A20/TNFAIP3 Regulates the DNA Damage Response and … · Cell culture and transfection HeLa, MCF7, and U2OS cells were purchased from ATCC and HEK293T was acquired from the National

Translational Science

A20/TNFAIP3 Regulates the DNA DamageResponse and Mediates Tumor Cell Resistance toDNA-Damaging TherapyChuanzhen 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 competentDNAdamage response (DDR)helps prevent cancer,but once cancer has arisen, DDR can blunt the efficacy of chemo-therapy and radiotherapy that cause lethal DNA breakage in cancercells. Thus, blockingDDRmay improve the efficacy of thesemodal-ities. Here, we report a new DDR mechanism that interfaces withinflammatory signalingandmightbeblockedto improveanticanceroutcomes. Specifically, we report that the ubiquitin-editing enzymeA20/TNFAIP3 binds and inhibits the E3 ubiquitin ligase RNF168,which is responsible for regulating histoneH2A turnover critical forproper DNA repair. A20 induced after DNA damage disruptedRNF168–H2A interaction in amanner independent of its enzymaticactivity. Furthermore, it inhibited accumulation of RNF168 anddownstream repair protein 53BP1 duringDNA repair. A20was alsorequired for disassembly of RNF168 and 53BP1 from damage sites

after repair. Conversely, A20 deletion increased the efficiency oferror-pronenonhomologousDNAend-joininganddecreasederror-free DNA homologous recombination, destablizing the genomeand increasing sensitivity to DNA damage. In clinical specimens ofinvasivebreast carcinoma,A20waswidelyoverexpressed, consistentwith its candidacy as a therapeutic target. Taken together, ourfindings suggest that A20 is critical for proper functioning of theDDR in cancer cells and it establishes a new linkbetween thisNFkB-regulated ubiquitin-editing enzyme and the DDR pathway.

Significance: This study identifies the ubiquitin-editing enzymeA20 as a key factor in mediating cancer cell resistance to DNA-damaging therapy, with implications for blocking its function toleverage the efficacy of chemotherapy and radiotherapy. Cancer Res;78(4); 1069–82. �2017 AACR.

IntroductionMammalian cells are exposed to various physical and chem-

ical agents that induce DNA damage. A single cell is likely toencounter tens of thousands of DNA lesions per day (1, 2).DNA double strand breaks (DSB) are among the most danger-ous types of DNA damage, and unrepaired or incorrectlyrepaired DSBs lead to genome instability, cancer, and aging(3, 4). To maintain genomic integrity, cells have evolved a set ofcomplex signaling cascades known as the DNA damageresponse (DDR; ref. 5). In response to DSBs, checkpoint kinaseATM (Ataxia Telangiectasia Mutated) phosphorylates H2AX(also designated as gH2AX) near the damage sites, leading torecruitment and phosphorylation of MDC1 (6, 7). In addition

to kinase signaling, ubiquitination also plays an important rolein the DDR. E3 ligase RNF8 is recruited by phosphorylatedMDC1 and ubiquitinates histone H1 (8). Next, another E3ligase, RNF168, is recruited to catalyze monoubiquitination ofH2A and H2AX at Lys13 and Lys15, which initiates the sub-sequent formation of a lysine 63–linked polyubiquitin chain.The ubiquitin signaling catalyzed by the RNF8/RNF168 cascadepromotes recruitment of downstream repair proteins such as53BP1 (9, 10). Error-free homologous recombination (HR) anderror-prone nonhomologous end-joining (NHEJ) are the majorpathways to repair DSBs (1, 3). 53BP1 is a crucial effector thatpromotes DSB repair through NHEJ (3, 11). NHEJ is importantfor maintaining genome stability; however, overuse of NHEJ forrepair leads to chromosomal translocation and genome insta-bility (12, 13). Therefore, a proper cellular response to DNAdamage is crucial for the maintenance of normal cell function.For instance, defective DNA repair results in a human immu-nodeficiency disorder called RIDDLE (radio sensitivity, immu-nodeficiency dysmorphic features, and learning difficulties)syndrome and enhanced DNA repair capacity renders cancercells resistant to radiotherapy and chemotherapy (14, 15). Thisconnection reveals the importance of delicate regulation of theDDR at DSBs.

In addition to E3 ligases, deubiquitinating enzymes (DUB)also participate in the DDR pathway through regulating H2Aubiquitination. The human genome encodes approximately100 DUBs, which are divided into five families: UCH (ubiqui-tin C-terminal hydrolases), USP (ubiquitin specific proteases),OTU (ovarian tumor proteases), Josephin, and JAMM (JAB1/

1State Key Laboratory of Protein and Plant Gene Research, School of LifeSciences, Peking University, Beijing, China. 2Department of Biochemistry andMolecular Biology, School of Life Sciences, Peking University, Beijing, China.3Biodynamic Optical Imaging Center, School of Life Sciences, Peking University,Beijing, China. 4State Key Laboratory of Molecular Oncology, Cancer Instituteand Hospital, Chinese Academy of Medical Sciences and Peking Union MedicalCollege, Beijing, China.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Corresponding Author:Xiaofeng Zheng, Department of Biochemistry and Molec-ular Biology, School of Life Sciences, Peking University, Beijing 100871, China.Phone: 8610-6275-5712; Fax: 86-10-62757924; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-17-2143

�2017 American Association for Cancer Research.

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MPN/Mov34 metalloenzyme). So far, most of the identifiedDUBs that antagonize DSB-induced H2A ubiquitinationbelong to the USP family. USP3 and USP44 abolish accumu-lation of RNF168 and 53BP1 at DNA damage sites (16, 17).Recently, USP51 was demonstrated to specifically deubiquiti-nate H2A at Lys13 and Lys15 and fine-tune the DDR (18).USP16 deubiquitinates H2A at Lys 119 and represses genetranscription (19). OTUB1 is the only reported deubiquitinat-ing enzyme in the OTU family that inhibits RNF168-mediatedH2A ubiquitination. Independent of its DUB catalytic activity,OTUB1 antagonizes H2A ubiquitination via direct bindingto and inhibition of E2 UBC13 (20). Although recent studieshave revealed the importance of deubiquitinating enzymes intightly controlling histone ubiquitination during the DDR, itremains unclear whether other DUBs from the OTU family canregulate DSB-induced H2A ubiquitination and the DDR.

TNFAIP3/A20, a member of the OTU deubiquitinase family,is a primary protein expressed in human venous endothelialcells in response to TNF, IL1, and LPS. Recent studies revealthat A20 is also expressed in other cell types in response tostimuli such as H2O2 and TPA (21). The most well-studiedfunction of A20 is negative regulation of inflammation andimmunity (22). In mice, knocking out A20 results in severeinflammation and cachexia, followed by death two weeks afterbirth (23). Moreover, several studies have identified somaticmutations, deletion, and aberrant expression of the TNFAIP3/A20 gene in various kinds of tumors (24–26). These studiesreveal the importance of fully exploring the functions of A20and its connection with tumors.

