atm-mediated kdm2a phosphorylation is required for the...

ORIGINAL ARTICLE

ATM-mediated KDM2A phosphorylation is required for theDNA damage repairL-L Cao1,2,5, F Wei1,2,5, Y Du1,2, B Song1,2, D Wang1,2, C Shen1,2, X Lu1,2, Z Cao1,2, Q Yang1,2, Y Gao3, L Wang1,2, Y Zhao1,2, H Wang1,2,Y Yang1,2 and W-G Zhu1,2,4

The ataxia-telangiectasia mutated (ATM) protein is a key signaling molecule that modulates the DNA damage response. However,the exact mechanism by which ATM regulates DNA damage repair has not yet been elucidated. Here, we report that ATM regulatesthe DNA damage response by phosphorylating lysine-specific demethylase 2A (KDM2A), a histone demethylase that acts at sites ofH3K36 dimethylation. ATM interacts with KDM2A, and their interaction significantly increases in response to DNA double-stranded,but not single-stranded, breaks. ATM specifically phosphorylates KDM2A at threonine 632 (T632) following DNA damage, asdemonstrated by a mutagenesis assay and mass spectrometric analysis. Although KDM2A phosphorylation does not alter its owndemethylase activity, T632 phosphorylation of KDM2A largely abrogates its chromatin-binding capacity, and H3K36 dimethylationnear DNA damage sites is significantly increased. Consequently, enriched H3K36 dimethylation serves as a platform to recruit theMRE11 complex to DNA damage sites by directly interacting with the BRCT2 domain of NBS1, which results in efficient DNAdamage repair and enhanced cell survival. Collectively, our study reveals a novel mechanism for ATM in connecting histonemodifications with the DNA damage response.

Oncogene advance online publication, 30 March 2015; doi:10.1038/onc.2015.81

INTRODUCTIONAtaxia-telangiectasia mutated (ATM) is a Ser/Thr protein kinasethat has a crucial role in the cellular response to double-strandedbreaks (DSBs).1 Following DSB induction, a large heterogeneousgroup of proteins forms several complexes at DSB sites. Proteinkinase activation, including ATM, ATR (ATM- and Rad3-related) orDNA-PK (DNA-dependent serine/threonine protein kinase) thenoccurs to regulate cellular responses to DNA damage.2 ATM hasbeen reported to primarily influence the DNA damage responseby phosphorylating and/or recruiting several effector proteins,such as 53BP1 (p53 binding-protein 1), BRCA1 (breast cancergene 1) or Abl tyrosine kinase, at DNA damage sites to promoteefficient DNA repair or induce other cellular processes.2,3 Inaddition, ATM can modulate histone modifications to regulatethe DNA damage response. For example, upon DSB induction,ATM rapidly phosphorylates the histone H2A variant H2AX toform γH2AX foci in the megabase chromatin domains flankingthe sites of DNA damage, which is considered to be a classicalmarker for DSB sites.4,5 It has also been reported that histone H1is markedly, but transiently, dephosphorylated in an ATM-dependent manner following low doses of ionizing radiation.6

In addition, histone acetylation is linked to the ATM-modulatedDNA damage response, as ATM regulates chromatin organizationby associating with the histone deacetylase HDAC1 and,subsequently, enhancing its deacetylase activity to regulatechromosomal integrity.7 Moreover, ATM was recently shown tomodulate histone methylation. For example, ATM phosphory-lates H4K20 methyltransferase multiple myeloma SET domain in

response to ionizing radiation, which is important for H4K20methylation, 53BP1 recruitment and the DNA damage responseat damage sites.8

It is becoming increasingly clear that H3K36 methylation is animportant modification for DNA damage repair. For example,H3K36me3 is required for the recruitment of the mismatchrecognition protein hMutSα (hMSH2-hMSH6) onto chromatin andfor the regulation of DNA mismatch repair.9 In addition, theSETD2-dependent histone H3K36me3 is required for homologousrecombination repair and genome stability.10–12 However, theprimary immediate methylation change after DSB is H3K36me2, asH3K36me2 increases at the DNA damage sites soon after γ-radiation, defining a histone methylation event that enhancesDNA damage repair.13

H3K36me2 is mainly catalyzed by nuclear receptor-binding SETdomain-containing protein 1 (NSD1), Metnase,14 and is primarilydemethylated by lysine-specific demethylase 2A (KDM2A, alsoknown as FBXL11 or JHDM1A), which contains the well-knownchromatin-binding domains PHD-type zinc-finger, CxxC-type zinc-finger and an enzymatic jmjc domain.15,16 The demethylationactivity of KDM2A is associated with the transcriptional repressionof ribosomal RNA,17 satellite repeats18 or DUSP3.19 In addition,KDM2A demethylates a non-histone protein, the p65 subunit ofnuclear factor-κB at K218 and K221, to inhibit nuclear factor-κBactivity.20,21 Furthermore, KDM2A overexpression reduces theaccumulation of H3K36me2 at the DSB site and therefore inhibitsDSB repair.13 Although the importance of H3K36 dimethylation inthe DNA damage response is recognized, how the H3K36

1Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), State Key Laboratory of Natural and Biomimetic Drugs, Beijing Key Laboratory of ProteinPosttranslational Modifications and Cell Function, Beijing, China; 2Department of Biochemistry and Molecular Biology, Peking University Health Science Center, Beijing, China;3Protein Chemistry Facility, School of Biological Sciences, Tsinghua University, Beijing, China and 4Peking University-Tsinghua University Joint Center for Life Sciences,Beijing, China. Correspondence: Professor W-G Zhu, Department of Biochemistry and Molecular Biology, Peking University Health Science Center, Xueyuan Road 38#, Beijing,100191, China.E-mail: [email protected] authors contributed equally to this work.Received 1 October 2014; revised 21 January 2015; accepted 22 February 2015

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modification enzymes, such as KDM2A, are themselves regulatedis completely unknown.Based on the critical role of both ATM and H3K36 methyla-

tion in modulating the DNA damage response, we hypothe-sized that ATM may interact with H3K36 methylation tocoordinate this response. In this study, we present evidencethat ATM interacts with and phosphorylates KDM2A atthreonine 632 (T632) in response to DSBs, which impairs thechromatin-binding capacity of KDM2A. Consequently, H3K36dimethylation at the DNA damage sites is increased andfacilitates the recruitment of the MRE11 complex near the DNAdamage sites to aid DNA damage repair through a directinteraction with the BRCT2 domain of NBS1. Overall, this studyis the first to demonstrate that KDM2A is a novel ATM substrateand KDM2A phosphorylation is critical to the DNA damageresponse.

