Histone modifications in response to DNA damage

Mutation Research 618 (2007) 81–90

Histone modifications in response to DNA damage Mohammed Altaf, Nehm´e Saksouk, Jacques Cˆot´e ∗ Laval University Cancer Research Center, Hˆotel-Dieu de Qu´ebec (CHUQ), 9 McMahon Street, Quebec City, Que. G1R 2J6, Canada Received 6 September 2006; accepted 16 September 2006 Available online 21 January 2007

Abstract The packaging of the eukaryotic genome into highly condensed chromatin makes it inaccessible to the factors required for gene transcription, DNA replication, recombination and repair. Eukaryotes have developed intricate mechanisms to overcome this repressive barrier imposed by chromatin. Histone modifying enzymes and ATP-dependent chromatin remodeling complexes play key roles here as they regulate many nuclear processes by altering the chromatin structure. Significantly, these activities are integral to the process of DNA repair where histone modifications act as signals and landing platforms for various repair proteins. This review summarizes the recent developments in our understanding of histone modifications and their role in the maintenance of genome integrity. © 2007 Elsevier B.V. All rights reserved. Keywords: H2AX; NuA4; Tip60; Ino80; Swr1; H2AZ

1. Introduction The eukaryotic genome is maintained as a nucleoprotein complex known as chromatin, which consists of positively charged histone proteins in addition to DNA. The basic unit of chromatin is the nucleosome which consists of 146 bp of DNA wrapped around an octamer containing two copies each of core histones H2A, H2B, H3 and H4. Histone H1, also called linker histone, locks the DNA at the entry and exit points from the nucleosome and further condenses chromatin. The packaging of eukaryotic DNA into chromatin solves the problem of accommodating the enormous length of DNA in the small nuclear space.

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Each core histone in the nucleosome contains a globular domain and a highly dynamic N-terminal tail rich in basic residues, which protrudes out from the nucleosome. In addition, H2A also possesses a protruding C-terminal domain. Recent findings have shown that these tails do not contribute either to the structure or stability of nucleosomes but play an important role in folding of nucleosomal arrays into higher order chromatin structures [1]. The histone tails are the sites for a number of post-translational modifications like acetylation and ubiquitination of lysine (K) residues, phosphorylation of serines (S) and threonines (T), and methylation of lysines and arginines (R). These modifications can regulate each other and are recognized by specific protein modules [2]. Thus, different combinations of these modifications dictate specific biological readouts, which form the basis of the histone code hypothesis. The packaging of DNA into chromatin affects all DNA-related processes such as replication, transcrip-

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tion, recombination and repair. The cell has developed various mechanisms by which chromatin structure can be manipulated to regulate access to DNA. These include (i) ATP-dependent chromatin remodeling, (ii) incorporation of histone variants into nucleosomes, and (iii) covalent histone modifications [3]. Chromatin remodeling by multisubunit complexes utilizes the energy from ATP hydrolysis to affect histone–DNA interactions. These complexes can slide nucleosomes on the DNA molecule, regulating access to specific sequences. Histone variants possess biophysical properties distinct from those of canonical core histones, and their substitution into nucleosomes can bring about alterations into the higher order chromatin structure. Covalent modifications of histones can alter the charge of specific residues, affecting the histone–histone and histone–DNA interactions, and can act as signals for binding of various protein complexes. For instance, bromodomains present in several transcriptional coactivators associate with specific acetylated lysine residues, while chromodomain-containing proteins bind to methylated lysines [2]. This review discusses such histone modifications, specifically focusing on their involvement in the repair of DNA double-strand breaks. 2. Histone modifications and DNA repair Each day the cell is exposed to a number of agents both extrinsic (chemical agents, UV radiation, ionizing radiation) and intrinsic (reactive oxygen species, endogenous alkylating agents), which cause DNA damage. Breaks in DNA also result from collapsed DNA replication forks or from oxidative destruction of deoxyribose residues. Failure to repair such lesions leads to genomic instability and cancer. Among the different types of damage, DNA double-strand breaks (DSBs) are the most deleterious since they affect both strands of DNA and can lead to loss of genetic material. DSBs are mainly repaired by two pathways: (i) homologous recombination (HR) and (ii) non-homologous end-joining (NHEJ), both of which are highly conserved in eukaryotes. Homologous recombination uses undamaged sister chromatid or homologous chromosome as a template to repair the break, whereas non-homologous end-joining involves direct ligation of two ends of broken DNA. There is an increasing body of evidence about the role of histone modifications in DNA repair [4–6]. Recently it became clear that specific histone modifications provide an essential function by acting as a landing platform for necessary repair/signaling proteins. The modifications not only signal the presence of damage, but particular