Here, we identify A20/TNFAIP3 as a negative regulator ofRNF168-mediated ubiquitination of H2A Lys13 and Lys15(H2AK13, 15ub). We find that NFkB is activated in response toDNA damage and binds to the A20 promoter, leading to upre-gulation of A20 expression. Subsequently, more A20 binds tochromatin and regulates the DDR. Deletion of A20 increases thepersistence of RNF168 and 53BP1 foci at DNA damage sites andgenome instability. Importantly, A20 is often upregulated ininvasive breast carcinomas, and knockout of A20 increases thesensitivity of cancer cells to radiotherapy and chemotherapy,suggesting that A20 is a potential target for cancer therapy.

Materials and MethodsAntibodies, reagents, and plasmids

The antibodies and reagents used in this studywere listed in theSupplementary Methods.OTUD3, OTUD5, andOTUD6B cDNAswere kindly provided by Dr. Lingqiang Zhang at the BeijingInstitute of Radiation Medicine. Human wild-type TNFAIP3(A20) and mutants, H2A-K118,119R mutant, RNF168 and dele-tion mutants, TAX1BP1, ITCH, and RNF11 were inserted into the3Flag-pcDNA vector. A20, RNF8, and RNF168 were inserted intothe 3Myc-pcDNA vector. A20-1-370 (A20-N) and A20-440-790(A20-C) were amplified by PCR and cloned into the pET-28avector. HumanRNF168 and deletionmutantsRNF168-1-249 andRNF168-249-571 were cloned into the pCMV-3HA vector.RNF168-1-249 was cloned into the pGEX-4T-1 vector. All expres-sion plasmids were verified by DNA sequencing.

Cell culture and transfectionHeLa, MCF7, and U2OS cells were purchased from ATCC

and HEK293T was acquired from the National Infrastructure

of Cell Line Resource in 2014. The identities of all celllines were authenticated by short tandem repeat analysis in2016. Cell lines were tested for mycoplasma contaminationby PCR. All cell lines were passaged for fewer than 2 monthsafter resuscitation and were used at the fourth throughtwelfth passage in culture for this study. The cell lines werecultured in DMEM (Gibco) supplemented with 10% FBS(Gibco). HEK293T cells were transfected with PEI accordingto the manufacturer's instructions (Polyscience). HeLacells were transfected with X-tremeGENE HP DNA Transfec-tion Reagent according to the manufacturer's instructions(Sigma).

His-ubiquitin pull-down assayHEK293T cells were transfected with his-ubiquitin and the

indicated plasmids. Cells were harvested 48 hours after transfec-tion. His-ubiquitin pull-down assays were performed following amethod described in a previous study (27).

Mononucleosome purificationMononucleosome purification was performed as described

previously (28) with the following modifications: anti-Flag M2beads binding with mononucleosomes were washed three timeswith washing buffer (10mmol/LHEPES-KOH, pH 7.5; 1mmol/LEDTA; 10mmol/L KCl; 10%glycerol; protease inhibitors). Mono-nucleosomes were eluted using 400 mg/mL Flag peptide for 2hours at 4�C.

Acid chromatin fractionationPreparation of chromatin fractions was carried out following a

procedure described in a previous study (29). Cell pellets wereresuspended in NP-40 lysis buffer and incubated at 4�C for 30minutes and nuclei were collected and resuspended in 0.2 mol/LHCl. The soluble fraction was neutralized with 1 mol/L Tris-HCl(pH 8.0).

IR treatmentIR treatment was performed following procedures described

previously (30). After irradiation at 10 Gy, cells were incubated at37�C for indicated time.

Immunofluorescence microscopyHeLa cells were transfected with the indicated plasmids using

PEI and treated with 10 Gy IR. At 24 hours after transfection, cellswere collected and fixed in precooled methanol for 8 minutes at�20�C following a procedure described previously (30). Imageswere obtained using a confocal microscope (Zeiss LSM-710 NLOand DuoScan) using a 40 � or 63 � oil objective lens. Quanti-fication analysis was performed using Imaris 7.6 software(Bitplane).

RT-PCR and quantitative real-time PCRMCF7 cells were treated with 40 mmol/L etoposide (VP16) at

different time points. Total RNA was extracted using TRIzolReagent (Invitrogen) and subjected to reverse transcription tosynthesize cDNA using the FastQuant RT Kit (TIANGEN). Theprimers used for the target genes are shown in SupplementaryTable S1. Quantitative real-time PCR was performed using FastStart Essential DNA Green Master (Roche).

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Cell fractionation assayThe cell fractionation assay was performed as described

previously (30) with modifications. Briefly, cells were lysed inbuffer A on ice for 30 minutes. The supernatant was ultracen-trifuged and collected as the cytosolic fraction. Cell pellets werewashed in buffer A and resuspended in NP-40 lysis buffer asnuclei samples.

Chromatin immunoprecipitationChromatin immunoprecipitation (ChIP) was performed fol-

lowing procedures described previously (31) with modifica-tions. MCF7 cells (1 � 107) were treated with DMSO oretoposide (VP16) for 1 hour before cross-linked. The inputDNA and DNA from ChIP complexes were purified using theQIAquick PCR Purification Kit, and analyzed by quantitativereal-time PCR. The primers used for the A20 promoter areshown in Supplementary Table S1. The detailed method wasdescribed in Supplementary information.

Chromatin extraction assayCells were lysed in chromatin extraction buffer A (10 mmol/L

PIPES, pH6.8; 100mmol/LNaCl; 300mmol/L sucrose; 3mmol/LMgCl2; 1 mmol/L EGTA; 0.2% Triton X-100) on ice for 30minutes and centrifuged at 3,000 � g for 5 minutes. The super-natant was removed, and the cell pellets were lysed in chromatinextraction buffer B (3 mmol/L EDTA, 0.2 mmol/L EGTA,1 mmol/L DTT) and centrifuged at 3,000 � g for 5 minutes.The supernatant was completely removed, and the sediment wasresuspended in buffer C (50 mmol/L Tris, pH 8.0; 150 mmol/LNaCl; 1 mmol/L EDTA; 0.1% SDS; 1% Triton X-100) anddenatured with 2� SDS loading buffer.

Laser microirradiationLaser microirradiation was carried out following procedures

described previously (32). U2OS cells were grown on thin glass-bottom plates and irradiated with an ultraviolet laser (16 Hzpulse, 41% laser output). Images were taken using a Nikon A1confocal imaging system every 30 seconds for 10 minutes.