RESULTSATM and KDM2A interact in vivoAs both ATM and H3K36 methylation are related to DSB, it wasexpected that ATM regulates H3K36-related histone methyltrans-ferases or demethylases. To investigate whether ATM and H3K36methyltransferases or demethylases interacted, a co-immuno-precipitation (co-IP) assay was performed in HEK293T (trans-formed human embryonic kidney) cells with overexpressedFlag-tagged ATM (Flag-ATM). As shown in Figure 1a, Flag-ATMdid not interact with the two H3K36 demethylases JHDM1B andJMJD2B or the H3K36 methyltransferase NSD1. Interestingly, ATMdid clearly interact with endogenous KDM2A, which is anH3K36me1/2 demethylase. To confirm this result, HEK293T cellswere co-transfected with yellow fluorescent protein (YFP)-taggedATM (YFP-ATM) and Flag-KDM2A, and the extracts were subjectedto a co-IP assay. As shown in Figure 1b, YFP-ATM and Flag-KDM2A

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Figure 1. Interaction between ATM and KDM2A. (a) Whole-cell lysates of HEK293T cells transfected with Flag-ATM were precipitated with anti-Flag antibody, and probed with the indicated antibodies. (b) HEK293T cells were transfected with YFP-ATM and Flag-KDM2A. At 48 h aftertransfection, the whole-cell lysates were precipitated with anti-GFP antibody, and probed with anti-GFP or anti-Flag antibody. (c) Whole-celllysates of HEK293 cells were precipitated with anti-ATM or anti-KDM2A antibody, and the interactive components were then analyzed byimmunoblotting. (d) HEK293 cells were treated with 5-aza-CdR at 5 μM for 48 h, adriamycin at 1 μM for 12 h, etoposide at 20 μM for 4 h, ultravioletradiation (UV) for 5min. The cell extracts were then precipitated with anti-ATM antibody and probed with anti-KDM2A antibody. The relativeintensity of the interaction between ATM and KDM2A was quantified and is shown as a histogram. The ‘NC’ indicates negative control withoutany treatment. Student’s t-test (two-tailed): 5-aza-CdR versus NC, Po0.01; adriamycin versus NC, Po0.05; etoposide versus NC, Po0.01; UVversus NC, P40.05. (e) HEK293 cells were treated with etoposide at concentration of 5 or 20 μM for 4 h. A co-IP assay was performed as describedin d. Data are shown as means± s.d.; n=3. Student’s t-test (two-tailed): 5 μM versus 0 μM, Po0.05; 20 μM versus 0 μM, Po0.01.

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clearly interacted. In addition, endogenous ATM and KDM2Ainteracted with each other as well, as demonstrated by co-IP inHEK293 and HCT116 cells (Figure 1c, Supplementary Figure 1a).To investigate whether the interaction between ATM and

KDM2A has a biological function within the cell, HEK293 cells weretreated with different DNA damage reagents, such as the DSBinducers, adriamycin,22 etoposide23 or 5-aza-2′-deoxycytidine (5-aza-CdR),24 and the DNA single-stranded break inducer ultravioletradiation.25 As shown in Figure 1d, the interaction betweenendogenous ATM and KDM2A markedly increased after treatmentwith adriamycin, etoposide or 5-aza-CdR, but not followingultraviolet radiation. This finding suggests that the interactionbetween ATM and KDM2A may be specifically associated withDSBs. In addition, the interaction between ATM and KDM2A wasdose dependent when treated with etoposide (Figure 1e).However, neither the interaction between KDM2A and ATR northe interaction between KDM2A and PARP1 was detected(Supplementary Figures 2a and c), which further suggests thatthe ATM–KDM2A interaction is a specific response to DSBs.

KDM2A is a novel ATM substrate that responds to DNA DSBsAs ATM interacted with KDM2A, it is hypothesized that KDM2A is anovel ATM substrate. In general, ATM kinase has a high substratespecificity, as it recognizes the Ser-Gln (SQ) or Thr-Gln (TQ) motif andphosphorylates Ser/Thr in these motifs.26,27 To investigate whetherKDM2A is phosphorylated by ATM, we used a specific anti-phospho-Ser/Thr ATM/ATR substrate antibody28 that recognizes the particularphosphorylation of S/TQ motifs (p-S/TQ) in ATM/ATR substrates todetect S/TQ phosphorylation in KDM2A. As shown in Figures 2a andb, KDM2A phosphorylation significantly increased after etoposidetreatment. This effect was abolished by λ phosphatase (λ-PPase)treatment, indicating that KDM2A is phosphorylated at its serine orthreonine in the S/TQ motifs in response to DNA damage. Consistentwith the previous result (Supplementary Figure 2c), we did notdetect any PARylation of KDM2A following etoposide treatment(Supplementary Figure 2d). To further identify which of the four S/TQmotifs (S11Q, T115Q, S180Q and T632Q) was phosphorylated withinKDM2A, in vitro phosphorylation assays were performed withendogenous activated (S1981-phosphorylated) ATM29,30 and twoglutathione S-transferase (GST)-fused truncation mutants of KDM2A(1-560aa/560-680aa). The catalyzed KDM2A fragments were thensubjected to mass spectrometry analysis, which indicated that T632,but not S11, T115 or S180, was phosphorylated by ATM(Supplementary Figure 3). In addition, cells expressing mutatedKDM2A (T632A) (T632 to alanine) exhibited reduced levels of S/TQphosphorylation compared with cells expressing wild-type KDM2A(Figure 2c). Furthermore, an in vitro phosphorylation assay using 32P-labeled ATP revealed that the KDM2A (T632A) mutant blockedortho-32P incorporation into KDM2A (Figure 2d). These findingssuggest that T632 is a bona fide predominant site in KDM2A for DSB-inducible phosphorylation.To analyze the role of T632-phosphorylated KDM2A in

