Plant cH2AX foci are required for proper DNA DSB repairresponses and colocalize with E2F factors

Julien Lang1*, Ondrej Smetana1*, Lenin Sanchez-Calderon2, Frederic Lincker1, Julie Genestier1,

Anne-Catherine Schmit1, Guy Houlne1 and Marie-Edith Chaboute1

1Institut de Biologie Moleculaire des Plantes, laboratoire propre du CNRS, (UPR 2357) conventionne avec l’Universite de Strasbourg 12, rue du General Zimmer, 67084 Strasbourg Cedex,

France; 2Laboratorio de Biologıa Molecular de Plantas Unidad Academica de Biologıa Experimental Universidad Autonoma de Zacatecas, Av. Revolucion S ⁄ N Col. Tierra y Libertad CP, 98615

Guadalupe, Zacatecas, Mexico

Author for correspondence:Marie-Edith Chaboute

Tel: +33 388 417 297Email: marie-edith.chaboute@ibmp-cnrs.

unistra.fr

Received: 13 September 2011

Accepted: 22 December 2011

New Phytologist (2012) 194: 353–363doi: 10.1111/j.1469-8137.2012.04062.x

Key words: Arabidopsis, BY-2 cells, DNAdouble-strand breaks (DSBs), DNA repair,E2F factor, H2AX, repair foci.

Summary

• Cellular responses to DNA double-strand breaks (DSBs) are linked in mammals and yeasts

to the phosphorylated histones H2AX (cH2AX) repair foci which are multiproteic nuclear com-

plexes responsible for DSB sensing and signalling. However, neither the components of these

foci nor their role are yet known in plants.

• In this paper, we describe the effects of cH2AX deficiency in Arabidopsis thaliana plants

challenged with DSBs in terms of genotoxic sensitivity and E2F-mediated transcriptional

responses.

• We further establish the existence, restrictive to the G1 ⁄ S transition, of specific DSB-

induced foci containing tobacco E2F transcription factors, in both A. thaliana roots and BY-2

tobacco cells. These E2F foci partially colocalize with cH2AX foci while their formation is

ataxia telangiectasia mutated (ATM)-dependent, requires the E2F transactivation domain

with its retinoblastoma-binding site and is optimal in the presence of functional H2AXs.

• Overall, our results unveil a new interplay between plant H2AX and E2F transcriptional acti-

vators during the DSB response.

Introduction

Exposure to genotoxins (i.e. DNA-damaging agents) triggers awide range of biological signalling pathways leading to DNAdamage repair, cell cycle arrest or cell death in the presence ofexcessive DNA damage. Proper coordination of all theseresponses is sustained by the repair foci which are large proteiccomplexes forming in the vicinity of the DNA damage site.Descriptions of these repair foci according to the type of damagehave been largely documented over the last years in yeasts andanimals (Lisby et al., 2004; Niida & Nakanishi, 2006).

Since the seminal work by Rogakou et al., 1999 with insect,yeast and mammalian cells, the phosphorylated histones H2AX(cH2AX) foci have been considered as solid markers of DNAdouble-strand breaks (DSBs) although their precise functionremains a matter of debate (Lobrich et al., 2010). The formationof cH2AX repair foci occurs rapidly after DSB induction and hasbeen proved to be mainly dependent on the kinase ataxia telangi-ectasia mutated (ATM) (Burma et al., 2001), even if the ataxiatelangiectasia and Rad-3-related (ATR) kinase might also con-tribute, to a lesser extent, to this process, especially in the context

of replicative stress (Ward & Chen, 2001). The loss of H2AX inmammals compromises genomic stability and cH2AX-deficientmice are radiation-sensitive as well as growth-retarded (Celesteet al., 2003a; Franco et al., 2006). In human cells, the directinteraction between cH2AX and Mediator of DNA damageCheckpoint protein 1 (MDC1) is demonstrated as critical forDNA damage checkpoint activation (Stewart et al., 2003; Stuckiet al., 2005), by promoting correct accumulation of other repairproteins such as 53BP1 and BRCA1 to sites of DSBs (Bassing &Alt, 2004; Kolas et al., 2007). In addition to phosphorylation,ubiquitination and acetylation of H2AX have important effects,as they facilitate the DNA damage responses (Vissers et al., 2008;Ikura et al., 2007). The spatiotemporal dynamics of cH2AX focihas also been discussed recently, considering that these structuresmight vary their composition and fulfil different functionsaccording to the cell cycle progression (Iliakis, 2010; Nakamuraet al., 2010).

In plants, several components of the DSB repair network areconserved. Orthologues of ATM and H2AX have notablybeen characterized in Arabidopsis (Garcia et al., 2003; Friesneret al., 2005). Besides its involvement in cH2AX foci formation(Friesner et al., 2005), ATM plays a pivotal role in the robust tran-scriptional response induced by DSB in Arabidopsis (Chen et al.,2003; Culligan et al., 2006; Ricaud et al., 2007). Recently, the*These authors contributed equally to this work.

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Suppressor Of Gamma response 1 (SOG1) was described as a keymediator in this response (Yoshiyama et al., 2009). For our part,we have shown that the specific DSB induction of RAD51 andTSO2, encoding proteins involved, respectively, in homologousrecombination repair and dNTP supply, is dependent on E2Fa – amember of the E2F transcription factors family (Roa et al., 2009).

