays were performed following a is a primary protein expressed in human venous endothelial method described in a previous study (27). cells in response to TNF, IL1, and LPS. Recent studies reveal that A20 is also expressed in other cell types in response to Mononucleosome purification stimuli such as H O and TPA (21). The most well-studied 2 2 Mononucleosome purification was performed as described function of A20 is negative regulation of inflammation and previously (28) with the following modifications: anti-Flag M2 immunity (22). In mice, knocking out A20 results in severe beads binding with mononucleosomes were washed three times inflammation and cachexia, followed by death two weeks after with washing buffer (10 mmol/L HEPES-KOH, pH 7.5; 1 mmol/L birth (23). Moreover, several studies have identified somatic EDTA; 10 mmol/L KCl; 10% glycerol; protease inhibitors). Mono- mutations, deletion, and aberrant expression of the TNFAIP3/ nucleosomes were eluted using 400 mg/mL Flag peptide for 2 A20 gene in various kinds of tumors (24–26). These studies hours at 4C. reveal the importance of fully exploring the functions of A20 and its connection with tumors. Here, we identify A20/TNFAIP3 as a negative regulator of Acid chromatin fractionation RNF168-mediated ubiquitination of H2A Lys13 and Lys15 Preparation of chromatin fractions was carried out following a (H2AK13, 15ub). We find that NFkB is activated in response to procedure described in a previous study (29). Cell pellets were DNA damage and binds to the A20 promoter, leading to upre- resuspended in NP-40 lysis buffer and incubated at 4 C for 30 gulation of A20 expression. Subsequently, more A20 binds to minutes and nuclei were collected and resuspended in 0.2 mol/L chromatin and regulates the DDR. Deletion of A20 increases the HCl. The soluble fraction was neutralized with 1 mol/L Tris-HCl persistence of RNF168 and 53BP1 foci at DNA damage sites and (pH 8.0). genome instability. Importantly, A20 is often upregulated in invasive breast carcinomas, and knockout of A20 increases the IR treatment sensitivity of cancer cells to radiotherapy and chemotherapy, IR treatment was performed following procedures described suggesting that A20 is a potential target for cancer therapy. previously (30). After irradiation at 10 Gy, cells were incubated at 37C for indicated time. Materials and Methods fl Antibodies, reagents, and plasmids Immuno uorescence microscopy The antibodies and reagents used in this study were listed in the HeLa cells were transfected with the indicated plasmids using Supplementary Methods. OTUD3, OTUD5, and OTUD6B cDNAs PEI and treated with 10 Gy IR. At 24 hours after transfection, cells fi were kindly provided by Dr. Lingqiang Zhang at the Beijing were collected and xed in precooled methanol for 8 minutes at À Institute of Radiation Medicine. Human wild-type TNFAIP3 20 C following a procedure described previously (30). Images (A20) and mutants, H2A-K118,119R mutant, RNF168 and dele- were obtained using a confocal microscope (Zeiss LSM-710 NLO Â Â tion mutants, TAX1BP1, ITCH, and RNF11 were inserted into the and DuoScan) using a 40 or 63 oil objective lens. Quanti- fi 3Flag-pcDNA vector. A20, RNF8, and RNF168 were inserted into cation analysis was performed using Imaris 7.6 software the 3Myc-pcDNA vector. A20-1-370 (A20-N) and A20-440-790 (Bitplane). (A20-C) were amplified by PCR and cloned into the pET-28a vector. Human RNF168 and deletion mutants RNF168-1-249 and RT-PCR and quantitative real-time PCR RNF168-249-571 were cloned into the pCMV-3HA vector. MCF7 cells were treated with 40 mmol/L etoposide (VP16) at RNF168-1-249 was cloned into the pGEX-4T-1 vector. All expres- different time points. Total RNA was extracted using TRIzol sion plasmids were verified by DNA sequencing. Reagent (Invitrogen) and subjected to reverse transcription to synthesize cDNA using the FastQuant RT Kit (TIANGEN). The Cell culture and transfection primers used for the target genes are shown in Supplementary HeLa, MCF7, and U2OS cells were purchased from ATCC Table S1. Quantitative real-time PCR was performed using Fast and HEK293T was acquired from the National Infrastructure Start Essential DNA Green Master (Roche).

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A20 Regulates the DNA Damage Response

