Chromatin modifications and chromatin-modifying enzymes are believed to play a major role in the process of DNA repair. The histone acetyl transferase Tip60 is physically recruited to DNA DSBs (double-strand breaks) where it mediates histone acetylation. In the present study, we show, using a reporter system in mammalian cells, that Tip60 expression is required for homology-driven repair, strongly suggesting that Tip60 participates in DNA DSB repair through homologous recombination. Moreover, Tip60 depletion inhibits the formation of Rad50 foci following ionizing radiation, indicating that Tip60 expression is necessary for the recruitment of the DNA damage sensor MRN (Mre11–Rad50–Nbs1) complex to DNA DSBs. Moreover, we found that endogenous Tip60 physically interacts with endogenous MRN proteins in a complex which is distinct from the classical Tip60 complex. Taken together, our results describe a physical link between a DNA damage sensor and a histone-modifying enzyme, and provide important new insights into the role and mechanism of action of Tip60 in the process of DNA DSB repair.
All processes requiring access to DNA have to deal with DNA compaction in chromatin. Chromatin modifications and chromatin-modifying machineries have been extensively studied in transcriptional control. However, it is now clear that they are also critical in other processes, such as DNA repair. In particular, DNA DSBs (double-strand breaks) are extremely deleterious, which require rapid and efficient repair. Chromatin modifications are known to be important for signalling and the dynamics of the DNA DSB repair machineries. In yeast, chromatin remodelling following DNA DSBs has been extensively studied and the machineries involved characterized .
In mammals, although far less is known, the involvement of the HAT (histone acetyl transferase) Tip60 has been clearly demonstrated . Tip60 belongs to the MYST family of HATs, which is conserved from yeast to human . In agreement with its role in histone acetylation, Tip60 has been widely described as a transcriptional co-activator. Its expression is required for the transcription of Myc- and p53-dependent genes [4,5].
Tip60 belongs to a multimolecular complex, called the Tip60 complex . The Tip60 complex is considered as the human orthologue of two yeast complexes, the NuA4 HAT complex and the SWR1 chromatin-remodelling complex, both known to be involved in DNA DSB repair . In mammals, a protein belonging to the Tip60 complex, TRRAP (transactivation/transformation-domain-associated protein), has been shown to be important for both HR (homologous recombination) and NHEJ (non-homologous end-joining), the two main pathways of DNA DSB repair [2,8]. Tip60 itself is recruited around DNA DSBs, where it mediates histone acetylation and subsequent recruitment to repair sites of DNA repair factors, such as Rad51 . In addition to its role in local histone acetylation, Tip60 activity is important for DNA DSB signalling: following DNA DSBs, it can acetylate the transducer kinase ATM (ataxia telangiectasia mutated), this acetylation being critical for ATM autophosphorylation and activation [9,10].
In the present study, we investigate the role of the Tip60 complex in DNA DSB repair. We identify a new multimolecular complex containing Tip60 and the MRN (Mre11–Rad50–Nbs1) complex, a sensor of DNA DSBs. Altogether, our results strongly suggest that this Tip60–MRN complex is important for the repair of DSBs by HR.
Jurkat cells were cultured in RPMI 1640 medium supplemented with 10% FCS (fetal calf serum). All other cells were cultured in DMEM (Dulbecco's modified Eagle's medium) supplemented with 10% FCS. The HeLa cell line stably overexpressing HA (haemagglutinin)–Tip60 was provided by Dr V. Ogryzko (Institute Gustave Roussy, CNRS UMR 8126, Villejuif, France). The U2OS cell line containing an integrated reporter substrate to measure HDR (homology-directed repair) of a single DSB has been described previously . IR (irradiation) was performed using a X-ray device (Faxitron X-Ray). Calicheamicin was from by Wyeth. HeLa cell nuclear extracts were purchased from the Computer Cell Culture Center (Seneffe, Belgium).