Protein purification and in vitro assaysRecombinant proteins were purified and in vitro assays were

performed following previously described procedures (20). For invitro pull-down assays, GST-fusion proteins were incubated withHis-tagged A20 in PBS buffer at 4�C for 1 hour. The beads werewashed with PBS buffer and boiled with 2� SDS loading buffer,followed by immunoblotting. For in vitro ubiquitination assays,0.0125 mmol/L UBE1 (E-305, Boston Biochem), 0.4 mmol/LUBC13/UEV1a (E2-664, Boston Biochem), 40mmol/L ubiquitin(U-100H, Boston Biochem), 50 mmol/L Tris-HCl (pH 8.0), 5mmol/L MgCl2, 2 mmol/L ATP, and 1 mmol/L DTT were incu-bated with recombinant OTUB1 or A20 for 16 hours at 37�C.

CoimmunoprecipitationCell lysate preparation, immunoprecipitation, and immuno-

blotting were performed as described previously (30).

Generation of RNF168�/� and A20�/� HEK293T cells byconventional CRISPR-Cas9 system

To generate a vector expressing pgRNA, two single-guideRNAs (sgRNAs 1–2) targeting different regions in the first exonof the human TNFAIP3/A20 and RNF168 genes were designed

(Supplementary Table S2) and cloned into a lentiviral sgRNAvector containing the mCherry selection marker using theGolden Gate method (33). Cells cotransfected with the sgRNAvector and a Cas9 vector were selected by FACS (MOFLO,Cytomation). Single clones were obtained after 10 days ofselection. Knockout efficiency was confirmed by immunoblot-ting. Mutations in the RNF168 and A20 genes were verified byPCR and sequencing.

A20 linear donor construction and knockout HeLa cellselection

The linear donor was constructed following proceduresdescribed previously (34) using primers containing sgRNA-targeting regions (Supplementary Table S2) and protectionsequences. HeLa cells were transfected with the purified A20linear donor, A20 pgRNA, and Cas9. Two weeks after transfec-tion, cells were treated with 1 mg/mL puromycin to obtainpuromycin-resistant single clones. Knockout efficiency was con-firmed by immunoblotting. A20 mutations were verified by PCRand sequencing.

Neutral comet assayNeutral comet assayswere performedusing the TrevigenComet

Assay kit (Trevigen). Images were obtained using a fluorescencemicroscope (Olympus I �73) with a 10� objective lens. Quan-tification was performed using Casp Lab software v1.2.2 (Uni-versity of Wroclaw, Wroclaw, Poland). Approximately 100 cellswere analyzed in each group.

NHEJ assay and HR assayNHEJ assays were performed following a procedure described

previously (35). For HR assays, A20WT and A20�/� cells werecotransfected with DR-GFP, an I-SceI expression vector, and apCherry plasmid. At indicated time after transfection (36 hoursfor NHEJ assays and 48 hours for HR assays), cells were harvestedand washed with 1� PBS. Green (EGFP) and red (Cherry) fluo-rescence was measured by FACS on an LSRFortessa instrument(BD Biosciences). The percentage of EGFP and pCherry doublepositive cells versus the percentage of pCherry-positive cells wastaken as the repair efficiency. The results are normalized to thoseof the A20WT cells.

Ethics statement and tissue specimensThe study was approved by the Ethics Committee of the

Chinese Academy of Medical Sciences and Peking University'sEthics Committee. Written informed consent was obtained fromeach individual based on the Declaration of Helsinki. Specimensfrom 60 breast invasive ductal carcinomas and 23 samples ofadjacent normal tissue were analyzed. None of the patients hadreceived radiotherapy or chemotherapy before surgery. Clinicalspecimens were obtained at the time of surgery. The specimenswere immediately fixed in 4% polyformaldehyde and completelyembedded in paraffin.

Tissue microarray and IHCTissue microarrays (10 mm tissue cores for each tissue) were

constructed. IHC staining was carried out following the standardstreptavidin–biotin–peroxidase complex method. Tissue micro-arrays were treated as described previously (36), followed byincubation with primary antibodies (anti-A20) overnight at 4�Cin a humid chamber (1:50 dilution). For the negative controls, the

A20 Regulates the DNA Damage Response

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primary antibody was replaced by nonimmune serum. Afterimmunostaining, the sections were scanned by a single investi-gator who was not informed of their clinical characteristics. Thevalue of the integral intensity was measured by Aperio ImageScope software (Aperio).

Statistical analysisThe experiments were repeated at least three times. Statistical

analysis was performed using Student t test (two-tailed) or one-way ANOVA. All results are presented as mean � SEM unlessotherwise stated. (�, P < 0.05; ��, P < 0.01; ��, P < 0.001).

Additional methods used in this study are described in theSupplementary Material.

ResultsA20 inhibits DNA damage–induced H2AK13,15ub and53BP1 focal accumulation

To identify new negative regulators of H2A ubiquitinationupon DNA damage, we explored the effects of variousDUBs from the OTU deubiquitinase family on H2A ubiquiti-nation. HEK293T cells expressing his-ubiquitin and the indi-cated DUBs were treated with etoposide (VP16) to triggerDSBs, after which his-tagged ubiquitinated proteins wereenriched through in vivo his-ubiquitin pull-down. Among theDUBs detected, a dramatic decrease in H2A ubiquitination wasobserved with overexpression of A20 or positive controlOTUB1 (Fig. 1A). Histone H2A can be ubiquitinated atLys118,119 or Lys13,15, and H2AK13,15ub induced by DNAdamage triggers the recruitment of downstream repair proteins(10, 18). We therefore tested whether A20 inhibited DNAdamage–induced H2AK13,15ub. We first purified H2AK118,119R-containing mononucleosomes using cells expres-sing a Flag-H2A (K118,119R) mutant, and examined the effectof A20 on H2AK13,15ub. Overexpression of A20 inhibitedDNA damage–induced H2AK13,15ub (Fig. 1B). Furthermore,a recently generated H2AK15ub-specific antibody that recog-nizes monoubiquitinated H2A at Lys 15 was used to assess theinhibitory effect of A20 on H2AK15ub. HEK293T cells weretreated with ionizing radiation (IR) and subjected to histoneacid extraction. H2AK15ub abundance increased in responseto IR, but this effect was significantly inhibited by A20 over-expression (Fig. 1C). Immunofluorescence assays were alsoperformed using IR-treated HeLa cells to confirm this obser-vation. Consistently, A20 suppressed IR-induced H2AK15ub(Fig. 1D). These observations suggest that A20 is a negativeregulator of H2AK15ub.

H2A/H2AX ubiquitination is a crucial step in the DDR, andDDR protein 53BP1 is recruited to DNA damage sites byrecognizing H2AK15ub (9, 10). As A20 is a negative regulatorof H2AK15ub, we detected the effect of A20 on IR-induced53BP1 accumulation at DNA damage sites by performingimmunofluorescence assays. A20 significantly abrogated IR-induced 53BP1 foci (Fig. 1E) to a degree similar to that ofpositive control OTUB1, whereas CYLD did not (Supplemen-tary Fig. S1A). Moreover, it has been reported that A20 nega-tively regulates NFkB signaling in a complex with TAX1BP1,ITCH, and RNF11 (37). Thus, we assessed whether these threesubunits are also involved in regulation of the DDR. In contrastwith A20, TAX1BP1, ITCH, and RNF11 did not affect 53BP1foci (Supplementary Fig. S1B); moreover, A20 inhibited 53BP1

foci independently of these three proteins (Supplementary Fig.S1C). These data demonstrate that A20 negatively regulates53BP1 accumulation at DNA damage sites.