Cell fractionation assay (Supplementary Table S2) and cloned into a lentiviral sgRNA The cell fractionation assay was performed as described vector containing the mCherry selection marker using the previously (30) with modifications. Briefly, cells were lysed in Golden Gate method (33). Cells cotransfected with the sgRNA buffer A on ice for 30 minutes. The supernatant was ultracen- vector and a Cas9 vector were selected by FACS (MOFLO, trifuged and collected as the cytosolic fraction. Cell pellets were Cytomation). Single clones were obtained after 10 days of washed in buffer A and resuspended in NP-40 lysis buffer as selection. Knockout efficiency was confirmed by immunoblot- nuclei samples. ting. Mutations in the RNF168 and A20 genes were verified by PCR and sequencing. Chromatin immunoprecipitation Chromatin immunoprecipitation (ChIP) was performed fol- A20 linear donor construction and knockout HeLa cell lowing procedures described previously (31) with modifica- selection tions. MCF7 cells (1 Â 107)weretreatedwithDMSOor The linear donor was constructed following procedures etoposide (VP16) for 1 hour before cross-linked. The input described previously (34) using primers containing sgRNA- DNA and DNA from ChIP complexes were purified using the targeting regions (Supplementary Table S2) and protection QIAquick PCR Purification Kit, and analyzed by quantitative sequences. HeLa cells were transfected with the purified A20 real-time PCR. The primers used for the A20 promoter are linear donor, A20 pgRNA, and Cas9. Two weeks after transfec- shown in Supplementary Table S1. The detailed method was tion, cells were treated with 1 mg/mL puromycin to obtain described in Supplementary information. puromycin-resistant single clones. Knockout efficiency was confirmed by immunoblotting. A20 mutations were verified by PCR Chromatin extraction assay and sequencing. Cells were lysed in chromatin extraction buffer A (10 mmol/L PIPES, pH 6.8; 100 mmol/L NaCl; 300 mmol/L sucrose; 3 mmol/L Neutral comet assay MgCl2; 1 mmol/L EGTA; 0.2% Triton X-100) on ice for 30 Neutral comet assays were performed using the Trevigen Comet Â minutes and centrifuged at 3,000 g for 5 minutes. The super- Assay kit (Trevigen). Images were obtained using a fluorescence natant was removed, and the cell pellets were lysed in chromatin microscope (Olympus I Â73) with a 10Â objective lens. Quan- extraction buffer B (3 mmol/L EDTA, 0.2 mmol/L EGTA, tification was performed using Casp Lab software v1.2.2 (Uni- Â 1 mmol/L DTT) and centrifuged at 3,000 g for 5 minutes. versity of Wroclaw, Wroclaw, Poland). Approximately 100 cells The supernatant was completely removed, and the sediment was were analyzed in each group. resuspended in buffer C (50 mmol/L Tris, pH 8.0; 150 mmol/L NaCl; 1 mmol/L EDTA; 0.1% SDS; 1% Triton X-100) and NHEJ assay and HR assay denatured with 2Â SDS loading buffer. NHEJ assays were performed following a procedure described À À previously (35). For HR assays, A20WT and A20 / cells were Laser microirradiation cotransfected with DR-GFP, an I-SceI expression vector, and a Laser microirradiation was carried out following procedures pCherry plasmid. At indicated time after transfection (36 hours described previously (32). U2OS cells were grown on thin glass- for NHEJ assays and 48 hours for HR assays), cells were harvested bottom plates and irradiated with an ultraviolet laser (16 Hz and washed with 1Â PBS. Green (EGFP) and red (Cherry) fluo- pulse, 41% laser output). Images were taken using a Nikon A1 rescence was measured by FACS on an LSRFortessa instrument confocal imaging system every 30 seconds for 10 minutes. (BD Biosciences). The percentage of EGFP and pCherry double positive cells versus the percentage of pCherry-positive cells was fi in vitro Protein puri cation and assays taken as the repair efficiency. The results are normalized to those fi Recombinant proteins were puri ed and in vitro assays were of the A20WT cells. performed following previously described procedures (20). For in vitro pull-down assays, GST-fusion proteins were incubated with Ethics statement and tissue specimens His-tagged A20 in PBS buffer at 4 C for 1 hour. The beads were The study was approved by the Ethics Committee of the washed with PBS buffer and boiled with 2Â SDS loading buffer, Chinese Academy of Medical Sciences and Peking University's followed by immunoblotting. For in vitro ubiquitination assays, Ethics Committee. Written informed consent was obtained from 0.0125 mmol/L UBE1 (E-305, Boston Biochem), 0.4 mmol/L each individual based on the Declaration of Helsinki. Specimens UBC13/UEV1a (E2-664, Boston Biochem), 40 mmol/L ubiquitin from 60 breast invasive ductal carcinomas and 23 samples of (U-100H, Boston Biochem), 50 mmol/L Tris-HCl (pH 8.0), 5 adjacent normal tissue were analyzed. None of the patients had mmol/L MgCl2, 2 mmol/L ATP, and 1 mmol/L DTT were incu- received radiotherapy or chemotherapy before surgery. Clinical bated with recombinant OTUB1 or A20 for 16 hours at 37 C. specimens were obtained at the time of surgery. The specimens were immediately fixed in 4% polyformaldehyde and completely Coimmunoprecipitation embedded in paraffin. Cell lysate preparation, immunoprecipitation, and immunoblotting were performed as described previously (30). Tissue microarray and IHC Tissue microarrays (10 mm tissue cores for each tissue) were À À À À Generation of RNF168 / and A20 / HEK293T cells by constructed. IHC staining was carried out following the standard conventional CRISPR-Cas9 system streptavidin–biotin–peroxidase complex method. Tissue micro- To generate a vector expressing pgRNA, two single-guide arrays were treated as described previously (36), followed by RNAs (sgRNAs 1–2) targeting different regions in the first exon incubation with primary antibodies (anti-A20) overnight at 4C of the human TNFAIP3/A20 and RNF168 genes were designed in a humid chamber (1:50 dilution). For the negative controls, the