patterns of modification are used to signal the damage type and control the recruitment of specific subset of factors. 2.1. Histone phosphorylation and DNA damage response Among the different histone modifications, phosphorylation plays a primary role in DNA damage response by facilitating access of different repair proteins to DNA breaks, as well as signaling to the cell and promoting chromosome stability. All canonical histones and some histone variants undergo phosphorylation at serine and threonine residues in vivo [1]. A specific histone H2A variant, H2AX, is evenly distributed throughout the genome and constitutes around 10% of total mammalian H2A. In contrast, 90% of budding yeast H2A is of the H2AX class. H2AX has a unique conserved SQE motif in the C-terminal tail, which is a consensus sequence for phosphatidylinositol 3-kinase-related kinases (PIKKs) [5]. In higher eukaryotes, there are three PIKKs, ataxia-telangiectasia mutated (ATM), ATM- and Rad3-related (ATR) and DNA-dependent protein kinase (DNA-PK), while yeast contains two, Tel1 and Mec1. All of these phosphorylate the serine residue in the SQE motif (serine 139 in mammals and serine 129 in yeast) [7–9]. In mammals, Xenopus laevis, Drosophila melanogaster, and Saccharomyces cerevisiae one of the first events in response to DNA damage is the phosphorylation of the serine residue in the SQE motif of H2AX. This modification, referred to as ␥-H2AX, has been found in the vicinity of the DNA DSBs by chromatin immunoprecipitation assays (ChIP), and extends up to megabase domains in mammals and more than 50 kb in yeast [10–12], with highest levels at 3–5 kb from the break site [12]. Intriguingly, the H2AX phosphorylation levels detected by ChIP adjacent to the DNA break are not high [11,12]. This could be due to histone loss or, more likely, epitope masking due to the high density of repair factors (some of which directly bind to the mark, see below) and/or the local presence of additional covalent modifications [12–16]. In fact, there is evidence that yeast H2A is also phosphorylated at the nearby serine 122 (threonine 119 in mammals) in response to DNA damage [17,18]. The important role of ␥-H2AX in DNA repair has been demonstrated through knockout studies. H2AX−/− cells or cells with a H2AX–S139A mutation show sensitivity to ionizing radiation (IR) or camptothecin and genomic instability [19,20]. However, mutation of H2AX phosphorylation site shows only a moderate sensitivity to DNA damage in compar-

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ison to alterations of DNA damage checkpoint proteins [8,21,22]. This suggests that ␥-H2AX contributes to but is not essential for DNA repair and genome maintenance. Celeste et al. demonstrated that ␥-H2AX was not required for the initial formation of DNA repair foci in vivo but was essential to their stability and structure, arguing for a role in retaining DNA repair factors near the DNA damage site rather than recruiting [23]. Other studies in mammals show that H2AX is involved in both NHEJ and HR, regulates homologous recombination between sister chromatids and suppresses single-strand annealing, and prevents DNA breaks from progressing to chromosome breaks and translocations [24,25]. DSBs in DNA lead to an accumulation of a number of DNA damage response proteins at the site of damage. The rapid occurrence of H2AX phosphorylation following damage suggests that this modification may act as a signal for the binding of several proteins involved in repair and signaling, and there are a number of reports in favor of this view. For instance, DNA end-binding Mre11–Rad50–Nbs1 (MRN) complex is required for recognition, signaling and repair of DSBs and contains a subunit, Nbs1, that specifically binds ␥-H2AX [22]. MDC1 is another early ␥-H2AX binding protein that assists Nbs1 during the cellular response to DNA damage [16]. 53BP1 and its yeast homologs ScRad9 and SpCrb2 are also critical during damage response as they act as adaptor proteins between the sensing kinase (e.g. ATM) and the effector kinases (Chk1) [26]. These proteins were all shown to interact with ␥-H2AX and depend on it for checkpoint function [21,27–29]. However the initial recruitment of 53BP1 to the damaged sites is normal in H2AX−/− cells and only the subsequent accumulation is impaired [23]. There is the possibility of redundant/cooperative recruitment mechanisms, and recent reports are in favor of this view (see below). On the other hand, the MRN complex, which functions both in homologous and non-homologous recombination, is recruited to damaged sites but is unable to form sustained foci in the absence of ␥-H2AX [19]. This suggests that the MRN complex localizes to breaks initially in a manner independent of ␥-H2AX, but the subsequent retention at the damaged site requires ␥-H2AX–Nbs1 interaction [22]. These findings suggest that H2AX phosphorylation acts to stabilize the association of different repair proteins at the damaged site and serves the function of an assembly platform rather than a recruitment signal. Another important function of H2AX phosphorylation for DNA repair is the recruitment of cohesins to DSBs [30,31]. Cohesin complexes form an important physical link between sister chromatids in S phase