responding to DNA damage, a rabbit polyclonal antibody wasgenerated that specifically recognizes the phosphorylated T632residue of KDM2A (pT632) (Supplementary Figure 4). Levels ofpT632 significantly increased in response to the 5-aza-CdR,adriamycin and etoposide treatments but not in response to theultraviolet radiation treatment (Figure 2e). In addition, the increasein pT632 levels following etoposide treatment was dose- andtime-dependent, and it appeared that the increases in γH2AX,pATM and pT632 occurred nearly simultaneously (Figures 2f and g).Consistently, pT632 levels also significantly decreased followingtreatment with the ATM-specific inhibitor KU55933 (ref. 31) whenresponding to DNA damage (Figure 2h). In addition, no pT632 wasdetected in the ATM-mutated A-T cells,32 but pT632 level wasinduced in A-T cells overexpressing ATM in response to DNAdamage (Figure 2i). These data further support the idea that ATMis the kinase that phosphorylates KDM2A at T632 after DSBs.

KDM2A phosphorylation at Thr632 abrogates its chromatin-binding capacitySubsequently, to detect whether T632 phosphorylation of KDM2Aaffects its demethylase activity, an in vitro demethylation assaywas performed. It is unlikely that KDM2A phosphorylation at T632is related to its demethylase activity because wild-type KDM2A,the unphosphorylated KDM2A (T632A) mutant and the phospho-mimetic KDM2A (T632E) (T632 to glutamate) mutant had similarin vitro demethylation efficiencies (Supplementary Figure 5). Asthe PHD zinc-finger domains of nuclear proteins are able to bindhistone lysines33 and because T632 is located within KDM2A’s PHDdomain (Figure 3a), we examined whether T632 phosphorylationaffected KDM2A’s ability to bind chromatin. Interestingly, KDM2Alevels were markedly decreased in the chromatin fraction and,accordingly, an increase in KDM2A appeared in the solublefraction following treatment in HEK293 (Figure 3b) or HCT116 cells(Supplementary Figure 1b). However, KDM2A pT632 was notdetected in the chromatin fraction and was only enriched in thesoluble fraction of HEK293 (Figure 3b) or HCT116 cells(Supplementary Figure 1b). In addition, HEK293 cells wereseparately transfected with different KDM2A constructs, includingwild-type KDM2A and T632A or T632E mutants (SupplementaryFigure 6). The enrichment of exogenous KDM2A was detected inthe chromatin fraction. As shown in Figure 3c, the strongestbinding capacity of KDM2A to chromatin was detected in the cellextracts with the overexpressed T632A mutant, whereas a veryweak chromatin-binding capacity was observed in cell extractswith the overexpressed phospho-mimetic T632E mutant. More-over, the interaction of the T632A mutant and histone H3increased, and the T632E mutant failed to interact with H3, asassayed by co-IP (Figure 3d). To further investigate the influence ofKDM2A phosphorylation on localization of H3K36me2 andKDM2A, an immunofluorescence assay was performed. As shownin Figures 3e and f, most of the HEK293 cells that overexpressedthe T632E mutant exhibited obvious H3K36me2 staining, butH3K36me2 did not fully colocalize with the overexpressed T632Emutant. However, H3K36me2 staining was not detected in most ofthe cells overexpressing wild-type KDM2A or the T632A mutant(Figures 3e and f). Together, these data demonstrate that thephosphorylation of KDM2A at T632 abrogates its chromatin-binding capacity but does not affect its demethylase activity.

An ATM-KDM2A signaling pathway is required for H3K36me2increase in response to DNA damageAs T632 phosphorylation of KDM2A decreased its chromatin-binding capacity, we hypothesized that H3K36me2 would increasein response to DNA damage. This prediction is consistent withthe evidence that H3K36me2 was induced at DSBs, but H3K36me3induction was absent following ionizing radiation and I-SceI orAsiSI-induced DNA double-strand break induction.8,10,12,13

Immunoblotting assays were performed to detect H3K36 methyla-tion changes in HEK293 cells after etoposide treatment.H3K36 dimethylation significantly increased at the early stage ofDNA damage response, which is consistent with a previousstudy,13 and H3K36 monomethylation correspondingly decreasedin a time- and dose-dependent manner (Figure 4a, SupplementaryFigures 7a and b). However, similar changes at other typicallysine sites were not observed (Supplementary Figure 7c). Inaddition, these changes of H3K36 dimethylation were furtherconfirmed in HEK293 cells treated with adriamycin, etoposide or5-aza-CdR. By contrast, ultraviolet radiation did not significantlyaffect H3K36 dimethylation (Figure 4b). Similar changes of H3K36dimethylation were also observed in HCT116 and HeLa cellsin response to etoposide treatment (Supplementary Figure 1c),suggesting that the increase in H3K36me2 is a universal responseto DSBs.


