A)
Question posée et démarches expérimentales
Les résultats décrits plus haut ont mis en évidence le recrutement par le complexe nucléoprotéique TIP49b/ADN simple-brin de l’acétylase TIP60 et du complexe MRN. Cette observation, ajoutée au fait qu’à la fois MRN et TIP60 activent la kinase ATM (Jiang et al., 2006; Lee and Paull, 2005; Sun et al., 2005), suggère que ces protéines pourraient agir au sein d’un même complexe.
En collaboration avec l’équipe de Didier Trouche, par une double approche biochimique et cellulaire, nous avons caractérisé l’existence d’un complexe TIP60/MRN in
vivo impliqué dans la réparation des CDB.
B)
Résultats et Discussions
Nos résultats sont présentés dans la publication ci-après : « A MRN/Tip60 complex involved in DNA double strand breaks repair ».
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A MRN/Tip60 complex involved in DNA double strand breaks repair
Sandrine Tyteca*, Catherine Chailleux*, Céline Courilleau*, Christophe Papin§, Nadine Puget¶, Mikhaïl Grigoriev§, Yvan Canitrot*, Didier Trouche*†
*LBCMCP, CNRS and University of Toulouse, 31062 Toulouse, France § LBME, CNRS and University of Toulouse, 31062 Toulouse, France ¶ IPBS, CNRS and University of Toulouse, 31062 Toulouse, France
†Corresponding author : Didier Trouche, LBCMCP UMR 5088 CNRS/UPS, 118
Route de Narbonne, 31062 Toulouse Cedex 9. Phone : +33 5 61 55 85 79 ; Fax : +33 5 61 55 81 09 ; E-mail : dtrouche@cict.fr
99 Abstract
Chromatin modifications and chromatin modifying enzymes are believed to play a major role in the process of DNA repair. The Tip60 histone acetyl transferase is physically recruited to DNA double strand breaks (DSB) where it mediates histone acetylation. Here, we show that Tip60 expression is required for DNA DSB repair through both Homology-Directed Recombination and Non Homologous End Joining. Tip60 is involved in the formation of Rad50 foci following ionizing radiations, suggesting that Tip60 expression is necessary for recruitment of the DNA damage sensor MRN (Mre11, Rad50, Nbs1) complex to DNA DSB. Moreover, Tip60 physically interacts with MRN proteins and the Tip60/MRN complex is rapidly mobilized following DNA DSB induction, suggesting that the effects of Tip60 on DNA repair are mediated within this complex. Taken together, our results provide important new insights into the role and mechanism of action of Tip60 in the process of DNA DSB repair.
Key words: DNA double strand breaks repair / Tip60 / MRN / Homologous Recombination / Non Homologous End-Joining
100 Introduction
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 double strand breaks (DSB) are extremely deleterious damages, which require rapid and efficient repair. Chromatin modifications are known to be important for signaling and dynamic of the DNA DSB repair machineries. In yeast, chromatin remodeling following DNA DSB has been extensively studied and the machineries involved characterized (Downs and Cote, 2005).
In mammals, although far less is known, the involvement of the histone acetyl transferase Tip60 has been clearly demonstrated (Murr et al., 2006). Tip60 belongs to the MYST family of histone acetyl transferases, which is conserved from yeast to human (Utley and Cote, 2003). In agreement with its role on histone acetylation, Tip60 has been widely described as a transcriptional co-activator. Its expression is required for myc and p53-dependent genes transcription (Frank et al., 2003b ; Tyteca et al., 2006).
Tip60 belongs to a multimolecular complex, called the Tip60 complex (Ikura et al., 2000). The Tip60 complex is considered as the human orthologue of two yeast complexes, the NuA4 histone acetyl transferase complex and the SWR1 chromatin remodeling complex, both known to be involved in DNA DSB repair (Doyon et al., 2004). In mammals, a protein belonging to the Tip60 complex, TRRAP, has been shown to be important for both Homologous Recombination (HR) and Non- Homologous End Joining (NHEJ), the two main pathways of DNA DSB repair (Murr et al., 2006; Robert et al., 2006). Tip60 itself is recruited around DNA DSB, where it mediates histone acetylation and subsequent recruitment to repair sites of DNA repair factors, such as Rad51 (Murr et al., 2006). In addition to its role in local histone acetylation, Tip60 activity is important for DNA dsb signaling: following DNA dsb, it can acetylate the transducer kinase ATM, this acetylation being critical for ATM autophosphorylation and activation (Sun et al., 2005; Sun et al., 2007).
