A critical role for histone H2AX in recruitment of repair factors to nuclear foci after DNA damage

BACKGROUND: The response of eukaryotic cells to double-strand breaks in genomic DNA includes the sequestration of many factors into nuclear foci. Recently it has been reported that a member of the histone H2A family, H2AX, becomes extensively phosphorylated within 1-3 minutes of DNA damage and forms foci at break sites. RESULTS: In this work, we examine the role of H2AX phosphorylation in focus formation by several repair-related complexes, and investigate what factors may be involved in initiating this response. Using two different methods to create DNA double-strand breaks in human cells, we found that the repair factors Rad50 and Rad51 each colocalized with phosphorylated H2AX (gamma-H2AX) foci after DNA damage. The product of the tumor suppressor gene BRCA1 also colocalized with gamma-H2AX and was recruited to these sites before Rad50 or Rad51. Exposure of cells to the fungal inhibitor wortmannin eliminated focus formation by all repair factors examined, suggesting a role for the phosphoinositide (PI)-3 family of protein kinases in mediating this response. Wortmannin treatment was effective only when it was added early enough to prevent gamma-H2AX formation, indicating that gamma-H2AX is necessary for the recruitment of other factors to the sites of DNA damage. DNA repair-deficient cells exhibit a substantially reduced ability to increase the phosphorylation of H2AX in response to ionizing radiation, consistent with a role for gamma-H2AX in DNA repair. CONCLUSIONS: The pattern of gamma-H2AX foci that is established within a few minutes of DNA damage accounts for the patterns of Rad50, Rad51, and Brca1 foci seen much later during recovery from damage. The evidence presented strongly supports a role for the gamma-H2AX and the PI-3 protein kinase family in focus formation at sites of double-strand breaks and suggests the possibility of a change in chromatin structure accompanying double-strand break repair.     

Focusing on Foci: H2AX and the Recruitment of DNA-Damage Response Factors

No abstract available.     

Critical role of monoubiquitination of histone H2AX protein in histone H2AX phosphorylation and DNA damage response

DNA damage response is an important surveillance mechanism used to maintain the integrity of the human genome in response to genotoxic stress. Histone variant H2AX is a critical sensor that undergoes phosphorylation at serine 139 upon genotoxic stress, which provides a docking site to recruit the mediator of DNA damage checkpoint protein 1 (MDC1) and DNA repair protein complex to sites of DNA breaks for DNA repair. Here, we show that monoubiquitination of H2AX is induced upon DNA double strand breaks and plays a critical role in H2AX Ser-139 phosphorylation (γ-H2AX), in turn facilitating the recruitment of MDC1 to DNA damage foci. Mechanistically, we show that monoubiquitination of H2AX induced by RING finger protein 2 (RNF2) is required for the recruitment of active ataxia telangiectasia mutated to DNA damage foci, thus affecting the formation of γ-H2AX. Importantly, a defect in monoubiquitination of H2AX profoundly enhances ionizing radiation sensitivity. Our study therefore suggests that monoubiquitination of H2AX is an important step for DNA damage response and may have important clinical implications for the treatment of cancers.     

Role of H2AX in DNA damage response and human cancers

H2AX, the evolutionarily conserved variant of histone H2A, has been identified as one of the key histones to undergo various post-translational modifications in response to DNA double-strand breaks (DSBs). By virtue of these modifications, that include acetylation, phosphorylation and ubiquitination, H2AX marks the damaged DNA double helix, facilitating local recruitment and retention of DNA repair and chromatin remodeling factors to restore genomic integrity. These modifications are essential for effective DSB repair, so is their removal for cell, to recover from checkpoint arrest. Because of these vital roles during DSB signaling and also its activation during early cancer stages, H2AX is emerging as an intriguing gene in tumor biology, supported further by frequent deletion of the region harboring this gene. This review focuses on the insights gained from recent studies on dynamic regulation of H2AX in DSB repair. Also, posing future challenges in the area of chromatin reorganization and retention of epigenetic signature post-DSB-repair with implication of its haploinsufficiency in human cancers.     

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     

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.     

Photobleaching of GFP-labeled H2AX in chromatin: H2AX has low diffusional mobility in the nucleus

The Ser-139 phosphorylated form of replacement histone H2AX (gamma-H2AX) is induced within large chromatin domains by double-strand DNA breaks (DSBs) in mammalian chromosomes. This modification is known to be important for the maintenance of chromosome stability. However, the mechanism of gamma-H2AX formation at DSBs and its subsequent elimination during DSB repair remains unknown. gamma-H2AX formation and elimination could occur by direct phosphorylation and dephosphorylation of H2AX in situ in the chromatin. Alternatively, H2AX molecules could be phosphorylated freely in the nucleus, diffuse into chromatin regions containing DSBs and then diffuse out after DNA repair. In this study we show that free histone H2AX can be efficiently phosphorylated in vitro by nuclear extracts and that free gamma-H2AX can be dephosphorylated in vitro by the mammalian protein phosphatase 1-alpha. We made N-terminal fusion constructs of H2AX with green fluorescent protein (GFP) and studied their diffusional mobility in transient and stable cell transfections. In the absence or presence of DSBs, only a small fraction of GFP-H2AX is redistributed after photobleaching, indicating that in vivo this histone is essentially immobile in chromatin. This suggests that gamma-H2AX formation in chromatin is unlikely to occur by diffusion of free histone and gamma-H2AX dephosphorylation may involve the mammalian protein phosphatase 1alpha.     