So far, no functional role has been ascribed to the cH2AX fociin plants, and no other DSB-induced foci-forming proteins havebeen mentioned. Here, through genetic, molecular and cellularapproaches, we established that cH2AX foci were instrumentalboth in efficient DSB repair and in transcriptional responses inArabidopsis. We also demonstrated that E2Fa and H2AX contrib-uted additively to the global DSB repair system. Additionally, weobserved – in Arabidopsis and BY-2 cells – some unprecedentedE2F foci which formed specifically at the G1 ⁄ S transition inresponse to DSBs, as long as ATM sensing and cH2AX foci for-mation were not impaired. Besides, these E2F foci colocalizedwith cH2AX foci. Altogether, our results provide new data con-cerning the interplay between cH2AX and E2F foci in DNADSB repair responses.

Materials and Methods

Plant material, growth and treatment conditions

Wildtype (WT) and transgenic Arabidopsis thaliana (L.) Heynh.plants are of the Columbia (Col-0) ecotype. They were sterilizedand grown in vitro as described previously (Roa et al., 2009).The BY-2 tobacco cell suspension was maintained by weeklysubculture as described previously (Chaboute et al., 2002). Treat-ments were carried out either by sowing seeds on an MS-agarmedium supplemented with genotoxins or by incubating theplantlets or cells in a liquid MS medium supplemented withthe chemicals. Drug concentrations were 10)5 M (unless other-wise mentioned) for bleomycin (BLM; Laboratoire Thissen,Belgium), 5 nM for camptothecin (CPT; Sigma), 10 mM forhydroxyurea (HU; Sigma) and 5 mM for caffeine (JohnsonMatthey Company, Karlsruhe, Germany).

Generation of transgenic cells and plants

The sequences of the NtE2F constructs were amplified via PCRwith specific primers (3 and 4 for NtE2F; 5 and 6 for the trun-cated version of NtE2FMU; Supporting Information, Table S1)and total cDNA from tobacco as a template. Using the Gateway�

technology (Invitrogen), the amplicons were then cloned into thepK7WGF2 vector (Karimi et al., 2002) to be fused to the EGFP(Enhanced Green Fluorescent Protein) under the control ofthe 35S promoter. The constructs were then introduced intoAgrobacterium LBA4404 for transformation of BY-2 cells as pre-viously described (Chaboute et al., 2000) and into GV3101 totransform Arabidopsis, using the floral-dip method (Clough &Bent, 1998). NtE2F:GFP line was crossed with previouslycharacterized atm) ⁄ ) and e2fa ) ⁄ ) lines (Garcia et al., 2003;Roa et al., 2009). Subsequently, all transgenic plants werescreened for the selection of homozygous lines.

The premiH2AX construct was amplified using the pre-miR171cDNA as a template (Parizotto et al., 2004) and specific primers(1–2; Table S1). It was then cloned into the pBin61 vector at theSpe I ⁄ Xho I sites. After transformation, homozygous lines wereselected. The premiH2AX construct was also introgressed into thee2fa as well as NtE2F:GFP lines, and homozygous lines were usedin the experiments.

Immunolabelling

Plant fixation and immunostaining were performed as describedpreviously (Friesner et al., 2005). The primary antibodies wererabbit anti-cH2AX (Friesner et al., 2005) and polyclonal chickenanti-GFP (Molecular Probes, Eugene, OR, USA), diluted at1 : 500. The secondary antibodies for cH2AX detection were,depending on the experiments, Alexa 546 goat anti-rabbit conju-gate (Molecular Probes) for red signals and Hilyte Fluor 488 goatanti-rabbit (AnaSpec, San Jose, CA, USA) for green signals, bothapplied at 1 : 500. For colocalization analyses, the secondaryantibody for GFP detection was Alexa 488 goat anti-chicken(Molecular Probes) applied at 1 : 200.

Microscopy

Images of Arabidopsis root tip cells and BY-2 cells were cap-tured using a Zeiss LSM510 laser scanning microscope with aC-Apochromat (63X; v1.2 W Korr) water objective lens. Excita-tion ⁄ emission wavelengths were 488 nm ⁄ 505–545 nm and543 ⁄ long pass 560 nm according to the fluorophores. Imageswere processed using the Zeiss LSM510 version 2.8 and ImageJv.1.43 (Rasband, W.S., NIH, Bethesda, MD, USA, http://imagej.nih.gov/ij/, 1997–2011).

Histone protein extraction

Plants were ground in nuclear isolation buffer (0.25 M sucrose,60 mM KCl, 15 mM NaCl, 5 mM MgCl2, 1 mM CaCl2,15 mM Pipes pH 6.8, 0.8% Triton X-100) plus 0.1% of a prote-ase inhibitor cocktail (Roche Diagnostics) and 50 mM of phos-phatase inhibitor sodium ortho-vanadate (P9599, Sigma). Theplant extract was filtered twice through miracloth (Calbiochem,Darmstadt, Germany). After centrifugation at 10 000 g for20 min at 4�C, the pellet was submitted to acid extractionthrough resuspension in 0.4 M sulphuric acid and incubation onice for 1 h. After centrifugation at 15 000 g for 5 min at 4�C,the soluble proteins from the supernatant were precipitated over-night with acetone at –20�C, then spun down at 7000 g for15 min at 4�C, and resuspended in 4 M urea.

Western blots

Protein extracts were analysed on 10 or 15% sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) andelectro-transferred onto a PVDF membrane (Millipore). Immuno-blotting for the detection of cH2AX was carried out as previouslydescribed (Friesner et al., 2005) with some modifications: PBS was

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used instead of TBS and the secondary anti-rabbit antibodywas linked to the anti-rabbit alkaline phosphatase (Bio-Rad). Theimmunodetection of GFP:E2F proteins was performed using apolyclonal rabbit anti-GFP (kindly provided by J.L. Evrard,IBMP) diluted to 1 : 5000 and the secondary anti-rabbit antibodywas linked to the alkaline phosphatase. Revelation of the blotswas performed with the BCIP ⁄ NBT system (Roche AppliedScience).