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© 2015 Macmillan Publishers Limited Oncogene (2015) 1 – 13

We next determined whether the signaling pathway of ATM-KDM2A is required to induce H3K36me2. As shown in Figure 4c,H3K36me2 did not increase in ATM-mutated A-T cells butsignificantly increased in A-T cells that overexpressed ATM inresponse to etoposide treatment. In addition, the H3K36me2

increase was significantly alleviated in HEK293 cells treated with theATM-specific inhibitor KU55933 (Figure 4d). However, ATR knock-down did not affect H3K36me2 levels after etoposide treatment(Supplementary Figure 2b). Moreover, H3K36me2 significantlydecreased in HEK293 cells that overexpressed wild-type KDM2A

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Figure 2. Induction of KDM2A phosphorylation by ATM at Thr632 in response to DNA damage. (a) HEK293T cells transfected with Flag-KDM2Awere treated with 40 μM etoposide for 2 h. The cell extracts were precipitated with anti-Flag antibody, and subjected to immunoblotting.(b) Flag-KDM2A immunoprecipitates from untreated or etoposide-treated HEK293T cells in the absence or presence of lambda phosphatase(λ PPase) were subjected to immunoblotting. (c) HEK293T cells transfected with wild-type Flag-KDM2A, S11A, or T632A mutant were treatedwith etoposide and an immunoprecipitation assay was performed as described in a. (d) KDM2A fragments were incubated with pATMATMand [γ-32P] ATP, and subsequently separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), stained by Coomassiebrilliant blue (CBB) or exposed by autoradiography. The arrow indicates phosphorylation of KDM2A. # Represents specific protein bands. The‘NC’ here indicates negative control without KDM2A fragment. (e) HEK293 cells were treated with 5-aza-CdR at 5 μM for 48 h, adriamycin at1 μM for 12 h, etoposide at 40 μM for 2 h, ultraviolet radiation (UV) for 5min. The cell extracts were then subjected to immunoblotting. The ‘NC’indicates negative control without any treatment. (f) The cell extracts of HEK293 cells treated with etoposide for 2 h at the indicated doseswere subjected to immunoblotting. (g) Immunoblotting of pT632 after treating HEK293 cells with etoposide at 20 μM for the indicated timeperiods. (h) ATM inhibitor KU55933 was introduced into HEK293 cells in the absence or presence of etoposide at 40 μM for 2 h, and pT632 wasdetected by immunoblotting. (i) A-T cells transfected with pcDNA or Flag-ATM separately were treated with etoposide at 40 μM for 2 h andsubjected to immunoblotting.


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but not in cells that overexpressed the T632E mutant (Figure 4e).This finding indicates that KDM2A phosphorylation is responsiblefor the increase in H3K36me2. Collectively, these findings suggestthat the ATM-KDM2A signaling pathway is essential for the increaseof H3K36me2 in response to DNA damage.

H3K36me2 interacts with the MRE11 complex by directly bindingto NBS1As H3K36me2 increase and recruitment of the MRE11 complexonto chromatin occur during an early stage of the DNA damageresponse,13,34 it is possible that that H3K36me2 and the MRE11

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Figure 3. Decreased binding capacity of KDM2A to chromatin by phosphorylation of KDM2A at Thr632. (a) Schematic representation ofthe KDM2A protein. KDM2A contains a PHD zinc finger, a CxxC-type zinc finger and a Jmjc domain. Thr632 is located in the PHD zinc finger.(b) HEK293 cells were treated with etoposide at 40 μM for 2 h, and were fractionated into soluble fraction and chromatin fraction.Immunoblotting of KDM2A was performed. Equal amount of KDM2A was loaded, and immunoblotted with anti-pT632 antibody. (c) HEK293cells were transfected with wild-type KDM2A, the T632A or T632E mutant. At 48 h after transfection, the chromatin fractions were separatedand subjected to immunoblotting. Data are also shown as means± s.d.; n= 3. Student’s t-test (two-tailed): T632A versus WT, Po0.01; T632Eversus WT, Po0.01; T632E versus T632A, Po0.01. (d) Whole-cell lysates of HEK293T cells transfected with wild-type KDM2A, the T632A andT632E mutant were precipitated with anti-Flag antibody, and then the interactive components were analyzed by immunoblotting. (e) HEK293cells were transfected as described in c. The cells were fixed and subjected to confocal fluorescent microscopy with Flag and H3K36me2antibodies. The merge channel was the combination of the two colors. The arrows indicate representative positively KDM2A-overexpressedcells, and the symbols ‘#’ indicate the normal untransfected cells. (f) The quantification of H3K36me2-positive cells from the experiment forwhich results are shown in e. H3K36me2-positive cells refer to the cells with the comparable H3K36me2 staining to that of the normaluntransfected cells inside. The percentage of H3K36me2-positive cells for each condition was calculated from a minimum of 100 cells. Data areshown as means± s.d. from three independent experiments.


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complex interact when responding to DNA damage. To test thispossibility, HEK293T cells were transfected with Flag-tagged MRE11,RAD50 or NBS1, and the cell extracts were subjected to co-IP assays.As shown in Figures 5a and c, all exogenous members of the MRE11complex interacted with H3K36me2, and this interaction signifi-cantly increased in response to etoposide treatment. In addition, anendogenous reciprocal co-IP also indicated an increased interactionbetween H3K36me2 and the MRE11 complex members after DSBinduction (Figure 5d). However, the mRNA and protein levels of theMRE11 complex components were not obviously changed afteretoposide treatment (Supplementary Figure 8). To determinewhether the interaction between H3K36me2 and the MRE11complex is direct, in vitro GST pull-down assays were performedusing GST-fused MRE11, RAD50 and NBS1 proteins and biotin-tagged H3 peptides. It was identified that H3K36me2 interactedwith NBS1 directly, but not with MRE11 or Rad50. NBS1 did notinteract with other H3 peptides (Figures 5e and f). Furthermore, theinteraction between H3K36me2 and NBS1 was dependent onpeptide concentration (Figure 5g).To further determine which functional domain of NBS1 is

required for the interaction of H3K36me2 and NBS1, a peptidepull-down assay was then performed, using deleted mutants ofNBS1 and biotin-tagged H3K36me2. NBS1 contains a FHA (fork-head-associated) domain and a BRCT (BRCA1 C-terminus) domain inits N terminus, a BRCT domain in its central region, and a MRE11-interacting region at the C-terminus (Figure 6a, upper).35,36 All thedeleted NBS1 mutants, except for NBS1(d219-460), clearly