NFkB binds to the A20 promoter and upregulates A20 uponinduction of the DDR

Previous studies have demonstrated that A20 is transcrip-tionally upregulated by NFkB (p50/p65 dimer) after treatmentwith TNF, IL1, LPS, or other stimuli (21). However, littleattention has been paid to whether A20 senses DNA damage,especially DNA DSBs. To investigate the capacity of A20 tosense DNA damage, we measured the abundance of A20 mRNAand protein in MCF7 cells treated with VP16 by quantitativereal-time PCR and Western blot analyses, respectively. A20transcription was upregulated after VP16 treatment (Fig. 2A).In contrast, the level of OTUB1 showed no obvious change(Supplementary Fig. S2). In addition, increased abundance ofA20 protein and decreased protein abundance of IkBa, anNFkB inhibitor, were observed (Fig. 2B, lane 1–4). To clarifywhether DNA damage–induced upregulation of A20 transcrip-tion is dependent on NFkB, cells were treated with NFkBinhibitor PDTC before VP16 treatment. In the presence ofPDTC, A20 expression was no longer increased, and IkBa wasnot degraded after VP16 treatment (Fig. 2B). Moreover, bothfractionation and immunofluorescence assays showed thatVP16 treatment induced translocation of NFkB subunit p65from the cytosol to the nucleus (Fig. 2C and D), and PDTCinhibited translocation of p65 and upregulation of A20 (Sup-plementary Fig. S3A). We also found that ATM kinase inhib-itor Ku55933 blocked translocation of p65 (SupplementaryFig. S3B), suggesting that NFkB activation upon DNA damage isATM-dependent. Next, we performed ChIP-qPCR to determinewhether NFkB binds to the A20 promoter and induces aug-mented expression of A20 in response to DSBs. Indeed, VP16induced binding of p65 to the A20 promoter (Fig. 2E), indi-cating that p65 upregulates A20 expression at the transcrip-tional level in response to DSBs. Furthermore, to examinewhether A20 associates with chromatin in response to DNAdamage, cells were treated with VP16 for the indicated timeperiods, after which the chromatin fraction was extracted.Consistent with the results shown in Fig. 2B, A20 proteinabundance increased in the whole-cell lysates. More impor-tantly, a more obvious time-dependent increase in chromatin-bound A20, but not TAX1BP1, ITCH, or RNF11, was observedafter VP16 treatment (Fig. 2F; Supplementary Fig. S1D). Fur-thermore, we examined the subcellular location of endogenousA20, which revealed that DNA damage promoted expressionand nuclear localization of A20 (Fig. 2G). These observationsdrove us to investigate whether A20 is recruited to DSB sites. Weobserved moderate accumulation of A20 at DSBs by perform-ing laser microirradiation assays (Fig. 2H). This phenomenoncould be a result of the transient presence of A20 at damagesites, which is in accordance with observations regardingUSP16 and OTUB2 (38, 39). Taken together, these resultssuggest that NFkB-induced expression of A20 is involved inthe DDR.

A20 affects the DDR independently of its DUB catalytic activityA20 contains an N-terminal OTU domain and seven zinc

finger (ZnF) domains in the C-terminus mediating the inter-action between A20 and its substrates/partners (40). A catalytic

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triad (Asp70, Cys103, and His256) within the OTU domain ofA20 is responsible for its deubiquitinating activity, amongwhich Cys103 is a critical residue and forms a region respon-sible for interacting with other proteins (21). In addition,zinc finger 4 of A20 binds ubiquitin and possesses E3 ligaseactivity (41). To detect whether the inhibitory effect of A20 onH2A ubiquitination is dependent on its DUB catalytic activity,

we generated a series of A20 point mutation constructs, includ-ing D70A, C103A, H256A, and the catalytic triad mutant D70A/C103A/H256A (designated as 3A; Fig. 3A). HEK293T cellsexpressing the Flag-H2A (K118,119R) mutant and A20 pointmutants were treated with VP16 and subjected to chromatinextraction. VP16 treatment induced an increase in H2AK13,15ub, and, interestingly, all of the tested mutants showed a

Figure 1.

A20 inhibits H2AK13,15ub and 53BP1 focal accumulation upon DNA damage. A, HEK293T cells transfected with his-ubiquitin and the indicated DUBswere treated with 40 mmol/L VP16 for 2 hours, after which his-tagged ubiquitinated proteins were enriched by his-ubiquitin pull-down assay andanalyzed by immunoblotting using an anti-H2A antibody. WCL, whole-cell lysate. B, HEK293T cells expressing Flag-H2A K118,119R were transfected withMyc-empty vector or Myc-A20. After 48 hours, cells were treated with 40 mmol/L VP16 for 2 hours, after which mononucleosomes were extractedand analyzed by immunoblotting using an anti-Flag antibody. � , nonspecific bands. C, HEK293T cells transfected with Flag-empty vector or Flag-A20were treated with or without IR (25 Gy). Histones were extracted by acid chromatin fractionation assay after 1 hour of incubation. The level of endogenous H2Aubiquitination at lysine 15 was detected using an anti-H2AK15ub antibody. D and E, HeLa cells transfected with Flag-empty vector or Flag-A20 weretreated with or without IR (10 Gy). One hour later, immunofluorescence assays were performed using antibodies against Flag and H2AK15ub (D) or Flag and53BP1 (E). The percentage of cells with�10 H2AK15ub foci or�15 53BP1 foci are shown. Data are shown as the mean� SEM of three independent experiments.Statistical analysis was performed using Student t test (��, P < 0.01; �� , P < 0.001). Scale bar, 10 mm. Approximately 200 cells in each group were counted.






decrease in H2AK13,15ub (Fig. 3B). Moreover, the results ofthe immunofluorescence assays showed that A20-C103A andA20-3A mutants inhibited 53BP1 foci accumulation as effi-ciently as did wild-type A20 (Fig. 3C and D).

To explore the defective functionality of mutant A20, wemade N-terminal or C-terminal truncation mutants of A20and tested their effects on H2A ubiquitination. Surprisingly,both the N-terminal and C-terminal were critical for inhibi-tion of H2A ubiquitination by A20 (Supplementary Fig. S4A).Next, we constructed more mutants with altered N-terminaland C-terminal regions (Supplementary Fig. S4B and S4C).The catalytic triad mutant in the N-terminal, A20 N-3A, lost itsinhibitory effect on H2A ubiquitination. However, the mutantlacking ZnF4 in the C-terminus did not completely lose itsfunction (Supplementary Fig. S4B and S4C). Assessmentsusing additional constructed mutants showed that, in addi-tion to ZnF4, ZnF5 and ZnF7 were also responsible for H2A

deubiquitination (Supplementary Fig. S4D). Moreover, theresults in Supplementary Fig. S4E and Fig. 3D show that onlyA20 mutant 3A-DZnF4–7, lacking ZnFs 4–7 and the catalytictriad, revealed complete abolition of the normal inhibitoryeffect of A20 on H2A ubiquitination and accumulation of53BP1 foci.