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primary antibody was replaced by nonimmune serum. After foci independently of these three proteins (Supplementary Fig. immunostaining, the sections were scanned by a single investi- S1C). These data demonstrate that A20 negatively regulates gator who was not informed of their clinical characteristics. The 53BP1 accumulation at DNA damage sites. value of the integral intensity was measured by Aperio Image Scope software (Aperio). NFkB binds to the A20 promoter and upregulates A20 upon induction of the DDR Statistical analysis Previous studies have demonstrated that A20 is transcrip- The experiments were repeated at least three times. Statistical tionally upregulated by NFkB (p50/p65 dimer) after treatment analysis was performed using Student t test (two-tailed) or one- with TNF, IL1, LPS, or other stimuli (21). However, little way ANOVA. All results are presented as mean Æ SEM unless attention has been paid to whether A20 senses DNA damage, otherwise stated. (Ã, P < 0.05; ÃÃ, P < 0.01; ÃÃÃ, P < 0.001). especially DNA DSBs. To investigate the capacity of A20 to Additional methods used in this study are described in the sense DNA damage, we measured the abundance of A20 mRNA Supplementary Material. and protein in MCF7 cells treated with VP16 by quantitative real-time PCR and Western blot analyses, respectively. A20 transcription was upregulated after VP16 treatment (Fig. 2A). Results In contrast, the level of OTUB1 showed no obvious change A20 inhibits DNA damage–induced H2AK13,15ub and (Supplementary Fig. S2). In addition, increased abundance of 53BP1 focal accumulation A20 protein and decreased protein abundance of IkBa,an To identify new negative regulators of H2A ubiquitination NFkB inhibitor, were observed (Fig. 2B, lane 1–4). To clarify upon DNA damage, we explored the effects of various whether DNA damage–induced upregulation of A20 transcrip- DUBs from the OTU deubiquitinase family on H2A ubiquiti- tion is dependent on NFkB, cells were treated with NFkB nation. HEK293T cells expressing his-ubiquitin and the indi- inhibitor PDTC before VP16 treatment. In the presence of cated DUBs were treated with etoposide (VP16) to trigger PDTC, A20 expression was no longer increased, and IkBa was DSBs, after which his-tagged ubiquitinated proteins were not degraded after VP16 treatment (Fig. 2B). Moreover, both enriched through in vivo his-ubiquitin pull-down. Among the fractionation and immunofluorescence assays showed that DUBs detected, a dramatic decrease in H2A ubiquitination was VP16 treatment induced translocation of NFkB subunit p65 observed with overexpression of A20 or positive control from the cytosol to the nucleus (Fig. 2C and D), and PDTC OTUB1 (Fig. 1A). Histone H2A can be ubiquitinated at inhibited translocation of p65 and upregulation of A20 (Sup- Lys118,119 or Lys13,15, and H2AK13,15ub induced by DNA plementary Fig. S3A). We also found that ATM kinase inhib- damage triggers the recruitment of downstream repair proteins itor Ku55933 blocked translocation of p65 (Supplementary (10, 18). We therefore tested whether A20 inhibited DNA Fig. S3B), suggesting that NFkB activation upon DNA damage is damage–induced H2AK13,15ub. We first purified H2A ATM-dependent. Next, we performed ChIP-qPCR to determine K118,119R-containing mononucleosomes using cells expres- whether NFkB binds to the A20 promoter and induces aug- sing a Flag-H2A (K118,119R) mutant, and examined the effect mented expression of A20 in response to DSBs. Indeed, VP16 of A20 on H2AK13,15ub. Overexpression of A20 inhibited induced binding of p65 to the A20 promoter (Fig. 2E), indi- DNA damage–induced H2AK13,15ub (Fig. 1B). Furthermore, cating that p65 upregulates A20 expression at the transcrip- a recently generated H2AK15ub-specific antibody that recog- tional level in response to DSBs. Furthermore, to examine nizes monoubiquitinated H2A at Lys 15 was used to assess the whether A20 associates with chromatin in response to DNA inhibitory effect of A20 on H2AK15ub. HEK293T cells were damage, cells were treated with VP16 for the indicated time treated with ionizing radiation (IR) and subjected to histone periods, after which the chromatin fraction was extracted. acid extraction. H2AK15ub abundance increased in response Consistent with the results showninFig.2B,A20protein to IR, but this effect was significantly inhibited by A20 over- abundance increased in the whole-cell lysates. More impor- expression (Fig. 1C). Immunofluorescence assays were also tantly, a more obvious time-dependent increase in chromatin- performed using IR-treated HeLa cells to confirm this obser- bound A20, but not TAX1BP1, ITCH, or RNF11, was observed vation. Consistently, A20 suppressed IR-induced H2AK15ub after VP16 treatment (Fig. 2F; Supplementary Fig. S1D). Fur- (Fig. 1D). These observations suggest that A20 is a negative thermore, we examined the subcellular location of endogenous regulator of H2AK15ub. A20, which revealed that DNA damage promoted expression H2A/H2AX ubiquitination is a crucial step in the DDR, and and nuclear localization of A20 (Fig. 2G). These observations DDR protein 53BP1 is recruited to DNA damage sites by drove us to investigate whether A20 is recruited to DSB sites. We recognizing H2AK15ub (9, 10). As A20 is a negative regulator observed moderate accumulation of A20 at DSBs by perform- of H2AK15ub, we detected the effect of A20 on IR-induced ing laser microirradiation assays (Fig. 2H). This phenomenon 53BP1 accumulation at DNA damage sites by performing could be a result of the transient presence of A20 at damage immunofluorescence assays. A20 significantly abrogated IR- sites, which is in accordance with observations regarding induced 53BP1 foci (Fig. 1E) to a degree similar to that of USP16 and OTUB2 (38, 39). Taken together, these results positive control OTUB1, whereas CYLD did not (Supplemen- suggest that NFkB-induced expression of A20 is involved in tary Fig. S1A). Moreover, it has been reported that A20 nega- the DDR. tively regulates NFkB signaling in a complex with TAX1BP1, ITCH,andRNF11(37).Thus,weassessed whether these three A20 affects the DDR independently of its DUB catalytic activity subunits are also involved in regulation of the DDR. In contrast A20 contains an N-terminal OTU domain and seven zinc with A20, TAX1BP1, ITCH, and RNF11 did not affect 53BP1 finger (ZnF) domains in the C-terminus mediating the inter- foci (Supplementary Fig. S1B); moreover, A20 inhibited 53BP1 action between A20 and its substrates/partners (40). A catalytic

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A20 Regulates the DNA Damage Response

Figure 1. A20 inhibits H2AK13,15ub and 53BP1 focal accumulation upon DNA damage. A, HEK293T cells transfected with his-ubiquitin and the indicated DUBs were treated with 40 mmol/L VP16 for 2 hours, after which his-tagged ubiquitinated proteins were enriched by his-ubiquitin pull-down assay and analyzed by immunoblotting using an anti-H2A antibody. WCL, whole-cell lysate. B, HEK293T cells expressing Flag-H2A K118,119R were transfected with Myc-empty vector or Myc-A20. After 48 hours, cells were treated with 40 mmol/L VP16 for 2 hours, after which mononucleosomes were extracted and analyzed by immunoblotting using an anti-Flag antibody. Ã,nonspeciﬁcbands.C, HEK293T cells transfected with Flag-empty vector or Flag-A20 were treated with or without IR (25 Gy). Histones were extracted by acid chromatin fractionation assay after 1 hour of incubation. The level of endogenous H2A ubiquitination at lysine 15 was detected using an anti-H2AK15ub antibody. D and E, HeLa cells transfected with Flag-empty vector or Flag-A20 were treated with or without IR (10 Gy). One hour later, immunoﬂuorescence assays were performed using antibodies against Flag and H2AK15ub (D)orFlagand 53BP1 (E).Thepercentageofcellswith10 H2AK15ub foci or 15 53BP1 foci are shown. Data are shown as the mean Æ SEM of three independent experiments. Statistical analysis was performed using Student t test (ÃÃ, P < 0.01; ÃÃÃ, P < 0.001). Scale bar, 10 mm. Approximately 200 cells in each group were counted.