interacted with H3K36me2 (Figure 6a, lower), indicating that NBS1(219-460) is required for the interaction between H3K36me2 andNBS1. NBS1(219-460) contains the BRCT2 domain (218-327) and itsadjacent region (328-460). To determine which region is requiredfor the interaction between NBS1 and H3K36me2, in vitro GST pull-down assays were performed using GST-NBS1(218-327) and GST-NBS1(328-460) proteins, as well as biotin-tagged H3K36me2peptide. As shown in Figure 6b, NBS1(218-327), but not NBS1(328-460), interacted with H3K36me2, indicating that the BRCT2domain of NBS1 is required for the interaction between H3K36me2and NBS1. In addition, two deleted BRCT2 mutants wereconstructed, and peptide pull-down assays using the two mutantswere performed. As shown in Figure 6c, NBS1-BRCT2(d218-247) didnot interact with H3K36me2, indicating that the N-terminal of theBRCT2 domain, which contains an α helix (α1) and a β sheet (β1),37

is critical for the interaction between H3K36me2 and the BRCT2domain of NBS1. Together, these results suggest that H3K36me2interacts with NBS1 directly through the BRCT2 domain of NBS1.

Enriched H3K36me2 facilitates MRE11 complex recruitment toDNA damage sitesNext, to determine whether H3K36me2 is required for therecruitment of the MRE11 complex to damaged DNA, HEK293 cellswere transfected with pcDNA, wild-type KDM2A or KDM2A-T632Emutant. An immunofluorescence assay was then performed toobserve MRE11 foci formation. As shown in Figures 7a and b, MRE11

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Figure 4. Increased H3K36me2 in response to DNA DSB and the role of ATM-KDM2A signaling pathway in H3K36me2 increase. (a) HEK293 cellswere treated with etoposide at 40 μM for the indicated time periods and subjected to histone acid extraction assay. Immunoblotting wasperformed with the indicated antibodies. (b) HEK293 cells were treated with 5-aza-CdR at 5 μM for 48 h, adriamycin at 1 μM for 12 h, etoposideat 40 μM for 2 h, ultraviolet radiation (UV) for 5min. The acid-extracted histones were subjected to immunoblotting with H3K36me2 antibody.The ‘NC’ here indicates negative control without any treatment. (c) A-T cells were transfected with pcDNA or Flag-ATM separately and treatedwith etoposide at 40 μM for 2 h. The acid-extracted histones were subjected to immunoblotting. (d) The ATM-specific inhibitor KU55933 wasintroduced into HEK293 cells treated with etoposide at 40 μM for 2 h, and H3K36me2 was detected by immunoblotting. (e) HEK293 cells weretransfected with pcDNA, wild-type KDM2A or the T632E mutant separately, and treated with etoposide at 40 μM for 2 h. The whole-cell lysatesand acid-extracted histones were subjected to immunoblotting.


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foci formation was significantly inhibited in cells overexpressingwild-type KDM2A. However, MRE11 foci formation in cells thatoverexpressed the phospho-mimetic T632E mutant was similar tothat of cells that overexpressed nonspecific pcDNA. A U2OS DRGFP(direct repeat green fluorescent protein) cell line and an endonu-clease I-SceI were introduced to induce a targeted single DNAdamage site38 to further determine the role of KDM2A phosphor-ylation in MRE11 complex recruitment. As shown in Figures 7c andd, an increase in H3K36me2 and recruitment of the MRE11 complexto the DNA near the I-SceI site were observed upon I-SceItransfection. The relative enrichment of the MRE11 complexsignificantly decreased in U2OS cells that overexpressed wild-typeKDM2A, but not in cells that overexpressed the T632E mutant.However, KDM2A knockdown did not significantly affect MRE11recruitment in response to DNA damage (Supplementary Figure 9a).We also confirmed that the KDM2A phosphorylation that is

associated with the recruitment of the MRE11 complex is ATMdependent. As shown in Figures 7e and f, the enrichment of

H3K36me2 on chromatin increased in A-T cells that overexpressedATM in response to DNA damage, but not in A-T cells thatoverexpressed pcDNA. Although the enrichment of MRE11 onchromatin was upregulated in both types of A-T cells in responseto etoposide treatment, the MRE11 enrichment on chromatin inA-T cells that overexpressed ATM was higher than that of the cellsthat overexpressed pcDNA (Po0.05). Consistently, MRE11 recruit-ment in response to DNA damage also decreased after treatmentwith the ATM-specific inhibitor KU55933 (Supplementary Figure9b). These data clearly demonstrate that ATM-mediated KDM2Aphosphorylation is essential for MRE11 complex recruitment toDNA damage sites.

KDM2A phosphorylation is required for efficient DNA damagerepair and enhanced cell survivalIt has been clearly demonstrated that the MRE11 complex has acrucial role in DNA damage repair.35,39–41 A comet assaywas performed to directly measure the repair efficiency of

Input IgG IP: H3K36me2

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Figure 5. The direct interaction between H3K36me2 and NBS1. HEK293T cells were transfected with Flag-MRE11 (a), Flag-Rad50 (b) or Flag-NBS1 (c), separately, and treated with etoposide at 40 μM for 2 h. The whole-cell lysates were precipitated with anti-Flag antibody, and probedwith anti-H3K36me2 or anti-Flag antibody. (d) HEK293 cells were treated with etoposide at 40 μM for 2 h, whole-cell lysates were precipitatedwith anti-H3K36me2 antibody, and the interactive components were then analyzed by immunoblotting. (e) Biotin-tagged H3 peptides wereincubated with GST-fused MRE11, Rad50 or NBS1, separately. (Upper) Bound H3 peptides were analyzed by immunoblotting with anti-biotinantibody. (Lower) GST, GST-MRE11, GST-Rad50 or GST-NBS1 proteins were stained by Coomassie brilliant blue (CBB). (f) HEK293T cells weretransfected with Flag-NBS1, and then the whole-cell lysates were subjected to peptide pull-down by the indicated biotin-tagged H3 peptides,separately. H3 peptides were stained by CBB and the bound Flag-NBS1 was subjected to immunoblotting. (g) HEK293T cells were transfectedwith Flag-NBS1, and then the whole-cell lysates were subjected to peptide pull-down by biotin-H3K36me2 peptide at different peptideconcentrations.