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which is maintained until mitosis. Recent studies in yeast have demonstrated the recruitment of cohesin complexes to endonuclease-induced DSBs. It is detectable within 45 min of the break formation, extends up to 50 kb, and is dependent on ␥-H2AX phosphorylation [30,31]. Taken together with their known role in sister chromatid attachment, these results could explain the formation of such a large domain of ␥-H2AX-containing chromatin around DSBs. By promoting association of cohesins, ␥-H2AX can prevent loss of entire chromosome regions due to the DSBs and keep the DNA ends in close proximity for efficient repair. This would explain also the abovementioned role of ␥-H2AX in preventing DNA breaks from progressing to chromosome breaks and translocations [24,25]. Beside H2AX, other histone phosphorylation events take place during DNA damage response. Yeast H2A is also phosphorylated at serine 122 (threonine 119 in higher eukaryotes) upon DNA damage and this residue is essential for cell survival in presence of DNA damaging agents [17,18]. Phosphorylated Ser122 may provide a binding surface to repair/signaling proteins like ␥H2AX. On the other hand it is surprising to note that mutation of the Ser122 residue shows stronger DNA repair defect phenotype than the Ser129 mutant (␥H2AX), arguing for an even more important role in DNA damage response [17]. Histone H2B also undergoes phosphorylation at serine 14 in response to DNA damage and this event is independent of ␥-H2AX. However, the radiation induced foci formation is dependent on ␥-H2AX [32]. One possibility is that ␥-H2AX may be required to retain the H2B Ser14 kinase after its initial recruitment to DSBs. An alternative view is that ␥-H2AX has a direct effect on chromatin structure around the DSB. Greater condensation of chromatin around the break could be induced by H2B phospho-Ser14 in a manner similar to nuclear condensation during apoptosis, and ␥-H2AX may lead to accumulation of the H2B kinase [32]. This phosphorylation is mediated by sterile 20 kinase (Mst1) and is well known for its role during apoptosis [33]. In budding yeast the sterile 20 kinase (Ste20) phosphorylates H2B serine 10 residue instead and this is important in a hydrogen peroxide-induced cell death pathway [34]. Recently, a new phosphorylation event in the Nterminal tail of histone H4 has been found to occur in response to DNA damage. This phosphorylation at serine 1 is performed by casein kinase 2 in response to DSBs [35,36]. It was also shown to inhibit histone H4 acetylation by the NuA4 acetyltransferase complex and to be induced during gene transcription [36]. Chromatin immunoprecipitation experiments have shown that this