Reverse transcription-polymerase chain reaction (RT-PCR)analysis

RNA was extracted and processed for RT-PCR reactions asdescribed (Roa et al., 2009). Amplicons were analysed by semi-quantitative PCR, using agarose gel electrophoresis and DNAfragments were quantified with the Quantity One software pro-gram (Bio-Rad). 18S and actin were used as standards. Theresults are presented as relative mRNA levels compared with stan-dards. Alternatively, quantitative Real-Time PCR was performedusing Qr evaluation as previously described (Roa et al., 2009).

Neutral COMET assays

Assays were performed on 8-d-old plantlets according to (Roaet al., 2009). The quantification of the comet figures was carriedout on nontreated plants and BLM-treated plants, and wasrelated to an arbitrary scoring of the comet figures as describedpreviously (Collins, 2004). In each assay, 200 comets were scoredand the results represent the mean values ± SDs from three inde-pendent experiments.

Synchronization experiments

BY-2 cells were synchronized using aphidicolin as described pre-viously (Chaboute et al., 2002). The mitotic index was evaluatedby counting the mitotic figures in DAPI stained nuclei. In paral-lel, S-phase was monitored through expression analyses using thetobacco S-phase marker H3 gene as described (Proust et al.,1999). The ribosomal gene 18S was used as a standard.

Results

AtH2AXa-b genes are constitutively expressed andAtH2AXa-b proteins form foci upon BLM exposure

In Arabidopsis, two H2AX orthologues, H2AXa and H2AXb(Friesner et al., 2005), have been identified. Both proteins arevery similar to yeast and mammalian H2AXs (68–70% ofidentity) as well as to each other (98.6% of identity). In theirC-terminal part, they share the same SQE motif which representsa consensus phosphorylation site for ATM, or alternatively otherkinases (Friesner et al., 2005).

While in mammals several functional studies had alreadydescribed the relevance of H2AX in the DSB responses (Bassinget al., 2002; Celeste et al., 2003b), such an analysis had not beenperformed in plants so far.

We first characterized the expression of H2AXa and H2AXband noticed that the two genes were expressed similarly in differ-ent organs, albeit with a lower level in the cauline leaves (Fig. 1a).During plant development, we also observed that the abundancefor both transcripts remained almost constant even if a slightincrease in H2AXa expression could be noted 15 d after germina-tion (Fig. 1a).

Furthermore, and consistently with results obtained upongamma irradiation (Friesner et al., 2005), the amount of cH2AXproteins was shown to increase after treatment with the DSB-inducer drug BLM in a time-dependent manner (Fig. 1b). In par-allel, cH2AX foci were also detected in Arabidopsis root tips uponexposure to BLM, using immunocytological approaches (Fig. 1c).

Characterization of the miH2AX line

To assess the effect of cH2AX deficiency in Arabidopsis, it wasimportant to obtain a decreased expression for both H2AX genes.Since no KO or knocked-down T-DNA lines were available, anRNAi approach was used. We mutated the Arabidopsis pre-mir171 cDNA (Parizotto et al., 2004) using PCR elongationwith primers harbouring mismatches, in order to generatethe pre-miH2AX, which we placed under the control of a 35Spromoter. After transitory expression in Nicotiana benthamianaleaves, where constitutive H2AX-GFP expression was down-regulated in the presence of the pre-miH2AX (data not shown),an Arabidopsis stably transformed homozygous line (named

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Fig. 1 Characterization of Arabidopsis H2Axs. (a) Analysis of H2AXa andH2AXb expression by semiquantitative RT-PCR in different plant organs(R, roots; Fl, flowers; Cl, cauline leaves) and at different developmentalstages (7, 12 and 15 d after germination). (b) Time-course analysis usingwestern blot of the cH2AX levels in 8-d-old plantlets upon exposure tobleomycin (BLM). The antibody used was raised against the phosphory-lated C-terminal tail common to H2AXa and H2AXb. As a loading control(LC), Coomassie blue staining of the immunoblot is presented. (c) Immu-nolocalization of cH2AX (green) in Arabidopsis root tip cells in nontreatedplants (control) and after a 2 h BLM treatment, using the same antibody asin (b). Eight-day-old plantlets were used in the experiments. Images werecaptured through confocal microscopy. Bars, 5lm.

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miH2AX in the following) was selected because of its decreasedH2AXa and H2AXb expression levels (of 85 and 48%, respec-tively, in comparison with WT) (Fig. 2a). In addition, the levelof cH2AX proteins in this line was below the threshold of detec-tion in our experimental conditions (Fig. 2b), suggesting that,besides the degradation of the H2AXa and H2AXb mRNAs, the

miH2AX construct might also cause the inhibition of their trans-lation. Finally, the percentage of cells presenting cH2AX fociwere also drastically reduced in miH2AX upon BLM exposure(Fig. 2c), confirming that the miH2AX line was affected incH2AX functions.

Phenotypically, in the absence of genotoxins, root growth wassimilar between miH2AX and WT plants (Fig. 3a), but appearedto be mildly delayed in miH2AX in the presence of the DSBinducers – CPT and BLM (Fig. 3b–c).