triad (Asp70, Cys103, and His256) within the OTU domain of we generated a series of A20 point mutation constructs, includ- A20 is responsible for its deubiquitinating activity, among ing D70A, C103A, H256A, and the catalytic triad mutant D70A/ which Cys103 is a critical residue and forms a region respon- C103A/H256A (designated as 3A; Fig. 3A). HEK293T cells sible for interacting with other proteins (21). In addition, expressing the Flag-H2A (K118,119R) mutant and A20 point zinc ﬁnger 4 of A20 binds ubiquitin and possesses E3 ligase mutants were treated with VP16 and subjected to chromatin activity (41). To detect whether the inhibitory effect of A20 on extraction. VP16 treatment induced an increase in H2AK13, H2A ubiquitination is dependent on its DUB catalytic activity, 15ub, and, interestingly, all of the tested mutants showed a

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Figure 2. p65 upregulates A20 expression in response to DNA damage. A, MCF7 cells were treated with 40 mmol/L VP16 for the indicated time periods. The mRNA level of A20 was analyzed by qRT-PCR. Data are shown as the mean Æ SEM of three independent experiments. Statistical analysis was performed using Student t test (ÃÃÃ, P < 0.001). B, MCF7 cells were treated with DMSO or 300 nmol/L of NFkB inhibitor PDTC before VP16 treatment. Whole- cell lysates were analyzed by immunoblotting using the indicated antibodies. C, MCF7 cells were treated with 40 mmol/L VP16 for the indicated time periods, after which cytoplasmic and nuclear fractions were extracted and analyzed by immunoblotting using the indicated antibodies. D, MCF7 cells were treated with DMSO or 20 mmol/L VP16 for 1 hour, after which immunofluorescence assays were performed using an anti-p65 antibody. Scale bar, 10 mm. E, MCF7 cells were treated with DMSO or 40 mmol/L VP16 for 1 hour, after which ChIP assays were performed to detect the recruitment of p65 to the A20 promoter using an anti-p65 antibody. IgG served as a negative control. The A20 promoter sequences in the input DNA and DNA from ChIP complexes were detected by quantitative PCR. The results were normalized using the IgG abundance of cells treated with DMSO. The results are shown as the mean Æ SEM of three experimental replicates. Statistical analysis was performed using Student t test (ÃÃÃ, P < 0.001). F, MCF7 cells were treated with 40 mmol/L VP16 for the indicated time periods, after which chromatin was isolated and analyzed using the indicated antibodies. The intensity of A20 was normalized against that of H3. G, MCF7 cells were treated with or without IR (10 Gy) and after 24 hours of incubation, immunofluorescence assays were performed using antibodies against A20. Quantification analysis was performed using Volocity software. Statistical analysis was performed using Student t test (ÃÃÃ, P < 0.001). About 100 cells were counted in each group. H, U2OS cells transfected with GFP-A20 were subjected to a laser microirradiation assay. Accumulation of GFP-A20 was detected by fluorescent microscopy at different time points. The red line indicates the positions for laser microirradiation.

decrease in H2AK13,15ub (Fig. 3B). Moreover, the results of deubiquitination (Supplementary Fig. S4D). Moreover, the the immunofluorescence assays showed that A20-C103A and results in Supplementary Fig. S4E and Fig. 3D show that only A20-3A mutants inhibited 53BP1 foci accumulation as effi- A20 mutant 3A-DZnF4–7, lacking ZnFs 4–7 and the catalytic ciently as did wild-type A20 (Fig. 3C and D). triad, revealed complete abolition of the normal inhibitory To explore the defective functionality of mutant A20, we effect of A20 on H2A ubiquitination and accumulation of made N-terminal or C-terminal truncation mutants of A20 53BP1 foci. À À and tested their effects on H2A ubiquitination. Surprisingly, Next, we constructed A20 / HeLa cells using a recently both the N-terminal and C-terminal were critical for inhibi- reported linear donor insertion system (Fig. 3E; ref. 34) and tion of H2A ubiquitination by A20 (Supplementary Fig. S4A). performed immunofluorescence assays to examine the effect of À À Next, we constructed more mutants with altered N-terminal A20 deletion on the DDR. The results showed that A20 / cells and C-terminal regions (Supplementary Fig. S4B and S4C). contained more H2AK15ub and 53BP1 foci than did wild-type The catalytic triad mutant in the N-terminal, A20 N-3A, lost its cells, and reintroducing wild-type A20 inhibited foci accumula- inhibitory effect on H2A ubiquitination. However, the mutant tion significantly, while A20-deficient mutant 3A-DZnF4–7 lacking ZnF4 in the C-terminus did not completely lose its showed no effect (Fig. 3F and G). Together, these observations function (Supplementary Fig. S4B and S4C). Assessments indicate that zinc fingers 4–7 and the integrity of the OTU domain, using additional constructed mutants showed that, in addi- rather than deubiquitinating activity, are crucial for the negative tion to ZnF4, ZnF5 and ZnF7 were also responsible for H2A regulatory effect of A20 on the DDR.

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A20 Regulates the DNA Damage Response

Figure 3. A20 affects the DNA damage response independently of its DUB catalytic activity. A, Schematic of A20 point mutants. B, HEK293T cells expressing the Flag-H2A (K118,119R) mutant were transfected with the indicated A20 mutants. At 48 hours after transfection, cells were treated with 40 mmol/L VP16 for 2 hours and subjected to chromatin extraction. Ã,nonspecificbands.C, Schematic for the A20 point and deletion mutants. D, HeLa cells were transfected with different A20 mutants. At 24 hours after transfection, cells were irradiated at 10 Gy and incubated for 1 hour. Immunofluorescence assays were performed using the indicated antibodies. Immunofluorescence images and the percentage of cells with 15 53BP1 foci are shown. The results are shown as the mean Æ SEM of three independent experiments. Statistical analysis was performed using Student t test (ÃÃÃ, P < 0.001). Scale bar, 10 mm. Approximately 200 cells in each group were counted. E, The partial coding sequences of human A20 exon 1 and the sequencing results for the mutated alleles of A20À/À clone1areshown.Knockoutefficiency was determined using an anti-A20 antibody. F and G, A20WT, A20À/À HeLa cells (clone 1), and A20À/À cells, to which the A20 or A20 mutant were reintroduced, were treated with IR (10 Gy). Immunofluorescence assays were performed at the indicated time points after DNA damage (12 hours for H2AK15ub and 24 hours for 53BP1). The percentages of cells with 10 H2AK15ub foci or 15 53BP1 foci are shown. Data are shown as the mean Æ SEM of three independent experiments. Statistical analysis was performed using Student t test (ÃÃÃ, P < 0.001). Scale bar, 10 mm. Approximately 200 cells in each group were counted. n.s., nonsignificant.