7


etoposide-induced DNA damage. By comparing the length andthe area of DNA tails, more serious DNA damage was retained inHEK293 cells that overexpressed wild-type KDM2A compared withcells that overexpressed pcDNA or the phospho-mimetic T632Emutant (Figures 8a and b). In addition, the γH2AX level (a markerof DNA damage) was detected during DNA damage recovery. Asshown in Figures 8c and e, the γH2AX level and the number ofγH2AX foci in HEK293 cells that overexpressed wild-type KDM2Awere much higher at later time points than that in cells thatoverexpressed pcDNA or the T632E mutant. Finally, a colonyformation assay was performed to quantify cell survival rate. Cellsthat overexpressed wild-type KDM2A exhibited a decreasedsurvival rate in response to DNA damage, whereas cells thatoverexpressed the KDM2A (T632E) mutant did not experience asimilar effect (Figure 8f). These results suggest that KDM2Aoverexpression inhibits DNA damage repair, which is consistentwith a previous study13 and that ATM-mediated KDM2Aphosphorylation is critical for DNA damage repair and cell survival.

DISCUSSIONIn this study, KDM2A was identified as a novel substrate of ATM.DSB enhanced the interaction between ATM and KDM2A, whichinduced ATM-mediated phosphorylation of KDM2A at T632. Thespecific KDM2A T632 phosphorylation compromised itschromatin-binding capacity and thus alleviated its ability todemethylate H3K36me2. Consequently, H3K36 dimethylationlevel, especially at DNA damage sites, significantly increased.High levels of H3K36me2 recruited the MRE11 complex to theDNA damage sites via a direct interaction with NBS1 and thuscontributed to the ability of cells to repair DSBs.It has recently been reported that histone methylation has a

critical role in the DNA damage response. For example, the DSBrepair component 53BP1 and its fission yeast homolog Crb2 arerecruited to the sites of DNA damage by the methylated histoneH3 lysine 79 (H3K79) or methylated histone H4 lysine 20(H4K20).8,42,43 Previous studies9–13 have also demonstrated thatH3K36 dimethylation/trimethylation mediates the DNA damage

1. FL FHA BRCT1

2.197-C3.197-4604.d24-1085.d108-1966.d219-460

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Figure 6. The BRCT2 domain of NBS1 directs the interaction between H3K36me2 and NBS1. (a) Schematic diagrams of plasmids encodingFlag-tagged full-length (FL) or deleted mutants of NBS1 are shown. FHA, the forkhead-associated domain; BRCT, BRCA1 C-terminus domain;MIR, MRE11-interacting region. HEK293T cells were transfected with Flag-tagged full-length (FL) or deleted mutants of NBS1, separately. Thewhole-cell lysates were subjected to peptide pull-down by biotin-tagged H3K36me2 (21-44aa) peptide. (Upper) Input NBS1 or deletionmutants of NBS1 proteins. (Lower) H3K36me2 were stained by Coomassie brilliant blue (CBB) and the bound NBS1 was subjected toimmunoblotting. (b) Biotin-H3K36me2 peptide was incubated with GST, GST-NBS1 (218-327aa), GST-NBS1 (328-460aa), GST-NBS1 full-lengthseparately. (Upper) Input GST, GST-NBS1 (218-327aa), GST-NBS1 (328-460aa) or GST-NBS1 full length. (Lower) H3K36me2 were stained by CBBand the bound NBS1 was subjected to immunoblotting. (c) Biotin-H3K36me2 peptide was incubated with GST, GST-BRCT2 full length, GST-BRCT2 (d218-247aa), GST-BRCT2 (d268-291aa) separately. (Left) Input GST, GST-BRCT2, GST-BRCT2 (d218-247aa) or GST-BRCT2 (d268-291aa).(Right) H3K36me2 were stained by CBB and the bound fragments were subjected to immunoblotting.


8


response. H3K36me2 level is increased in response to DNA DSBs,whereas H3K36me3 level does not change. The increase inH3K36me2 level is associated with the early response to DNA DSBsand promotes the recruitment of Ku70 or NBS1 to DSB sites.13

However, H3K36me3 constitutively recruits LEDGF (lensepithelium-derived growth factor p75) to chromatin. FollowingDNA DSBs, LEDGF recruits CtIP to promote end resection and thenfacilitates RPA and RAD51 recruitment.10 Therefore, bothH3K36me2 and H3K36me3 are critical for DNA DSB, but they actin different stages of the DNA damage response. Our studycontributes evidence that H3K36 dimethylation is critical in the

DNA damage repair. Distinct from other reports,9–13 our studyelucidated a mechanism by which the ATM-KDM2A signalingpathway modulated H3K36me2 changes, MRE11 complex recruit-ment and DNA damage repair.The PHD-type zinc-finger domain, one of the main domains of

KDM2A, has been demonstrated to bind to histone lysines. Forexample, the PHD fingers of inhibitor of growth 2 andbromodomain and PHD domain transcription factor can bind totri-methylated histone H3K4 (H3K4me3),44,45 whereas the PHDfinger of Jumonji A-T-rich interactive domain 1C can bind to tri-methylated histone H3K9 (H3K9me3).46 By contrast, the LSD1

