H2AX post-translational modifications in the ionizing radiation response and homologous recombination

Histone H2AX phosphorylation on a C-terminal serine residue to form “γ-H2AX” is a critical early event in the chromatin response to chromosomal DNA double strand breaks in eukaryotes. In mammalian cells, γ-H2AX is formed when H2AX is phosphorylated on serine 139 by ATM or by other DNA damage response kinases. H2AX prevents genomic instability and tumorigenesis, and supports class-switch recombination at immunoglobulin heavy chain loci in mammals. We showed previously that H2AX controls double strand break repair by homologous recombination (HR) between sister chromatids. the HR functions of H2AX are mediated by interaction of γ-H2AX with the chromatin-associated adaptor protein MDC1. H2AX is potentially subject to additional post-translational modifications associated with the DNA damage response and with other chromatin functions. To test this idea, we used mass spectroscopy to identify H2AX residues additional to serine 139 that are post-translationally modified following exposure of cells to ionizing radiation (IR) and identified several new IR-responsive residues of H2AX. We determined the impact of IR-responsive H2AX residues on cellular resistance to IR and on H2AX-dependent HR, and also analyzed the contribution to HR of other known or potential post-translationally modified residues of H2AX. The results suggest that the HR and IR-resistance functions of H2AX are controlled in large part by specific MDC1-interacting residues of H2AX, but that additional H2AX residues modulate these core functions of H2AX.Key words: H2AX, homologous recombination, ionizing radiation, double strand break repair, histone, histone code, post-translational modification, chromatin, DNA repair     

Acetylation of Histone H2AX at Lys 5 by the TIP60 Histone Acetyltransferase Complex Is Essential for the Dynamic Binding of NBS1 to Damaged Chromatin

The association and dissociation of DNA damage response (DDR) factors with damaged chromatin occurs dynamically, which is crucial for the activation of DDR signaling in a spatiotemporal manner. We previously showed that the TIP60 histone acetyltransferase complex acetylates histone H2AX, to facilitate H2AX exchange at sites of DNA damage. However, it remained unclear how the acetylation of histone H2AX by TIP60 is related to the DDR signaling. We found that the acetylation but not the phosphorylation of H2AX is essential for the turnover of NBS1 on damaged chromatin. The loss of H2AX acetylation at Lys 5 by TIP60 in cells disturbed the accumulation of NBS1 at sites of DNA damage. Although the phosphorylation of H2AX is also reportedly required for the retention of NBS1 at damage sites, our data indicated that the acetylation-dependent NBS1 turnover by TIP60 on damaged chromatin restricts the dispersal of NBS1 foci from the sites of DNA damage. These findings indicate the importance of the acetylation-dependent dynamic binding of NBS1 to damaged chromatin, created by histone H2AX exchange, for the proper accumulation of NBS1 at DNA damage sites.     

MST1 promotes apoptosis through phosphorylation of histone H2AX

MST1 (mammalian STE20-like kinase 1) is a serine/threonine kinase that is cleaved and activated by caspases during apoptosis. Overexpression of MST1 induces apoptotic morphological changes such as chromatin condensation, but the mechanism is not clear. Here we show that MST1 induces apoptotic chromatin condensation through its phosphorylation of histone H2AX at Ser-139. During etoposide-induced apoptosis in Jurkat cells, the cleavage of MST1 directly corresponded with strong H2AX phosphorylation. In vitro kinase assay results showed that MST1 strongly phosphorylates histone H2AX. Western blot and kinase assay results with a mutant S139A H2AX confirmed that MST1 phosphorylates H2AX at Ser-139. Direct binding of MST1 and H2AX can be detected when co-expressed in HEK293 cells and was also confirmed by an endogenous immunoprecipitation study. When overexpressed in HeLa cells, both the MST1 full-length protein and the MST1 kinase domain (MST1-NT), but not the kinase-negative mutant (MST1-NT-KN), could induce obvious endogenous histone H2AX phosphorylation. The caspase-3 inhibitor benzyloxycarbonyl-DEVD-fluoromethyl ketone (Z-DEVD-fmk) attenuates phosphorylation of H2AX by MST1 but cannot inhibit MST1-NT-induced histone H2AX phosphorylation, indicating that cleaved MST1 is responsible for H2AX phosphorylation during apoptosis. Histone H2AX phosphorylation and DNA fragmentation were suppressed in MST1 knockdown Jurkat cells after etoposide treatment. Taken together, our data indicated that H2AX is a substrate of MST1, which functions to induce apoptotic chromatin condensation and DNA fragmentation.     