The polyclonal anti-HA Ab (Y-11) was purchased from Santa Cruz Biotechnology. The anti-Rad50 (13B3), anti-Nbs1 (1C3) and anti-Mre11 (12D7) Abs were purchased from GeneTex. The anti-phospho-ATM (S1981), anti-γH2AX (phosphorylated histone H2AX) and anti-HDAC3 (histone deacetylase 3) Abs were purchased from Cell Signaling Technologies. The anti-phospho-Nbs1 Ab (S343) was purchased from Interchim. We used two anti-ATM Abs (AB-3, Calbiochem; ab17995, Abcam). The anti-p400 Ab was purchased from Abcam. The anti-Myc Ab (9E10) was purchased from Roche Diagnostics. The anti-TTRAP Ab (2TRR1B3) was a gift from Dr L. Tora (IGBMC, CNRS UMR 7104, Illkirch, France). The anti-Tip60-DT Ab has been described previously . The anti-Tip60-CLHF, -RLPV and -CLGT Abs were gifts from Dr B. Amati (European Institute of Oncology, Milan, Italy) and have been described previously . The anti-Tip60-LM Ab was raised and purified using the CLGT and CLHF peptides described previously . Secondary Abs were purchased from Amersham (horseradish-peroxidase-conjugated) or Molecular Probes (dye-conjugated). Details on the Ab dilutions used are available from the authors upon request.
Plasmids, siRNAs (small interfering RNAs) and transfection methods
All siRNAs were purchased at Eurogentec. The C1, C2 and Tip60 siRNAs have been described previously . The sequence of the top strands of the Rad50, Mre11 and TRRAP siRNAs were GCUAAUGACUCUGAUGAUA-dTdT, GGAGAAGGAAAUACCAGAA-dTdT and GAAGUGUAAGCCUCAGUCA-dTdT respectively. siRNAs were transfected by the electroporation of 5×106 cells in 200 μl of serum-free OptiMEM® (Invitrogen) with 15 μl of siRNA (100 μM) using a Bio-Rad electroporation device set to 250V and 950 μF for 70 ms or using an Amaxa device according to the manufacturer's instructions. For transfection and expression of the I-SceI endonuclease, cells were transfected by calcium-phosphate co-precipitation with the pcDNA3βmycNLS-I-SceI plasmid. As a control for the transfection efficiency, a plasmid encoding GFP (green fluorescent protein) was used. The plasmid expressing untagged Tip60 (pcDNA3 Tip60; details of construction are available upon request) or empty vector (pcDNA3) were transfected by calcium-phosphate co-precipitation.
U2OS cells electroporated with the various siRNAs were plated in T25 flasks at a density of 103 or 2×103 cells per flask, then irradiated. For mitomycin C (Sigma) cytotoxicity assays, cells were treated with increasing concentrations of the drug for 1 h. After 10 days, cells were stained with Crystal Violet and colonies containing more than 50 cells were counted.
Cells were seeded at 2×105 in six-well plates. Recombination was induced by transient transfection of cells with 2 μg of pcDNA3β-NLS-I-SceI plasmid. Transfection efficiency was measured by the co-transfection of a mixture of the GFP plasmid (0.2 μg) with the pBlueScript plasmid (1.8 μg) to obtain equivalent amounts of transfected DNA. At 3 days after transfection, cells were washed with PBS and treated with trypsin. Cells were resuspended in PBS and HR was measured by the quantification of the GFP-positive cells by flow cytometry (FACScalibur; Becton Dickinson). Quantification was performed on 2.5×104 sorted events. The relative fraction of DSB-induced HR was normalized to the transfection efficiency obtained for each cell treatment condition.