%In

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Figure 7. Regulation of MRE11 complex recruitment by KDM2A phosphorylation at DNA damage sites. (a) HEK293 cells were transfected withpcDNA, wild-type Flag-KDM2A or the T632E mutant and treated with etoposide at 40 μM for 2 h. The cells were fixed and subjected to confocalfluorescent microscopy. The merge channel was the combination of the two colors. The arrows indicate representative cells. (b) Quantificationof MRE11-positive cells from the experiment for which results are shown in a. MRE11-positive cells refer to cells with ⩾ 5 MRE11 foci inside. Thepercentage of MRE11-positive cells for each condition was calculated from a minimum of 100 cells. Data are shown as means± s.d. from threeindependent experiments. (c) The DRGFP U2OS cells were transiently transfected with the I-SceI expression construct and subjected tochromatin immunoprecipitation (Chip) assay 24 h later with the indicated antibodies. The ‘Control’ indicates the genomic control far from theI-SceI site and the ‘NC’ indicates empty vector. Student’s t-test (two-tailed): H3K36me2, NC versus I-SceI, Po0.001; MRE11, NC versus I-SceI,Po0.001; pNBS1, NC versus I-SceI, Po0.01. (d) The DRGFP U2OS cells were first transiently transfected with pcDNA, wild-type Flag-KDM2A orthe T632E mutant, and then the I-SceI expression construct. Chip assay was performed on the transfectants. Student’s t-test (two-tailed):H3K36me2, pcDNA versus WT, Po0.01, pcDNA versus T632E, Po0.05, WT versus T632E, Po0.001; MRE11, pcDNA versus WT, Po0.01, pcDNAversus T632E, P40.05, WT versus T632E, Po0.01; pNBS1, pcDNA versus WT, Po0.01, pcDNA versus T632E, Po0.05, WT versus T632E, Po0.01.(e) A-T cells were transfected with pcDNA or Flag-ATM, separately, and treated with etoposide at 40 μM for 2 h. The chromatin fraction wassubjected to immunoblotting. (f) The bands from immunoblotting of MRE11 and H3K36me2 in e were scanned and quantified, as shown in ahistogram (means± s.d., n= 3). Student’s t-test (two-tailed): MRE11, ATM versus ATM+etoposide, Po0.05; ATM versus pcDNA, Po0.05; ATM+etoposide versus pcDNA+etoposide, Po0.05; pcDNA versus pcDNA+etoposide, Po0.01.


9


pcDNA WT T632E

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Figure 8. Regulation of DNA repair by KDM2A phosphorylation. (a) HEK293 cells were transfected with pcDNA, wild-type Flag-KDM2A or theT632E mutant, and treated with etoposide at 40 μM for 2 h. Soon after 2-h etoposide treatment, cells were washed free of drug and thenincubated for 18 h. Cells were collected for comet assays. A representative image of cells under each condition is presented. (b) Quantificationof the tail moments from the experiment for which results are shown in a. The tail moment for each condition was calculated from a minimumof 100 cells. Data are shown as means± s.d. (n⩾ 100). Student’s t-test (two-tailed): pcDNA versus WT, Po0.001, pcDNA versus T632E, P40.05,WT versus T632E, Po0.001. (c) HEK293 cells were transfected with the indicated expression constructs and treated with etoposide asdescribed in a. Cells were washed free of drug, incubated for the indicated time periods and whole-cell lysates were subjected toimmunoblotting. (d) HEK293 cells were transfected with the indicated expression constructs and treated with etoposide as described in a.Cells were washed free of drug, incubated for the indicated time periods and fixed for microscopic imaging of γH2AX after etoposidetreatment. (e) Quantification of γH2AX-positive cells from the experiment for which results are shown in d. γH2AX-positive cells refer to cellswith ⩾ 10 γH2AX foci inside. The percentage of γH2AX-positive cells for each condition was calculated from a minimum of 100 cells. Data areshown as means± s.d. from three independent experiments. (f) HeLa cells stably expressing pcDNA, wild-type Flag-KDM2A or the T632Emutant were treated with etoposide at 20 or 40 μM for 2 h and washed free of drug. Then cells were trypsinized and equally plated into six-wellplates. Ten days later, the cell colonies were stained and counted. Data are shown as means± s.d. from three independent experiments.Student’s t-test (two-tailed): 20 μM, pcDNA versus WT, Po0.05, pcDNA versus T632E, P40.05, WT versus T632E, Po0.01; 40 μM, pcDNA versusWT, Po0.001, pcDNA versus T632E, P40.05, WT versus T632E, Po0.05.


10


complex component BHC80 can bind to unmethylated H3K4 viaits PHD finger to stabilize the complex at its targeted promoters torepress transcription.47 It has not yet been reported that the PHDdomain of KDM2A binds to methylated histones, and KDM2A isrecruited to CpG islands by CxxC-type zinc finger rather than byPHD zinc finger;48,49 however, the PHD domain of KDM2A may stillaffect its chromatin-binding ability. In this study, T632 of KDM2Awas located just within the PHD domain, and the phosphorylationof KDM2A at T632 affected its chromatin-binding capacity, whichfurther strengthens the importance of PHD zinc-finger domains inthe chromatin-binding ability. However, thr632 is located at theN-terminal of the PHD domain, which is near to the CxxC-type zincfinger, and it is possible that phosphorylation of KDM2A at Thr632may have affected its chromatin-binding ability by influencing theCxxC-type zinc finger. It remains unknown how phosphorylationof Thr632 influences the chromatin-binding ability of KDM2A, andKDM2A phosphorylation may exert allosteric effects that influenceits chromatin-binding activity.Histone modification enzymes are often regulated in response

to DNA damage. Modification of the enzymes themselves isassociated with their enzymatic activity. For example, in ourprevious study, the histone methyltransferase SET7/9 interactedwith and methylated histone methyltransferase SUV39H1 inresponse to DNA damage, resulting in decreased SUV39H1methyltransferase activity and genome instability.50 However, itis unlikely that the modifications of all histone methyltransferases/demethylases are related to their enzymatic activity. For example,it has been recently reported that PARP1-mediated PARylation ofhistone demethylase KDM4D is essential for the recruitment ofKDM4D to damaged DNA but does not influence demethylaseactivity.51,52 In addition, histone demethylase KDM4A is ubiquiti-nated by RNF8 and RNF168 and then degraded to promote 53BP1accumulation at damaged sites in response to DNA damage.53,54

