Leukemia (2007) 21, 195–200 & 2007 Nature Publishing Group All rights reserved 0887-6924/07 $30.00 www.nature.com/leu REVIEW

Chromatin modifications and DNA double-strand breaks: the current state of play

TC Karagiannis1,2 and A El-Osta3

1Molecular Radiation Biology, Trescowthick Research Laboratories, Peter MacCallum Cancer Centre, East Melbourne, Victoria, Australia; 2Department of Pathology, The University of Melbourne, Parkville, Melbourne, Victoria, Australia and 3Epigenetics in Human Health and Disease, Baker Medical Research Institute, The Alfred Medical Research and Education Precinct, Prahran, Victoria, Australia

The packaging and compaction of DNA into is mutated (ATM) and ataxia telangiectasia-related (also known as important for all DNA-metabolism processes such as transcrip- Rad3-related, ATR), which are members of the phosphoinositide tion, replication and repair. The involvement of chromatin 4,5 modifications in transcriptional regulation is relatively well 3-kinase (PI(3)K) superfamily. They catalyse the phosphoryla- characterized, and the distinct patterns of chromatin transitions tion of numerous downstream substrates that are involved in 4,5 that guide the process are thought to be the result of a code on cell-cycle regulation, DNA repair and apoptosis. the proteins (histone code). In contrast to , In mammalian cells, DSBs are repaired by one of two distinct the intricate link between chromatin and responses to DNA and complementary pathways – damage has been given attention only recently. It is now (HR) and non-homologous end-joining (NHEJ).6,7 Briefly, NHEJ emerging that specific ATP-dependent involves processing of the broken DNA terminii to make them complexes (including the Ino80, Swi/Snf and RSC remodelers) 3 and certain constitutive ( of 79 of ) compatible, followed by a ligation step. The core components and DNA damage-induced covalent histone modifications of NHEJ are the Ku70/Ku80 heterodimer, which binds the (the most well characterized being the rapid broken DNA ends, DNA-dependent protein kinase catalytic of ) facilitate responses to double-strand breaks. subunit (DNA-PKcs), a PI(3)K member that activates and forms a Indeed, evidence is already accumulating for a DNA repair- complex with the nuclease Artemis, and the XRCC4/DNA ligase specific histone code. In this review, the recent advances in 3 our understanding of the relationship between chromatin IV complex, which ligates the processed DNA terminii. modifications and double-strand break signaling and repair Recently, Cernunnos-XLF, a protein that is defective in a rare is discussed. inherited human syndrome characterized by severe immuno- Leukemia (2007) 21, 195–200. doi:10.1038/sj.leu.2404478; deficiency, developmental delay and hypersensitivity to agents published online 7 December 2006 that cause DSBs, has been found to be implicated in NHEJ.8,9 It Keywords: double-strand break; chromatin modification; histone is thought that Cernunnos-XLF interacts with the XRCC4/DNA ; histone phosphorylation; ; chromatin- 8,9 remodeling complex ligase IV complex. Owing to nucleolytic processing, the NHEJ pathway usually results in the loss of a few nucleotides at each broken end and is typically error-prone. The HR pathway relies on extensive sequence homology between the recombining ends and essentially involves copying Introduction the missing information from an undamaged homologous .1,2 In contrast to NHEJ, it is typically error-free DNA double-strand breaks (DSBs), which can arise from normal and is the pathway that is predominantly used by the simple (e.g. V(D)J and meiotic recombination) and pathological (stalled eucaryotes such as the yeasts Saccharomyces cerevisiae and replication forks and reactive oxygen species) endogenous Schizosaccharomyces pombe to repair DSBs.1,2 Genetic ana- metabolic processes or from exogenous agents (e.g. ionizing lysis of S. cerevisiae indicated that proteins coded by the RAD52 radiation and radiomimetic drugs), are the most severe lesions epistasis group of genes – RAD50, RAD51, RAD52, RAD54, with respect to cell survival and preservation of genomic RAD55, RAD57, RAD59, MRE11 and XRS2 – are important in integrity.1,2 Therefore, cells have evolved complex mechanisms HR.1,2,10 Sequentially, the HR repair process that involves the that include cell cycle arrest, activation and increased expres- generation of a single-stranded region of