Here we investigate the role of the Tip60 complex in DNA DSB repair. We identify a new multimolecular complex containing Tip60 and the MRN complex, a sensor of
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DNA DSB. Altogether, our results strongly suggest that this Tip60/MRN complex is important both for HR and NHEJ.
102 Materials and Methods
Cell culture
Jurkat cells were cultured in RPMI supplemented with 10% FCS. All other cells were cultured in DMEM supplemented with 10% FCS. The HeLa cell line stably overexpressing HA-Tip60 was kindly provided by V. Ogryzko. The U2OS cell line containing an integrated reporter substrate to measure Homology-Directed Repair of a single DSB has been previously described (Puget et al., 2005). The CHO cell line containing an integrated chromosomal NHEJ substrate designed to allow the measurement of NHEJ events by targeting breaks to this substrate has been previously described (Guirouilh-Barbat et al., 2004). Irradiation was performed using a Xray Faxytron device. Calicheamicin was provided by Wyeth. HeLa Nuclear Extracts were purchased from Computer Cell Culture Center (Belgium).
Antibodies
The polyclonal anti-HA Ab (Y-11) was purchased from Santa Cruz. The anti-Rad50 (13B3), anti-Nbs1 (1C3) and anti-Mre11 (12D7) Ab were purchased from GeneTex. The anti-phosphoATM (S1981), g-H2AX and anti-HDAC3 Ab were purchased from Cell Signaling Technologies. The anti-phosphoNbs1 (S343) was purchased from Interchim. We used two anti-ATM Ab (AB-3, Calbiochem; ab17995, Abcam). The anti-Tag myc Ab (9E10) was purchased from Roche Diagnostics. Secondary Ab were purchased from Amersham (peroxydase-conjugated) or Molecular Probes (dye- conjugated). Details on the Ab dilutions used are available upon request.
Plasmids, siRNAs and transfection methods
The c1 and Tip60 siRNAs are described in (Tyteca et al., 2006). siRNAs were transfected by electroporating 5.106 cells in 200!L of serum-free OptiMEM with 15!L siRNA (100!M) on a BioRad electroporation device set to 250V, 950!F. For transfection and expression of the I-SceI endonuclease, cells were transfected by calcium phosphate co-precipitation with the pcDNA3bmycNLS-I-SceI plasmid. As a control for the transfection efficiency, a GFP-encoding plasmid was used. The plasmid expressing DN Tip60 was constructed by inserting the DN Tip60 cDNA described in (Ikura et al., 2000) into the pCMV 2N3T vector.
103 Clonogenic assay
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 cells were treated with increasing concentrations of drug for 1 h. Ten days later, cells were stained with crystal violet and colonies containing more than 50 cells were counted.
NHEJ assay
Recombination was induced by transfection of 5 x105 cells with 1!g of the pcDNA3b- NLS-I-SceI plasmid plus 1 !g of pBlueScript plasmid or transfection of 1!g of the pcDNA3b-NLS-I-SceI plasmid plus 1 !g DN-Tip60 coding plasmid. Transfection efficiency was measured by the cotransfection of a mixture of GFP plasmid (0.2!g) with the pBlueScript plasmid (1.8!g) to obtain equivalent amount of transfected DNA. Three days after transfection cells were dissociated with PBS/EDTA (25mM) and fixed in PBS/formaldehyde 3.7% for 20 min at room temperature. Cells were then stained with anti-H2Kd (1/30 mouse isotype, SF1-1.1, Pharmingen), or anti-CD4 (1/30 mouse isotype, H129.19, Pharmingen) or anti-CD8 (1/30 rat isotype, 53-6.7, Pharmingen) for 30 min at room temperature. Cells were then incubated with anti- mouse-alexafluor488 (1/500 mouse isotype, Molecular Probes) for 30 min at room temperature. Scoring of the NHEJ events affecting CD4 and CD8 was performed by flow cytometry using FACScalibur (Becton Dickinson). Quantification was performed on 2.5x104 sorted events.