Preparation of nuclear extracts, immunoprecipitation and immunodepletion
Preparation of Jurkat cell nuclear extracts, immunoprecipitation and immunodepletion were performed as described previously . Transfected U2OS cells were harvested using trypsin, resuspended in five cell pellet vol. of lysis buffer [10 mM Tris, pH 8.0, 10 mM NaCl and 2 mM MgCl2, supplemented with Complete EDTA-free protease inhibitors (Roche)]. After a 5 min incubation on ice, Nonidet P40 was added (0.5% of final volume) and the cells were incubated on ice for 5 min. After centrifugation at 2000 g for 5 min, the supernatant was discarded and the pellet was resuspended in buffer 3 (equal volume to the cell pellet) (20 mM Hepes, pH 7.9, 420 mM NaCl, 1.5 mM MgCl2 and 0.2 mM EDTA). After a 30 min incubation at 4 °C, samples were centrifugated at 20000 g and supernatants (nuclear extracts) were collected.
For Western blot analysis, samples were separated either by SDS/PAGE or on a NuPAGE® Novex 3–8% Tris/acetate gel (Invitrogen). Proteins were then transferred on to nitrocellulose membranes or PVDF membranes respectively. Specific primary Abs, as well as horseradish-peroxidase-conjugated secondary Abs, were used according to standard Western blotting procedures and horseradish-peroxidase activity was then detected using LumiLight Plus reagent (Roche).
Cells, seeded on to glass coverslips, were fixed with formaldehyde (3.7%) for 20 min at room temperature (20 °C) then permeabilized with Triton X-100 (0.1%) for 5 min at room temperature. Coverslips were mounted with the Vectashield anti-fade solution containing DAPI (4′,6-diamidino-2-phenylindole) (Molecular Probes). Images were collected using a digital camera attached to a DM5000 fluorescence microscope (Leica). Digital images were prepared for publication using MetaMorph software (Molecular Devices).
Nuclear extracts (2 ml, 3 mg) were clarified by centrifugation at 10600 g and was loaded on to a 10/300 GL Superdex 200 column (GE Healthcare) pre-equilibrated in 120 ml of buffer (20 ml Tris, pH 7.5, 100 mM KCl, 10% glycerol and 1 mM dithiothreitol). The column was run at a flow rate of 0.2 ml/min, and the proteins in 1.5 ml fractions were analysed by measuring the absorbance at 280 nm, and for the presence of Tip60, p400 and MRN by Western blotting.
Tip60 is required for DSB repair by HR
To test whether Tip60 is important for DSB repair, we transfected U2OS cells with an siRNA directed against Tip60 (described in ). This siRNA specifically decreased the expression of Tip60 (Figure 1A). We next performed a clonogenic assay after IR. We found that cells transfected by the Tip60 siRNA were more sensitive to IR than cells transfected by the control siRNA, indicating that Tip60 influences DSB repair (Figure 1A). Cells defective in the HR pathway are known to display increased sensitivity to IR . To examine a potential defect in the HR pathway, we treated the cells with mitomycin C, as sensitivity to this drug is a hallmark of a defective HR pathway . Again, we found that transfection of an siRNA against Tip60 leads to increase sensitivity to mitomycin C (Figure 1A). Altogether, these drug sensitivity studies suggest that Tip60 is important for HR.
Tip60 is required for repair of DSBs by HR
To confirm this finding directly, we used U2OS cells containing a reporter system (see Supplementary Figure S1 at http://www.BiochemJ.org/bj/426/bj4260365add.htm) for HDR , a process highly related to HR. The HDR of a unique DNA DSB induced at an I-SceI site on a transgene leads to the appearance of GFP-positive cells. As positive controls, we transfected U2OS cells with siRNAs against two components of the MRN complex, Rad50 or Mre11 (see Supplementary Figure S2 for Western blots demonstrating the efficiency of Rad50 and Mre11 siRNAs http://www.BiochemJ.org/bj/426/bj4260365add.htm). As described previously , we observed a decrease in HDR after transfection with the Mre11 siRNA , as well as with the Rad50 siRNA (compared with the control siRNA) (see Supplementary Figure S3 for representative FACS data for each sample at http://www.BiochemJ.org/bj/426/bj4260365add.htm), therefore demonstrating that our assay allows the adequate assessment for the involvement of proteins in the HR pathway. Strikingly, the transfection of two Tip60 siRNAs also significantly decreased the number of GFP-positive cells (Figure 1B). Since the expression of I-SceI was not affected by Tip60 knockdown (Figure 1B), this result indicates that Tip60 expression is required for HDR.