To relate this sensitivity to genotoxins with possible DSBrepair defects, we evaluated DSB repair efficiency in miH2AXplants using neutral COMET assays (Fig. 3d). We first noticedthat in the absence of genotoxins the basal amounts of DSBs inthe miH2AX line and in the WT were similar, which a prioriexcluded the possibility that deficiency in H2AXs could result inhigher genomic instability. Surprisingly, we also observed thatthe amounts of DSBs after a 6h BLM treatment were not signifi-cantly different between the miH2AX and the WT lines, possiblybecause of the length of the treatment or ⁄ and the high BLM con-centration used in the experiment (10)5M), which may lead tosaturating effects. However, when plants were submitted to a30 min recovery after BLM treatment, the amount of remainingDSBs was twice as high in miH2AX plants as in the WT forwhich the level of DNA DSBs dropped significantly (Fig. 3d).Altogether, these findings strongly suggested that miH2AX plantswere not more susceptible than WT to generate DSBs in responseto genotoxins, but were rather impaired in their ability to repairnewly emerging DSBs.

Since previous results showed that deficiency in DSB repairmay be correlated to defects in transcriptional response, we furtheranalysed the response of the DSB transcriptional marker TSO2(Roa et al., 2009). Without genotoxins, the TSO2 mRNA level inmiH2AX plantlets was not affected compared with the WT.However, when miH2AX plants faced a 2h BLM exposure, theTSO2 mRNA level was reduced by more than five times (Fig. 3e).

Overall, these results showed that the miH2AX plants wereimpaired not only in repair efficiency but also in the TSO2 tran-scriptional response induced by BLM.

Interplay between E2Fa and cH2AXs in the DSB repairresponse

As the DSB-induced accumulation of TSO2 mRNAs wasAtE2Fa-dependent (Roa et al., 2009), we explored the geneticinteraction between AtE2Fa and AtH2AXa-b in response toDSBs. To do so, the miH2AX construct was introgressed intoe2fa, which is completely deficient in the DSB-induced TSO2up-regulation (Roa et al., 2009). The resulting e2fa · miH2AXline exhibited a similar decrease in H2AXa and H2AXb expressionas observed in the miH2AX line (data not shown). Singularly,the e2fa x miH2AX line was affected about twice as strongly asthe single mutants miH2AX and e2fa, regarding sensitivity toCPT (measured by root growth) as well as DNA repair efficiency(measured in COMET assays) (Fig. 4a–b). Consequently, weinferred that AtE2Fa and AtH2AXa-b acted in an additive way inthe DSB response.

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Fig. 2 Characterization of the Arabidopsis miH2AX line. Experiments wereperformed on 8-d-old plantlets. (a) Expression levels of H2AXa andH2AXb in the miH2AX line (closed bars) compared with the wildtype (WT;open bars). Semiquantitative reverse transcription-polymerase chain reac-tion (RT-PCR) experiments were repeated three times, SDs are indicated.Actin was used as a standard. (b) Detection through western blot ofcH2AX levels in the WT and in the miH2AX line after a 2 h bleomycin(BLM) treatment. As a loading control (LC), Coomassie blue staining of theimmunoblot is presented. (c) Comparison of the percentage of cells pre-senting cH2AX foci (CPcF) in WT and miH2AX plants in response to BLMafter a 2 h BLM treatment. Two hundred cells from 20 different root tipswere analysed randomly in two independent experiments. Error bars indi-cate SD. In the lower part, representative confocal images are presented;arrows indicate foci. Bar, 5lm.

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DSB-induced cellular response of a tobacco E2Ffactor includes foci formation and requires itsC-terminal part

To further unravel the interplay between plant E2Fs and H2AXs,we considered their subcellular localization upon exposure togenotoxins. One crucial aspect could be the possible relocaliza-tion of some E2F factors after generation of DSBs.

The Arabidopsis E2F family includes six members split intotwo classes. AtEFa, b and c belong to the first one and AtDEL1, 2and 3 to the second one. Unlike AtDEL1-3, AtEFa-c possesses atransactivation (TA) domain containing a conserved retinoblas-toma (Rb)-binding site which is critical for the G1 ⁄ S checkpointregulation (Mariconti et al., 2002). In addition, AtE2Fa and bare transcriptional activators, while AtE2Fc and all AtDEL factorsare transcriptional repressors. In the animal E2F family, asalready shown, the misexpression of one member could inducederegulation of the other members (Kong et al., 2007). To avoidsuch a complication in Arabidopsis with the overexpression of onemember, we chose to focus on NtE2F which is the only charac-terized E2F factor from tobacco (Sekine et al., 1999). This factoris induced in response to high doses of UV-C in BY-2 cells(Lincker et al., 2004), possesses an operative NLS (Fig. S1a–d)and harbours structural similarities with AtE2Fa-b, including aTA domain and an Rb-binding site in its C-terminal part

(Lincker et al., 2008). Moreover, the GFP-NtE2F fusion, drivenby a 35S promoter, is able to transactivate the E2F-regulatedRNR gene in BY-2 cells (Fig. S2a). In the Arabidopsis e2fa mutant(Roa et al., 2009), it can also restore the specific TSO2 inductionin response to DSBs (Fig. S2b). We therefore concluded that theGFP:NtE2F construct was functional and that NtE2F could beconsidered as a typical representative of the plant E2F transcrip-tional activators.

When constitutively expressed in tobacco BY-2 cells, GFP-NtE2Fs were mainly nuclear in the absence of BLM (Fig. 5a).Remarkably, upon exposure to BLM, part of the GFP-NtE2Fsrelocalized into discrete subnuclear bright foci (Fig. 5b). As acontrol, in a line constitutively expressing the single GFP, thepattern of fluorescent protein was not affected by the addition ofgenotoxins (Fig. 5a–b, GFP).