Next, we constructed A20�/� HeLa cells using a recentlyreported linear donor insertion system (Fig. 3E; ref. 34) andperformed immunofluorescence assays to examine the effect ofA20 deletion on the DDR. The results showed that A20�/� cellscontained more H2AK15ub and 53BP1 foci than did wild-typecells, and reintroducing wild-type A20 inhibited foci accumula-tion significantly, while A20-deficient mutant 3A-DZnF4–7showed no effect (Fig. 3F and G). Together, these observationsindicate that zincfingers 4–7and the integrity of theOTUdomain,rather than deubiquitinating activity, are crucial for the negativeregulatory effect of A20 on the DDR.

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. ThemRNA level of A20 was analyzed by qRT-PCR. Data are shown as the mean � SEM of three independent experiments. Statistical analysis wasperformed using Student t test (�� , 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 timeperiods, after which cytoplasmic and nuclear fractions were extracted and analyzed by immunoblotting using the indicated antibodies. D, MCF7 cellswere 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 theA20 promoter using an anti-p65 antibody. IgG served as a negative control. The A20 promoter sequences in the input DNA and DNA from ChIP complexeswere detected by quantitative PCR. The results were normalized using the IgG abundance of cells treated with DMSO. The results are shown as themean � SEM of three experimental replicates. Statistical analysis was performed using Student t test (��, P < 0.001). F, MCF7 cells were treated with40 mmol/L VP16 for the indicated time periods, after which chromatin was isolated and analyzed using the indicated antibodies. The intensity of A20was normalized against that of H3. G, MCF7 cells were treated with or without IR (10 Gy) and after 24 hours of incubation, immunofluorescence assayswere performed using antibodies against A20. Quantification analysis was performed using Volocity software. Statistical analysis was performed usingStudent t test (�� , P < 0.001). About 100 cells were counted in each group. H, U2OS cells transfected with GFP-A20 were subjected to a laser microirradiationassay. Accumulation of GFP-A20 was detected by fluorescent microscopy at different time points. The red line indicates the positions for lasermicroirradiation.

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A20does not affect UBC13 stability in response toDNAdamageIt has been reported that A20 interacts with UBC13 (E2) and

mediates the degradation of UBC13 upon stimulation by TNF,IL1, and LPS (42). As RNF8–UBC13 complex-catalyzed H1ubiquitination is important for recruitment of RNF168 andactivation of downstream repair signaling (8), we assessedwhether A20-triggered UBC13 degradation followed VP16treatment for the indicated time periods. We did not observedegradation of UBC13 associated with the DNA damage–induced increase in A20 protein abundance (Fig. 4A). Next,we treated cells with DMSO or proteasome inhibitor MG132before VP16 treatment and prolonged the VP16 exposure timeto 240 minutes. However, no degradation of UBC13 wasobserved in cells exposed to VP16 (Fig. 4B). Moreover, toexplore the direct effect of A20 on UBC13 stability, A20WT and

A20�/� HeLa cells were treated with or without IR (10 Gy),harvested at the indicated times, and subjected to measurementof UBC13 abundance. A20 deletion did not affect the stabilityof UBC13 following IR treatment (Fig. 4C). Furthermore, toinvestigate whether A20 inhibits the E2 activity of UBC13 asOTUB1 does, we performed in vitro ubiquitination assays andfound that A20 did not affect UBC13-dependent ubiquitina-tion (Fig. 4D; Supplementary Fig. S5A). These results suggestthat UBC13 stability is not affected by A20 in response to DNAdamage.

A20 directly interacts with RNF168 and abrogates RNF168accumulation at DNA damage sites

As A20 does not affect the stability of UBC13, we next deter-mined whether A20 inhibits H2A ubiquitination by interacting

Figure 3.

A20 affects the DNA damage response independently of its DUB catalytic activity. A, Schematic of A20 point mutants. B, HEK293T cells expressingthe Flag-H2A (K118,119R) mutant were transfected with the indicated A20 mutants. At 48 hours after transfection, cells were treated with 40 mmol/LVP16 for 2 hours and subjected to chromatin extraction. � , nonspecific bands. C, Schematic for the A20 point and deletion mutants. D, HeLa cellswere transfected with different A20 mutants. At 24 hours after transfection, cells were irradiated at 10 Gy and incubated for 1 hour. Immunofluorescenceassays were performed using the indicated antibodies. Immunofluorescence images and the percentage of cells with �15 53BP1 foci are shown. Theresults are shown as the mean � SEM of three independent experiments. Statistical analysis was performed using Student t test (�� , 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 forthe mutated alleles of A20�/� clone 1 are shown. Knockout efficiency was determined using an anti-A20 antibody. F and G, A20WT, A20�/� HeLacells (clone 1), and A20�/� cells, to which the A20 or A20 mutant were reintroduced, were treated with IR (10 Gy). Immunofluorescence assayswere 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 � SEM of three independent experiments. Statistical analysis wasperformed using Student t test (��, P < 0.001). Scale bar, 10 mm. Approximately 200 cells in each group were counted. n.s., nonsignificant.






with the E3 ligases responsible forH2Aubiquitination uponDNAdamage, with the goal of elucidating the mechanism underlyingthe effect of A20 on H2A ubiquitination. HEK293T cells weretransfected with Flag-A20 and E3 ligases RNF8 or RNF168, andcoimmunoprecipitation (co-IP) analysis was performed. A20interacted only with RNF168, which catalyzes H2A and H2AXmonoubiquitination at Lys13 and Lys15 (Fig. 5A). Moreover, weconfirmed the endogenous interaction betweenA20 andRNF168,and found that IR- or VP16-induced DNA damage enhanced thisinteraction (Fig. 5B), which is dependent on ATM (Supplemen-tary Fig. S3C). We also found that wild-type A20, but not A20-deficient mutant 3A-DZnF4–7, interacted with RNF168 (Fig. 5C).

To map the critical domain of RNF168 responsible for itsinteraction with A20, we constructed truncations, includingRNF168 1–249 and RNF168 249–571, and assessed their inter-actions with Flag-A20. The N-terminus, rather than the C-termi-nus, of RNF168 was essential for its binding to A20 (Fig. 5D).Furthermore, the co-IP results showed that both the RING and theUDM1 domain at the N-terminus of RNF168 are important for itsinteractionwithA20 (Fig. 5E; Supplementary Fig. S6A).Moreover,the results of in vitro pull-down assays using purified proteins(Supplementary Fig. S5B) demonstrated that RNF168 is a directtarget of A20, while both the N-terminus and C-terminus of A20bind to RNF168 (Fig. 5F).