Tip60 expression is required for induction of γH2AX and Rad50 foci in U2OS cells
We then intended to characterize the molecular event controlled by Tip60. We first investigated the role of Tip60 in γH2AX foci formation in U2OS cells. Indeed, conflicting results have been reported previously, as two studies found that depleting Tip60 leads to increased γH2AX levels [17,18], whereas two others describe the opposite [19,20]. We thus transfected U2OS cells with an Tip60 siRNA, the cells were irradiated for 1 and 6 h and we analysed the γH2AX immunofluorescence. IR induces the appearance of a strong punctuate γH2AX staining on most of the cells (Figure 2A and see Supplementary Figure S4 for enlargement of typical cells at http://www.BiochemJ.org/bj/426/bj4260365add.htm). To quantify precisely and unequivocally the effect of the Tip60 siRNA, we applied a threshold on γH2AX staining using the ImageJ software and calculated the percentage of cells which stained positive for γH2AX (total of 250 independent cells). We found that transfection of the Tip60 siRNA decreased the amount of γH2AX-positive cells (Figure 2B). Moreover, the effects of Tip60 on γH2AX induction were also observed when the total γH2AX fluorescence levels per cell were averaged in 500 independent cells (see Supplementary Figure S5 at http://www.BiochemJ.org/bj/426/bj4260365add.htm). Tip60 knockdown inhibited γH2AX induction as soon as 1 h following IR, indicating that Tip60 expression is required for efficient phosphorylation of H2AX in U2OS cells. We then tested the effects of Tip60 knockdown on one of the first events observed following DSB induction, which is the recruitment of the MRN complex to DSBs. MRN recruitment to damaged DNA can be visualized by the appearance of foci following induction of DNA DSBs . We thus transfected U2OS cells with the Tip60 siRNA, then irradiated and stained the cells with an anti-Rad50 Ab (Figure 3A and see Supplementary Figure S4 for enlargement of a typical cell). We performed the quantification of foci formation by blind counting of the percentage of foci-presenting cells from 250 independent cells [although most cells presented multiple foci (see Figure 3A), cells were recorded as positive as soon as they had one foci]. Quantification is shown in Figure 3(B). We found that, 6 h following IR, approx. 80% of control cells harboured Rad50 foci in cells transfected by the control siRNA (Figures 3A and 3B). In cells transfected with the Tip60 siRNA, the number of cells presenting foci was significantly reduced. Again, this effect was also observed at an early time point (1 h), indicating that Tip60 controls the formation of MRN foci, rather than their maintenance. Since Tip60 down-regulation did not significantly affect Rad50 expression or the expression of other members of the MRN complex (Figure 3C), this result indicates that Tip60 expression is important for the efficient recruitment of Rad50 and probably of the MRN complex to damaged DNA.
