NR4A2 Promotes DNA Double-strand Break Repair Upon ... · DNA Damage and Repair NR4A2 Promotes DNA Double-strand Break Repair Upon Exposure to UVR Kelvin Yin1,Yash Chhabra2,3, Romain

DNA Damage and Repair

NR4A2 Promotes DNA Double-strand BreakRepair Upon Exposure to UVRKelvin Yin1, Yash Chhabra2,3, Romain Trop�ee2, Yi Chieh Lim4, Mitchell Fane1,Eloise Dray2,5,6, Richard A. Sturm3, and Aaron G. Smith3,5

Abstract

Exposure of melanocytes to ultraviolet radiation (UVR)induces the formation of UV lesions that can produce dele-terious effects in genomic DNA. Encounters of replicationforks with unrepaired UV lesions can lead to several complexphenomena, such as the formation of DNA double-strandbreaks (DSBs). The NR4A family of nuclear receptors aretranscription factors that have been associated with mediatingDNA repair functions downstream of the MC1R signalingpathway in melanocytes. In particular, emerging evidenceshows that upon DNA damage, the NR4A2 receptor cantranslocate to sites of UV lesion by mechanisms requiringpost-translational modifications within the N-terminaldomain and at a serine residue in the DNA-binding domainat position 337. Following this, NR4A2 aids in DNA repair byfacilitating chromatin relaxation, allowing accessibility for

DNA repair machinery. Using A2058 and HT144 melanomacells engineered to stably express wild-type or mutant forms ofthe NR4A2 proteins, we reveal that the expression of func-tional NR4A2 is associated with elevated cytoprotectionagainst UVR. Conversely, knockdown of NR4A2 expressionby siRNA results in a significant loss of cell viability after UVinsult. By analyzing the kinetics of the ensuing 53BP1 andRAD51 foci following UV irradiation, we also reveal that theexpression of mutant NR4A2 isoforms, lacking the ability totranslocate, transactivate, or undergo phosphorylation, displaycompromised repair capacity.

Implications: These data expand the understanding of the mech-anism by which the NR4A2 nuclear receptor can facilitate DNADSB repair. Mol Cancer Res; 15(9); 1184–96. �2017 AACR.

IntroductionThe NR4A proteins are a subgroup of the nuclear hormone

receptor (NR) family of transcriptional regulators. This subgroupof NRs is comprised of NR4A1 (Nur77), NR4A2 (Nurr1), andNR4A3 (Nor1). Like classical NRs, NR4A receptors have an N-terminus domain (NTD), a highly conserved zinc-finger DNA-binding domain (DBD), and a carboxyl-terminal ligand-bindingdomain (LBD) (1). It has been suggested that the NR4A receptorsare true orphan receptors as no endogenous ligands have beenidentified to date. Furthermore, crystal structure analysis suggeststhe NR4A receptors may be unable to accommodate binding of

molecules due to the presence of bulky hydrophobic amino acidside chains in the ligand-binding cavity of the LBD (2).

The NR4A receptors have been identified as immediate or earlyresponse genes that are rapidly induced upon various acutestimuli. They have been implicated in regulating a wide range ofbiological processes including cell proliferation, differentiation,and apoptosis, in addition to other important pathophysiologicroles such as neurologic disorders, inflammation, and cardiovas-cular disease (3–7). More recent studies have identified NR4Aproteins as tumor suppressors and as mediators of DNA repairresponses in various cell types (8–10). Interestingly, we havepreviously reported the rapid induction of the NR4A subfamilyfollowing Melanocortin-1-receptor activation (MC1R) in mela-nocytes (11). MC1R is a G-protein–coupled receptor that is thekey regulator of melanogenesis in melanocytes and has also beenshown to augmentDNA repair in response to ultraviolet radiation(UVR) (12). This further implies the importance of NR4A genes aspotential mediators constituting the MC1R-coordinated DNAdamage response (DDR) cascade.

UVR exposure, particularly the UVB spectrum (280–320 nmwavelength) is the main etiologic factor for both melanomaand non-melanoma skin cancer development. UVB radiationresults in the formation of cyclobutane pyrimidine dimer (CPD)and 6-4-photoproduct (6-4PP) lesions in the DNA (13, 14),which distort the normal helical structure and hinder the pro-gression of DNA replication and transcription by polymerases.Inability to restore UVB damage will result in subsequentreplication fork collapse, giving rise to DNA double-strandbreaks (DSBs).

DSBs are one of the most deleterious forms of DNA damage,and can cause genomic instability if left unrepaired. Briefly, to

1School of Biomedical Sciences, University of Queensland, Brisbane, Queens-land, Australia. 2Queensland University of Technology, Translational ResearchInstitute, Brisbane, Queensland, Australia. 3Dermatology Research Centre, TheUniversity of Queensland-Diamantina Institute, Translational Research Institute,Brisbane, Queensland, Australia. 4Translational Brain Cancer Research, QIMRBerghofer Medical Research Institute, Herston, Queensland, Australia. 5Queens-land University of Technology, Institute of Health and Biomedical Innovation,Kelvin Grove, Queensland, Australia. 6Mater Research - The University ofQueensland, Translational Research Institute, Brisbane, Queensland, Australia.

Note: Supplementary data for this article are available at Molecular CancerResearch Online (http://mcr.aacrjournals.org/).

K. Yin and Y. Chhabra contributed equally to this work.

CorrespondingAuthor:AaronG. Smith, School of Biomedical Sciences, Institutefor Health and Biomedical Innovation, Queensland University of Technology,Brisbane, Queensland 4000, Australia. Phone: 6104-1006-6200; Fax: 617-3176-7440; E-mail: [email protected]

doi: 10.1158/1541-7786.MCR-17-0002

�2017 American Association for Cancer Research.