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phosphorylation is abundant near endonuclease-induced double-strand breaks [35,36]. Studies have also shown that histone H1 modifications play a role in DNA repair. H1, which seals the nucleosome at the DNA entry and exit points, is important for the stability of higher order chromatin structure. Since H1 stabilizes chromatin structure, it is known to have an inhibitory activity on transcription. In yeast, linker histone Hho1 inhibits DNA repair by homologous recombination (HR), as well as recombination-mediated telomere maintenance [37]. Incorporation of linker histone into nucleosomes inhibits DNA end-joining by DNA ligase IV/XRCC4 (LX) in vitro. Since LX is important for NHEJ, the linker histone may inhibit NHEJ-mediated DNA repair. However this inhibition is compromised by the phosphorylation of histone H1 by DNA-PK, which reduces its affinity for DNA and decreases its capacity to inhibit end-joining, and thus facilitates NHEJ-mediated repair [38]. 2.2. Cross-talk between histone modifications and chromatin remodelers Since access to DNA damage sites is important for efficient repair it was expected that chromatin modifiers and ATP-dependent remodelers would be important and locally recruited to facilitate repair. On the other hand it was somewhat surprising to find that ␥-H2AX was again important in that process. The NuA4 histone acetyltransferase complex, the Ino80 and Swr1 Swi2family ATP-dependent remodelers were all found to be recruited to DSBs and to directly interact with ␥-H2AX [11,39,40]. Each of these multisubunit complexes has been shown to specifically interact with ␥-H2AX peptides in vitro [11]. NuA4 is a histone acetyltransferase which acetylates histones H4 and H2A [41]. NuA4 plays an important role in DNA repair since mutation of its key subunits or the four acetylatable lysine residues of H4 causes hypersensitivity to DSB-inducing agents [11,42]. The NuA4 complex interacts directly with ␥H2AX through its Arp4 subunit which is also present in Ino80 and Swr1 chromatin remodelers [11]. The Ino80 and Swr1 complexes, which belong to SWI/SNF family of chromatin remodelers, also have a role in DNA repair. ChIP studies have shown that NuA4 and Ino80 complexes are recruited to endonuclease-induced DSBs at least in part through association with ␥-H2AX [11,39,40]. Prolonged recruitment of Ino80 to the DSBs is greatly impaired in yeast strains lacking Tel1 and Mec1 kinases, in strains containing mutated H2A S129, as well as in strains lacking the Nhp10 and Ies3, two subunits of Ino80 [39,40]. This indicates that Ino80 interacts

with ␥-H2AX through Nhp10 and/or Ies3, in contrast to Arp4’s role in NuA4 interaction with ␥-H2AX [11]. It is possible that Arp4 and Nhp10 subunits cooperate for Ino80 interaction, Nhp10 for binding to the octamer and Arp4 for retention at the break [43]. Ino80 recruitment to DSBs also seems dependent on the MRN complex, and cells lacking Ino80 show a defect in DNA end resection, nucleosome remodeling and recruitment of repair factors [40,44]. Furthermore, several reports show that Ino80 is also involved in homologous recombination [45,46]. The Ino80 complex is highly conserved from yeast to human as a single multisubunit assembly [47]. Interestingly, Ino80 and Swr1 complexes contains Rvb1 and Rvb2 helicase subunits, which are similar to the bacterial ATPase RuvB required for movement of the Holiday junction during recombination [48]. Another remodeling complex, Swr1, which is related to Ino80 and shares several subunits with NuA4 including Arp4, also has a role in DNA repair. In S. cerevisiae, Swr1 is responsible for the incorporation of the histone H2AZ variant into chromatin (10% of total H2A) [49–51]. Like Ino80, yeast strains lacking the Swr1 complex show increased sensitivity to DNA damaging agents [49]. Additionally, Swr1 physically and functionally interacts with ␥-H2AX [11,39]. In higher eukaryotes, the NuA4 complex is also called Tip60. Interestingly, this multisubunit assembly corresponds to an almost exact physical merge of the yeast NuA4 and Swr1 complexes and represents a unique complex in the sense that it encompasses acetyltransferase, ATPdependent chromatin remodeling and helicase activities (Fig. 1). It is involved in transcription, cell cycle control, DNA damage signaling and repair, and apoptosis [41,52–54]. The Tip60/NuA4 complex is also recruited to DSBs in higher eukaryotes and is required for efficient recruitment of DNA repair factors and for DNA damage signaling [52,54]. A key finding demonstrating the role of Tip60/NuA4 in DNA DSB repair was obtained in Drosophila [55]. In that organism histone variant H2Av cumulates the roles of H2AX and H2AZ in other systems. Kusch et al. found that the purified Tip60/NuA4 complex catalyzes the exchange of nucleosomal phospho-H2Av (␥-H2AX) with an unmodified H2Av in vitro and in vivo during the response to DNA damage [55]. The authors further show that phosphoH2Av is acetylated by Tip60 before the exchange reaction and that phospho-H2Av accumulates after DNA damage in cells lacking Tip60. Based on these findings it is suggested that, in budding yeast, acetylation of ␥H2AX-containing chromatin by NuA4 near DSBs may create a mark recognized by a bromodomain-containing subunit (Bdf1) of Swr1, thus stimulating replacement of