Tip60 is important for γH2AX foci formation
Tip60 is involved in MRN foci formation
Endogenous Tip60 and MRN proteins physically interact
Interestingly, a molecular link between Tip60 and the MRN complex has been described previously, as MRN proteins interact with TRRAP, a component of the Tip60 complex , suggesting that the effects described above could be due to a physical interaction between Tip60 and MRN. To test this possibility, we immunoprecipitated endogenous Tip60 from HeLa cell nuclear extracts and we tested for the presence of endogenous MRN proteins and TRRAP in the immunoprecipitates by Western blotting (Figure 4A). As expected, immunoprecipitation of Tip60 led to the co-immunoprecipitation of TRRAP protein. Moreover, we found that all of the three MRN proteins were detected in the Tip60 immunoprecipitate and not the control immunoprecipitate (Figure 4A). Detection of endogenous Tip60 in the MRN immunoprecipitates was impossible, since endogenous Tip60 co-migrates with the immunoglobulin heavy chains (results not shown). Thus to rule out the possibility that the presence of MRN proteins in the Tip60 immunoprecipitate shown in Figure 4(A) was due to a cross-reaction of the anti-Tip60 Ab with another protein, we performed immunoprecipitation of endogenous Tip60 with three other unrelated anti-Tip60 Abs (anti-Tip60-CLHF, -RLPV and -CLGT Abs). We detected endogenous MRN proteins in all four immunoprecipitates (Figure 4B). Taken together, these data indicate that Tip60 and the MRN complex interact at endogenous levels.
Identification of a Tip60–MRN complex
Note that Robert et al.  failed to detect such a complex between Tip60 and MRN proteins following immunoprecipitation of Nbs1 or Mre11 proteins, perhaps because the presence of Tip60 masks the epitopes of the Abs. Alternatively, since Robert et al.  tested the presence of Tip60 using a HAT assay, Tip60 bound by MRN may not be able to acetylate histones (see the Discussion section).
Also MRN proteins were not found associated with overexpressed tagged Tip60 . We did not detect any MRN protein that co-immunoprecipitated with Tip60 from the cell line expressing the tagged Tip60 used previously to purify the Tip60 complex (see Supplementary Figure S6 at http://www.BiochemJ.org/bj/426/bj4260365add.htm), strongly suggesting that the addition of a tag at the N-terminus of Tip60 is detrimental for its association with MRN proteins.
MRN does not interact with the classical Tip60 complex
The next question we addressed was whether the whole Tip60 complex associates with MRN proteins. For this aim, we immunodepleted endogenous p400 using an Ab directed against p400, a component of the classical Tip60 complex , or with an anti-HA Ab as a control. Endogenous p400 protein was efficiently immunodepleted from Jurkat cell nuclear extracts (Figure 5A). Moreover, immunodepletion of p400 led to the depletion of the endogenous Tip60 complex, since Tip60 was also depleted by p400 immunodepletion (Figure 5A). As expected, the amount of p400 immunoprecipitated by Tip60 were decreased upon p400 immunodepletion (Figure 5B), confirming that the classical Tip60 complex was indeed depleted. In contrast, the amount of endogenous Rad50 and Nbs1 proteins in the Tip60 immunoprecipitate was not decreased (Figure 5C), suggesting that the Tip60 protein which interacts with MRN proteins is not physically associated with p400.
Tip60–MRN complex does not contain p400
To confirm this finding, we prepared Jurkat cell nuclear extracts and fractionated them by size-exclusion chromatography. Using Western blot experiments, we found that p400 was eluted in fractions 5, 6 and 7, whereas the MRN proteins were observed in fractions 5–9 (Figure 5D). A band migrating at 60 kDa is observed in fractions 5–11, probably corresponding to endogenous Tip60. Thus this experiment suggests that MRN and Tip60 are both present in fractions in which there is no detectable p400 (fractions 8 and 9). To test whether they are physically associated in these fractions, we performed immunoprecipitation experiments using fractions 5 and 6 (which contain Tip60, MRN and p400) and 8 and 9 (which contain MRN and Tip60, but not p400). Immunoprecipitation of Tip60 led to the co-immunoprecipitation of p400 from fractions 5 and 6, and of Rad50 from fractions 5, 6 and 8 (Figure 5E). This result thus indicates that the classical Tip60 complex elutes in fractions 5 and 6, whereas the Tip60–MRN complex elutes in fractions 5, 6 and 8. Importantly, since p400 is not detected in direct Western blotting from fraction 8 or in Tip60 immunoprecipitate from fraction 8, this result indicates that the Tip60–MRN complex can be devoid of p400. Taken together, the results in Figure 5 thus indicate that the MRN-associated Tip60 protein is not embedded within the classical p400-containing Tip60 complex.