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repair DNA DSBs, DNA sensors are first recruited to the site ofstrand breakage followed by DNA mediators such as gH2AX,MDC1, and p53-binding protein 1 (53BP1) to facilitate down-stream cascade activation of other DSB-repair factors and check-point arrest. These DNA DSB sites where DDR proteins gatherare commonly known as foci and can be identified as early as15 minutes following genotoxic insults (15). The recovery ofDNA strand breakage is predominantly performed by two majorDNA DSB pathways: homologous recombination (HR) or non-homologous end joining (NHEJ; 16–19). To carry out HR, theMRN (MRE11, RAD50, andNBS1) complex is first recruited to thesite of DSB, which together with CtBP-interacting protein (CtIP)and exonuclease1 (EXO1) resect the DNA ends, generating a 30

ssDNA overhang for strand exchange (20, 21). TheMRN complexalso recruits and activates ataxia-telangiectasia mutated (ATM)that phosphorylates and stimulates MRE11, NBS1, CtIP, andEXO1 activities. In addition, ATM also phosphorylates histoneH2AX, which subsequently assists in the recruitment of 53BP1and BRCA1 (22). To prevent strand degradation, the ssDNAoverhang is rapidly coated with replication protein A (RPA), andis phosphorylated by ataxia-telangiectasia and RAD3-related(ATR)-ATR interacting protein (ATRIP) complex. The binding ofATR-ATRIP to RPA-coated ssDNA initiates checkpoint signalingvia the phosphorylation of kinase CHK1 (23), which in turnphosphorylates RAD51. To catalyze the strand exchange reaction,RPA is displaced in exchange for RAD51, forming aRAD51-coatednucleoprotein filament that can invade the complementaryduplex DNA, thus completing the HR reaction. In contrast, toinitiate NHEJ repair, the DSB ends are recognized by the Ku70/Ku80 heterodimer, followed by the recruitment of the DNA-dependent protein kinase catalytic subunit (DNA-PKcs). DNA-PKcs is subsequently autophosphorylated upon the interactionbetween two DNA-PKcs positioned at each DSB terminus, allow-ing the activation of Artemis to create terminal overhangs (24).NHEJ repair is then completed by gap filling and end joiningcarried out by XRCC4/LIG4 and XLF complexes.

One of the major roadblocks that DNA repair mechanismsmust confront is the tight packaging ofDNA into nucleosomes. Toinitiate repair, chromatin surrounding the damaged site needs tobe modified and remodeled to provide accessibility to DNAlesions and binding interfaces for the recruitment of repair factors(25). Interestingly, recentfindings have suggested that in responseto UVR-induced DNA damage, the NR4A2 protein is translocatedto nuclear foci at the sites of DNA damage by mechanismsrequiring the PARP-1 activity and p38–MAPK signaling pathways(9, 10). It was shown that the NR4A2 foci colocalize with knownDNA repair proteins of the NER pathway such as DDB2 and XPC;however, a LBD-mutant form of NR4A2 lacking the C-terminalhelix 12 does not colocalize with the DNA repair factors despiteretaining the ability to form UV-induced foci (10). Furthermore,in the context of DNA DSBs, an interaction between DNA-PKand NR4A2 via phosphorylation of the Ser337 residue in theNR4A2 DBD is necessary for determining the repair efficiencyof DSBs in cells; however, the molecular events that underpinthis process have not been fully characterized (9). Here, weexamine DNA repair efficiency by depleting or overexpressingwild-type or mutant versions of NR4A2, potentially affecting itspost-translational events (K91A, S337A) and DNA binding(C266R), in melanoma cells. Our data suggest NR4A2 expressionis essential for functional DNA DSB repair and subsequentcell survival.

Materials and MethodsCell culture and transfection analysis

The human melanoma cell lines HT144 (ATCC, HTB-63),MM96L (26), and A2058 (ATCC, CRL-11147) (27) were culturedin RPMI1640 media supplemented with 3% FBS, 2% serumsupreme, 2 mmol/L L-glutamine, and 50 mg/mL penicillin/strep-tomycin. Cell line identity was confirmed by short tandem repeat(STR) profiling (Queensland Institute of Medical Research, Bris-bane, Australia) in addition to frequent Western blot profiling ofmelanoma-specific antigen expression to verify their authenticity.All cells were maintained in 5% CO2 incubator at 37�C. Forfluorescence studies, cells were grown on glass coverslips in 12-well tissue culture dishes. Transfection was carried out usingLipofectamine 2000 (Invitrogen) as per manufacturer's protocol.NR4A2 siRNA transfection in MM96L and A2058 cells wereperformed in 6-well dishes.

Plasmid constructionHuman NR4A2 WT and mutant proteins were engineered by

PCR into the pEYFP-C1 (HindIII; BamHI sites) and pLVX-Puro(XhoI; XbaI sites) plasmid vectors (Clontech Laboratories). Pointmutations were engineered using a two-fragment PCR approach,initially introducing the mutation by PCR, with the respectivefragments subsequently fused together using an In-fusion-HDcloning kit (Clontech Laboratories), the mutated product wasthen used as a PCR template to generate the clones required.Primer sequences used to generate the clones are listed in Sup-plementary Table S1.

Lentivirus transductionWild-type and mutant forms of NR4A2 lentivirus were gener-

ated using the Lenti-X HT packaging system (Clontech Labora-tories) according to the manufacturer's protocol. A2058 andHT144 melanoma cells were subjected to media containingtitrated lentiviral particles containing 4 mg/mL polybrene, andcentrifuged at 1,200 RCF for 75minutes. Oldmedia was replaced24 hours post-centrifugation and cells were selected in mediacontaining 0.75 mg/mL puromycin and confirmed by Westernblotting prior to performing experiments.

Irradiation and fluorescent imagingCells were seeded on coverslips, where on day of treatment, the

normal growth medium was replaced with warm PBS, and thenexposed to either UVR or ionizing radiation (IR). The source ofUVB was a GL20SE lamp (Sankyo Denki) with the UVB dosagemeasured using aDigital Ultraviolet Radiometer (Model 6.0UVB,Solarmeter, Solartech Inc.), and determined to fall within theUVBspectrum only (280–320 nm). The doses of UVB that are con-sidered physiologically relevant in the context of UV-inducedmelanoma vary according to different skin types, and is measuredon the basis of the minimum UVB dose that is required forinducing erythema. In melanoma cells, 70 mJ/cm2 UVB is con-sidered to be the minimum erythema dose (MED). For thisreason, 25 mJ/cm2 and 50 mJ/cm2 of UVB were selected in ourexperiments (28). For IR, cells were exposed to 5 Gy IR using theFaxitron RX650 X-ray Cabinet. Following irradiation, cells wereincubated in media until fixation with 4% paraformaldehyde inPBS at various time points postirradiation. For visualization offoci formation, coverslipsweremounted onto slides withDABCOmounting media. Images were obtained on an Olympus BX-51epifluorescence microscope with a DP-70 12 MP Color camera.

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For foci analysis, at least ninety cells were analyzed and tenrepresentative pictures per coverslip were taken.