When constitutively expressed in an Arabidopsis Col-0(GFP:NtE2F) background, GFP:NtE2F proteins were mainlydetected in the highly dividing cells of the root tips; but they wereconsiderably less expressed in differentiated root cells, suggestinga negative control of GFP:NtE2F in these non-proliferative cells.Moreover, in the absence of BLM, the subcellular localization ofthe GFP:NtE2F proteins was homogeneously nuclear, while inthe presence of BLM, part of them relocalized into foci (Fig. 5c).For a 2h long treatment with BLM, we recorded about 2.3 fociper cell in the GFP:NtE2F line. This result was congruent with

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Fig. 3 Phenotypic analyses of the Arabidopsis miH2AX line in response to double-strand breaks (DSBs). (a, b) Measurement of root growth in wildtype(WT, closed bars) and miH2AX (open bars) plants within 21 d after germination on medium without genotoxins (a) or in the presence of camptothecin(CPT, b). (c) Measurement of root growth within 10 d after germination on medium containing 10)6 bleomycin (BLM). (d) Evaluation of DSBs in COMETassays in the WT and without genotoxins (no BLM), after a single 6 h BLM treatment (BLM) and after a 6 h BLM treatment followed by a 30 min recoveryperiod (BLM + recovery). WT, closed bars; miH2AX, open bars. Eight-day-old plantlets were used in the experiments. Each comet figure was given an arbi-trary score as presented in the upper inset. The results were obtained from three independent experiments. Error bars indicate SD. (e) Evaluation of TSO2

expression by quantitative reverse transcription-polymerase chain reaction (RT-PCR) in WT and miH2AX plantlets. Relative mRNA levels correspond tostandardization with 18S. Analyses were performed on total RNA extract from 8-d-old plantlets either treated with BLM (open bars, 2 h) or untreated(closed bars). The results were obtained from three independent experiments. Error bars indicate SD.

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our independent quantification of cH2AX foci in the same geno-toxic conditions, suggesting that the two processes might berelated. In a second experiment, the GFP:NtE2F construct wasintrogressed into the e2fa background (e2fa x GFP:NtE2F) and aline was selected, in which the GFP:NtE2F proteins were presentat a level similar to that in Col-0 (Fig. 5d). In this line, as in Col-0, we observed that the GFP:NtE2F proteins were mainly nuclearin the absence of BLM but relocalized into foci upon BLM treat-ment (Fig. 5e).

Altogether, using both homologous and heterologous systems,these findings tended to confirm that the formation of E2F fociwas an aspect of the DSB cellular response conserved in plants.

Interestingly, a truncated version of GFP:NtE2F (calledGFP:NtE2FMU), lacking the TA domain of NtE2F, exhibitedno foci at all in response to BLM (Fig. 5f), although this con-struct was expressed at a level similar to GFP:NtE2F (Fig. 5g).We therefore concluded that the sequence which encompassesthe TA domain as well as the regulating Rb-binding site was criti-cal for GFP:NtE2F accumulation within foci.

Cellular response of E2F is cell cycle-regulated

Curiously, in both Arabidopsis and tobacco cells, the GFP-NtE2Ffoci were mainly observed in dividing cells (mid-log phase cells

for BY-2 and meristematic tissue of the root tip for Arabidopsis).More precisely, only 7% of the observed Arabidopsis root meriste-matic cells and only 15% of the observed BY-2 mid-log phasecells presented GFP-NtE2F foci in response to BLM. Moreover,in Arabidopsis root meristem, the percentage of cells presentingfoci (CPF) increased with the time of exposure to BLM (twice asmany cells when the BLM treatment rises from 2 to 6 h)(Fig. 6a). Likewise, the foci number per cell (FNC) increasedby c. 34% between 2 and 6 h (Fig. 6b). By contrast, when a 4 hperiod of recovery was allowed after a 2 h BLM treatment, thepercentage of CPF as well as the FNC decreased significantly(Fig. 6a,b). As a whole, these findings were interesting becausethey did not only substantiate the fact that the temporal dimen-sion of the GFP:NtE2F foci formation matched the typicaldynamics of repair foci, but they also implied that the formationof these foci might be cell cycle-regulated.

To test the latter hypothesis, we analysed the GFP:NtE2F fociformation during the cell cycle in synchronized BY-2 cellsexpressing GFP:NtE2F. Cell cycle progression was monitoredthrough mitotic index evaluation as well as the analysis of theS-phase H3 gene expression. For each cell cycle stage, a sample ofthe synchronized cells was treated with BLM for 2 h, then thenumber of GFP:NtE2F foci was determined. Strikingly, these fociformed preferentially in the G1 phase or at the G1 ⁄ S transition.They were also present at a significantly lower level in the S phasebut were practically absent at the G2 ⁄ M transition (Fig. 6c). Thisclearly showed that the E2F foci were part of the DSB responseessentially in the cellular context of the G1 ⁄ S transition.

NtE2F foci are ATM-dependent and colocalize withcH2AX foci

Ataxia telangiectasia mutated and ATR are known to be the keysensors of the DSB signalling. Using caffeine as an inhibitor ofboth ATM and ATR kinases (Sarkaria et al., 1999), we could nolonger detect GFP:NtE2F foci in Arabidopsis root tips uponBLM exposure (Fig. 7a-caffeine). Interestingly, with anothergenotoxin – hydroxyurea (HU), triggering an ATR-dependentDNA damage signalling (Culligan et al., 2004) as well as in thecrossed line atm · GFP:NtE2F challenged with BLM – we couldnot observe any GFP:NtE2F foci (Fig. 7a-HU and 7a-atm).Altogether, these results strongly suggested that GFP:NtE2F fociformation was mainly ATM-dependent.

In other organisms, several ATM-dependent mediators andtransducers of the DSB signalling were identified through theirability to colocalize and ⁄ or interact with cH2AX foci, and so weinvestigated a possible colocalization of cH2AX foci with NtE2Ffoci, using immunolocalization experiments on Arabidopsis roottips exposed to BLM.