We next investigated how Tip60 contacts the MRN complex. We could not detect any interaction between Tip60 and MRN proteins in GST (glutathione transferase) pull-down experiments (results not shown), suggesting that the interaction is not direct. Since TRRAP is known to be physically associated with Tip60 on one hand  and with the MRN complex on the other hand , we tested whether TRRAP mediates the Tip60–MRN interaction. We transfected U2OS cells with an siRNA directed against TRRAP or with a control siRNA and with an untagged Tip60 expression vector or the corresponding empty vector. As expected TRRAP expression was decreased in cells transfected with the TRRAP siRNAs (Figure 6). We then prepared nuclear extracts and subjected these extracts to immunoprecipitation with an anti-Tip60 Ab (or without Ab as a control). In cells transfected with the control siRNA, overexpression of untagged Tip60 led to a large increase in the amount of p400 and Rad50 co-immunoprecipitating with Tip60 (Figure 6) respectively, reflecting formation of the classical Tip60 complex and of the Tip60–MRN complex. Depletion of TRRAP does not affect the amount of p400 co-immunoprecipitating with Tip60 (Figure 6), indicating that TRRAP expression is not required for the Tip60–p400 interaction. However, the amount of Rad50 co-immunoprecipitating with Tip60 was strongly decreased upon knock-down of TRRAP, although TRRAP siRNA had no effect on Rad50 expression. Thus these data indicate that TRRAP expression is required for the Tip60–Rad50 interaction, strongly suggesting that TRRAP physically bridges Tip60 and MRN proteins through direct or indirect interactions.
TRRAP mediates the interaction between Tip60 and MRN proteins
In conclusion, we show in the present study that Tip60 expression is required for a very early step of DNA DSB repair, as Tip60 depletion affects the earliest known molecular events occurring following DSB induction, which are γH2AX foci formation and MRN complex recruitment. In addition, this role of Tip60 is likely to be direct, since we demonstrate that endogenous Tip60 and the sensor MRN complex belongs to the same multimolecular complex (named ‘the Tip60–MRN complex’, see below). We thus uncover a physical interaction between a sensor complex and a chromatin-modifying enzyme. We further characterize this Tip60–MRN complex by demonstrating that: (1) it does not involve the whole Tip60 complex; and (2) it requires endogenous TRRAP expression, strongly suggesting that TRRAP physically forms a bridge between Tip60 and the MRN complex. It is important to note that the Tip60–MRN complex probably contains other proteins, as it elutes in size-exclusion chromatography experiments as a complex of approx. 1 MDa. These uncharacterized proteins may participate, together with TRRAP, in structuring the Tip60–MRN complex.
The interaction between Tip60 and MRN proteins requires TRRAP. As such, we provide insights into the mechanism by which TRRAP participates in DNA repair. Interestingly, TRRAP regulates the transcription of Mad1 and Mad2 genes through the recruitment of Tip60, which acetylates histone H4, and GCN5 (general control non-derepressible 5), which acetylates H3 . It would thus be important to test whether TRRAP can also target the GCN5 acetyl transferase to MRN proteins, and thus potentially to DNA DSBs. In addition, we demonstrate that the Tip60–MRN complex is different from the classical complex. Interestingly, our overexpression experiments suggest that Tip60 expression is rate limiting for the formation of these two complexes, since we immunoprecipitated more p400 and Rad50 in the presence of exogenous Tip60. This suggests that the two Tip60-containing complexes could be competing for limiting amounts of Tip60. This possibility underlines the importance of understanding what regulates the presence of Tip60 in one complex or the other. Moreover, disturbing the formation of one complex could indirectly affect the function of the other complex.