Moreover, phosphorylation of the yeast KDM4 ortholog Rph1occurs upon ultraviolet irradiation and induces the release of Rph1from chromatin.55,56 Similarly, this study reports that ATM-mediated KDM2A phosphorylation at T632 did not affect itsdemethylase activity but significantly decreased its chromatin-binding capacity in response to DNA damage.The MRE11 complex participates in DNA damage repair and

coordinates the response to broken chromosomes.35 However, themechanism of MRE11 complex recruitment to DSBs is still unclear.For example, the MRE11 complex may recognize DSBs and directlytarget the damaged sites.57 Alternatively, a previous study hasshown that PARP1 accumulates at DSB sites and is essential for therapid accumulation of MRE11 and NBS1 at DNA damage sitesthrough its direct interaction with MRE11.58 However, NBS1appears to be much more critical to the recruitment of theMRE11 complex in response to DNA damage. MDC1 and γH2AXhave been shown to promote MRE11 complex recruitment afterDNA damage by directly binding to the FHA/BRCT domains ofNBS1.59–62 In addition, Rad17 is required for the early recruitmentof the MRE11 complex to DSB sites through direct interaction withthe FHA domain of NBS1.63 In our study, we identified thatH3K36me2 is also required for MRE11 complex recruitment to DSBsites by interacting with NBS1, such that H3K36me2 binds directlyto the BRCT2 domain of NBS1, but not the FHA or BRCT1 domains.This novel finding explains a mechanism of how the MRE11complex binds to damaged DNA. Consistent with our study,H3K36me2 was shown to interact with NBS1 upon ionizingradiation by co-IP,13 which further confirms the recruitment of theMRE11 complex by H3K36me2. Although the structural basis ofthis H3K36me2-NBS1 interaction is not yet clear, our study furthersupports the essential role of the MRE11 complex in the DNAdamage response.It has been described that KDM2A is frequently overexpressed

in non-small cell lung cancer tumors and gastric cancers and canpromote the growth and motility of cancer cells.19,64 This study is

the first to report that KDM2A phosphorylation is critical for DNAdamage response and cell survival. Therefore, KDM2A may be anew therapeutic target for cancer treatment. This study provides auseful strategy with which to design anticancer therapies and,eventually, cure cancer by manipulating histone methylation anddemethylation at specific sites upon DNA damage-relatedchemical therapy.

MATERIALS AND METHODSChromatin immunoprecipitation assayCells were fixed with formaldehyde and lysed with lysis buffer (50mM

Tris·HCl pH 8.0, 5 mM EDTA, 1% sodium dodecyl sulfate). After sonication,the supernatant was collected by centrifugation and precleaned in dilutionbuffer (20mM Tris·HCl pH 8.0, 2 mM EDTA, 150mM NaCl, 1% Triton X-100)with protein G or A sepharose and salmon DNA. The precleaned sampleswere incubated with the indicated antibody and protein G or A sepharose.The beads were washed and heated at 65 °C to reverse the cross-link. DNAwas purified and real-time PCR was performed with primers as follows:5'-TCTTCTTCAAGGACGACGGCAACT-3' (sense) and 5'-TTGTAGTTGTACTCCAGCTTGTGC-3' (antisense); genomic control: 5'-CCTGAGCAAAGACCCCAA-3'(sense) and 5'-TTACTTGTACAGCTCG-3' (antisense).

Peptide pull-down assayBiotin-tagged H3 peptides were immobilized on streptavidin beads inphosphate-buffered saline with rotation at 4 °C overnight. Then the beadswere washed with phosphate-buffered saline and incubated with celllysates. After three washes with washing buffer (20mM Tris·HCl pH 7.5,1 mM EDTA, 500mM NaCl), the beads were subjected to Coomassie brilliantblue staining and immunoblotting.

Histone acid extraction assayCells were lysed in hypotonic lysis buffer (10mM Tris·HCl pH 8.0, 1mM KCl,1.5mM MgCl2 and 1mM DTT, protease inhibitors), then pellet the intact nucleiby spinning. Discard supernatant, re-suspend nuclei in 0.4 N H2SO4 andincubate for at least 30min. Spin samples and transfer the supernatantcontaining histones. Add trichloroacetic acid and incubate on ice for 30min.Collect histone pellet by spinning, wash with acetone and dissolve in ddH2O.

Comet assayComet assay has been described before.65 Briefly, cells were mixed gentlywith pre-melted low-temperature-melting agarose at a volume ratio of 1 to1 (v/v) and spread on glass slides. The slides were then submerged inprecooled lysis buffer at 4 °C for 90min. After rinsing, the slides wereelectrophoresed at 1.0 V/cm for 20min, and then stained with propidiumiodide. Fluorescence images for at least 100 nuclei were captured using anOlympus FV1000-IX81 Confocal Microscope (Tokyo, Japan). The imageswere analyzed by Cometscore Version 1.5 software (TriTek Corp.,Sumerduck, VA, USA) for tail moment.

Colony formation assayCells were plated and exposed to 40 μM etoposide for 1 h. Etoposide wasremoved by three rinses using phosphate-buffered saline. Cells weretrypsinized and plated into six-well plates in an equal number. After10 days, methanol fixation and staining with methylene blue wasundertaken to identify visible colonies.

CONFLICT OF INTERESTThe authors declare no conflict of interest.

ACKNOWLEDGEMENTSWe thank Dr David J Chen at University of Texas Southwestern Medical Center forkindly providing YFP-ATM plasmid. We also thank Drs Jun Huang at ZhejiangUniversity and Jiadong Wang at Peking University Health Science Center for kindlyproviding NBS1 deleted mutants. This work was supported by National Key BasicResearch Program of China grant 2011CB504200, 2013CB911000; National NaturalScience Foundation of China grants 31070691, 81321003, 91319302; Minister ofEducation of China ‘111 Project’.


11


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Supplementary Information accompanies this paper on the Oncogene website (http://www.nature.com/onc)


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atm-mediated kdm2a phosphorylation is required for the...

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