We first noticed that only a fraction of the cells displayingcH2AX foci also presented GFP:NtE2F foci. This was actually inagreement with the results establishing that NtE2F foci occurredexclusively in the G1 phase and at the G1 ⁄ S transition, whilecH2AX foci were detectable in all phases of the cell cycle. Then,in this fraction of cells presenting GFP:NtE2F and cH2AX foci,we showed that both colocalized in response to BLM (Fig. 7b).

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Fig. 4 Phenotypic comparisons between Arabidopsis miH2AX, e2fa andmiH2AX · e2fa lines. (a) Measurement of root growth within 21 d aftergermination on medium containing camptothecin. (b) Evaluation of dou-ble-strand breaks (DSBs) in COMET assays after a 6 h bleomycin treatmentfollowed by a 30 min recovery period. Eight-day-old plantlets were used inthe experiments. Maximum damage was normalized as 100% at t = 0.Three independent experiments were performed. Error bars indicate SD.

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However, this colocalization was only partial (80% of the foci),suggesting that the formations of NtE2F and cH2AX foci werenot completely synchronous.

To check if such colocalization might be related to a directinteraction between GFP:NtE2F and AtH2AXs, we performedyeast-two hybrid (Y2H) assays as well as immunoprecipita-tions using the NBS1 ⁄ Mre11 interaction as a positive control(Waterworth et al., 2007). However, no direct interactionbetween GFP:NtE2F and AtH2AXs could be detected (data notshown). Since in mammals NBS1 had been described as directlyinteracting with cH2AX (Kobayashi et al., 2002), we also testedpossible interactions between AtH2AXs and the AtNBS1 candi-date (Waterworth et al., 2007). But again, no interaction wasdetected (data not shown).

Overall these results suggested either that the interactions mayrequire intermediate factors or post-translational modificationsunavailable in our experimental conditions (for instance, wecould not determine if AtH2AX proteins were proficiently phos-phorylated upon genotoxic exposure in yeasts) or, alternatively,that the mechanisms of interactions within repair foci in plantswere distinguishable from other eukaryotes.

The NtE2F · miH2AX line displays fewer E2F foci butrescues the TSO2 transcriptional dampening

As NtE2F foci colocalized with cH2AX foci, we wonderedwhether H2AX was important for the recruitment of NtE2F inthe DSB-induced foci. So we introgressed the miH2AX construct

into Arabidopsis GFP:NtE2F and selected a homozygous line(named NtE2F · miH2AX in the following) for its H2AXa-bdown-regulation, comparable to the one in miH2AX (data notshown). Even if the level of GFP:NtE2F was not affected inNtE2F · miH2AX (Fig. 8a), we observed an 80% decrease inthe number of cells presenting NtE2F foci after a 2 h BLM treat-ment (Fig. 8b). It was thus clear that AtH2AXa-b were necessaryfor proper GFP:NtE2F foci formation.

Intriguingly the overexpression of GFP-NtE2F in the Col-0background did not impact the TSO2 response to BLM in compar-ison to WT. However, in the NtE2F · miH2AX line, the BLM-induced TSO2 expression, which was dampened in the miH2AXline, was partially restored (Fig. 8c). These results suggested thatthe overexpression of the NtE2F transcriptional activator was suffi-cient to overcome the TSO2 transcriptional decrease in miH2AXand that this did not need NtE2F foci formation.

Discussion

Relevance of H2AX in the plant DSB responses

The first objective of this study was to investigate the effects ofH2AX’s deficiency in Arabidopsis. AtH2AX genes exist in twohighly redundant copies and are more or less constitutivelyexpressed in all plant organs (Fig. 1a). Even if this might be inter-preted as a clue to their importance, we clearly established thatthe down-regulation of their expression levels (85% for H2AXa,48% for H2AXb) had only mild impacts on the plant’s ability to

(b)(a)

(c)

(d) (f) (g)

(e)

Fig. 5 Characterization of the GFP:NtE2F foci. All pic-tures were captured using confocal microscopy. Bars,5 lm. Eight-day-old Arabidopsis plantlets and mid-logphase BY-2 cells were used in the experiments (a, b).Localization of single GFP and of GFP:NtE2F expressed inBY-2 cells, without genotoxins (a) and after a 2 h bleo-mycin (BLM) treatment (b). (c) Localization of GFP:NtE2Fin Arabidopsis thaliana Col-0 background, without geno-toxins and after a 2 h BLM treatment. Root tip cells wereanalysed. (d) Western blot showing levels of GFP:NtE2Fexpressed in Col-0 and in e2fa backgrounds. As a loadingcontrol (LC), Coomassie blue staining of the immunoblotis presented. (e) Localization of GFP:NtE2F in Arabidopsise2fa background, without genotoxins and after a 2 hBLM treatment. Root tip cells were analysed. (f, g) A trun-cated version of the NtE2F gene lacking the C-termdomain was fused to the GFP and expressed in Col-0plants. (f) The percentage of cells presenting foci (CPF)was evaluated in the GFP:NtE2FMU line vs theGFP:NtE2F line for both control (black bar) and BLM (greybar) treatments. A total of 200 cells from 10 different roottips were randomly screened in two independent experi-ments. Error bars indicate SD. (g) Western blot showinglevels of GFP:NtE2F and GFP: NtE2FMU. As a LC, Coo-massie blue staining of the immunoblot is presented.

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cope with DNA DSBs. Indeed, without stress, miH2AX plantsgrew like the WT and, in the presence of DSB-inducer drugssuch as BLM or CPT, they met moderate growth delay (Fig. 3a–c).Further COMET assays also showed that impaired recovery fromDSBs, rather than higher genomic instability, might account forthis phenotype (Fig. 3d). These results do not help to distinguishindividual AtH2AXa from AtH2AXb, and the study of KO linesmay be necessary for a complete understanding of the AtH2AXs

functions; yet, they are consistent with reports stating that inmammals, H2AX contributes to DSB repair in a moderate wayonly (Pinto & Flaus, 2010).