MTT and crystal violet assayAll cell lines were seeded into 24-well plates, treated with 50

mJ/cm2 UVB. Cell viability was measured on the basis of thereduction of 3-[4,5-dimethyltiazol-2-yl]-2,5-diphenyltetrazo-lium bromide (MTT) assay (Sigma). MTT solution was preparedin serum-freemedium at a final concentration of 0.5mg/mL, thenadded to wells, and incubated at 37�C for 60 minutes 24 and48 hours following the initial UVB treatment. MTT medium wasthen removed and replaced with isopropanol and absorbancemeasured near 570 nm (29). For crystal violet (CV) assay, cellswere washed in PBS and fixed in ice-cold methanol for 5minutes,stained for 5 minutes with 0.2% (w/v in methanol) crystal violetsolution. The wells were washed at least 5 times in water. Thestained cells were solubilized in 1% SDS solution and absorbancewas measured at 570 nm.

Immunofluorescence analysisFixed cells were permeabilized in 0.1% Triton X-100 diluted in

PBS and blocked with 5% BSA diluted in PBS. Primary antibodieswere diluted in 0.5% BSA and Alexa Fluor 594 or 488–conjugatedsecondary antibodies (Invitrogen) were used at a 1:400 dilutionwith 0.5% BSA. For analysis of the 53BP1 kinetics, stable A2058and HT144 cells were irradiated and the nuclei were preextractedwith nuclear extraction buffer (10 mmol/L PIPES pH 6.8, 100mmol/L NaCl, 300 mmol/L sucrose, 3 mmol/L MgCl2, 1 mmol/LEGTA pH 8, 0.5% Triton X-100) at 30minutes, 1, 3, 6, 12, and 24hours postirradiation and fixed in 4% PFA. Cells were thenblocked in 5% FCS/0.1% Triton X-100 and stained with rabbitanti-53BP1 (1:1,000; #4937, Cell Signaling Technology), or rab-bit anti-RAD51 (1:200; #ab63801, Abcam). All antibodies werediluted in 5%FCS/0.05% Triton X-100 solution. All nuclei werecounterstained with 10 mg/mL DAPI.

BrdU incorporation assayA2058-stable cells grown on coverslips (at equal seeding den-

sities) were subjected to mock and UVB (50 mJ/cm2) and treatedwith 20 mmol/L BrdU (5-Bromo-20-deoxyuridine, Sigma-Aldrich)24 hours posttreatment, for 1 hour. Cells were washed with PBSand fixed in 70% ethanol and coverslips were stained with BrdUantibody (1:1,400; #5292, Cell Signaling Technology) as permanufacturer's guidelines, followed by Alexa Fluor-488 antibodyand counterstained with 10 mg/mL DAPI. Random fields of viewswere first imaged for DAPI and the same field of view was imagedfor BrdU and quantified blind and data represented as percentageof BrdU-positive cells.

Apoptosis assayA2058 cells mock and UVB-treated (50 mJ/cm2) were trypsi-

nized 24 hours post-treatment and stained with Annexin V (FITC-Annexin V/Propidium Iodide staining) according to the manu-facturer's protocol (BD Biosciences). A total of 10,000 events wereanalyzed byflow cytometry onCytoFLEX (BeckmanCoulter)withappropriate controls.

Western blot analysisCells were seeded in 6-well plates (Nunc, Thermo Scientific)

and irradiated with 50 mJ/cm2 of UVB. Whole-cell lysates wereharvested in RIPA buffer [50 mmol/L Tris (pH 7.5), 150 mmol/LNaCl, 1% NP-40, 0.25% sodium deoxycholate, 0.1% SDS,

1% Triton-X], supplied with protease inhibitor (Roche) andsodium orthovanadate. Antibodies used include; rabbit anti-MRE11 (1:2,000; #4847, Cell Signaling Technology), rabbitanti-RAD50 (1:1,000; #3427, Cell Signaling Technology), rabbitanti-NBS1 (1:1,000; #HPA001429, Sigma-Aldrich), rabbit anti-pNBS1 (1:1,000; #3001, Cell Signaling Technology), rabbit anti-pRPA (1:2,000; A300-245A, Bethyl Laboratories), rabbitanti-Ku80 (1:2,000; #2180, Cell Signaling Technology), andrabbit b-tubulin (1:5,000; #2128, Cell Signaling Technology),and mouse anti-GAPDH (1:5,000; #2275-PC-100, Trevigen).

Quantitative real-time PCRRNA was extracted from a panel of metastatic melanoma cell

lines and human foreskin-derived primary melanoblast andmelanocyte cells using TRIzol reagent (Life Technologies) as perthe manufacturer's guidelines. cDNA was synthesized usingiScript RT Supermix (Bio-Rad) and transcripts levels werequantified using Sybr Green Mix (Applied Biosystems) onViiA7 machine (Applied Biosystems). TaqMan-validated primersets were obtained from Applied Biosystems NR4A2(Hs01117527_g1); b-2-microglobulin (Hs99999907_m1).

Clonogenic assayA2058- and HT144-stable cells were seeded in duplicate in 6-

well dishes at 100 cells and 300 cells per well, respectively. Cellswere cultured for 14–21 days. Colonies were permeabilized withmethanol and stained with crystal violet solution (0.2% w/v inmethanol). Excess stain was removed and plates were dried andimaged on Bio-Rad Chemi-Doc Imager.

Statistical analysisStatistical analyses were performed using GraphPad PRISM

software based on a minimum of three independent experimentsusing one-way ANOVA followed by a Tukey post-test or two-wayANOVA followed by a Bonferroni post-test. Significance wasscored as ��, P < 0.0001; ��, P < 0.001; ��, P < 0.01; �, P < 0.05.

ResultsNR4A2 expression confers cytoprotection against UVB-inducedcell death in melanoma cells

We have previously observed that NR4A2 is a vital factor forDNA UV-photolesion clearance (10). However, we have yet todetermine whether the absence of NR4A2 expression will affectcell viability following treatment of cells with 50 mJ/cm2 of UVB.Here, we transfected negative control (NC) or pooled NR4A2siRNA into MM96L melanoma cells, which have been reportedto express high levels of NR4A2 mRNA (Supplementary Fig.S1B) (10). MTT assay revealed that following treatment with50 mJ/cm2 of UVB, a reduction in cell viability was not evidentin the NC-siRNA–transfected cells, whereas a 9-fold reduction incell viability was observed when NR4A2 levels are depleted (Fig.1A). To further study the cytoprotective effects of NR4A2 incutaneous melanoma cell lines, we employed A2058 and HT144cell lines that were found to express low and moderate levels ofNR4A2, respectively, by RT-PCR (Supplementary Fig. S1B). Usinga lentivirus-mediated delivery system, we engineered A2058melanoma cells that express high levels of MYC-tagged-NR4A2WT

(NR4A2WT), previously shown to be more resistant to UV irradi-ation (10).We transfected theNR4A2WT-overexpressing cells withnegative or pooled siRNA targeting NR4A2. The efficiency of

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exogenous NR4A2 knockdown in the stable lines was verified byWestern blot analysis (Fig. 1B). Following treatment with UVB,cell viability was determined by MTT assay 24 hours postirradi-ation. The NR4A2WT-expressing cells transfected with NC-siRNAonly showed a modest decrease in the percentage of cell viability,whereas cells treated with NR4A2-siRNA displayed significantlyreduced viability (�7-fold reduction; Fig. 1C). Taken together,these observations suggest that elevated NR4A2 expression mayresult in improved cell survival after UVB insult.