À À A20 does not affect UBC13 stability in response to DNA damage A20 / HeLa cells were treated with or without IR (10 Gy), It has been reported that A20 interacts with UBC13 (E2) and harvested at the indicated times, and subjected to measurement mediates the degradation of UBC13 upon stimulation by TNF, of UBC13 abundance. A20 deletion did not affect the stability IL1, and LPS (42). As RNF8–UBC13 complex-catalyzed H1 of UBC13 following IR treatment (Fig. 4C). Furthermore, to ubiquitination is important for recruitment of RNF168 and investigate whether A20 inhibits the E2 activity of UBC13 as activation of downstream repair signaling (8), we assessed OTUB1 does, we performed in vitro ubiquitination assays and whether A20-triggered UBC13 degradation followed VP16 found that A20 did not affect UBC13-dependent ubiquitina- treatment for the indicated time periods. We did not observe tion (Fig. 4D; Supplementary Fig. S5A). These results suggest degradation of UBC13 associated with the DNA damage– that UBC13 stability is not affected by A20 in response to DNA induced increase in A20 protein abundance (Fig. 4A). Next, damage. we treated cells with DMSO or proteasome inhibitor MG132 before VP16 treatment and prolonged the VP16 exposure time A20 directly interacts with RNF168 and abrogates RNF168 to 240 minutes. However, no degradation of UBC13 was accumulation at DNA damage sites observed in cells exposed to VP16 (Fig. 4B). Moreover, to As A20 does not affect the stability of UBC13, we next deter- explorethedirecteffectofA20onUBC13stability,A20WT and mined whether A20 inhibits H2A ubiquitination by interacting

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Figure 4. A20 does not affect UBC13 stability in response to DNA damage. A, MCF7 cells were treated with 40 mmol/L VP16 for the indicated time periods. Whole-cell lysates were analyzed by immunoblotting using the indicated antibodies. B, MCF7 cells were treated with DMSO or 20 mmol/L MG132 before VP16 treatment, after which whole-cell lysates were analyzed by immunoblotting. C,A20WT and A20À/À HeLa cells (clone 1) were treated with or without IR (10 Gy) and harvested at the indicated time. Whole-cell lysates were analyzed by immunoblotting using the indicated antibodies. D,In vitro ubiquitination assays were performed using combinations of UBE1, UBC13/UEV1a, and ubiquitin with recombinant OTUB1 or A20. The reaction mixtures were analyzed by immunoblotting using an anti-ubiquitin antibody.

with the E3 ligases responsible for H2A ubiquitination upon DNA The RING domain of RNF168 is important for recognizing damage, with the goal of elucidating the mechanism underlying its target H2A (Supplementary Fig. S6B and S6C; ref. 43), the effect of A20 on H2A ubiquitination. HEK293T cells were while the UDM1 domain is responsible for identification of transfected with Flag-A20 and E3 ligases RNF8 or RNF168, and ubiquitinated H1 (8). As both the RING and UDM1 domains coimmunoprecipitation (co-IP) analysis was performed. A20 of RNF168 are essential for its binding to A20 (Fig. 5E), we interacted only with RNF168, which catalyzes H2A and H2AX speculated that A20 might disrupt the binding of RNF168 to monoubiquitination at Lys13 and Lys15 (Fig. 5A). Moreover, we H2A and ubiquitinated H1. Indeed, co-IP assays showed that confirmed the endogenous interaction between A20 and RNF168, A20deletionpromotedtheRNF168–H2A interaction, which and found that IR- or VP16-induced DNA damage enhanced this was inhibited by reexpression of wild-type A20, but not by interaction (Fig. 5B), which is dependent on ATM (Supplemen- reexpression of the A20-deficient mutant (Fig. 5G; Supplemen- tary Fig. S3C). We also found that wild-type A20, but not A20- tary Fig. S7A). In addition, A20 disrupted the interaction deficient mutant 3A-DZnF4–7, interacted with RNF168 (Fig. 5C). between RNF168 and ubiquitinated H1 (Fig. 5H), but it did To map the critical domain of RNF168 responsible for its not affect H1 ubiquitination (Supplementary Fig. S8). More- interaction with A20, we constructed truncations, including over, we determined whether A20 abrogated the accumulation RNF168 1–249 and RNF168 249–571, and assessed their inter- of RNF168 at DNA damage sites. Immunofluorescence assays actions with Flag-A20. The N-terminus, rather than the C-termi- showed that A20 significantly reduced the number of IR- nus, of RNF168 was essential for its binding to A20 (Fig. 5D). induced RNF168 foci (Fig. 5I). To exclude the possibility that Furthermore, the co-IP results showed that both the RING and the A20 might affect upstream regulators of RNF168, we examined UDM1 domain at the N-terminus of RNF168 are important for its the effect of A20 on MDC1 and RNF8 foci by immunofluo- interaction with A20 (Fig. 5E; Supplementary Fig. S6A). Moreover, rescence assays. The results showed that A20 did not affect the results of in vitro pull-down assays using purified proteins MDC1 and RNF8 foci (Fig. 5J; Supplementary Fig. S9). Overall, (Supplementary Fig. S5B) demonstrated that RNF168 is a direct these results suggest that A20 regulates the DDR by directly target of A20, while both the N-terminus and C-terminus of A20 binding to RNF168 and impeding accumulation of RNF168 at bind to RNF168 (Fig. 5F). damage sites.