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Fig. 1. Conservation of NuA4 and Swr1 complexes from yeast to human. Homologous protein complexes are depicted with colors and arrangements pointing to functionally equivalent subunits. Protein domains are also indicated (BrD; bromodomain; CHD: chromodomain; SWI2/SNF2: Swi2family ATPase domain). It is thought that only a subset of yeast Swr1 complexes contain the bromodomain-containing Bdf1 subunit. The homologous human protein is found in Tip60/NuA4 but not the SRCAP complex. Yeast Swr1 has two homologs in human, p400/Domino and SRCAP. The Ino80 complex is also highly conserved from yeast to human and seems to be kept as a single multisubunit assembly (not shown).

␥-H2A(X) with H2AZ (see Figs. 1 and 2). It is important to point out that NuA4-dependent histone acetylation can affect H2AZ incorporation in yeast chromatin, that H2AZ itself can be acetylated by NuA4 and that NuA4 and Swr1 have been shown to cooperate in the regulation of chromosome stability [56–61]. This intricate chromatin cross-talk is certain to have a critical role in how the cell responds to the DNA damage at the level of DNA repair and/or cell cycle control. 2.3. Step-wise chromatin modifications during repair of DNA DSBs Taken as a whole, results in the literature help to build a simplified model for the different stages of

chromatin modifications/remodeling during the repair of DNA DSBs (Fig. 2). Phosphorylation of H2AX by damage sensing-kinase of the ATM family is one of the first events to occur after damage. Even though NuA4, Ino80 and Swr1 complexes all interact with ␥-H2AX, it was shown that the NuA4 complex was the first to appear at the break [11]. Furthermore, the same report showed that a mutation that cripples NuA4 HAT activity also affects recruitment of Ino80/Swr1 complexes. This is arguing that chromatin needs to be acetylated first before these remodelers can be efficiently recruited to the break. Once Ino80 is present, nucleosomes near the break can be remodeled and resection can occur [44]. Again, recruitment per se cannot be solely through ␥-H2AX and other factors are likely involved (e.g. MRN). We speculate

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Fig. 2. Chromatin dynamics during the repair of DNA double-strand breaks. Simplified model showing the different steps of chromatin modifications/remodeling during the process of DSB repair, mostly based on work in yeast. While Ino80 and Swr1 are depicted as recruited at the same time, it is thought that Swr1 acts after Ino80 to remove ␥-H2AX after repair is done and replace it with H2A(Z) (see text).

that Swr1 could act subsequently to the repair in order to remove ␥-H2AX from the surrounding chromatin, as part of a signal that repair has been completed. This is supported by a recent publication by the Peterson group that indicates an interplay of Ino80 and Swr1 complexes during cell cycle checkpoint adaptation in response to DNA damage [62]. When a DSB cannot be repaired cells can adapt and bypass the cell cycle checkpoint. This study demonstrates that Ino80 and Swr1 function antagonistically at chromatin surrounding a DSB, Ino80 maintaining high levels of ␥-H2AX while Swr1 trying to replace it with H2AZ. When Ino80 is mutated, ␥-H2AX signal drops and H2AZ accumulates at the break [62]. As just mentioned above, when the damage is repaired, ␥-H2AX must be removed from the chromatin in order to prevent sequestration of repair factors and release from the cell cycle checkpoint. We have already discussed the role of the Tip60/NuA4/Swr1 complexes in the removal of ␥-H2AX from chromatin (see above). A simpler mechanism would suggest the presence of phosphatases in the cell that can dephosphorylate ␥H2AX. Two recent reports identified such phosphatases. A report by Keogh et al. describes a three-protein com-

plex (HTP-C) in yeast containing the Pph3 phosphatase and regulating the phosphorylation status of H2AX in vivo [63]. The authors further show that Pph3 targets ␥-H2AX but only after its displacement from chromatin and that this displacement is independent of HTP-C [63]. This again favors the view that after repair ␥-H2AX is first acetylated by NuA4 and then removed from chromatin (by NuA4/Tip60 in higher eukaryotes; by Swr1 in yeast; see Figs. 1 and 2). Another report shows that, in mammals, protein phosphatase 2A (PP2A) dephosphorylates ␥-H2AX in vitro and that the recruitment of PP2A to DSB sites is ␥-H2AX-dependent [64]. Inhibition of PP2A by siRNA or by inhibitors leads to the accumulation of ␥-H2AX and inefficient repair of DNA damage. After repair has been performed, chromatin has to be restored to its initial state. Removal of ␥-H2AX is part of this process. Histone tails that have been acetylated by NuA4 also need to be deacetylated. Accordingly, NuA4-dependent acetylation near DSBs is transient and decreases with time, along with ␥-H2AX [11,65]. Local binding and function of the Sin3/Rpd3 histone deacetylase complex has also been documented [66,67]. The