Characterization of foci recruitment in NR4A2 mutantsAs previous reports have demonstrated that the ability of

NR4A2 to augment DNA repair requires its ability to translocateto sites ofDNA lesions (9, 10), examiningwhethermutantNR4A2proteins exhibit defects in foci formation may provide vitalinsights into the mechanism of NR4A2 conferred cytoprotection.A2058, HT144, and MM96L melanoma cells were transfectedwith constructs expressing wild-type or mutant forms of NR4A2proteins with an N-terminal EYFP fusion, which were then irra-diated with 25mJ/cm2 of UVB. Cells were fixed 4 hours post-UVBand examined by fluorescence microscopy for changes in thenuclear sublocalization of EYFP-NR4A2 fusion proteins. Asexpected, NR4A2WT and NR4A2DH12 proteins translocated intodistinct nuclear foci, whereas the NR4A2DNT mutant remainsdiffused throughout the nucleus 4 hours post-UVB (Fig. 2A;Supplementary Fig. S1A). In addition, a sumoylation site muta-tion NR4A2K91A (30), mutation of a conserved cysteine residuewithin the first zinc-finger of DNA-binding domain NR4A2C266R

and phosphorylation-defectivemutationNR4A2S337A (9)mutantall retained the ability to form nuclear foci upon UVR, albeitwith variable efficiency. Our findings suggest that functionalDNA binding and other post-transcriptional events such assumoylation and phosphorylation are unlikely to be essential

for the recruitment ofNR4A2 toUV-induced lesions.However, wecannot rule out the possibility that their respective effect on DNArepair capacities may be dependent on the efficiency of fociformation. Intriguingly, we also observed that NR4A2C266R isable to form distinct foci in the absence of UV irradiation.

Cells expressing mutant forms of NR4A2 exhibit increasedsusceptibility to UV-induced death

Our previous findings showed that the NR4A2 protein isrecruited to nuclear foci at sites of DNA lesions, and colocalizeswith DNA repair proteins known to mediate DNA repair inresponse to UVR, which is dependent on the integrity of boththe NTD and Helix 12 (H12) domains of the receptor (10).Supporting evidence from independent studies also suggests thatphosphorylation at the S337 residue is likely to play a role in itsDDR signaling (31). Accordingly,we generatedA2058 andHT144melanoma cell lines stably expressing a S337A-mutant form ofNR4A2 receptor (NR4A2S337A) in addition to two truncatedversions with deleted NTD (NR4A2DNT) or H12 (NR4A2DH12)domains (Fig. 2B). The expression levels of these MYC-taggedNR4A2 proteins in cells were verified via immunoblotting (Fig.2C). These cell lines were irradiated with UVB and the ability ofthe WT or mutant NR4A2 proteins to confer cytoprotection wasassessed using BrdU incorporation, clonogenic assay, apoptosis,and viability assays.

A2058 cells stably expressing NR4A2 isoforms and emptycontrol cells subjected to BrdU pulse indicated NR4A2WT over-expression significantly increases DNA replication as comparedwith the cells transduced with the empty virus and all mutantisoforms (Fig. 2D). The improved survival of WT NR4A2–expres-sing cells compared with empty vector or mutant NR4A2-expres-sing cells was further corroborated using clonogenic assays (Sup-plementary Fig. S1C). Moreover, FACS analysis demonstrated a

Figure 1.

Overexpression of NR4A2 in melanoma cells is associated with increased cytoprotection after exposure to UVB. A, MM96L cells were transfected with eitherscrambled (NC) or NR4A2 siRNA (1, pooled 2þ3). Forty-eight hours post-transfection, cells were treated with mock or 50 mJ/cm2 UVB. MTT assays wereconducted 24 hours post-UVB treatment. B, Whole-cell extracts were prepared 48 hours post-transfection and were probed for MYC-tag expression byimmunoblot to verify the siRNA knockdown efficiency. b-tubulin was used as loading control. C, A2058 melanoma cells stably expressing GFP or MYC-tagged-NR4A2 were transfected with either scrambled (NC) or NR4A2 siRNA (1, pooled 2/3). Cells were treated with mock or 50 mJ/cm2 UVB at 48 hourspost-transfection, followed by MTT assay at 24 hours postirradiation. Statistical analyses were performed using two-way ANOVA with Tukey post-test(�� , P < 0.0001; �� , P < 0.01).






reduced percentage of both apoptotic (Annexin V-positive) anddead (propidium iodide-positive) cells in NR4A2WT cell follow-ing UVR (Supplementary Fig. S1D), suggesting that expression ofthe WT NR4A2 receptor allows the cells to recover faster andovercome the UV-mediated growth arrest.

This observation was supported by both CV and MTT analysis24 and 48 hours postirradiation as overexpression of NR4A2WT

resulted in higher percentage of viable cells when compared withempty control cells after exposure toUVR across both the cell linestested (Fig. 2E; Supplementary Fig. S2A). Interestingly, an approx-imately 2.5-fold reduction in cell viability was detected in cellsexpressing NR4A2DNT and a 2-fold reduction was recorded inNR4A2S337A- and NR4A2DH12-expressing cells at 24 hours by CVassay (Fig. 2E). This difference in the level of cytoprotection acrossNR4A2 mutants was exacerbated 48 hours post-UVR across bothcell lines (Supplementary Fig. S2B and S2C).Given the percentageof viable cells in UV-irradiated NR4A2DNT-, NR4A2S337A-, andNR4A2DH12-expressing cells was lower than empty control cells,

these data imply that protection against UV insult is moreimpaired in these lines. In addition, theNR4A2DNTmutant, whichwas unable to form foci upon UVR exposure, exhibited themaximal decrease in cell viability in response to UVR.