Monoubiquitination of H2AX protein regulates DNA damage response signaling

Double strand breaks (DSBs) are the most deleterious of the DNA lesions that initiate genomic instability and promote tumorigenesis. Cells have evolved a complex protein network to detect, signal, and repair DSBs. In mammalian cells, a key component in this network is H2AX, which becomes rapidly phosphorylated at Ser(139) (γ-H2AX) at DSBs. Here we show that monoubiquitination of H2AX mediated by the RNF2-BMI1 complex is critical for the efficient formation of γ-H2AX and functions as a proximal regulator in DDR (DNA damage response). RNF2-BMI1 interacts with H2AX in a DNA damage-dependent manner and is required for monoubiquitination of H2AX at Lys(119)/Lys(120). As a functional consequence, we show that the H2AX K120R mutant abolishes H2AX monoubiquitination, impairs the recruitment of p-ATM (Ser(1981)) to DSBs, and thereby reduces the formation of γ-H2AX and the recruitment of MDC1 to DNA damage sites. These data suggest that monoubiquitination of H2AX plays a critical role in initiating DNA damage signaling. Consistent with these observations, impairment of RNF2-BMI1 function by siRNA knockdown or overexpression of the ligase-dead RNF2 mutant all leads to significant defects both in accumulation of γ-H2AX, p-ATM, and MDC1 at DSBs and in activation of NBS1 and CHK2. Additionally, the regulatory effect of RNF2-BMI1 on γ-H2AX formation is dependent on ATM. Lacking their ability to properly activate the DNA damage signaling pathway, RNF2-BMI1 complex-depleted cells exhibit impaired DNA repair and increased sensitivity to ionizing radiation. Together, our findings demonstrate a distinct monoubiquitination-dependent mechanism that is required for H2AX phosphorylation and the initiation of DDR.     

The complexity of phosphorylated H2AX foci formation and DNA repair assembly at DNA double-strand breaks

The maintenance of genome stability requires efficient DNA double-stranded break (DSB) repair mediated by the phosphorylation of multiple histone H2AX molecules near the break sites. The phosphorylated H2AX (γ-H2AX) molecules form foci covering many megabases of chromatin. The formation of g-H2AX foci is critical for efficient DNA damage response (DDR) and for the maintenance of genome stability, however, the mechanisms of protein organization in foci is largely unknown. To investigate the nature of γ-H2AX foci formation, we analyzed the distribution of γ-H2AX and other DDR proteins at DSB sites using a variety of techniques to visualize, expand and partially disrupt chromatin. We report here that γ-H2AX foci change composition during the cell cycle, with proteins 53BP1, NBS1 and MRE11 dissociating from foci in G2 and mitosis to return at the beginning of the following G1. In contrast, MDC1 remained colocalized with g-H2AX during mitosis. In addition, while γ-H2AX was found to span large domains flanking DSB sites, 53BP1 and NBS1 were more localized and MDC1 colocalized in doublets in foci. H2AX and MDC1 were found to be involved in chromatin relaxation after DSB formation. Our data demonstrates that the DSB repair focus is a heterogeneous and dynamic structure containing internal complexity.     

Computational Methods for analysis of foci: validation for radiation-induced gamma-H2AX foci in human cells

Observation and counting of gamma-H2AX foci in untreated cells as well as in cells exposed to cytotoxic agents is a widely used method for documenting the presence of double-strand breaks (DSBs) in the DNA and for analysis of their repair. Similar methods are employed to analyze formation of foci by a variety of proteins implicated in the cellular responses to DNA damage. Despite the wide application of the approach, the manual counting that is frequently used is prone to inaccuracies and investigator-related biases and artifacts. To alleviate this limitation, we developed and describe here personal computer-based algorithms, operating as utilities on available software, that allow an objective and quantitative analysis of foci from confocal images. The algorithms allow focus counting as well as size definition and correct for focus coincidence due to the overlap normally occurring with an increasing number of foci per nucleus. Furthermore, the software allows measurement of the integrated optical density (IOD) of each individual focus, which enables analysis of properties of foci as a function of time. Finally, the information generated by the above analysis algorithms can be employed to evaluate colocalization between foci formed by different proteins. A validation of the software is presented for radiation-induced gamma-H2AX foci in three widely used human cell lines and colocalization tested with RAD51 and gamma-H2AX foci. The computational methods presented extend to images generated by digital cameras.     