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Sin3/Rpd3 complex has been shown to interact with casein kinase 2, and histone deacetylation correlates temporally with phosphorylation of histone H4 serine 1 near DNA damage sites. Such late phosphorylation of H4 could insure that NuA4 cannot reacetylate chromatin once the break has been repaired and histone deacetylated by Sin3/Rpd3 (see Fig. 2). 2.4. Cross-talk and combinatory histone modifications in DNA repair Methylation is another important post-translational modification that plays an important role in gene regulation and chromatin structure. It is carried out by histone methyltransferases (HMTs) which covalently modify the lysine and arginine residues by transferring methyl groups from S-adenosyl-l-methionine (SAMe) to the ␧amino group on histones [68]. Methylation of H3 and H4 plays an important role in DNA repair through the association of DNA damage response proteins. Disruptor of telomeric silencing-1 (Dot1), which methylates nucleosomal histone H3 at lysine 79, has a role in DNA damage induced cell cycle checkpoints. H3 K79 methylation is required for recruitment of mammalian 53BP1 and its budding yeast homolog Rad9 to DNA damage sites in vivo [69]. 53BP1/ScRad9 binds H3 methylated at K79 through its Tudor domain. Dot1 deletion or mutations in the Tudor domain of 53BP1 abolish the recruitment of 53BP1 to DSBs [69,70]. In yeast, H3 K79 methylation plays an important role in the activation of the G1 and intra S-phase DNA damage checkpoints, but not the Rad9-dependent G2 /M checkpoint. Deletion of Dot1 or mutations of H3 K79 result in a defect in activation of the central checkpoint kinase Rad53 [70,71]. Since H3 K79 methylation levels do not change in response to DNA damage, it was suggested that DSBs probably affect the accessibility of 53BP1 to methylated K79 of histone H3 through changes in higher order chromatin structure. Recent findings suggest that there is a requirement of two distinct histone modifications for 53BP1/Rad9 binding to DNA damage sites. 53PB1/Rad9 interacts with both ␥-H2AX and H3 MeK79 in vitro and both modifications are required for its recruitment to DSBs in vivo [28,29]. This cooperative/dual binding of two different histone modifications could explain why H3 methylation is not regulated in response to DNA damage since ␥-H2AX would provide such function. Trimethylation of H3 lysine 79 by Dot1 depends on previous transient ubiquitination of H2B at lysine 123 [72,73]. Accordingly, yeast Rad9 recruitment/signaling also requires ubiquitination of H2B by the Bre1/Rad6 complex [70,71]. Surprisingly, in fission yeast, it is methylation of histone

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H4 at lysine 20 that is important for the recruitment of the 53BP1/Rad9-related adaptor protein Crb2 [74]. Like H3 K79 methylation, the levels of H4 K20 methylation do not change in response to DNA damage and Crb2 has been shown to require ␥-H2AX as well [21]. Finally, it was recently shown that Dot1/Rad9 and NuA4 have opposite functions during G1 DNA damage checkpoint [28]. Along the same lines, 53BP1 was found to interact with histone deacetylase-4, an interaction required for DNA damage response [75]. 2.5. Other histone modifications involved in genome integrity While the model presented in Fig. 2 depicts a picture of the different chromatin modifiers/remodelers implicated in DSB repair, it is far from being complete and is clearly simplified. Reports in the literature have also described the recruitment of yeast Gcn5 and Hat1 histone acetyltransferases to an endonuclease-induced DNA break in vivo during recombinational repair [66,76]. It was initially thought that these could be involved in the chromatin assembly step after repair [77,78]. Histone H4 lysine 16-specific deacetylase Sir2 has also been detected near DSBs in vivo and this might be related to the recently described role of hMof, an acetyltransferase with the same specificity, in ATM function during DNA damage response [66,79–81]. Acetylation of lysine 56 on newly synthesized histone H3 is an abundant modification that plays an important role in DNA repair. Mutation of K56 leaves cells sensitive to genotoxic agents. This modification disappears in the G2 phase, but upon DNA damage it persists in a manner dependent on DNA damage checkpoint proteins [82]. It was also independently shown to regulate recruitment of the SWI/SNF remodeler in transcription [83]. The SWI/SNF complex and the related RSC complex have also been shown to be recruited to DSBs during repair [84,85]. Recruitment of mammalian SWI/SNF to DSB regions has been shown to be in part through an interaction with ␥-H2AX and was shown to be required for stimulation of ␥-H2AX signal upon DNA damage [86]. Interestingly, mammalian SWI/SNF shares a subunit with the Tip60/NuA4 complex, Baf53a, a protein homologous to the ␥-H2AX-binding Arp4 subunit of NuA4/Ino80/Swr1 in yeast (Fig. 1). 3. Future directions As mentioned above, it is tempting to draw a simplified model of chromatin dynamics during the repair of DNA breaks in eukaryotes. Nevertheless, things are