Functional NR4A2 promotes DSB repair followingexposure to UVB

Recent studies have presented compelling evidence suggestingthat NR4A2 is involved in the DNA DSB repair pathway uponexposure to IR (31). Given that UVR-induced transcription/replication blockage induces replication fork collapse, an indirectsource of DNA DSBs, we investigated the role of NR4A2 in DSBrepair under UVB-induced DNA damage. We have previouslyreported the colocalization of NR4A2 foci with gH2AX, a markerfor DNADSBs (10), but the role of these foci for the repair ofDNADSBs was not explored. Accordingly, to assess whether NR4A2is involved in the early DSB repair process, we examined thekinetics of 53BP1 foci in A2058-stable lines by monitoring the

Figure 2.

Nuclear foci characterization of wild-type and mutant NR4A2 proteins inmock and UVB conditions. A,Representative images in A2058 andHT144 melanoma cells indicatingnuclear localization of EYFP-WT, DNT,K91A, C266R, S337A, and DH12 NR4A2proteins following mock or 25 mJ/cm2

UVB treatment for 4 hours.B, Schematic diagram showing theNR4A2 illustration. Asterisk (�)indicates the site of point mutationintroduced for each NR4A2 mutants.NTD, N-terminal domain; DBD, DNA-binding domain; LBD, ligand-bindingdomain; helix 12, H12. C, Expressionlevels of wild-type and mutant NR4A2isoforms in stably transduced A2058melanoma cells were verified byimmunoblotting for MYC-tag. GAPDHwas used as loading control. D, BrdUincorporation assay in A2058 stablytransduced cells 24 hours followingmock or 50 mJ/cm2 UVB treatment.E, Crystal violet viability assay inA2058 and HT144 stably transducedcells at 24 hours following 50 mJ/cm2

UVB treatment. Each þUV values arenormalized to the respective mock(-UV) treated values. Statisticalanalyses were performed usingtwo-wayANOVAwith Tukey post-test(�� , P < 0.0001; �� , P < 0.001;�� , P < 0.01; �, P < 0.05; ###, P < 0.001;##, P < 0.01).

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recruitment and disappearance of 53BP1 foci in melanoma cellsexpressing control, wild-type, or mutant forms of NR4A2 atdifferent time intervals after an initial exposure to 50 mJ/cm2

UVB (Fig. 3A), an experimental approach that is widely utilizedto examine DSB repair kinetics (32). On the basis of 90 ran-domly chosen cells at each time point, we assessed the numberof 53BP1 foci per nucleus. On average, the number of 53BP1foci per one nucleus in unirradiated cells was not significantlydifferent among cell lines (P > 0.05). In contrast, irradiated cellsfixed 3 hours after UVB showed a striking increase in thenumber of 53BP1 foci in all cell lines (Fig. 3B; SupplementaryTable S2). Thereafter, the number of foci declined. Supplemen-tary Fig. S3 shows the number of 53BP1 foci per nucleus at 30minutes or 1 hour post-UVB, with no discernable difference inthe number of foci between the lines evident. Notably, thenumber of 53BP1 foci per nucleus in NR4A2WT-expressing cellsat 12 hours postirradiation was significantly lower than theempty control cells at the same timepoint (P < 0.05). We alsoquantified the population of cells that were positive for 53BP1foci (Fig. 3C; Supplementary Table S3). We observed a signif-icantly higher population of cells still carrying DSBs at 24 hourspostirradiation in NR4A2DNT-, NR4A2S337A-, and NR4A2DH12-expressing cells (P < 0.05). As the persistence of 53BP1 fociallows an interpretation for the presence of DSBs, these results

indicate that overexpression of NR4A2WT in melanoma cellsimproves the DSB repair efficiency. The prolonged presence ofDSBs in NR4A2DNT-, NR4A2S337A-, and NR4A2DH12-expressingcells suggest a diminished repair capacity. A similar result wasalso observed in HT144-stable lines with foci levels peaking at12 hours (Supplementary Fig. S4). The NR4A2WT cells showedthe least percentage of 53BP1 foci and were clearedmore rapidlythan empty control- and mutant isoform–expressing cells (Sup-plementary Fig. S4B). Interestingly, the NR4A2DH12 cellsshowed a significantly higher percentage of 53BP1 foci beforeUV treatment (Supplementary Fig. S4B).

IR-induced 53BP1 foci suggests for a direct involvement ofNR4A2 in DNA DSB repair following UVB irradiation

As UVR leads to the formation of DSBs in the genomicDNA viareplication fork collapse, it prompted us to investigate whetherthe 53BP1 foci kinetics in cells treated with UVR is an accuraterepresentation of DSB repair kinetics. We exposed cells to IR(5 Gy) to directly induce DSBs, and monitored the appearanceand disappearance of 53BP1 foci (Fig. 4A). In contrast to UV-induced 53BP1 foci, the maximal recruitment of 53BP1 fociinduced by IR occurred almost immediately and was maintaineduntil 3 hours postirradiation after which the foci numberdecreased. The number of 53BP1 foci per nucleus at 30 minutes

Figure 3.

NR4A2 expression alters 53BP1 foci kinetics after UVB radiation. A, A2058 cells stably expressing Empty, NR4A2WT, NR4A2DNT, NR4A2S337A, or NR4A2DH12 proteinswere exposed to 50 mJ/cm2 UVB, prenuclear extracted and fixed at 3, 6, 12, and 24 hours postirradiation. Immunofluorescence was performed to detect53BP1 proteins. Representative images taken from three independent experiments are shown.B,Quantitative assessment of number of foci per nucleus from90 cellsat each time point of each cell lines. C, Quantitative assessment on the percentage of cells showing positive 53BP1 foci (cells displaying 4 or more fociwere scored as positive) from 90 cells at each time point of each cell lines. Statistical analyses performed in graph B and C are normalized againstvalues recorded in the respective time points from the empty control cell line. Statistical analyses were performed using two-way ANOVA with Tukeypost-test (�� , P < 0.0001; ��, P < 0.001; �� , P < 0.01; �, P < 0.05).






or 1 hour post-IR is presented in Supplementary Fig. S5. By 6hours postirradiation, the number of 53BP1 foci in NR4A2WT-expressing cells had decreased to a level significantly lower thanthe empty cell line (P < 0.05; Fig. 4B; Supplementary Table S4).Furthermore, comparative analyses of the percentage of cells withpositive 53BP1 foci revealed that a significant amount of damagehad been resolved in NR4A2WT cells by 12 hours postirradiationin comparison to empty (P < 0.05). In contrast, NR4A2DNT-,NR4A2S337A-, and NR4A2DH12-mutant lines still retained a higherpopulation of cells carrying DSBs even after 12 hours of recovery(P < 0.05; Fig. 4C; Supplementary Table S5).