H2AX: the histone guardian of the genome

At close hand to one's genomic material are the histones that make up the nucleosome. Standing guard, one variant stays hidden doubling as one of the core histones. But, thanks to its prime positioning, a variation in the tail of H2AX enables rapid modification of the histone code in response to DNA damage. A role for H2AX phosphorylation has been demonstrated in DNA repair, cell cycle checkpoints, regulated gene recombination events, and tumor suppression. In this review, we summarize what we have learned about this marker of DNA breaks, and highlight some of the questions that remain to be elucidated about the physiological role of H2AX. We also suggest a model in which chromatin restructuring mediated by H2AX phosphorylation serves to concentrate DNA repair/signaling factors and/or tether DNA ends together, which could explain the pleotropic phenotypes observed in its absence.     

H2AX: functional roles and potential applications

Upon DNA double-strand break (DSB) induction in mammals, the histone H2A variant, H2AX, becomes rapidly phosphorylated at serine 139. This modified form, termed γ-H2AX, is easily identified with antibodies and serves as a sensitive indicator of DNA DSB formation. This review focuses on the potential clinical applications of γ-H2AX detection in cancer and in response to other cellular stresses. In addition, the role of H2AX in homeostasis and disease will be discussed. Recent work indicates that γ-H2AX detection may become a powerful tool for monitoring genotoxic events associated with cancer development and tumor progression.     

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.     

ATM phosphorylates histone H2AX in response to DNA double-strand breaks

A very early step in the response of mammalian cells to DNA double-strand breaks is the phosphorylation of histone H2AX at serine 139 at the sites of DNA damage. Although the phosphatidylinositol 3-kinases, DNA-PK (DNA-dependent protein kinase), ATM (ataxia telangiectasia mutated), and ATR (ATM and Rad3-related), have all been implicated in H2AX phosphorylation, the specific kinase involved has not yet been identified. To definitively identify the specific kinase(s) that phosphorylates H2AX in vivo, we have utilized DNA-PKcs-/- and Atm-/- cell lines and mouse embryonic fibroblasts. We find that H2AX phosphorylation and nuclear focus formation are normal in DNA-PKcs-/- cells and severely compromised in Atm-/- cells. We also find that ATM can phosphorylate H2AX in vitro and that ectopic expression of ATM in Atm-/- fibroblasts restores H2AX phosphorylation in vivo. The minimal H2AX phosphorylation in Atm-/- fibroblasts can be abolished by low concentrations of wortmannin suggesting that DNA-PK, rather than ATR, is responsible for low levels of H2AX phosphorylation in the absence of ATM. Our results clearly establish ATM as the major kinase involved in the phosphorylation of H2AX and suggest that ATM is one of the earliest kinases to be activated in the cellular response to double-strand breaks.     

Replication protein A and gamma-H2AX foci assembly is triggered by cellular response to DNA double-strand breaks

Human replication protein A (RPA p34), a crucial component of diverse DNA excision repair pathways, is implicated in DNA double-strand break (DSB) repair. To evaluate its role in DSB repair, the intranuclear dynamics of RPA was investigated after DNA damage and replication blockage in human cells. Using two different agents [ionizing radiation (IR) and hydroxyurea (HU)] to generate DSBs, we found that RPA relocated into distinct nuclear foci and colocalized with a well-known DSB binding factor, gamma-H2AX, at the sites of DNA damage in a time-dependent manner. Colocalization of RPA and gamma-H2AX foci peaked at 2 h after IR treatment and subsequently declined with increasing postrecovery times. The time course of RPA and gamma-H2AX foci association correlated well with the DSB repair activity detected by a neutral comet assay. A phosphatidylinositol-3 (PI-3) kinase inhibitor, wortmannin, completely abolished both RPA and gamma-H2AX foci formation triggered by IR. Additionally, radiosensitive ataxia telangiectasia (AT) cells harboring mutations in ATM gene product were found to be deficient in RPA and gamma-H2AX colocalization after IR. Transfection of AT cells with ATM cDNA fully restored the association of RPA foci with gamma-H2AX illustrating the requirement of ATM gene product for this process. The exact coincidence of RPA and gamma-H2AX in response to HU specifically in S-phase cells supports their role in DNA replication checkpoint control. Depletion of RPA by small interfering RNA (SiRNA) substantially elevated the frequencies of IR-induced micronuclei (MN) and apoptosis in human cells suggestive of a role for RPA in DSB repair. We propose that RPA in association with gamma-H2AX contributes to both DNA damage checkpoint control and repair in response to strand breaks and stalled replication forks in human cells.