DNA by a process sion of various genes including those associated with DNA which involves nucleolytic resection by the Mre11–Rad50–Xrs2 repair and in certain cases induction of the apoptotic pathway, (MRE11-RAD50-NBS1(nibrin) in mammalian cells) complex, to respond to DSBs.1–3 The overall cellular response to DSBs has strand invasion mediated by the replication protein A (RPA)- been considered as a classical signal-transduction cascade coated Rad51 nucleoprotein filament, formation of a Holliday where the lesions (signals) are detected by sensor proteins that junction, DNA synthesis by polymerases, branch migration and activate protein kinase (transduction) cascades, which result in resolution.7 Studies have identified that mammalian homologs amplification and diversification of the signal through a series of exist for all known gene products involved in yeast HR, downstream effector molecules (response).1,2 The central DSB indicating that the basic HR pathway is conserved in higher signaling kinases in mammalian cells are ataxia telangiectasia eucaryotes.11–13 Common sense dictates that the molecular responses to DBSs Correspondence: Dr A El-Osta, in Human Health and must occur within the constraints of chromatin. In eucaryotic Disease Laboratory, Baker Medical Research Institute, The Alfred cells, the primary repeating unit of chromatin is the , Medical Research and Education Precinct (AMREP), Second Floor, a nucleoprotein complex that consists of DNA wrapped around Commercial Road, Prahran, Victoria 3181, Australia. an octameric structure, which is constructed by two copies of E-mail: [email protected] 14,15 Received 10 October 2006; accepted 18 October 2006; published each of the four core , H2A, H2B, H3 and H4. online 7 December 2006 Strings of thousands of are organized onto a Chromatin modifications and DSB response TC Karagiannis and A El-Osta 196 continuous DNA helix (typically separated by 10–60 base pairs catalytically active monomers (a radiation dose of 0.5 Gy, of linker DNA).16 Nucleosomes are further compacted into which induces about 18 DSBs, resulted in the phosphorylation 30 nm chromatin fibers via linker histones (H1) and looped of approximately 50% of ATM molecules within 5 min) may be domains and these are assembled into so-called higher order due to changes in chromatin structure and/or histone modifica- chromatin structures that are not well defined.16 Although tions, which are detected by ATM.21 This model is supported by nucleosomes have been traditionally known simply as stable the findings from a very recent report in which changes in DNA packaging units, it is now evident that they are dynamic chromatin structure following DSB induction were monitored.22 structures that can be altered by at least three different The findings indicated that chromatin undergoes an energy- processes. The composition of nucleosomes may be modified dependent local expansion immediately following the induction by the incorporation of histone variants, which are thought to of DSBs.22 It was found that the conformational change in have specialized functions.17 Additionally, the organization of chromatin occurs independently of ATM and g-H2AX (discussed chromatin can be altered by the replacement, repositioning or in below).22 Furthermore, in support of the initial ATM autopho- certain cases the ejection of nucleosomes by ATP-dependent sphorylation hypothesis,21 the data demonstrate that local chromatin remodeling complexes.18 Finally, chromatin struc- activation of ATM occurs with the same spatio-temporal ture can be modified by coordinated post-translational mod- dynamics as the changes in chromatin architecture.22 For a ifications on histone residues such as acetylation of , balanced perspective, it should be noted that other recent phosphorylation, methylation of lysines and arginines, observations may question some aspects of the chromatin polyribosylation and ubiquitylation.19,20 It is not unexpected modification-induced ATM autophosphorylation model. The that numerous studies have begun to demonstrate that specific ATP-dependent chromatin remodeling complexes and certain constitutive and DNA damage-induced covalent histone mod- ifications facilitate the DNA-damage response. The remaining of the article will focus on the relationships between chromatin modifications and DSB signaling and repair that have been identified to date (Figure 1).