VRK1 chromatin kinase phosphorylates H2AX and is required for foci formation induced by DNA damage

All types of DNA damage cause a local alteration and relaxation of chromatin structure. Sensing and reacting to this initial chromatin alteration is a necessary trigger for any type of DNA damage response (DDR). In this context, chromatin kinases are likely candidates to participate in detection and reaction to a locally altered chromatin as a consequence of DNA damage and, thus, initiate the appropriate cellular response. In this work, we demonstrate that VRK1 is a nucleosomal chromatin kinase and that its depletion causes loss of histones H3 and H4 acetylation, which are required for chromatin relaxation, both in basal conditions and after DNA damage, independently of ATM. Moreover, VRK1 directly and stably interacts with histones H2AX and H3 in basal conditions. In response to DNA damage induced by ionizing radiation, histone H2AX is phosphorylated in Ser139 by VRK1. The phosphorylation of H2AX and the formation of γH2AX foci induced by ionizing radiation (IR), are prevented by VRK1 depletion and are rescued by kinase-active, but not kinase-dead, VRK1. In conclusion, we found that VRK1 is a novel chromatin component that reacts to its alterations and participates very early in DDR, functioning by itself or in cooperation with ATM.     

A cooperative activation loop among SWI/SNF, γ‐H2AX and H3 acetylation for DNA double‐strand break repair

Although recent studies highlight the importance of histone modifications and ATP‐dependent chromatin remodelling in DNA double‐strand break (DSB) repair, how these mechanisms cooperate has remained largely unexplored. Here, we show that the SWI/SNF chromatin remodelling complex, earlier known to facilitate the phosphorylation of histone H2AX at Ser‐139 (S139ph) after DNA damage, binds to γ‐H2AX (the phosphorylated form of H2AX)‐containing nucleosomes in S139ph‐dependent manner. Unexpectedly, BRG1, the catalytic subunit of SWI/SNF, binds to γ‐H2AX nucleosomes by interacting with acetylated H3, not with S139ph itself, through its bromodomain. Blocking the BRG1 interaction with γ‐H2AX nucleosomes either by deletion or overexpression of the BRG1 bromodomain leads to defect of S139ph and DSB repair. H3 acetylation is required for the binding of BRG1 to γ‐H2AX nucleosomes. S139ph stimulates the H3 acetylation on γ‐H2AX nucleosomes, and the histone acetyltransferase Gcn5 is responsible for this novel crosstalk. The H3 acetylation on γ‐H2AX nucleosomes is induced by DNA damage. These results collectively suggest that SWI/SNF, γ‐H2AX and H3 acetylation cooperatively act in a feedback activation loop to facilitate DSB repair.     

H2AX post-translational modifications in the ionizing radiation response and homologous recombination

Histone H2AX phosphorylation on a C-terminal serine residue to form "γ-H2AX" is a critical early event in the chromatin response to chromosomal DNA double strand breaks in eukaryotes. In mammalian cells, γ-H2AX is formed when H2AX is phosphorylated on serine 139 by ATM or by other DNA damage response kinases. H2AX prevents genomic instability and tumorigenesis, and supports class-switch recombination at immunoglobulin heavy chain loci in mammals. We showed previously that H2AX controls double strand break repair by homologous recombination (HR) between sister chromatids. The HR functions of H2AX are mediated by interaction of γ-H2AX with the chromatin-associated adaptor protein MDC1. H2AX is potentially subject to additional post-translational modifications associated with the DNA damage response and with other chromatin functions. To test this idea, we used mass spectroscopy to identify H2AX residues additional to serine 139 that are post-translationally modified following exposure of cells to ionizing radiation (IR) and identified several new IR-responsive residues of H2AX. We determined the impact of IR-responsive H2AX residues on cellular resistance to IR and on H2AX-dependent homologous recombination, and also analyzed the contribution to HR of other known or potential post-translationally modified residues of H2AX. The results suggest that the HR and IR-resistance functions of H2AX are controlled in large part by specific MDC1-interacting residues of H2AX, but that additional H2AX residues modulate these core functions of H2AX.     