Functional NR4A2 is required for DNA DSB repairHR and NHEJ are two major independent repair pathways

found in eukaryotic cells that are capable of counteracting thedeleterious effects of DNA DSBs. Having shown that theexpression of different functional mutants of the NR4A2 canalter the kinetics of DSB repair at an early stage, we nextdetermined whether NR4A2 is involved in the events down-stream of 53BP1 recruitment. We exposed A2058 and HT144stably transduced cells to mock or 50 mJ/cm2 of UVB and celllystates harvested 4 and 8 hours following irradiation wereanalyzed by immunoblotting for sensor and effector proteinsfrom both DSB repair pathways. First, we examined the acti-

vation of HR by detecting the expression of MRE11-RAD50-NBS1 (MRN) complex and observed modest to no change inthe basal, or UV-induced, level of these proteins between therespective lines (Fig. 5A). The MRN complex has been reportedto act as a sensor of DSBs and facilitate the resection of 50 endsto produce a 30 ssDNA overhang to initiate HR-mediated repair.Moreover, the phosphorylation of MRN proteins is recognizedas a crucial step in activating MRN function that facilitatessubsequent signaling activities required for lesion resolution(33). Accordingly, we assessed NBS1 phosphorylation status inresponse to UVR in our cell lines as a proxy for activation of theMRN complex. Immunoblotting revealed that the level of p-NBS1 increased at 8 hours post-UVR in the NR4A2S337A com-pared with the 4-hour timepoint in both A2058 and HT144cells, although the NBS1 activation was of much lower intensitythan that observed at the corresponding timepoint in NR4A2WT cells (Fig. 5B). Interestingly, while a robust increase in NBS1phosphorylation was evident in the NR4AWT cells, only a minorincrease above basal levels was observed at 4 hours in theNR4A2S337A and NR4A2DH12 cells and was barely detectable by8 hours post-UVR in the NR4A2DH12 cells (Fig. 5B). As MRNactivation is an important early event in DNA repair, the delayin p-NBS1 foci indicated that DNA repair could be perturbed inthe cells expressing mutant forms of NR4A2.

Figure 4.

NR4A2 expression alters 53BP1 foci kinetics after ionizing radiation. A, A2058 cells stably expressing empty, NR4A2WT, NR4A2DNT, NR4A2S337A, or NR4A2DH12

proteins were exposed to 5 Gy IR, prenuclear extracted and fixed at 3, 6, 12, and 24 hours postirradiation. Immunofluorescence was performed to detect53BP1 proteins. Representative images taken from three independent experiments are shown.B,Quantitative assessment of number of foci per nucleus from90 cellsat each time point of each cell lines. C, Quantitative assessment on the percentage of cells showing positive 53BP1 foci (cells displaying 4 or more fociwere scored as positive) from 90 cells at each time point of each cell lines. Statistical analyses performed in graph B and C are normalized against valuesrecorded in the respective time points from the empty control cells. Statistical analyses were performed using two-way ANOVA with a Tukeypost-test (�� , P < 0.0001;�� , P < 0.001; �� , P < 0.01; � , P < 0.05).

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Functional NR4A2 protein is required for RAD51 resolutionHaving shown that HR activation may be affected by NR4A2

overexpression, we examined the kinetics of the subsequent HR-mediated DSB repair pathway by visualizing RAD51 foci. RAD51is the key facilitator of HR, downstream of the activation of MRNsignaling. We monitored the RAD51 foci formation and disap-pearance in melanoma cell lines at 6, 12, and 24 hours afterexposure to 50mJ/cm2UVB inA2058 cells (Fig. 6A). Comparativeanalyses of foci number per nucleus in 90 cells revealed that peakRAD51 foci recruitment occurred 6 hours postirradiation for allcell lines (Fig. 6B; Supplementary Table S6). Analysis of thepercentage of cells retaining RAD51 foci revealed a significantlyhigher proportion of positive nuclei in the NR4A2 mutant–expressing cells (Fig. 6C; Supplementary Table S7). Figure 6Ddetails the subpopulation of nuclei stratified for varying numbersof RAD51 foci per nucleus 6 hours postirradiation. We observedthat NR4A2S337A- and NR4A2DH12-expressing cells had the great-est number of nuclei with more than 10 RAD51 foci 6 hourspostirradiation. This was followed by a decrease of RAD51 foci inall cell lines. Most notably, the number of RAD51 foci per nucleusin NR4A2WT-expressing cells had reduced to a level significantlylower than the empty control cells by 24 hours postirradiation. Byassessing the percentage of cells with RAD51 foci, we also revealedthat RAD51 foci disappearance was highly efficient in NR4A2WT-expressing cells. However, NR4A2DNT-, NR4A2S337A-, andNR4A2DH12-stable lines showed a persistence in RAD51 foci forup to 24 hours in this analysis (Fig. 6C; Supplementary Table S7).

At the 24-hour post-UVR timepoint, the NR4A2S337A- andNR4A2DH12-expressing cells retained the greatest number ofnuclei with more than 10 RAD51 foci indicating an impairedDDR in these cell lines (Fig. 6E). In contrast, NR4A2WT-expressingcells had significantly fewer nuclei withmore than 10 RAD51 foci24 hours postirradiation, suggesting a more efficient DDR sig-naling and repair response in these cells. Thepersistence of RAD51foci following DNA damage provides an indication for repairduration, and our data suggest that overexpressionofNR4A2WT inmelanoma cells augments HR repair efficiency. Conversely,expressionofNR4A2DNT,NR4A2S337A, andNR4A2DH12may resultin the prolonged presence of DSBs after UVR, possibly due toattenuated HR repair capacity.

DiscussionWehave previously identified the crucial role ofMC1R-induced

NR4A2 expression in melanocytic cells in response to UVR DNAdamage (11). Studies have also shown that theNR4A2 is involvedin variousDNA repair pathways in response to different genotoxicinsults (9, 10). In light of these findings, it is tempting to speculatethat NR4A2 protein functions as an early response factor uponDNAdamage,with its role in promotingDNA repair implicated instudies using several different cell types (9, 10, 34). Interestingly,RXRa, a recognized dimerization partner of the NR4A proteins,has been reported to suppress UV-induced melanoma formationwhen expressed in keratinocytes (35) further implicating a

Figure 5.