What could be the role of the Tip60–MRN complex that we have uncovered here? Clearly, it is probably to play a major role in DNA DSB repair, since the five characterized components of this complex are known to favour HR (present study for Tip60 and Rad50,  for Mre11,  for TRRAP and  for Nbs1). Interestingly, we found that Tip60 expression is required for efficient formation of Rad50 foci. It is tempting to speculate that the Tip60–MRN complex we characterized in the present study is important for Rad50 foci formation. One could imagine, for example, that the physical interaction with Tip60 helps the recruitment of MRN proteins to DSBs. Alternatively, since Tip60 is an enzyme, it can acetylate an MRN protein in the context of the Tip60–MRN complex, therefore favouring its recognition of DNA DSBs. In this regard, it is important to note that Nbs1 has been shown to be acetylated .
In addition, since both Tip60 and MRN proteins are known to be required for ATM activation , the Tip60–MRN complex could participate in local ATM activation. In agreement with such an hypothesis, we showed recently that ATM activation following oncogenic stress requires Tip60, but not p400 , suggesting that this role of Tip60 is not mediated within the classical Tip60 complex. Along this line, it is tempting to speculate that the Tip60–MRN complex is the enzymatic complex which mediates ATM acetylation . Strikingly, Tip60 is involved in two different steps on repair of DNA DSBs, each of these steps being characterized by acetylation of a specific substrate. Through acetylation of ATM, Tip60 is involved in DNA damage signalling , and through nucleosome acetylation, it is required for the recruitment of repair proteins  or removal of H2AX [17,28]. These two distinct acetylation events could be mediated by two distinct enzymatic complexes, with the Tip60–MRN complex being the actual enzyme acetylating ATM and the classical Tip60 complex being involved in histone acetylation, as only this latter complex can acetylate nucleosomes . If this hypothesis is true, then Tip60 would be highly dynamic during DSB repair. First, it would be recruited very rapidly with MRN proteins to allow proper signalling through ATM acetylation. Then the classical Tip60 complex would be recruited to mediate histone acetylation. Interestingly, such a model is supported by our recent findings that knock-down of p400 favours the activation of Tip60-dependent DNA damage pathways . One could imagine that in cells in which p400 levels are decreased, Tip60 is redistributed to the Tip60–MRN complex, therefore allowing a stronger response to DNA damage. Studies of the recruitment dynamic and interdependence of the various Tip60-containing complexes are required to validate such a model.
ataxia telangiectasia mutated
fetal calf serum
general control non-derepressible
green fluorescent protein
histone acetyl transferase
phosphorylated histone H2AX
- MRN complex
small interfering RNA
Catherine Chailleux, Sandrine Tyteca, Christophe Papin, François Boudsocq, Celine Courilleau and Yvan Canitrot performed the experiments. Nadine Puget provided the U2OS cell line and the conditions of use. Mikhaïl Grigoriev supervised the biochemical experiments. Catherine Chailleux, Yvan Canitrot and Didier Trouche designed the research and analysed the data. Didier Trouche wrote the paper.
We thank Dr B. Amati, Dr V. Ogryzko, Dr L. Tora and Dr B.S. Lopez (I-SceI plasmid; CEA/CNRS, UMR 217, Fontenay-aux-Roses, France) for materials, Dr B. S. Lopez for critical reading of the manuscript prior to submission, C. Lorenzo for assistance with fluorescence microscopy and M. Quaranta for assistance with flow cytometry. We acknowledge the use of the imaging facilities of CBD-IFR109 (Toulouse).
This work was supported by the Association pour la Recherche contre le Cancer (ARC) (‘subvention libre’ and studentship to S.T.); the Agence Nationale de la Recherche (ANR) [grant number ANR-06-3-13-7148]; the Ligue Nationale Contre le Cancer (‘équipe labellisée’); and Electricité De France.
These authors contributed equally to this work.