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Figure 5. A20 directly interacts with RNF168 and abrogates the accumulation of RNF168 at DNA damage sites. A, HEK293T cells transfected with Flag-A20 and the indicated Myc-tagged E3 ligases were subjectedtoco-IPusingananti-Mycantibody.B, HeLa cells were treated with VP16 (40 mmol/L) for 2 hours or IR (10 Gy), followed by 1 hour of incubation. Co-IP assays were then performed to examine the endogenous interaction between A20 and RNF168. C and D, HEK293T cells transfected with different plasmids were subjected to co-IP assays with the indicated antibodies. E, HEK293T cells transfected with Flag-RNF168 wild-type or mutants were treated with VP16 (40 mmol/L) for 2 hours before they were subjected to a co-IP assay using an anti-Flag antibody. F, The N-terminus (1–370 aa) and C-terminus (440–790 aa) of His-A20 and GST-RNF168 (1–249 aa) were purified from E. coli. In vitro pull-down analysis was performed with GST protein as a negative control. G,A20WT and A20À/À HEK293T cells were transfected with the indicated plasmids. At 48 hours after transfection, cells were treated with VP16 for 2 hours, followed by co-IP assays with the indicated antibodies. H, HEK293T cells transfected with HA-RNF168 (1–249 aa) with or without Myc-A20 were treated with VP16 for 2 hours and then subjected to a co-IP assay. I, HeLa cells transfected with the indicated plasmids were treated with or without IR (10 Gy) and subjected to immunofluorescence assays after 1 hour of incubation. Immunofluorescence images and the percentage of cells with 10 RNF168 foci are shown. The results are shown as the mean Æ SEM of three experimental replicates. Statistical analysis was performed using Student t test (ÃÃÃ, P < 0.001). Scale bar, 10 mm. Approximately 200 cells in each group were counted. J, HeLa cells transfected with the indicated plasmids were irradiated at 10 Gy. Immunofluorescence assays were performed using antibodies against Flag/MDC1 or Flag/RNF8 after 1-hour incubation. The percentage of cells with 10 MDC1 and RNF8 foci are shown. Data are shown as the mean Æ SEM of three independent experiments. Statistical analysis was performed using Student t test. Scale bar, 10 mm. Approximately 200 cells in each group were counted. n.s., nonsignificant.

Deletion of A20 results in persistent accumulation of DNA wild-type cells (Fig. 6A and B). This phenomenon is in accor- damage foci dance with previous reports that DDR proteins are recruited To further elucidate the effect of A20 on the dynamic regu- dramatically during the early phase of DNA damage to repair lation of DNA damage foci, the abundance of RNF168 and DNA lesions (18, 38). Consistently, the abundance of A20 was À À 53BP1 foci in A20WT and A20 / HeLa cells was assessed at slightly increased at 1 hour after IR treatment (Fig. 4C), sug- different time points following IR treatment. Knockout of A20 gesting that A20 moderately regulates IR-induced DNA damage increased the abundance of chromatin-bound RNF168 and foci during the early period of DNA repair (1 hour after IR). À À 53BP1 under normal conditions (Fig. 6A and B). The numbers Strikingly, at 24 hours after IR treatment, A20 / cells con- of RNF168 and 53BP1 foci were increased signiﬁcantly in wild- tained more RNF168 and 53BP1 foci than did wild-type À À À À type and A20 / cells 1 hour after IR treatment, and A20 / cells (Fig. 6A and B). In accordance with this observation, after cells contained slightly more RNF168 and 53BP1 foci than did DNA lesions were repaired (24 hours after IR treatment), the

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Figure 6. Deletion of A20 results in persistent accumulation of DNA damage foci. A and B,A20WT and A20À/À HeLa cells were treated with or without IR (10 Gy). Immunofluorescence assays were performed at the indicated time points after DNA damage. Immunofluorescence images and the percentages of cells with 10 RNF168 foci or 15 53BP1 foci from three independent experiments are shown. Data are shown as mean Æ SEM. Statistical analysis was performed using Student t test (Ã, P < 0.05; ÃÃ, P < 0.01; ÃÃÃ, P < 0.001). Approximately 400 cells in each group were counted. C and D, The partial sequences of human RNF168 exon 1 and the sequencing results for the mutated alleles are shown. Knockout efficiency was assessed using an anti-RNF168 antibody. E,RNF168WT and RNF168À/À HEK293T cells transfected with or without Flag-A20 were treated with VP16 for 2 hours and then subjected to an acid chromatin fractionation assay. The level of endogenous H2A ubiquitination was detected using an anti-H2A antibody. F,RNF168WT and RNF168À/À HEK293T cells transfected with or without Flag-A20 were irradiated at 10 Gy and subjected to a chromatin extraction assay.

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abundance of A20 protein increased significantly(Fig.4C),and genome instability (15). Thus, we performed neutral comet knockout of A20 resulted in RNF168 and 53BP1 retention at assays to investigate the effect of A20 on genome stability, which damage sites (Fig. 6A and B). Taken together, these results showed that A20 deletion slightly increased comet tail length À À indicate that A20 fine-tunes DNA damage–induced foci during under normal conditions. Moreover, A20 / cells possessed the early phase of the DDR and is essential for the disassembly more obvious comet tails than did wild-type cells at 24 hours of 53BP1 at DNA damage sites after repair. after IR treatment (Fig. 7B), suggesting that the loss of A20 resulted Moreover, to investigate whether the inhibitory effect of A20 in impaired DNA repair kinetics and increased genome instability. À À on DDR is dependent on RNF168, we generated RNF168 / These results suggest that A20 plays an important role in guaran- 293T cells using a CRISPR-Cas9 system (Fig. 6C and D; ref. 33) teeing proper DNA repair. and compared the level of H2A ubiquitination upon DNA In addition, to explore the effect of A20 on cell survival after À À damage in RNF168WT and RNF168 / cells with or without DNA damage, a clonogenic survival analysis was performed. À À À À A20. The results in RNF168 / cells showed that H2A ubi- A20WT and A20 / cells (Fig. 3E; Supplementary Fig. S7B) were quitination decreased obviously, while A20 was unable to treated with different doses of IR and allowed to grow for 12 days, À À inhibit H2A ubiquitination (Fig. 6E). In addition, RNF168WT after which the number of colonies was counted. A20 / cells À À and RNF168 / 293T cells transfected with or without Flag- were more sensitive to IR treatment than were wild-type cells A20 were irradiated at 10 Gy and subjected to chromatin (Fig. 7C). In addition, expression of wild-type A20 but not A20- À À separation. Deletion of RNF168 impeded recruitment of deficent mutant in A20 / cells rescued cell viability (Fig. 7D). À À 53BP1 to chromatin, which was consistent with a published Similarly, in response to VP16 treatment, A20 / cells also À À report (10); moreover, A20 no longer functioned in RNF168 / showed reduced survival in comparison with wild-type cells cells (Fig. 6F). These results suggest that A20 relies upon RNF168 (Supplementary Fig. S10A and S10B). Moreover, overexpression to finely regulate the DDR. of A20 rendered cancer cells resistant to IR (Fig. 7E) and VP16 treatment (Supplementary Fig. S10C), suggesting that increased A20 knockout cells exhibit increased sensitivity to ionizing A20 conferred resistance to DNA damage therapy in cancer cells. radiation and DNA-damaging agents These results suggest that A20 is required for cell survival follow- Next, we determined whether A20 affects HR and NHEJ effi- ing DNA damage. ciency by performing reporter assays. We found that A20 deletion resulted in decreased HR efficiency and increased NHEJ efficiency A20 is highly expressed in breast carcinoma (Fig. 7A). NHEJ is an error-prone repair mechanism with a Radiotherapy and chemotherapy resistance are obstacles of tendency to produce chromosome translocation, leading to cancer treatments. As increased A20 could confer resistance to