The RING domain of RNF168 is important for recognizingits target H2A (Supplementary Fig. S6B and S6C; ref. 43),while the UDM1 domain is responsible for identification ofubiquitinated H1 (8). As both the RING and UDM1 domainsof RNF168 are essential for its binding to A20 (Fig. 5E), wespeculated that A20 might disrupt the binding of RNF168 toH2A and ubiquitinated H1. Indeed, co-IP assays showed thatA20 deletion promoted the RNF168–H2A interaction, whichwas inhibited by reexpression of wild-type A20, but not byreexpression of the A20-deficient mutant (Fig. 5G; Supplemen-tary Fig. S7A). In addition, A20 disrupted the interactionbetween RNF168 and ubiquitinated H1 (Fig. 5H), but it didnot affect H1 ubiquitination (Supplementary Fig. S8). More-over, we determined whether A20 abrogated the accumulationof RNF168 at DNA damage sites. Immunofluorescence assaysshowed that A20 significantly reduced the number of IR-induced RNF168 foci (Fig. 5I). To exclude the possibility thatA20 might affect upstream regulators of RNF168, we examinedthe effect of A20 on MDC1 and RNF8 foci by immunofluo-rescence assays. The results showed that A20 did not affectMDC1 and RNF8 foci (Fig. 5J; Supplementary Fig. S9). Overall,these results suggest that A20 regulates the DDR by directlybinding to RNF168 and impeding accumulation of RNF168 atdamage sites.

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-celllysates were analyzed by immunoblotting using the indicated antibodies. B, MCF7 cells were treated with DMSO or 20 mmol/L MG132 before VP16treatment, after which whole-cell lysates were analyzed by immunoblotting. C,A20WT and A20�/� 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 assayswere performed using combinations of UBE1, UBC13/UEV1a, and ubiquitin with recombinant OTUB1 or A20. The reaction mixtures were analyzed byimmunoblotting using an anti-ubiquitin antibody.

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Deletion of A20 results in persistent accumulation of DNAdamage foci

To further elucidate the effect of A20 on the dynamic regu-lation of DNA damage foci, the abundance of RNF168 and53BP1 foci in A20WT and A20�/� HeLa cells was assessed atdifferent time points following IR treatment. Knockout of A20increased the abundance of chromatin-bound RNF168 and53BP1 under normal conditions (Fig. 6A and B). The numbersof RNF168 and 53BP1 foci were increased significantly in wild-type and A20�/� cells 1 hour after IR treatment, and A20�/�

cells contained slightly more RNF168 and 53BP1 foci than did

wild-type cells (Fig. 6A and B). This phenomenon is in accor-dance with previous reports that DDR proteins are recruiteddramatically during the early phase of DNA damage to repairDNA lesions (18, 38). Consistently, the abundance of A20 wasslightly increased at 1 hour after IR treatment (Fig. 4C), sug-gesting that A20 moderately regulates IR-induced DNA damagefoci during the early period of DNA repair (1 hour after IR).Strikingly, at 24 hours after IR treatment, A20�/� cells con-tained more RNF168 and 53BP1 foci than did wild-typecells (Fig. 6A and B). In accordance with this observation, afterDNA lesions were repaired (24 hours after IR treatment), the

Figure 5.

A20 directly interacts with RNF168 and abrogates the accumulation of RNF168 at DNA damage sites. A, HEK293T cells transfected with Flag-A20 andthe indicated Myc-tagged E3 ligases were subjected to co-IP using an anti-Myc antibody. B, HeLa cells were treated with VP16 (40 mmol/L) for2 hours or IR (10 Gy), followed by 1 hour of incubation. Co-IP assays were then performed to examine the endogenous interaction between A20 andRNF168. C and D, HEK293T cells transfected with different plasmids were subjected to co-IP assays with the indicated antibodies. E, HEK293T cellstransfected 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 ananti-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 vitropull-down analysis was performed with GST protein as a negative control. G,A20WT and A20�/� HEK293T cells were transfected with the indicatedplasmids. At 48 hours after transfection, cells were treated with VP16 for 2 hours, followed by co-IP assays with the indicated antibodies. H, HEK293T cellstransfected 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, HeLacells transfected with the indicated plasmids were treated with or without IR (10 Gy) and subjected to immunofluorescence assays after 1 hour ofincubation. Immunofluorescence images and the percentage of cells with �10 RNF168 foci are shown. The results are shown as the mean � SEM ofthree experimental replicates. Statistical analysis was performed using Student t test (��, P < 0.001). Scale bar, 10 mm. Approximately 200 cells in eachgroup were counted. J, HeLa cells transfected with the indicated plasmids were irradiated at 10 Gy. Immunofluorescence assays were performedusing antibodies against Flag/MDC1 or Flag/RNF8 after 1-hour incubation. The percentage of cells with �10 MDC1 and RNF8 foci are shown. Data areshown as the mean � SEM of three independent experiments. Statistical analysis was performed using Student t test. Scale bar, 10 mm. Approximately200 cells in each group were counted. n.s., nonsignificant.






Figure 6.

Deletion of A20 results in persistent accumulation of DNA damage foci. A and B,A20WT and A20�/� 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 cellswith �10 RNF168 foci or �15 53BP1 foci from three independent experiments are shown. Data are shown as mean � SEM. Statistical analysis wasperformed using Student t test (� , P < 0.05; �� , P < 0.01; �� , P < 0.001). Approximately 400 cells in each group were counted. C and D, The partialsequences of human RNF168 exon 1 and the sequencing results for the mutated alleles are shown. Knockout efficiency was assessed using an anti-RNF168antibody. E,RNF168WT and RNF168�/� HEK293T cells transfected with or without Flag-A20 were treated with VP16 for 2 hours and then subjected to an acidchromatin fractionation assay. The level of endogenous H2A ubiquitination was detected using an anti-H2A antibody. F,RNF168WT and RNF168�/� HEK293Tcells 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), andknockout of A20 resulted in RNF168 and 53BP1 retention atdamage sites (Fig. 6A and B). Taken together, these resultsindicate that A20 fine-tunes DNA damage–induced foci duringthe early phase of the DDR and is essential for the disassemblyof 53BP1 at DNA damage sites after repair.

Moreover, to investigate whether the inhibitory effect of A20on DDR is dependent on RNF168, we generated RNF168�/�

293T cells using a CRISPR-Cas9 system (Fig. 6C and D; ref. 33)and compared the level of H2A ubiquitination upon DNAdamage in RNF168WT and RNF168�/� cells with or withoutA20. The results in RNF168�/� cells showed that H2A ubi-quitination decreased obviously, while A20 was unable toinhibit H2A ubiquitination (Fig. 6E). In addition, RNF168WT

and RNF168�/� 293T cells transfected with or without Flag-A20 were irradiated at 10 Gy and subjected to chromatinseparation. Deletion of RNF168 impeded recruitment of53BP1 to chromatin, which was consistent with a publishedreport (10); moreover, A20 no longer functioned in RNF168�/�

cells (Fig. 6F). These results suggest that A20 relies upon RNF168to finely regulate the DDR.