Moreover, we demonstrated that the typical DSB induction ofTSO2 disappeared in miH2AX (Fig. 3e). Given that our previ-ous data showed the dependency of TSO2 up-regulation onAtE2Fa (Roa et al., 2009), this fact was of special interest becauseit entailed a possible epistatic pathway between AtE2Fa andAtH2AXs for the TSO2 phenotype. Strikingly, the study of ane2fa · miH2AX line revealed that, in addition to their commonaction for TSO2 regulation, AtE2Fa and AtH2AXs also contrib-uted additively to the global DSB responses (Fig. 4).

On the other hand, it is known that proper activity of TSO2 isrequired for correct checkpoint activation and DNA repair afterexposure to UV-C (Wang & Liu, 2006). The depletion of TSO2transcripts might thus explain the phenotypes observed inmiH2AX.

Overall, our results are evidence for a direct link betweenArabidopsis H2AX and E2Fa proteins, with repercussions on thetransactivation of an E2F-regulated gene. However, a more thor-ough survey of the transcriptional responses in the miH2AXline, targeting genes other than TSO2, should provide valuablesupplementary information about the role of plant H2AXs in thisdomain, especially regarding the SOG1 functions (Yoshiyamaet al., 2009).

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Fig. 6 GFP:NtE2F foci formation is cell cycle-regulated. (a) Percentage ofcells presenting foci (CPF) in the root meristem of 8-d-old GFP-NtE2FArabidopsis plantlets, after a 2 h bleomycin (BLM) treatment (2H), a 6 hBLM treatment (6H) and a 2 h BLM treatment followed by a 4 h recoveryperiod (4HR). A total of 200 cells from 10 different root tips were ran-domly screened in two independent experiments. Error bars indicate SD.(b) Evaluation of foci number per cell (FNC) in the root meristem of GFP-NtE2F, in the same conditions as (a). (c) Evolution of the percentage ofCPF after a 2 h BLM treatment in different fractions of a synchronizedtransgenic BY-2 cell culture (lower panel). As a control, the value of CPFfor a nontreated fraction is presented. Synchronization was monitored withthe percentage of cells in mitosis (upper panel) and with H3 expressionlevels (middle panel). AP, aphidicholin.

Merge

Anti-γH2AX

Anti-GFP

(a)

(b)

Fig. 7 GFP-NtE2F foci are ataxia telangiectasia mutated (ATM)-dependentand colocalize with cH2AX foci. All pictures were captured using confocalmicroscopy. Bars, 5 lm. Eight-day-old Arabidopsis plantlets were used inthe experiments (a) Localization of GFP-NtE2F, after a 2 h bleomycin(BLM) treatment (GFP-NtE2F BLM), after a 2 h BLM treatment in thepresence of caffeine (GFP-NtE2F BLM + caffeine), after a 2 h hydroxyureatreatment (GFP-NtE2F HU) and after a 2 h BLM treatment in the GFP-NtE2F · atm line (GFP-NtE2F · atm BLM). (b) Immunodetection ofGFP:NtE2F foci (green), cH2AX foci (red) and colocalization (merge). Leftand right panels are two examples from two independent experiments.

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GFP-NtE2F relocalization revealed the existence of plantE2F foci which are part of the plant DSB signalling

For a more extensive analysis of the interplay between plantH2AXs and E2Fs, we showed that a tobacco E2F factor fusedto GFP relocalized upon BLM exposure to nuclear foci inA. thaliana root tip cells as well as in BY-2 cells (Fig. 5a–e). Thenumber of these foci actually matched the number of cH2AXfoci we could independently observe in the same experimentalconditions. In comparison to previous results, the number ofthese foci corresponded to that obtained after a 2.5 Gy dose ofgamma irradiation (Friesner et al., 2005), although the latterresults were acquired on mitotic cells only, while ours were basedon interphasic cells.

We further demonstrated that in Arabidopsis, GFP-NtE2F fociformation was time-dependent (Fig. 6a,b) and mainly controlledby ATM (Fig. 7a). In a previous report, Friesner et al., 2005 hadshown that, in mitotic cells, ATR was responsible for 10% of thecH2AX foci, but our results here, with the atm mutant and theHU (Fig. 7a), made very unlikely any role of ATR in the NtE2Ffoci formation, even if we cannot totally exclude a possible impli-cation of ATR for the few NtE2F foci observed in the S phase(Fig. 6c).

Our results also revealed that functional cH2AX foci (Fig. 8b)as well as the C-terminal part of NtE2F were required for fociformation (Fig. 5f). These findings provide new insight into theE2F’s properties which are important for foci formation. Inmammals, for instance, it has not been demonstrated that theDNA repair-induced E2F1 foci are ATM- and H2AX-dependent, or that the TA domain of E2F1 is important for itsaccumulation within foci. Instead, it has been suggested thatE2F1 foci formation is dependent on interaction with TopBP1via its N-terminal part (Stevens & La Thangue, 2004). Here, bycontrast, we present results which strongly suggest that the abilityof plant E2F to form foci in response to DSBs is directly corre-lated to its C-terminal part, which includes the TA domain andthe Rb-binding site. In addition, we can also note that NtE2Fpossesses a canonical SQD ⁄ E motif in its TA domain, suggestingthat an ATM-dependent phosphorylation of the E2F factormight be a prerequisite for foci formation. As supporting infor-mation, two-dimensional gel showed that indeed, upon BLMexposure, the GFP-NtE2Fs underwent a post-translational modi-fication, which, however, could not be specified (Fig. S3,Methods S1).