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not as clear and straightforward as it seems. The model of interplay between Ino80, Swr1, NuA4, H2AZ and H2AX is still very speculative. Experiments need to be done to support or modify this model in both yeast and mammalian systems. The reported implication of other chromatin modifiers and remodelers also has to be investigated further in order to include them in the model. Most likely, different players and chromatin modifications will be used depending on the repair pathway used by the cell. Acknowledgements We are grateful to the editors for their understanding during preparation of this review. We also thank Nikita Avvakumov for critical reading of the manuscript and colleagues for stimulating discussions. Work in our lab is supported by grants from the Canadian Institutes of Health Research (CIHR). JC is a CIHR Investigator. References [1] C.L. Peterson, M.A. Laniel, Histones and histone modifications, Curr. Biol. 14 (2004) R546–R551. [2] K.L. Yap, M.M. Zhou, Structure and function of protein modules in chromatin biology, Results Probl. Cell Differ. 41 (2006) 1– 23. [3] A. Vaquero, A. Loyola, D. Reinberg, The constantly changing face of chromatin, Sci. Aging Knowl. Environ. 2003 (2003) RE4. [4] C.L. Peterson, J. Cˆot´e, Cellular machineries for chromosomal DNA repair, Genes Dev. 18 (2004) 602–616. [5] C. Thiriet, J.J. Hayes, Chromatin in need of a fix: phosphorylation of H2AX connects chromatin to DNA repair, Mol. Cell 18 (2005) 617–622. [6] H. van Attikum, S.M. Gasser, The histone code at DNA breaks: a guide to repair? Nat. Rev. Mol. Cell Biol. 6 (2005) 757–765. [7] T. Stiff, M. O’Driscoll, N. Rief, K. Iwabuchi, M. Lobrich, P.A. Jeggo, ATM and DNA-PK function redundantly to phosphorylate H2AX after exposure to ionizing radiation, Cancer Res. 64 (2004) 2390–2396. [8] J.A. Downs, N.F. Lowndes, S.P. Jackson, A role for Saccharomyces cerevisiae histone H2A in DNA repair, Nature 408 (2000) 1001–1004. [9] E.P. Rogakou, D.R. Pilch, A.H. Orr, V.S. Ivanova, W.M. Bonner, DNA double-stranded breaks induce histone H2AX phosphorylation on serine 139, J. Biol. Chem. 273 (1998) 5858– 5868. [10] E.P. Rogakou, C. Boon, C. Redon, W.M. Bonner, Megabase chromatin domains involved in DNA double-strand breaks in vivo, J. Cell Biol. 146 (1999) 905–916. [11] J.A. Downs, S. Allard, O. Jobin-Robitaille, A. Javaheri, A. Auger, N. Bouchard, S.J. Kron, S.P. Jackson, J. Cˆot´e, Binding of chromatin-modifying activities to phosphorylated histone H2A at DNA damage sites, Mol. Cell 16 (2004) 979–990. [12] R. Shroff, A. Arbel-Eden, D. Pilch, G. Ira, W.M. Bonner, J.H. Petrini, J.E. Haber, M. Lichten, Distribution and dynamics of chromatin modification induced by a defined DNA double-strand break, Curr. Biol. 14 (2004) 1703–1711.

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