HR assay
Cells were seeded at 2x105 in 6-well plates. Recombination was induced by transient transfection of cells with 2!g of the pcDNA3b-NLS-I-SceI plasmid. Transfection efficiency was measured by the cotransfection of a mixture of GFP plasmid (0.2!g) with the pBlueScript plasmid (1.8!g) to obtain equivalent amount of transfected DNA. Three days after transfection cells were washed with PBS and trypsinized. Cells were resuspended in PBS and recombination was measured by the quantification of GFP positive cells by flow cytometry (FACScalibur, Becton Dickinson). Quantification was performed on 2.5x104 sorted events. Relative fraction of DSB-induced Homologous Recombination was normalized to the transfection efficiency obtained for each cell treatment condition.
104 Preparation of Jurkat nuclear extracts
Jurkat cells were dispatched in order to have 250!L of pelleted cells per microtube. Cells were resuspended in 5 volumes of solution A (10mM Hepes pH 7.9, 1.5mM MgCl2, 10mM KCl, 0.5mM DTT, protease inhibitors), incubated for 10 minutes on ice and centrifuged. Pelleted cells were then resuspended in 2 volumes of solution A and centrifuged at full speed for 1 min. Supernatants were discarded and this step was repeated once. After centrifugation, the pellet was resuspended in 1 volume of solution C (20mM Hepes pH 7.9, 25% glycerol, 420mM NaCl, 1.5mM MgCl2, 0.2mM EDTA, 0.5mM DTT, protease inhibitors) and incubated for 30 min. After centrifugation at full speed for 10 min, supernatants were finally collected, mixed and frozen at -80°C.
Immunoprecipitation and Western Blotting
For total cell extracts, cell pellets were resuspended in lysis buffer (50mM Tris pH 8, 300mM NaCl, 0.4% NP-40, 10mM MgCl2, protease inhibitors), incubated on ice and
centrifuged at full speed for 15 minutes. Supernatants were diluted twice in dilution buffer (50mM Tris pH 8, 0.4% NP-40, 5mM CaCl2, DNase). Nuclear extracts were
diluted 10 times in wash buffer (" lysis + " dilution). Diluted extracts were then precleared by incubation for 1h at 4°C with 30!L of previously blocked protein-A and protein-G beads (Sigma). Blocking was achieved by incubating the agarose beads with 500!g of BSA and 200!g of herring sperm DNA for 3h at 4°C. Precleared samples were incubated overnight or 1h at 4°C with antibodies. Immune complexes were then recovered by incubating the samples with 20!L of blocked protein- A/protein-G beads for 2h at 4°C on a rotating wheel. Beads were washed four times in wash buffer and resuspended in Laemmli sample buffer or in NuPAGE® LDS Sample Buffer (Invitrogen).
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 respectively to nitrocellulose membrane or to PVDF membrane. Specific primary antibodies as well as peroxidase-conjugated secondary antibodies were used according to standard western blot procedures and peroxidase activity was then detected by using LumiLight Plus reagent (Roche).
105 Immunodepletions
In figure 4A, 500!L of SN2 supernatant prepared as described in (Robert et al., 2006) (kind gift from Dr L. Tora) were incubated for 1h (4°C) with 30!g anti-Tip60 DT antibody or anti-Mre11 antibody previously bound to 50!L protein-G sepharose beads in IP100 buffer (25mM TrisCl pH8, 10% glycerol, 0.1% NP-40, 0.5mM DTT, 5mM MgCl2, 100mM KCl). After centrifugation, supernatants were collected and
designated as “Id” (Immunodepletion).
In figure 5, precleared Jurkat nuclear extracts were subjected to immunoprecipitation with agarose beads previously coated with anti-p400 or anti-HA antibody in wash buffer (see above). After centrifugation, the supernatant was collected and subjected to 2 other rounds of immunoprecipitation. The last supernatant is collected and used as the “depleted extract”.