Intriguingly, the NtE2F foci were apparently not involved inthe regulation of TSO2 expression, as in NtE2F · miH2AX theTSO2 up-regulation was restored with no foci formation(Fig. 8b,c). This finding established, a priori, an independencebetween the E2F functions in transcriptional responses andwithin the foci. However, we could not exclude the possibilitythat the recruitment of other E2F factors to the repair foci mightaffect their transcriptional activities. It had been shown in mam-mals that the E2F1 foci formation at stalled replication forks cor-related with the repression of E2F1 functions, including S-phaseentry, transactivation and apoptosis (Liu et al., 2003). The sameprocess might occur in plants and, in this context, the majorG1 ⁄ S occurrence of the NtE2F foci in our experiments (Fig. 6c)could imply that the recruitment of E2F factors into repair focicoincides with the inhibition of the proliferative functions and istherefore instrumental in the implementation of the G1 ⁄ S check-point. Consistent with this hypothesis was the diminution, inmiH2AX and upon BLM exposure, of the TSO2 messengers,which might be indicative of a leaky G1 ⁄ S checkpoint inresponse to DSBs. Besides, S-phase cH2AX foci were recentlyobserved in a relationship with an ATR-dependent DNA replica-tion checkpoint (Amiard et al., 2010).

Additionally, we observed that the formation of NtE2F focirequired cH2AX foci (Fig. 8b) and that both significantlyoverlapped in Arabidopsis root tip cells (Fig. 7b). Similar colocal-ization was already observed in mammals with E2F1, which

(a)

(b)

(c)

Fig. 8 Analysis of the GFP:NtE2F · miH2AX line. Eight-day-old plantletswere used in the experiments. (a) Western blot showing GFP:NtE2F levelsin Arabidopsis GFP:NtE2F and NtE2F · miH2AX lines. As a loading control(LC), Coomassie blue staining of the immunoblot is presented. (b) Com-parison of the percentage of cells presenting GFP:NtE2F foci (CPF) after a2 h bleomycin (BLM) treatment in the GFP:NtE2F and the GFP:NtE2F ·miH2AX lines. A total of 200 cells from 10 different root tips were ran-

domly screened in two independent experiments. (c) TSO2 expressionafter a 2 h BLM treatment in the GFP:NtE2F · miH2AX line comparedwith the WT, GFP:NtE2F and miH2AX lines. Relative mRNA levelscorrespond to fold changes in gene expression in treated plantscompared with nontreated plants. Results were obtained from threeindependent semiquantitative reverse transcription-polymerase chainreaction (RT-PCR) experiments. Error bars indicate SD. 18s was used as astandard.

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promoted the recruitment to the DSBs of other repair factorssuch as NBS1 (Chen et al., 2011) known to directly interactwith cH2AX (Kobayashi et al., 2002). However, in ourexperimental conditions, we could not bring to light any interac-tions between NtE2F, AtNBS1 and AtH2AXs, suggesting somedifferences between plants and mammals in the setting of theDSB-induced repair foci machinery. It is also noteworthy that inmammals, the interaction between MDC1 and cH2AX is con-sidered a major process of the DSB response (Kinner et al.,2008), but that no homologue of MDC1 has yet been identifiedin plants.

Interestingly, Shechter et al. (2009) had already put forwardthe suggestion that the absence of GKK residues in the vicinity ofthe AtH2AXs SQE motif argued in favour of some differencesbetween plant cells and other eukaryotes regarding the mecha-nisms of interactions within DSB repair foci.

In conclusion, with the miH2AX and GFP:NtE2F lines, thisstudy has offered interesting tools to help untangle the complexinterplay between plant H2AXs and plant E2F factors encompass-ing a TA domain. It has emphasized the functions of ArabidopsisH2AXs in the context of genotoxin stress and demonstrated theexistence of DSB-induced E2F foci in plants. In future, the identi-fication of partners recruited to cH2AX and E2F foci, as well asthe elucidation of their post-translational modifications, shouldallow a wider understanding of the DSB cellular response in plantsand of its difference with other eukaryotes.

Acknowledgements

This research was supported in part by an Action Concertee(Biologie Moleculaire Cellulaire et Structurale), a grant from theMinistere de l’Education Nationale et de la Recherche. We thankDr D. Inze for providing GFP gateway vectors; Dr E. Herzog forthe PCK-GFP vector; Dr J. D. Friesner and Prof. A. B. Brittfor the anti-cH2AX antibody; Drs O. Voinnet and E. Parizotto fortheir advice on the miRNA strategy; and Dr C. E. West forproviding NBS1 clone. We are grateful to Dr. B. Winsor andL. Blech for their critical reading of the manuscript. The inter-institute confocal microscopy platform was co-financed by theCentre National de la Recherche Scientifique, the University ofStrasbourg, the Region Alsace, the Association de la Recherchesur le Cancer and the Ligue Nationale contre le Cancer.

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Supporting Information

Additional supporting information may be found in the onlineversion of this article.

Fig. S1 Nuclear localization of NtE2F is assumed by its N termi-nal NLS.

Fig. S2 The GFP:NtE2F fusion is functional.

Fig. S3 Post-translational modification of GFP:NtE2F in BY-2cells treated with BLM.

Table S1 Pairs of primers used in RT-PCR or cloning

Methods S1 Preparation of nuclear extracts and two-dimensionalelectrophoresis.

Please note: Wiley-Blackwell are not responsible for the contentor functionality of any supporting information supplied by theauthors. Any queries (other than missing material) should bedirected to the New Phytologist Central Office.

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