Mdb1, a Fission Yeast Homolog of Human MDC1, Modulates DNA Damage Response and Mitotic Spindle Function

During eukaryotic DNA damage response (DDR), one of the earliest events is the phosphorylation of the C-terminal SQ motif of histone H2AX (H2A in yeasts). In human cells, phosphorylated H2AX (γH2AX) is recognized by MDC1, which serves as a binding platform for the accumulation of a myriad of DDR factors on chromatin regions surrounding DNA lesions. Despite its important role in DDR, no homolog of MDC1 outside of metazoans has been described. Here, we report the characterization of Mdb1, a protein from the fission yeast Schizosaccharomyces pombe, which shares significant sequence homology with human MDC1 in their C-terminal tandem BRCT (tBRCT) domains. We show that in vitro, recombinant Mdb1 protein binds a phosphorylated H2A (γH2A) peptide, and the phospho-specific binding requires two conserved phospho-binding residues in the tBRCT domain of Mdb1. In vivo, Mdb1 forms nuclear foci at DNA double strand breaks (DSBs) induced by the HO endonuclease and ionizing radiation (IR). IR-induced Mdb1 focus formation depends on γH2A and the phospho-binding residues of Mdb1. Deleting the mdb1 gene does not overtly affect DNA damage sensitivity in a wild type background, but alters the DNA damage sensitivity of cells lacking another γH2A binder Crb2. Overexpression of Mdb1 causes severe DNA damage sensitivity in a manner that requires the interaction between Mdb1 and γH2A. During mitosis, Mdb1 localizes to spindles and concentrates at spindle midzones at late mitosis. The spindle midzone localization of Mdb1 requires its phospho-binding residues, but is independent of γH2A. Loss of Mdb1 or mutating its phospho-binding residues makes cells more resistant to the microtubule depolymerizing drug thiabendazole. We propose that Mdb1 performs dual roles in DDR and mitotic spindle regulation.     

Rac1 Protein Signaling Is Required for DNA Damage Response Stimulated by Topoisomerase II Poisons

To investigate the potency of the topoisomerase II (topo II) poisons doxorubicin and etoposide to stimulate the DNA damage response (DDR), S139 phosphorylation of histone H2AX (γH2AX) was analyzed using rat cardiomyoblast cells (H9c2). Etoposide caused a dose-dependent increase in the γH2AX level as shown by Western blotting. By contrast, the doxorubicin response was bell-shaped with high doses failing to increase H2AX phosphorylation. Identical results were obtained by immunohistochemical analysis of γH2AX focus formation, comet assay-based DNA strand break analysis, and measuring the formation of the topo II-DNA cleavable complex. At low dose, doxorubicin activated ataxia telangiectasia mutated (ATM) but not ATM and Rad3-related (ATR). Both the lipid-lowering drug lovastatin and the Rac1-specific inhibitor NSC23766 attenuated doxorubicin- and etoposide-stimulated H2AX phosphorylation, induction of DNA strand breaks, and topo II-DNA complex formation. Lovastatin and NSC23766 acted in an additive manner. They did not attenuate doxorubicin-induced increase in p-ATM and p-Chk2 levels. DDR stimulated by topo II poisons was partially blocked by inhibition of type I p21-associated kinases. DDR evoked by the topoisomerase I poison topotecan remained unaffected by lovastatin. The data show that the mechanisms involved in DDR stimulated by topo II poisons are agent-specific with anthracyclines lacking DDR-stimulating activity at high doses. Pharmacological inhibition of Rac1 signaling counteracts doxorubicin- and etoposide-stimulated DDR by disabling the formation of the topo II-DNA cleavable complex. Based on the data we suggest that Rac1-regulated mechanisms are required for DNA damage induction and subsequent activation of the DDR following treatment with topo II but not topo I poisons.     