Immunofluorescence
Cells seeded on glass coverslips were fixed with formaldehyde (3.7%) for 20 min at RT then permeabilized with Triton X100 (0.1%) for 5 min at RT. Coverslips were mounted with the Vectashield antifade solution containing DAPI (Molecular probes). Images were collected using a digital camera attached to a Leica (DM5000) fluorescence microscope. Digital images were prepared for publication using Metamorph software.
106 Results
Tip60 is required for DSB repair by HR and NHEJ
To test whether Tip60 is important for DSB repair, we transfected U2OS cells with a siRNA directed against Tip60 (described in (Tyteca et al., 2006)) and we performed a clonogenic assay after irradiation (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 (Fig. 1A).DSB repair mainly involves HR or NHEJ. Though NHEJ is the major pathway to repair DSB, cells defective in the HR pathway are also known to display increased sensitivity to IR, albeit to a lesser extent than NHEJ defective cells (van Gent et al., 2001). To examine a potential defect in the HR pathway we treated the cells with mitomycin C since sensitivity to this drug is a hallmark of a defective HR pathway (Hochegger et al., 2004). Again, we found that transfection of a siRNA against Tip60 leads to increase sensitivity to mitomycin C (Fig. 1A). Altogether, these drug sensitivity studies suggest that Tip60 is important for both NHEJ and HR.
To confirm this finding directly, we used artificial reporter systems. We first used U2OS cells containing a reporter system (described supplementary data 1) for homology-directed repair (HDR) (Puget et al., 2005), 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. We transfected U2OS cells with two siRNAs against Tip60 (previously described in (Tyteca et al., 2006)). The expression of I-SceI was not affected by Tip60 knockdown (Fig. 1B). The two Tip60 siRNAs significantly decreased the number of GFP-positive cells compared to cells transfected with the control siRNA (Fig. 1C), indicating that Tip60 expression is required for HDR.
To test the involvement of Tip60 in NHEJ, we used another reporter system (described in supplementary data 2) stably introduced in CHO cells (Guirouilh-Barbat et al., 2004). NHEJ is assessed by measuring the appearance of CD4 or CD8 positive cells after DSB induction by I-SceI. Since CHO are Chinese hamster cells (whose genome is not sequenced), we could not use a siRNA-based strategy. However, co-expression of a dominant negative mutant of Tip60 (described in (Ikura et al., 2000 ; Murr et al., 2006)) does not affect I-SceI expression but efficiently reduces NHEJ events (Fig. 1D). Thus, taken together, these data indicate that Tip60 is important for DNA DSB repair by promoting both HR and NHEJ.
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Tip60 expression is required for induction of !H2AX 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 phosphorylated H2AX (gH2AX) foci formation in U2OS cells. Indeed, conflicting results have been previously reported since two studies found that depleting Tip60 leads to increased gH2AX levels (Jha et al., 2008; Kusch et al., 2004), whereas two others describe the opposite (Eymin et al., 2006; Gorrini et al., 2007 ). In our hand Tip60 knockdown decreased gH2AX foci following irradiation (Fig. 2A and 2B) indicating that Tip60 expression is required for efficient formation of gH2AX foci 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 DSB. MRN recruitment to damaged DNA can be visualized by the appearance of foci following induction of DNA DSB (Mirzoeva and Petrini, 2001). Six hours following irradiation, about 80 % of control cells harbored Rad50 foci (Fig. 3A and B). In cells transfected by the Tip60 siRNA, the number of cells presenting foci was significantly reduced. Since Tip60 down-regulation did not significantly affect Rad50 expression, nor the expression of other members of the MRN complex (Fig. 3C), this result indicates that Tip60 expression is important for the formation of Rad50 foci, and thus for the efficient recruitment of Rad50 and likely of the MRN complex to damaged DNA.