Figure 7. Loss of A20 expression sensitizes cancer cells to ionizing radiation. A,A20WT and A20À/À cells were subjected to HR and NHEJ assays. The experiments were performed three times. The results were normalized to those of the A20 wild-type cells. Data are shown as mean Æ SEM. Statistical analysis was À À performed using Student t test (Ã, P < 0.05). B,A20WT and A20 / HeLa cells were subjected to the neutral comet assay. About 100 cells were counted in each group. Images and quantified data are shown. Statistical analysis was performed using Student t test (Ã, P < 0.05; ÃÃÃ, P < 0.001). n.s., nonsignificant. C–E, Cells were treated with the indicated IR doses and subjected to clonogenic survival assays. Endogenously and exogenously expressed A20 were confirmed by immunoblotting using an anti-A20 antibody. Statistical analysis was performed using Student t test (ÃÃ, P < 0.01; ÃÃÃ, P < 0.001). F, A model for the role of A20 in the DDR. In response to DNA damage, A20 is upregulated by NFkB and binds to chromatin, where it terminates H2A ubiquitination by disrupting the binding of RNF168 to H2A and ubiquitinated H1, thereby reducing the accumulation of RNF168 and facilitating disassembly of 53BP1 at DNA damage sites.

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IR- and DNA-damaging agents, we wanted to understand the (Fig. 4). Instead, we showed that A20 affects the DDR through biological relevance of A20 in cancer and to explore whether it direct interaction with RNF168 and attenuates the accumulation can serve as a therapeutic target. First, we analyzed A20 expres- of RNF168 at DNA damage sites, which is independent of sion in specimens from 60 breast invasive ductal carcinomas TAX1BP1, ITCH, or RNF11. These studies suggest that A20 utilizes and 23 samples of adjacent normal tissue by performing tissue different mechanisms to function in different pathways. microarrays and IHC. We found that A20 expression was Moreover, we demonstrated that the integrity of the A20 OTU significantly higher in tumor tissue in comparison with its domain and ZnFs 4–7, rather than its deubiquitinating activity, expression level in adjacent normal breast tissue (Supplemen- are important for its effect on the DDR. This observation is tary Fig. S11A and S11B). Moreover, we used a gene expression similar to previous studies, in which A20 has been shown to dataset (GSE70905) from the NCBI-GEO for differential bind target proteins either through the conserved surface patch expression analysis between the tumor group and adjacent formed by its catalytic triad (21) or via its seven zinc finger normal group, which showed that A20 was highly expressed domains (40). In addition to directly removing the ubiquitin in tumor tissue in comparison with its expression level in chain from its target, A20 also affects ubiquitination in an adjacent normal tissue (Supplementary Fig. S11C). Analysis indirect manner. For example, the OTU and ZnF4 domains of of dataset GSE65194 from the NCBI-GEO confirmed this A20 are important for its inhibitory effect on E3 ligase activity, conclusion (Supplementary Fig. S11D). A20 expression in which is accomplished by blocking the interaction between E2 breast cancer patients with different histologic grades was also and E3 enzymes (42). Other studies also demonstrate that the analyzed using the breast cancer dataset from the work by OTU domain collaborates with ZnF4 and ZnF7 at the C-termi- Calda and colleagues (44, 45). The results of this analysis nus of A20 to negatively regulate NFkB activation (47, 48). showed that A20 expression was increased significantly in Therefore, further research should focus on studying the struc- high-grade breast cancers relativetothatofthelow-gradegroup ture of full length A20 to explain the coordination between its (Supplementary Fig. S11E). These results demonstrate that A20 OTU domain and zinc finger domains. is highly expressed in breast carcinomas, particularly in patients Recently, several studies have shown that a number of DUBs afflicted with high-grade tumors. in the USP family may target H2AK15ub and thus affect the DDR, but these DUBs may function in different stages of the DDR. For instance, USP51 directly targets ubiquitinated H2A Discussion on K13 and K15 and modulates the DDR (18). USP3 and This study reveals that chromatin-bound A20 plays an USP44 affect recruitment of RNF168 at DNA damage sites and important role in connecting NFkB signaling and the DDR. inhibit DNA damage–induced H2A ubiquitination (16, 17). In Specifically, we show that, in response to DSBs, NFkBtranslo- this study, we demonstrated that A20 inhibited H2AK13,15ub cates from the cytosol to the nucleus and binds to the A20 and regulated the DDR. Independently of its deubiquitinase promoter. Once A20 is transcriptionally upregulated, more A20 activity, A20 directly interacts with RNF168 and disrupts the binds to chromatin, where it directly binds to RNF168 and binding of RNF168 to H2A and ubiquitinated H1. Unlike other disrupts the binding of RNF168 to H2A and ubiquitinated H1, deubiquitinating enzymes, the abundance of A20 is regulated thereby inhibiting accumulation of RNF168 at DNA damage by NFkB in response to DNA damage. A20 is significantly sites. Thus, A20 inhibits RNF168-mediated H2AK13,15ub and upregulated at 12 and 24 hours after IR treatment (Fig. 4C), impairs accumulation of downstream repair proteins at DNA suggesting that it mainly functions at the late stage of DNA damage sites (Fig. 7F). repair. Therefore, we conclude that A20 is essential for the Previous studies have shown that A20 exerts an anti-inflam- disassembly of RNF168 foci after DNA repair to avoid hyper- matory effect by downregulating NFkB signaling (22). In addi- accumulation of RNF168 at DNA damage sites and prevent tion, A20 is an NFkB-responsive gene upon various types of excessive ubiquitination. Loss of A20 leads to increased NHEJ stimulation (21). The role of A20 in the cytoplasm is well activity and decreased HR, which may be a result of persistent characterized, but little is known about its function in the nucleus. 53BP1 accumulation at DSB sites and disruption of the balance Although DNA damage can trigger NFkB activation, the conse- among DNA repair pathways, thereby contributing to impaired quences of NFkB activation for the DDR have not been elucidated. DNA repair kinetics and increased sensitivity of cancer cells to Here, we demonstrated that A20 expression is induced by NFkB DNA damage. These observations reveal the importance of an when DSBs occur. Moreover, we showed that, in the nucleus, A20 appropriate DNA damage response and repair process. inhibits RNF168-mediated H2AK13,15ub and accumulation of Interestingly, we also found that A20 deletion resulted in repair protein 53BP1 at DNA damage sites. This study reveals a persistent BRCA1 accumulation at DNA damage sites (Supple- connection between NFkB signaling and the DDR. mentary Fig. S12). However, A20 deletion decreased HR effi- It has been shown that OTUB1 inhibits RNF168-dependent ciency. Although these observations may seem contradictory, H2A ubiquitination and suppresses the DDR. OTUB1 binds to they can be explained by results from other studies. RAP80 has and inhibits E2 UBC13 independently of its DUB catalytic activity been shown to recognize RNF168-generated K63–ubiquitin (20). Interestingly, here we found that A20 negatively regulated chains and recruits the BRCA1-A complex (2, 49). A model H2A ubiquitination in a DUB activity–independent manner has been proposed in which BRCA1 functions together with (Fig. 3B). The noncatalytic role of DUBs such as Ubp6 has also RAP80 in the BRCA1-A complex to reduce HR by restricting been observed (46). In addition, it has been shown that, together DSB end processing, while it promotes resection when inter- with TAX1BP1, A20 interacts with UBC13 and triggers ubiquitin– acting with other complexes (50, 51). Moreover, other groups proteasome degradation in response to TNF, IL1, and LPS stim- have reported that accumulation of 53BP1 at DSBs promotes ulation (42). Distinct from this mechanism, here we found that NHEJ while suppressing HR (39), and RNF168 inhibits HR A20 does not affect UBC13 stability in response to DNA DSBs similar to 53BP1 (52). Accordingly, the results in this study