Recognition of double-strand breaks

The findings from a landmark study provided evidence for a link between changes in chromatin conformation and the initial detection of DSBs.21 The results from the study indicated that in response to genotoxic stress, inactive homodimeric or multi- meric ATM molecules are modified by autophosphorylation of the serine-1891 residue resulting in activation of the ATM protein kinase domain.21 It was rationalized that the rapid and widespread dissociation of inactive ATM complexes into

Figure 1 Key histone modifications and chromatin remodeling events implicated in the DSB response pathway in budding yeast. The Mre11–Rad50–Xrs2 (MRE11–RAD50–NBS1 in mammals) complex rapidly binds to the broken DNA ends and is thought to recruit Tel1 (ATM) and Mec1 (ATR), which phosphorylate the serine 129 residue of histone H2A. Phosphorylated H2A spreads within an B50-kb region around the DSB and is thought to be required for the concentration and stabilization of repair proteins. The histone variant H2AX is phosphorylated on serine 139 in mammals and H2Av is phosphory- lated on serine 137 in Drosophila melanogaster. Following the H2A phosphorylation event, the NuA4 histone acetyltransferase complex (homologue of the human TIP60 complex) binds directly to phospho- H2A and mediates acetylation of the first four lysine residues of . Presumably, histone acetylation results in a more relaxed chromatin conformation allowing efficient loading of repair factors. The topology of chromatin is also thought to be altered by the recruitment of chromatin remodeling complexes. The chromatin remodeling activity of Ino80 and Swr1 is required for access to end- processing enzymes and repair factors and the Swi/Snf and RSC complexes have been implicated in facilitating the loading of cohesins. The key DNA damage signaling protein Rad9 (which shares homologous regions with the human 53BP1 protein) binds to histone H3, which is methylated on lysine 79. Methylation of lysine 79 on histone H3 is a constitutive modification and it is proposed that DSBs expose the methylated lysines required for recruitment of the signaling proteins. Following DSB repair, phospho-H2A is eliminated by dephosphorylation, which is mediated by the histone phosphatase H2A complex (HPT-C) containing the phosphoserine phosphatase Pph3 (protein phosphatase H2A in mammals).

Leukemia Chromatin modifications and DSB response TC Karagiannis and A El-Osta 197 MRE11–RAD50-NBS1 (MRN) complex has traditionally been of cohesin complex in response to DSBs.40,47 Cohesins facilitate assigned a role downstream of ATM in the DSB response the repair of DSBs by maintaining the two sister chromatids in pathway. Paradoxically, new evidence indicates that the MRN close proximity for post-replicative repair by HR.40,47 Studies in complex is required for the activation of the ATM protein.23–25 S. cerevisiae indicate that cohesin complexes can be loaded Specifically, it has been shown that Nbs1 binds to ATM (the de novo in response to an HO-induced DSB, in a phospho-H2A- C-terminal 20 amino acids – FXF/Y domain – of Nbs1 have been dependent manner.40,47 Recent findings indicate that depho- shown to bind to the N-terminal HEAT repeats of ATM) and that sphorylation of g-H2AX following DNA repair is necessary for this interaction is required for activation and recruitment of ATM efficient recovery from the DNA damage checkpoint.48,49 to sites of DNA damage.23,26 Therefore, further research is Although the details of g-H2AX elimination and its role in the required to clarify the pathways involved in the initial completion of DSB repair are not yet fully understood, the recognition of DSBs. available data indicate that a three-protein complex, HTP-C (histone phosphatase H2A complex) containing the phospho- serine-specific phosphatase Pph3 and protein phosphatase 2A Recruitment and accumulation of repair factors regulate the phosphorylation status of g-H2AX in S. cerevisiae and mammalian cells, respectively.48,49 It has been speculated Phosphorylation of histone H2A is a relatively that g-H2AX may be dephosphorylated in yeast and mammalian well-characterized response to double-strand breaks cells following its displacement from chromatin, by