Functional NR4A2protein is required for effectiveDNADSB repair. A2058 andHT144 cells stably expressing empty, NR4A2WT, NR4A2DNT, NR4A2S337A, orNR4A2DH12

isoforms were exposed to mock or 50 mJ/cm2 UVB. Protein lysates were prepared 4 and 8 hours postirradiation. Total protein (20 mg) was loaded forSDS-PAGE and detected by Western blotting with antibodies against; MRE11, RAD50, total NBS1, RPA, and Ku80 (A). B, Activation of NBS1 [Phospho-NBS1(Ser343)] is delayed in NR4A2S337A- and NR4A2DH12-expressing cells. b-tubulin was used as loading control. Western blots representative of threeindependent experiments.






broader role for the nuclear receptor family in ameliorating thedamaging effects of UVR. However, we have not observed UV-induced foci formation with RXRa or RXRg (10) suggesting thefacilitation of DNA repair by NR4A2 that we report here isindependent of RXR dimerization.

Exposure to UVB (28–315 nm) induces CPD and 6-4PPphotolesions (36, 37). These premutagenic DNA lesions areshown to be detrimental to replication, capable of causingreplication fork collapse (38–40). In this study, we investigatedthe DSB repair kinetics by visualizing 53BP1 foci in melanoma

Figure 6.

NR4A2 expression affects RAD51 foci kinetics after UVR radiation.A,A2058 cells stably expressing empty, NR4A2WT, NR4A2DNT, NR4A2S337A, or NR4A2DH12 proteinswere exposed to 50 mJ/cm2 UVB, prenuclear extracted, and fixed at 6, 12, and 24 hours post-UVB. Immunofluorescence was performed to detect RAD51.Representative images taken from three independent experiments are shown. B, Quantitative assessment of number of foci per nucleus from 90 cells at eachtime point of each cell lines. C, Quantitative assessment on the percentage of cells showing positive RAD51 foci (cells displaying 4 or more foci were scoredas positive) from 90 cells at each time point of each cell lines. Statistical analyses performed in graph B and C are normalized against values recorded in therespective time points from the empty control cell line. D, Number of nuclei displaying varying number of foci; 0–4 (white), 5–9 (yellow), or �10 (red),per nucleus from 90 cells 6 hours post-UVB irradiation. E, Number of nuclei displaying varying number of foci; 0–4 (white), 5–9 (yellow), or �10 (red),per nucleus from 90 cells 6 hours post-UVB irradiation. Statistical analyses were performed using two-way ANOVA with a Tukey post-test (�� , P < 0.0001;�� , P < 0.001; ��, P < 0.01; � , P < 0.05).

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cells irradiated with both UVB and IR. 53BP1 has been shownto localize rapidly to foci, and mediate downstream DDR atdamage centres (41). As the persistence of 53BP1 foci reflectsthe amount of unrepaired DSBs, our findings demonstrate thatoverexpression of wild-type NR4A2 can significantly increasethe efficiency of DSB foci resolution in melanoma cells. Intrigu-ingly, our study revealed that while normal 53BP1 foci recruit-ment was observed postirradiation in cells engineered to over-express WT and mutant NR4A2 proteins, a slow resolution offoci was evident in melanoma cells expressing NR4A2DNT-,NR4A2S337A-, and NR4A2DH12-mutant forms. These resultssuggest that different NR4A2 isoforms likely influence thesubsequent recruitment of DSB repair factors.

End resection of DNA is essential for the repair of DNA DSBs.The MRN complex has emerged as a crucial player that mediatesDNA resection in conjunction with the ATM signaling kinase(42, 43). The resulting ssDNAs are coated by RPA (44, 45), whichsubsequently allows for the recruitment of RAD51 (46). Althoughthe exact mechanisms that underlie the formation of RAD51-ssDNA nucleo-filament are only partially understood, researchhas shown that RAD51 forms nuclear foci at DSB sites and isconsidered as the core element of theHR repair signaling pathway(47, 48). We have shown here that in NR4A2-stable melanomacell lines, the recruitment of 53BP1foci occurred as early as 3hoursfollowing exposure to UVB. Signaling pathways activating therepair of DNA DSBs in these cells may be activated 4 hours afterirradiation, and this was followed by the recruitment of RAD51foci 6 hours postirradiation. In contrast, NR4A2S337A- andNR4A2DH12-stable cells both showed recruitment of 53BP1 focibut a diminished HR repair activation. These results suggest thatend resection of DNA is potentially facilitated through otherunidentified signaling pathways that compensate for impairedMRN complex activity. Indeed, studies have found that CtIP andEXO1 facilitate DNA end resection independently of the MRNcomplex to prevent genomic instability (49–51). Depletion ofMRN, CtIP, or EXO1 results in the delay of HR-dependent DNADSB repair and reduced cell survival in response to DNA damage(52). This is consistent with our findings whereby we observedslow RAD51 foci resolution and decreased cell viability inNR4A2S337A- and NR4A2DH12-expressing cells.

The involvement of NR4A2 in DNA DSB repair has beenimplied due to the colocalization of NR4A2 with gH2AX and53BP1 foci identified in previous studies (9, 10). However, themechanism by which NR4A2 facilitates DNA DSB repair was notinvestigated. Current models of the DNA DSB response describethe activation of two independent repair pathways following therecruitment of 53BP1 foci at DSB sites (53–55). We tested for theintegrity of HR andNHEJ repair pathways, both of which proceedas a highly ordered sequential process involving the recruitmentofmultiple proteins (56–58).We reveal that the overexpression ofNR4A2 in cells has no effect on pathway choice as sensor proteins,MRN (Mre11-Rad50-Nbs1) and Ku80, in the HR and NHEJpathway, respectively, displayed normal activation 4 hours post-irradiation in NR4A2-stable cell lines. Interestingly, we observedthatNR4A2S337A- andNR4A2DH12-stable cell lines seem to exhibitdefective MRN activation as evidenced by the significantly dimin-ished or delayed phosphorylation of NBS1. The MRN complex isthought to play multiple roles early in the DDR, acting primarilyas a sensor of DNA DSBs (59), and launch a network of signalingcascades that form the DDR (60). Accordingly, it is reasonable tospeculate that phosphorylation and the H12 domain of NR4A2

protein play crucial role in the NR4A2-mediated DSB repair, andthat signaling events downstream of the MRN complex may beimpeded in these cell lines.