Identification of a Tip60/MRN complex
Interestingly, a molecular link between Tip60 and the MRN complex was recently described, since MRN proteins interact with TRRAP, a component of the Tip60 complex (Robert et al., 2006). To test whether Tip60 was present in this complex, we immunodepleted Tip60-containing complexes from the extracts used to identify the TRRAP/MRN complex and we analysed protein composition by SDS PAGE followed by silver staining (Fig. 4A). Four bands were specifically depleted: the band corresponding to TRRAP, as expected, but also three bands migrating at the expected size for MRN proteins. These bands were also immunodepleted by Mre11 antibodies, strongly suggesting that they correspond to MRN proteins. We confirmed this identification by western blot on the same samples (Fig. 4B). These data thus suggest that Tip60 is present in a large proportion of the TRRAP/MRN complexes
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previously identified (Robert et al., 2006). Moreover, immunoprecipitation (IP) of Tip60 from HeLa nuclear extracts led to the specific co-IP of the three MRN proteins (Fig. 4C). Finally, we detected the MRN proteins in the IP performed with four different anti-Tip60 antibodies (supplementary data 3A), ruling out the possibility that the co-IP we observed above was due to a cross-reactivity of our antibody with MRN proteins. Taken together, these data indicate that endogenous Tip60 interacts with the MRN complex.
Note that Robert et al. failed to detect such a complex between Tip60 and MRN proteins following IP of Nbs1 or Mre11 proteins (Robert et al., 2006), perhaps because the presence of Tip60 masks the antibodies epitopes. 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 discussion). Also, MRN proteins were not found associated with overexpressed tagged Tip60 (Ikura et al., 2000). We did not detect any MRN protein co-immunoprecipitating with Tip60 from the cell line expressing the tagged Tip60 previously used to purify the Tip60 complex (supplementary data 3B), strongly suggesting that the addition of a tag at the N- terminus of Tip60 is detrimental for its association with MRN proteins.
The next question we addressed was whether the entire Tip60 complex associates with the MRN complex or whether two independent Tip60-containing complexes can be present in cells. We depleted p400, a component of the classical Tip60 complex (Fuchs et al., 2001), from Jurkat nuclear extracts (Fig. 5A, upper panel). As expected, IP of Tip60 co-precipitated less p400 from p400-depleted extracts than from control extracts (Fig. 5A, lower panel), demonstrating that immunodepletion of p400 led to depletion of the classical Tip60 complex. However, the amount of Rad50 and Nbs1 proteins in the Tip60 IP was not decreased (Fig. 5B), indicating that the Tip60/MRN complex does not contain p400. This result indicates that two different Tip60-containing complexes co-exist in untreated Jurkat cells, the classical Tip60 complex which contains p400 and the Tip60/MRN complex devoid of p400. It is tempting to speculate that these two different complexes have different functions, with the Tip60/MRN complex being important in DNA repair.
Dynamic of the Tip60/MRN complex following DNA DSB
We next investigated the dynamic of the Tip60/MRN complex following DNA DSB. We treated Jurkat cells with calicheamicin, a DNA DSB-inducing drug (Ismail et al.,
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2005). DSB induction was efficient, since we rapidly observed increased ATM phosphorylation and ATM-dependent Nbs1 phosphorylation (Fig. 6, upper panel). We then immunoprecipitated Tip60 from extracts of calicheamicin treated cells. We found that, as early as 30 min, the Tip60/MRN complex could no longer be observed by co- precipitation (Fig. 6, lower panel). This result indicates that the Tip60/MRN complex is rapidly mobilized upon DNA DSB induction. This decrease of the Tip60/MRN co-IP could indicate that the Tip60/MRN complex dissociates following DSB induction. Alternatively, this decrease could reflect increased chromatin binding of the complex (for example through recruitment to DSB), the amount of the complex being consequently decreased in soluble nuclear extracts. Whatever the mechanism, our data suggest that the Tip60/MRN complex is involved in an early step of DNA DSB repair.
Discussion
In this work, we show that Tip60 expression is important for the two main pathways of DSB repair in mammals, HR and NHEJ. We demonstrate that Tip60 expression is required for the formation of Rad50 foci, and thus certainly for the recruitment of the MRN complex to DNA DSB. Strikingly, MRN proteins are important both for HR (Dupre et al., 2008; Yang et al., 2006 ), and for NHEJ (Huang and Dynan, 2002). Thus, the effect of Tip60 on MRN binding to DSB can probably explain its involvement in HR and NHEJ. What is then the mechanism by which Tip60 favors MRN recruitment to DSB? Since Rad50 foci are observed independently of ATM