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suggest that A20 may affect the DNA repair pathway choice by Disclosure of Potential Conflicts of Interest modulating DNA end resection. No potential conflicts of interest were disclosed. A20 has been reported to be a crucial regulator in many types of cancer. It plays an oncogenic role in some solid tu- Authors' Contributions mors including gliomas (26), hepatocellular carcinoma (53), Conception and design: C. Yang, X. Zheng poorly differentiated head and neck cancers, and undiffer- Development of methodology: C. Yang, W. Zang, Y. Ji, Z. Liu entiated nasopharyngeal carcinoma (54). Meanwhile, other Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): C. Yang, W. Zang, Z. Tang, Y. Ji, R. Xu, Y. Yang, studies have revealed that A20 is also a tumor suppressor that A. Luo, B. Hu, Z. Liu is frequently deleted and inactivated in B-cell lymphoma, Analysis and interpretation of data (e.g., statistical analysis, biostatistics, Hodgkin lymphomas, and non-Hodgkin lymphomas (24). computational analysis): C. Yang, W. Zang, Z. Tang, Z. Zhang, Z. Liu, X. Zheng These findings suggest that the function of A20 in tumors is Writing, review, and/or revision of the manuscript: C. Yang, Z. Zhang, cell-type–dependent. In our study, we found that the abun- X. Zheng dance of A20 protein is upregulated in invasive breast carci- Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): C. Yang, X. Zheng nomas (Supplementary Fig. S11A and S11B), which is in Study supervision: X. Zheng accordance with a previous study showing that the mRNA level of A20 is higher in more aggressive breast cancers (25), as Acknowledgments well as another recently published article (55). Moreover, This work was supported by the National Science Foundation of China À/À A20 cells are more sensitive to IR and VP16 treatment than (81730080, 31470754, 31670786) and the National Key Research and are wild-type cells, whereas wild-type cells with A20 over- Development Program of China (2016YFC1302401). We sincerely thank expression show resistance to IR and VP16 (Fig. 7C–E; Sup- Prof. Lingqiang Zhang for providing DUB plasmids, Prof. Wensheng Wei plementary Fig. S10). These findings indicate that A20 influ- for providing CRISPR/Cas9-related plasmids, Dr. Qinzhi Xu for assistance ences chemotherapy and radiation resistance, suggesting the with neutral comet assays, Prof. Huadong Pei for providing the HR and NHEJ systems, and Prof. Xingzhi Xu for helping with the laser microirradia- potential of A20 as a target in breast cancer treatment. tion assays. We also appreciate the assistance of Xiaochen Li, Guopeng In summary, our finding that A20 functions in the nucleus as Wang, Liying Du, and Hongxia Lv from the Core Facilities of Life Sciences at an inhibitor of DNA damage-induced H2A ubiquitination Peking University for their assistance with microscopic imaging and cell provides new insights into the connection between NFkB flow cytometry. signaling and the DDR. Our results indicate that A20 regulates the DDR by inhibiting the binding of RNF168 to H2A and The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked ubiquitinated H1, thereby playing an important role in guaran- advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate teeing proper DNA repair and maintaining genome stability. this fact. A20 might be a promising clinical target for new strategies to prevent resistance to conventional radiotherapy and chemo- Received July 18, 2017; revised November 2, 2017; accepted December 1, therapy in breast cancer. 2017; published OnlineFirst December 12, 2017.

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1082 Cancer Res; 78(4) February 15, 2018 Cancer Research

A20/TNFAIP3 Regulates the DNA Damage Response and Mediates Tumor Cell Resistance to DNA-Damaging Therapy

Chuanzhen Yang, Weicheng Zang, Zefang Tang, et al.

Cancer Res 2018;78:1069-1082. Published OnlineFirst December 12, 2017.

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