a mechan- The first histone modification that was discovered to be ism analogous to the TIP60-mediated exchange of acetylated associated specifically with DSBs is phosphorylation of the and phosphorylated H2Av with unmodified H2Av in chromatin histone variant H2AX, which represents 10–15% of nuclear D. melanogaster.48,49 However, further investigation is required H2A in mammals.27,28 Phosphorylation of the serine 139 to define the precise mechanism. residue in the carboxy-terminal SQ motif of H2AX, forming g-H2AX, occurs rapidly following the induction of DSBs and spreads over large chromatin domains (megabase-sized regions) Chromatin remodeling complexes are involved in surrounding the DNA lesions.27 Within minutes g-H2AX forms double-strand break repair discrete nuclear foci, containing DNA repair factors such as the With respect to DSB-related chromatin modifications, it is MRN complex, 53BP1, BRCA129 and MDC1,30–32 which are important to note that phosphorylated H2A has been shown to easily detectable by immunofluorescence microscopy.33–35 recruit chromatin remodelers to the sites of DNA damage.45,50 Parenthetically, the finding that g-H2AX foci act as sensitive Briefly, reports demonstrate that the Ino80 chromatin remodel- markers of DSBs has added a valuable experimental tool for ing complex functions in DSB repair in yeast.51–53 The findings the investigation of DSB induction and repair at relevant levels indicate that phosphorylation of H2A is required for recruitment of DNA damage. The H2AXS139 phosphorylation event is of the remodeler.51,52 Indeed, it was shown that the Ino80 dependent on the action of members of the PI(3)K family complex binds directly to H2A phosphorylated on serine 139.51 including ATM, ATR and DNA-PKcs.36–38 Findings indicate Separate studies suggest that the Ino80 subunits Arp453 and that ATM is the major kinase responsible for phosphorylating Nhp1051 are required for this interaction. Given that Swr1 has H2AX in response to DSBs,37 whereas ATR is also required for subunits such Arp4 and Rvb1 (which are recruited to HO- UV-induced damage and DNA damage occurring at stalled induced DSBs), in common with InoO80,53–55 this chromatin replication forks.38 remodeling complex has also been implicated in DSB Although both S. cerevisiae and S. pombe lack a separate repair.45,50 Notably, yeast cells lacking either a functional H2AX variant, an SQ motif analogous to the one in mammalian Ino80 or Swr1 complex are hypersensitive to DSB-inducing H2AX is present in histone H2A.35,39 The serine 129 residue of genotoxic agents.55–57 The finding that yeast strains lacking the SQ motif in H2A is phosphorylated by the ATM/ATR Ino80 convert double-stranded DNA into single-stranded DNA homologs Tel1 and Mec1 in budding yeast in response to DNA at DSBs ends less efficiently suggests that Ino80 chromatin damage forming phospho-H2A, which spreads within a B50-kb remodeling activity is required for allowing access to end- region around the site of the DSB.40–42 Furthermore, an H2AZ- processing enzymes such as the MRX complex.52 In addition, histone-like variant, H2Av, in Drosphila melanogaster becomes the Swi/Snf and RSC chromatin remodeling complexes have phosphorylated on its C-terminal tail serine 137 residue in been implicated in the DSB response pathway.58,59 The findings response to DNA damage.43 It has been shown that in response that subunits of the RSC complex interact with Mre11 and Ku80 to DNA damage, TIP60, which has both acetylation and ATPase suggest a role in both the NHEJ and HR repair pathways.58 activities, binds to and acetylates phosphorylated H2Av on the Interestingly, a recent study has indicated that the Swi/Snf and lysine 5 residue, and mediates the exchange of the modified RSC complexes have distinct roles in HR.59 Again, it has been histone with unmodified H2Av in chromatin.44 Importantly, the shown that mutations in the subunits of the Swi/Snf and RSC H2Av histone variant also contains the conserved SQ motif remodeling complexes result in hypersensitivity to agents that found in mammalian H2AX and yeast H2A. Although the induce DSBs.58–61 Overall, the role of chromatin remodelers in histone variants differ between species, the conserved