A20 knockout cells exhibit increased sensitivity to ionizingradiation and DNA-damaging agents

Next, we determined whether A20 affects HR and NHEJ effi-ciency by performing reporter assays. We found that A20 deletionresulted in decreased HR efficiency and increased NHEJ efficiency(Fig. 7A). NHEJ is an error-prone repair mechanism with atendency to produce chromosome translocation, leading to

genome instability (15). Thus, we performed neutral cometassays to investigate the effect of A20 on genome stability, whichshowed that A20 deletion slightly increased comet tail lengthunder normal conditions. Moreover, A20�/� cells possessedmore obvious comet tails than did wild-type cells at 24 hoursafter IR treatment (Fig. 7B), suggesting that the loss ofA20 resultedin impairedDNA repair kinetics and increased genome instability.These results suggest that A20 plays an important role in guaran-teeing proper DNA repair.

In addition, to explore the effect of A20 on cell survival afterDNA damage, a clonogenic survival analysis was performed.A20WT and A20�/� cells (Fig. 3E; Supplementary Fig. S7B) weretreated with different doses of IR and allowed to grow for 12 days,after which the number of colonies was counted. A20�/� cellswere more sensitive to IR treatment than were wild-type cells(Fig. 7C). In addition, expression of wild-type A20 but not A20-deficent mutant in A20�/� cells rescued cell viability (Fig. 7D).Similarly, in response to VP16 treatment, A20�/� cells alsoshowed reduced survival in comparison with wild-type cells(Supplementary Fig. S10A and S10B). Moreover, overexpressionof A20 rendered cancer cells resistant to IR (Fig. 7E) and VP16treatment (Supplementary Fig. S10C), suggesting that increasedA20 conferred resistance to DNA damage therapy in cancer cells.These results suggest that A20 is required for cell survival follow-ing DNA damage.

A20 is highly expressed in breast carcinomaRadiotherapy and chemotherapy resistance are obstacles of

cancer treatments. As increased A20 could confer resistance to

Figure 7.

Loss of A20 expression sensitizes cancer cells to ionizing radiation. A,A20WT and A20�/� cells were subjected to HR and NHEJ assays. The experimentswere performed three times. The results were normalized to those of the A20 wild-type cells. Data are shown as mean � SEM. Statistical analysis wasperformed using Student t test (� , P < 0.05). B,A20WT and A20�/� HeLa cells were subjected to the neutral comet assay. About 100 cells were countedin each group. Images and quantified data are shown. Statistical analysis was performed using Student t test (� , P < 0.05; �� , 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 wereconfirmed by immunoblotting using an anti-A20 antibody. Statistical analysis was performed using Student t test (�� , P < 0.01; �� , P < 0.001). F,Amodel for the roleof A20 in the DDR. In response to DNA damage, A20 is upregulated by NFkB and binds to chromatin, where it terminates H2A ubiquitination by disruptingthe binding of RNF168 to H2A and ubiquitinated H1, thereby reducing the accumulation of RNF168 and facilitating disassembly of 53BP1 at DNA damage sites.






IR- and DNA-damaging agents, we wanted to understand thebiological relevance of A20 in cancer and to explore whether itcan serve as a therapeutic target. First, we analyzed A20 expres-sion in specimens from 60 breast invasive ductal carcinomasand 23 samples of adjacent normal tissue by performing tissuemicroarrays and IHC. We found that A20 expression wassignificantly higher in tumor tissue in comparison with itsexpression level in adjacent normal breast tissue (Supplemen-tary Fig. S11A and S11B). Moreover, we used a gene expressiondataset (GSE70905) from the NCBI-GEO for differentialexpression analysis between the tumor group and adjacentnormal group, which showed that A20 was highly expressedin tumor tissue in comparison with its expression level inadjacent normal tissue (Supplementary Fig. S11C). Analysisof dataset GSE65194 from the NCBI-GEO confirmed thisconclusion (Supplementary Fig. S11D). A20 expression inbreast cancer patients with different histologic grades was alsoanalyzed using the breast cancer dataset from the work byCalda and colleagues (44, 45). The results of this analysisshowed that A20 expression was increased significantly inhigh-grade breast cancers relative to that of the low-grade group(Supplementary Fig. S11E). These results demonstrate that A20is highly expressed in breast carcinomas, particularly in patientsafflicted with high-grade tumors.

DiscussionThis study reveals that chromatin-bound A20 plays an

important role in connecting NFkB signaling and the DDR.Specifically, we show that, in response to DSBs, NFkB translo-cates from the cytosol to the nucleus and binds to the A20promoter. Once A20 is transcriptionally upregulated, more A20binds to chromatin, where it directly binds to RNF168 anddisrupts the binding of RNF168 to H2A and ubiquitinated H1,thereby inhibiting accumulation of RNF168 at DNA damagesites. Thus, A20 inhibits RNF168-mediated H2AK13,15ub andimpairs accumulation of downstream repair proteins at DNAdamage sites (Fig. 7F).

Previous studies have shown that A20 exerts an anti-inflam-matory effect by downregulating NFkB signaling (22). In addi-tion, A20 is an NFkB-responsive gene upon various types ofstimulation (21). The role of A20 in the cytoplasm is wellcharacterized, but little is known about its function in the nucleus.Although DNA damage can trigger NFkB activation, the conse-quences ofNFkB activation for theDDRhave not been elucidated.Here, we demonstrated that A20 expression is induced by NFkBwhen DSBs occur. Moreover, we showed that, in the nucleus, A20inhibits RNF168-mediated H2AK13,15ub and accumulation ofrepair protein 53BP1 at DNA damage sites. This study reveals aconnection between NFkB signaling and the DDR.

It has been shown that OTUB1 inhibits RNF168-dependentH2A ubiquitination and suppresses the DDR. OTUB1 binds toand inhibits E2UBC13 independently of its DUB catalytic activity(20). Interestingly, here we found that A20 negatively regulatedH2A ubiquitination in a DUB activity–independent manner(Fig. 3B). The noncatalytic role of DUBs such as Ubp6 has alsobeen observed (46). In addition, it has been shown that, togetherwith TAX1BP1, A20 interacts with UBC13 and triggers ubiquitin–proteasome degradation in response to TNF, IL1, and LPS stim-ulation (42). Distinct from this mechanism, here we found thatA20 does not affect UBC13 stability in response to DNA DSBs

(Fig. 4). Instead, we showed that A20 affects the DDR throughdirect interaction with RNF168 and attenuates the accumulationof RNF168 at DNA damage sites, which is independent ofTAX1BP1, ITCH, or RNF11. These studies suggest that A20 utilizesdifferent mechanisms to function in different pathways.