The VRK1 chromatin kinase regulates the acetyltransferase activity of Tip60/KAT5 by sequential phosphorylations in response to DNA damage

The regulation of histone epigenetic modifications mediates the adaptation of chromatin to different biological processes. DNA damage causes a local relaxation of chromatin associated to histone H4 acetylation in K16, mediated by Tip60/KAT5. In this work, we have studied the role that the VRK1 chromatin kinase plays on the activation of Tip60 during this process. In the DNA damage response induced by doxorubicin, VRK1 directly phosphorylates Tip60. However, the phosphorylated Tip60 residues and their functional roles are unknown. In DDR, we have identified these two Tip60 phosphorylated residues and the cooperation of the participating kinases. The T158 phosphorylation, mediated by VRK1, is early and transient, preceding that of S199, which is more sustained in time, and mediated by DNA-PK. The role of each phosphorylated residues was determined by using phosphomimetic and phosphonull mutants and their combination. T158 phosphorylation protects Tip60 from ubiquitin-mediated degradation, promotes its recruitment to chromatin from the nucleoplasm, and is necessary for its full trans-acetylase activity. The phosphorylation in S199 by DNA-PK directly facilitates Tip60 autoacetylation, but it is not enough for trans-acetylation of two of its targets, histone H4 and ATM, which requires a double phosphorylation of Tip60 in T158 and S199. DNA-PK inhibitors block the phosphorylation of S199. We propose a model in which the cooperation between VRK1 and DNA-PK mediates the sequential phosphorylation of Tip60/KAT5, and contributes to the recruitment of this protein to initiate the sequential remodeling of chromatin in DDR. Both proteins are candidates for novel synthetic lethality strategies in cancer treatment.     

Focus on histone variant H2AX: To be or not to be

Phosphorylation of histone variant H2AX at serine 139, named γH2AX, has been widely used as a sensitive marker for DNA double-strand breaks (DSBs). γH2AX is required for the accumulation of many DNA damage response (DDR) proteins at DSBs. Thus it is believed to be the principal signaling protein involved in DDR and to play an important role in DNA repair. However, only mild defects in DNA damage signaling and DNA repair were observed in H2AX-deficient cells and animals. Such findings prompted us and others to explore H2AX-independent mechanisms in DNA damage response. Here, we will review recent advances in our understanding of H2AX-dependent and independent DNA damage signaling and repair pathways in mammalian cells.     

Nucleosome Acidic Patch Promotes RNF168- and RING1B/BMI1-Dependent H2AX and H2A Ubiquitination and DNA Damage Signaling

Histone ubiquitinations are critical for the activation of the DNA damage response (DDR). In particular, RNF168 and RING1B/BMI1 function in the DDR by ubiquitinating H2A/H2AX on Lys-13/15 and Lys-118/119, respectively. However, it remains to be defined how the ubiquitin pathway engages chromatin to provide regulation of ubiquitin targeting of specific histone residues. Here we identify the nucleosome acid patch as a critical chromatin mediator of H2A/H2AX ubiquitination (ub). The acidic patch is required for RNF168- and RING1B/BMI1-dependent H2A/H2AXub in vivo. The acidic patch functions within the nucleosome as nucleosomes containing a mutated acidic patch exhibit defective H2A/H2AXub by RNF168 and RING1B/BMI1 in vitro. Furthermore, direct perturbation of the nucleosome acidic patch in vivo by the expression of an engineered acidic patch interacting viral peptide, LANA, results in defective H2AXub and RNF168-dependent DNA damage responses including 53BP1 and BRCA1 recruitment to DNA damage. The acidic patch therefore is a critical nucleosome feature that may serve as a scaffold to integrate multiple ubiquitin signals on chromatin to compose selective ubiquitinations on histones for DNA damage signaling.     

DNA damage response as a biomarker in treatment of leukemias

Early assessment of cancer response to the treatment is of great importance in clinical oncology. Most antitumor drugs, among them DNA topoisomerase (topo) inhibitors, target nuclear DNA. The aim of the present study was to explore feasibility of the assessment of DNA damage response (DDR) as potential biomarker, eventually related to the clinical response, during treatment of human leukemias. We have measured DDR as reported by activation of ATM through its phosphorylation on Ser 1981 (ATM-S1981P) concurrent with histone H2AX phosphorylation on Ser139 (γH2AX) in leukemic blast cells from the blood of twenty patients, 16 children/adolescents and 4 adults, diagnosed with acute leukemias and treated with topo2 inhibitors doxorubicin, daunomycin, mitoxantrone or idarubicin. Phosphorylation of H2AX and ATM was detected using phospho-specific Abs and measured in individual cells by flow cytometry. The increase in the level of ATM-S1981P and γH2AX, varying in extent between the patients, was observed in blasts from the blood collected one hour after completion of the drug infusion with respect to the pre-treatment level. A modest degree of correlation was observed between the induction of ATM activation and H2AX phosphorylation in blasts of individual patients. The number of the studied patients (20) and the number of the clinically non-responding ones (2) was too low to draw a conclusion whether the assessment of DDR can be clinically prognostic. The present findings, however, demonstrate the feasibility of assessment of DDR during the treatment of leukemias with drugs targeting DNA.     