feature is transcription (essentially their function in altering the topology the position of the SQ motif relative to the stop codon.39,45 of chromatin and increasing DNA accessibility) has predomi- Phosphorylation of H2AX is not essential for the initial nantly been used as a basis to speculate on the function of these recruitment of DSB response factors and checkpoint signaling; complexes in the DSB response pathway. Although intuitively however it is becoming apparent that the phosphorylation event this seems correct, further research is required to confidently is required for effective repair of DSBs in multiple ways.33,34,39 It assign specific roles for these chromatin- remodeling complexes has been proposed that H2AX phosphorylation is required for in the context of DSB repair. the concentration and stabilization of DNA repair proteins to The Rad54 protein is a chromatin remodeling enzyme that has damaged chromatin.33 Numerous studies implicate g-H2AX in specific functions in the HR repair pathway.62–66 Rad54 both the NHEJ and HR repair pathways.41,46 An example of a contains a DNA-dependent ATPase domain of the SWI2/SNF2 direct link of phosphorylated H2A and repair is the recruitment family and has been shown to possess ATP-dependent

Leukemia Chromatin modifications and DSB response TC Karagiannis and A El-Osta 198 chromatin remodeling activity both in vitro63–66 and in vivo.62 ionizing radiation-induced 53BP1 foci.72 Interestingly, it was Although the physical interaction of Rad54 with Rad51–DNA found that the levels of methylated lysine 79 of histone H3 do not nucleoprotein filament is well known, the precise role of Rad54 change following the induction of DNA damage. Therefore, it is in HR is controversial. A recent study indicates that Rad54 proposed that changes in chromatin conformation following DSB mediates Rad51 binding to single-stranded DNA nucleoprotein induction expose the methylated lysine residues resulting in the filaments by a process that does not require ATP hydrolysis.62 recruitment of 53BP1 to the lesions.72 Furthermore, this particular study identifies a role for Rad54 in The tandem Tudor domains in 53BP1 comprise residues that the later steps of HR which relies on the chromatin remodeling are conserved in S. cerevisiae Rad9 and S. pombe Crb2 activity of the enzyme.62 Specifically, the recent evidence orthologues.72–74 As with 53BP1, Rad9 also binds to lysine 79 suggests that ATP-dependent chromatin remodeling by Rad54 methylated histone H3 in vitro.72 Furthermore, it was shown in enhances the accessibility of chromatin and facilitates stable S. cerevisiae that deletion of DOT1 or loss of lysine 79 DNA joint formation that is extended by DNA polymerases.62 methylation of histone H3 inhibits Rad9-dependent activation of the key checkpoint kinase Rad53 following DNA damage.75 Notably, the same study identified that ubiquitylation of histone Modification of histone H4 in response to double-strand H2B on lysine 123 by the Rad–Bre1complex is necessary for breaks activation of the Rad53 kinase in response to DNA damage.75 It In addition to phosphorylation of H2AX and to the involvement is known that in S. cerevisiae, methylation of lysine 79 of histone of chromatin remodelers, numerous other histone tail modifica- H3 by Dot1 is dependent upon the ubiquitylation of tions have been implicated in the DSB response pathway. on lysine 123.76 This indicates that the ubiquitylation and A series of phosphorylation and acetylation events in the phosphorylation events may function together to activate the N-terminal tail of histone H4 have been implicated in the checkpoint in response to DNA damage in budding yeast. DNA damage response. In S. cerevisiae, the Arp4 subunit Fission yeast also use histone methylation to recruit the (which, as discussed above, is also part of the ATP-remodeling checkpoint adaptor protein Crb2 to sites of DNA damage. complexes Ino80 and Swr1) of the NuA4 histone acetyltransfer- However, methylation of lysine 20 of histone H4 (instead of ase complex binds directly to phosphorylated H2A and methylation of lysine 79 of histone H3) by the histone mediates acetylation of the first four lysine residues of histone methyltransferase Set9 controls