Moreover, we demonstrated that the integrity of the A20 OTUdomain and ZnFs 4–7, rather than its deubiquitinating activity,are important for its effect on the DDR. This observation issimilar to previous studies, in which A20 has been shown tobind target proteins either through the conserved surface patchformed by its catalytic triad (21) or via its seven zinc fingerdomains (40). In addition to directly removing the ubiquitinchain from its target, A20 also affects ubiquitination in anindirect manner. For example, the OTU and ZnF4 domains ofA20 are important for its inhibitory effect on E3 ligase activity,which is accomplished by blocking the interaction between E2and E3 enzymes (42). Other studies also demonstrate that theOTU domain collaborates with ZnF4 and ZnF7 at the C-termi-nus of A20 to negatively regulate NFkB activation (47, 48).Therefore, further research should focus on studying the struc-ture of full length A20 to explain the coordination between itsOTU domain and zinc finger domains.

Recently, several studies have shown that a number of DUBsin the USP family may target H2AK15ub and thus affect theDDR, but these DUBs may function in different stages of theDDR. For instance, USP51 directly targets ubiquitinated H2Aon K13 and K15 and modulates the DDR (18). USP3 andUSP44 affect recruitment of RNF168 at DNA damage sites andinhibit DNA damage–induced H2A ubiquitination (16, 17). Inthis study, we demonstrated that A20 inhibited H2AK13,15uband regulated the DDR. Independently of its deubiquitinaseactivity, A20 directly interacts with RNF168 and disrupts thebinding of RNF168 to H2A and ubiquitinated H1. Unlike otherdeubiquitinating enzymes, the abundance of A20 is regulatedby NFkB in response to DNA damage. A20 is significantlyupregulated at 12 and 24 hours after IR treatment (Fig. 4C),suggesting that it mainly functions at the late stage of DNArepair. Therefore, we conclude that A20 is essential for thedisassembly of RNF168 foci after DNA repair to avoid hyper-accumulation of RNF168 at DNA damage sites and preventexcessive ubiquitination. Loss of A20 leads to increased NHEJactivity and decreased HR, which may be a result of persistent53BP1 accumulation at DSB sites and disruption of the balanceamong DNA repair pathways, thereby contributing to impairedDNA repair kinetics and increased sensitivity of cancer cells toDNA damage. These observations reveal the importance of anappropriate DNA damage response and repair process.

Interestingly, we also found that A20 deletion resulted inpersistent BRCA1 accumulation at DNA damage sites (Supple-mentary Fig. S12). However, A20 deletion decreased HR effi-ciency. Although these observations may seem contradictory,they can be explained by results from other studies. RAP80 hasbeen shown to recognize RNF168-generated K63–ubiquitinchains and recruits the BRCA1-A complex (2, 49). A modelhas been proposed in which BRCA1 functions together withRAP80 in the BRCA1-A complex to reduce HR by restrictingDSB end processing, while it promotes resection when inter-acting with other complexes (50, 51). Moreover, other groupshave reported that accumulation of 53BP1 at DSBs promotesNHEJ while suppressing HR (39), and RNF168 inhibits HRsimilar to 53BP1 (52). Accordingly, the results in this study

Yang et al.





suggest that A20 may affect the DNA repair pathway choice bymodulating DNA end resection.

A20 has been reported to be a crucial regulator in manytypes of cancer. It plays an oncogenic role in some solid tu-mors including gliomas (26), hepatocellular carcinoma (53),poorly differentiated head and neck cancers, and undiffer-entiated nasopharyngeal carcinoma (54). Meanwhile, otherstudies have revealed that A20 is also a tumor suppressor thatis frequently deleted and inactivated in B-cell lymphoma,Hodgkin lymphomas, and non-Hodgkin lymphomas (24).These findings suggest that the function of A20 in tumors iscell-type–dependent. In our study, we found that the abun-dance of A20 protein is upregulated in invasive breast carci-nomas (Supplementary Fig. S11A and S11B), which is inaccordance with a previous study showing that the mRNAlevel of A20 is higher in more aggressive breast cancers (25), aswell as another recently published article (55). Moreover,A20�/� cells are more sensitive to IR and VP16 treatment thanare wild-type cells, whereas wild-type cells with A20 over-expression show resistance to IR and VP16 (Fig. 7C–E; Sup-plementary Fig. S10). These findings indicate that A20 influ-ences chemotherapy and radiation resistance, suggesting thepotential of A20 as a target in breast cancer treatment.

In summary, our finding that A20 functions in the nucleus asan inhibitor of DNA damage-induced H2A ubiquitinationprovides new insights into the connection between NFkBsignaling and the DDR. Our results indicate that A20 regulatesthe DDR by inhibiting the binding of RNF168 to H2A andubiquitinated H1, thereby playing an important role in guaran-teeing proper DNA repair and maintaining genome stability.A20 might be a promising clinical target for new strategies toprevent resistance to conventional radiotherapy and chemo-therapy in breast cancer.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: C. Yang, X. ZhengDevelopment of methodology: C. Yang, W. Zang, Y. Ji, Z. LiuAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): C. Yang, W. Zang, Z. Tang, Y. Ji, R. Xu, Y. Yang,A. Luo, B. Hu, Z. LiuAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): C. Yang, W. Zang, Z. Tang, Z. Zhang, Z. Liu, X. ZhengWriting, review, and/or revision of the manuscript: C. Yang, Z. Zhang,X. ZhengAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): C. Yang, X. ZhengStudy supervision: X. Zheng

AcknowledgmentsThis work was supported by the National Science Foundation of China

(81730080, 31470754, 31670786) and the National Key Research andDevelopment Program of China (2016YFC1302401). We sincerely thankProf. Lingqiang Zhang for providing DUB plasmids, Prof. Wensheng Weifor providing CRISPR/Cas9-related plasmids, Dr. Qinzhi Xu for assistancewith neutral comet assays, Prof. Huadong Pei for providing the HR andNHEJ systems, and Prof. Xingzhi Xu for helping with the laser microirradia-tion assays. We also appreciate the assistance of Xiaochen Li, GuopengWang, Liying Du, and Hongxia Lv from the Core Facilities of Life Sciences atPeking University for their assistance with microscopic imaging and cellflow cytometry.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received July 18, 2017; revised November 2, 2017; accepted December 1,2017; published OnlineFirst December 12, 2017.

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2018;78:1069-1082. Published OnlineFirst December 12, 2017.Cancer Res Chuanzhen Yang, Weicheng Zang, Zefang Tang, et al. Tumor Cell Resistance to DNA-Damaging TherapyA20/TNFAIP3 Regulates the DNA Damage Response and Mediates

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A20/TNFAIP3 Regulates the DNA Damage Response and … · Cell culture and transfection HeLa, MCF7, and U2OS cells were purchased from ATCC and HEK293T was acquired from the National