Malewicz and colleagues (2011) reported that in the pres-ence of DNA DSBs, NR4A2 is phosphorylated at Serine 337 byDNA-PK, which subsequently promotes DNA end ligation.Substitution of serine 337 for alanine results in prolongedassociation with DNA-PKcs that ultimately disrupts the repairprocess (31). Our current investigation provides further insightinto the functional nature of the DNA-PK and NR4A2 partner-ship. First, DNA-PK phosphorylation is dispensable for NR4A2foci recruitment as melanoma cells transfected with YFP-NR4A2S337A successfully formed nuclear foci in response toUVR. This observation is in corroboration with previous work,which found no significant changes in the mRNA expressionlevels of genes involved in NHEJ after NR4A2 or NR4A2S337A

overexpression in HEK 293 cells (9).In classical nuclear receptors, binding of a ligand to the LBD

triggers a conformational change in theH12domain, allowing thedisplacement of a corepressor in exchange for a coactivator (61).By engineering a NR4A2DH12 mutant, we created a dominant-negative version of NR4A2 protein that shows strong interactionwith corepressors. The use of dominant-negative constructs hasbeen well established in nuclear receptors, including PPARs (62).Jagirdar and colleagues (2011) proposed a model whereby theNR4A2DH12 mutant facilitates the deacetylation of chromatin,creating a closed chromatin environment, inaccessible to NERfactors after UVR. Treatment of cells with histone deacetylaseinhibitor blocks chromatin deacetylation, thereby permitting therecruitment of NER factors to sites of DNA damage, despite thepresence of the mutant NR4A2 protein. As a result, NR4A2appears to mediate chromatin remodeling, which is a criticalearly process in DDR. On the basis of the persistence of 53BP1foci and the reduced NBS1 phosphorylation in NR4A2DH12-expressing cells, we provide further evidence suggesting thatNR4A2 function in the UVR DNA damage response extendsbeyond the repair of direct photolesions by NER but also func-tions in the repair of DNA DSBs that can be indirectly caused as aresult of replication fork collapse. The prolonged or diminishedrepair observed with the NR4A2DH12 mutant may arise due toineffective clearance of photolesions which subsequently resultsin the sustained production of DSBs.

While ligand-dependent activation through the LBD is a widelyaccepted model of nuclear receptor superfamily function (63),other studies have demonstrated that the NTD facilitates ligand-independent activation (64). It has been suggested that binding ofcoactivators, including Pin-1 and PARP-1, to the NTD enhancesNR4A2's transcriptional activity (9, 65). Previous and currentstudies presented here revealed that melanoma cells transfectedwith YFP-NR4A2DNT fail to form nuclear foci despite beingexposed to UVB. Given we have previously demonstrated thatNR4A2 initiates DNA repair by foci formation (10), the inabilityof NR4A2DNT to be recruited to the site of UVR-mediated lesionswould likely result in a disrupted lesion clearance. Indeed, in theNR4A2DNT-stable cells, we observed prolonged 53BP1 persis-tence, despite the normal activation of the MRN complex andKu80 after exposure to UVB. As a result, we speculate that thereduced DSB clearance in the context of the NR4A2DNT mutant ispotentially a consequence of ineffective NER. In support of this,we show that the high percentage of cells with persistent 53BP1foci positively correlates with the low level of cell viability in






NR4A2DNT- and NR4A2DH12-stable cells after UVR, which mostlikely reflects the well-established role of DSBs as potent inducersof cell death (66).

As the deletion of the NTD can affect NR4A2's transcriptionalactivity (67), it is important to consider the possibility that DNADSB repair impairment may be the result of abolished transcrip-tional activity. Interestingly, the additional mutant, NR4A2C266R,which is unable to bind cognate DNA-binding sites, displayed theability to form nuclear foci and activate DSB repair pathways inresponse toUVR.Given thedeletionof theNTDalso inadvertentlydeletes a potential sumoylation site located at the lysine 91residue (30), we also generated a NR4A2K91A mutant for furtheranalysis. Sumoylation has been implicated to regulate transcrip-tion factors (68), histonemodifications (69), nuclear localization(70), and DNA repair (71). In our investigation, we observed thatYFP-NR4A2K91A exhibited normal foci formation upon UVR butits DNA repair ability has not been investigated.

In summary, we have demonstrated that the kinetics of DNADSB repair can be altered in an in vitro melanoma model stablyexpressing wild-type or mutant versions of NR4A2. It is assumedthat these DNA DSBs arise from UV-induced replication forkcollapse, upon which functional NR4A2 is translocated to thesite of DNA damage to facilitate DNA DSB repair. In contrast,mutant forms of NR4A2, lacking the ability to translocate, trans-activate, or undergo phosphorylation, exhibit compromisedrepair capacity. Moreover, our results clearly illustrate the criticalrequirement of NR4A2 for a functional HR-mediated DNA repair.Coupled with recent reports demonstrating the involvement ofNR4A2-mediated NER following UVR (10), and its role as anantioxidative stress factor via gene regulation (72), we revealmolecular events associated with the NR4A2 receptor that areresponsible for combating the deleterious effects of UV-inducedmelanocytic carcinogenesis.

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

Authors' ContributionsConception and design: K. Yin, Y. Chhabra, Y.C. Lim, E. Dray, A.G. SmithDevelopment of methodology: K. Yin, Y. Chhabra, Y.C. LimAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): K. Yin, Y. Chhabra, R. Trop�ee, A.G. SmithAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): K. Yin, Y. Chhabra, R. Trop�ee, Y.C. Lim, M. Fane,E. Dray, A.G. SmithWriting, review, and/or revision of the manuscript: K. Yin, Y. Chhabra,R. Trop�ee, M. Fane, E. Dray, R.A. Sturm, A.G. SmithAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): Y.C. Lim, R.A. Sturm, A.G. SmithStudy supervision: Y.C. Lim, E. Dray, A.G. Smith

AcknowledgmentsThe authors thank the Australian Cancer Research Foundation (ACRF)/

Institute forMolecular BioscienceDynamic Imaging Facility for Cancer Biology,which was established with the support of the ACRF; and the School ofBiomedical Sciences at the University of Queensland for technical assistancewith microscopy. We acknowledge the assistance of Dr. David Sester from TRIFlow Cytometry Facility with FACS analysis.

Grant SupportK. Yin is the recipient of a University of Queensland APA PhD scholarship. R.

Trop�ee is the recipient of a PA Research Foundation Fellowship. E. Dray is therecipient of a NBCF Fellowship ECF-13-04. This work was supported by theNational Health and Medical Research Council (#APP631510; APP1083612),and Cancer Council Queensland (#APP1065270).

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received January 13, 2017; revised April 7, 2017; accepted June 6, 2017;published OnlineFirst June 12, 2017.

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Yin et al.




2017;15:1184-1196. Published OnlineFirst June 12, 2017.Mol Cancer Res Kelvin Yin, Yash Chhabra, Romain Tropée, et al. to UVRNR4A2 Promotes DNA Double-strand Break Repair Upon Exposure

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NR4A2 Promotes DNA Double-strand Break Repair Upon ... · DNA Damage and Repair NR4A2 Promotes DNA Double-strand Break Repair Upon Exposure to UVR Kelvin Yin1,Yash Chhabra2,3, Romain