the recruitment of Crb2 in H4.53,54 The findings that mutation of lysine residues in the H4- fission yeast.77 It should be noted that there are alternative terminal tail, deletion of the N-terminus, or mutations in the pathways for recruitment of Crb2 to sites of DNA damage in Esa1 or Yng2 subunits of NuA4 increases the sensitivity of cells S. pombe. For example, phosphorylation of serine 129 on to DSBs are consistent with a role for NuA4 and histone histone H2A by the ATR and ATM homologues, Rad3 and Tel1, acetylation in the DSB response pathway.53,54,67 Recently, it is also known to control Crb2 activation.78 In addition, a histone was shown that the human TIP60 histone acetyltransferase modification-independent pathway for Crb2 recruitment, invol- complex (homologue of the S. cerevisiae NuA4) binds to ving an interaction with Cut5 (an essential BRCT protein with chromatin surrounding DSBs and mediates H4 acetylation and multiple roles in DNA replication and checkpoint signaling), in consequent chromatin relaxation, which is required for efficient response to DNA damage has been recently identified.79 loading of repair factors and DSB repair by HR.68 Furthermore, the differential acetylation of lysine 16 residue of histone H4 in S. cerevisiae has been identified as another important histone Conclusion modification following induction of DSBs.69 Findings indicate that deletion of the Sin3 component of the Sin3–Rpd3 histone In a manner analogous to gene transcription, which requires deacetylase complex results in hypersensitivity to DSBs due to a access to nuclear DNA, repair requires that chromatin structure defect in the NHEJ repair pathway.69 In addition to changes in is modified potentially for the initial recognition of DNA lesions, acetylation status, casein-kinase 2-mediated phosphorylation of as well as for the recruitment of DNA damage repair factors and serine 1 in the N-terminal tail of histone H4 has been shown to the efficient cellular response to DSBs. Apart from phosphoryla- occur in response to DSBs and to be important in the NHEJ tion of the histone variant H2AX, which is a relatively well- repair pathway.70 Another phosphorylation event, namely the characterized modification in response to DSBs, numerous other phosphorylation of serine 14 in the N-terminal tail of histone chromatin modifications, such as differential histone acetylation H2B at sites of DSBs, is thought to be involved in altering the and the recruitment of chromatin remodeling complexes, have chromatin structure within damage-induced foci.71 now been functionally related with the DNA damage response. Unfortunately, even though there has been a rapid increase in our knowledge regarding the relationship of chromatin mod- Constitutive lysine methylation is important for ifications and the DSB response, we are still a long way from double-strand break repair constructing a coherent picture. Further research is required to Another type of histone modification, lysine methylation, has clarify the specific role of the recently identified chromatin been identified to be important in the DSB response pathway. In modifications in facilitating DNA damage responses. Undoubt- mammalian cells, methylation of lysine 79 of histone H3 is edly, additional DNA repair associated histone modifications important for localization of the 53BP1 protein to DSBs.72 The and chromatin remodeling events will be characterized, and 53B1 protein interacts with p53 and functions in checkpoint these will help unravel the complex relationships between activation. It forms foci that colocalize with g-H2AX and are chromatin and DSB signaling and repair. detectable within minutes after exposure to ionizing radiation. The 53BP1 protein contains two tandem Tudor domains that are essential for binding to histone H3 which is methylated on lysine Acknowledgements 79 following induction of DBSs.72 Using RNA interference, it was shown that reduced levels of the histone methyltransferase (Dot1) We acknowledge the support of the Australian Institute of Nuclear responsible for lysine 79 methylation inhibited the formation of Science and Engineering. TCK was the recipient of AINSE awards.

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