Targeting the DNA damage response (DDR) by natural compounds

Natural compounds (NC) are an important source of anticancer drugs. The genomic DNA of tumor cells is a major target of conventional anticancer therapeutics (cAT). DNA damage elicits a complex stress response programme termed DNA damage response (DDR), with the PI3-like kinase ATM and ATR being the key regulators. Since the DDR coordinates mechanisms of DNA repair and apoptosis, hence regulating the balance between death and survival, it is an attractive target of novel anticancer strategies. The aim of the study was to identify natural compounds derived from endophytic fungi, lichens, marine sponges or plants that interfere with mechanisms of the DDR. To this end, the cytotoxic and DDR modulating potency of 296 natural compounds, used alone or in combination with the cAT cisplatin (Cis) and doxorubicin (Doxo) was investigated by fluorescence-based analysis of the ATM/ATR-catalyzed S139 phosphorylation of histone 2AX (γH2AX), a surrogate marker of DNA damage-triggered DDR. After initial screening, a total of ten natural compounds were identified that were toxic in pancreatic carcinoma cells and activated the DDR on their own and/or promoted the DDR if used in combination with cAT. Their mode of action was shown to be independent of drug transport mechanisms. Based on their chemical structures, DDR modulatory activity and published data we suggest the marine NC 5-epi-nakijiquinone Q and 5-epi-ilimaquinone as well as the fungal compound secalonic acid F as most promising NC-based drug candidates for future synthesis of DDR-modulating chemical derivatives and their preclinical in vitro and in vivo testing.     

Deregulation of DNA damage response pathway by intercellular contact

Deregulation of the DNA damage response (DDR) pathway could compromise genomic integrity in normal cells and reduce cancer cell sensitivity to anticancer treatments. We found that intercellular contact stabilizes histone H2AX and γH2AX (H2AX phosphorylated on Ser-139) by up-regulating N/E-cadherin and γ-catenin. γ-catenin and its DNA-binding partner LEF-1 indirectly increase levels of H2AX by suppressing the promoter of the RNF8 ubiquitin ligase, which decreases levels of H2AX protein under conditions of low intercellular contact. Hyperphosphorylation of DDR proteins is induced by up-regulated H2AX. Constitutive apoptosis is caused in confluent cells but is not further induced by DNA damage. This is conceivably due to insufficient p53 activation because ChIP assay shows that its DNA binding ability is not induced in those cells. Together, our results illustrate a novel mechanism of the regulation of DDR proteins by the cadherin-catenin pathway.     

Histone H2A phosphorylation in DNA double-strand break repair

DNA repair must take place within the context of chromatin, and it is therefore not surprising that many aspects of both chromatin components and proteins that modify chromatin have been implicated in this process. One of the best-characterized chromatin modification events in DNA-damage responses is the phosphorylation of the SQ motif found in histone H2A or the H2AX histone variant in higher eukaryotes. This modification is an early response to the induction of DNA damage, and occurs in a wide range of eukaryotic organisms, suggesting an important conserved function. One function that histone modifications can have is to provide a unique binding site for interacting factors. Here, we review the proteins and protein complexes that have been identified as H2AS129ph (budding yeast) or H2AXS139ph (human) binding partners and discuss the implications of these interactions.     

Baculoviruses Modulate a Proapoptotic DNA Damage Response To Promote Virus Multiplication

The baculovirus Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) initiates apoptosis in diverse insects through events triggered by virus DNA (vDNA) replication. To define the proapoptotic pathway and its role in antivirus defense, we investigated the link between the host''s DNA damage response (DDR) and apoptosis. We report here that AcMNPV elicits a DDR in the model insect Drosophila melanogaster. Replication of vDNA activated DDR kinases, as evidenced by ATM-driven phosphorylation of the Drosophila histone H2AX homolog (H2Av), a critical regulator of the DDR. Ablation or inhibition of ATM repressed H2Av phosphorylation and blocked virus-induced apoptosis. The DDR kinase inhibitors caffeine and KU55933 also prevented virus-induced apoptosis in cells derived from the permissive AcMNPV host, Spodoptera frugiperda. This block occurred at a step upstream of virus-mediated depletion of the cellular inhibitor-of-apoptosis protein, an event that initiates apoptosis in Spodoptera and Drosophila. Thus, the DDR is a conserved, proapoptotic response to baculovirus infection. DDR inhibition also repressed vDNA replication and reduced virus yields 100,000-fold, demonstrating that the DDR contributes to virus production, despite its recognized antivirus role. In contrast to virus-induced phosphorylation of Drosophila H2Av, AcMNPV blocked phosphorylation of the Spodoptera H2AX homolog (SfH2AX). Remarkably, AcMNPV also suppressed SfH2AX phosphorylation following pharmacologically induced DNA damage. These findings indicate that AcMNPV alters canonical DDR signaling in permissive cells. We conclude that AcMNPV triggers a proapoptotic DDR that is subsequently modified, presumably to stimulate vDNA replication. Thus, manipulation of the DDR to facilitate multiplication is an evolutionarily conserved strategy among DNA viruses of insects and mammals.     

Histone H2AX and Fanconi anemia FANCD2 function in the same pathway to maintain chromosome stability

Fanconi anemia (FA) is a chromosome fragility syndrome characterized by bone marrow failure and cancer susceptibility. The central FA protein FANCD2 is known to relocate to chromatin upon DNA damage in a poorly understood process. Here, we have induced subnuclear accumulation of DNA damage to prove that histone H2AX is a novel component of the FA/BRCA pathway in response to stalled replication forks. Analyses of cells from H2AX knockout mice or expressing a nonphosphorylable H2AX (H2AX(S136A/S139A)) indicate that phosphorylated H2AX (gammaH2AX) is required for recruiting FANCD2 to chromatin at stalled replication forks. FANCD2 binding to gammaH2AX is BRCA1-dependent and cells deficient or depleted of H2AX show an FA-like phenotype, including an excess of chromatid-type chromosomal aberrations and hypersensitivity to MMC. This MMC hypersensitivity of H2AX-deficient cells is not further increased by depleting FANCD2, indicating that H2AX and FANCD2 function in the same pathway in response to DNA damage-induced replication blockage. Consequently, histone H2AX is functionally connected to the FA/BRCA pathway to resolve stalled replication forks and prevent chromosome instability.     

Nucleolin Participates in DNA Double-Strand Break-Induced Damage Response through MDC1-Dependent Pathway

H2AX is an important factor for chromatin remodeling to facilitate accumulation of DNA damage-related proteins at DNA double-strand break (DSB) sites. In order to further understand the role of H2AX in the DNA damage response (DDR), we attempted to identify H2AX-interacting proteins by proteomics analysis. As a result, we identified nucleolin as one of candidates. Here, we show a novel role of a major nucleolar protein, nucleolin, in DDR. Nucleolin interacted with γ-H2AX and accumulated to laser micro-irradiated DSB damage sites. Chromatin Immunoprecipitation assay also displayed the accumulation of nucleolin around DSB sites. Nucleolin-depleted cells exhibited repression of both ATM-dependent phosphorylation following exposure to γ-ray and subsequent cell cycle checkpoint activation. Furthermore, nucleolin-knockdown reduced HR and NHEJ activity and showed decrease in IR-induced chromatin accumulation of HR/NHEJ factors, agreeing with the delayed kinetics of γ-H2AX focus. Moreover, nucleolin-knockdown decreased MDC1-related events such as focus formation of 53 BP1, RNF168, phosphorylated ATM, and H2A ubiquitination. Nucleolin also showed FACT-like activity for DSB damage-induced histone eviction from chromatin. Taken together, nucleolin could promote both ATM-dependent cell cycle checkpoint and DSB repair by functioning in an MDC1-related pathway through its FACT-like function.