Heterochromatin is refractory to gamma-H2AX modification in yeast and mammals.
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ABSTRACT:
PROVIDER: S-EPMC2064441 | BioStudies | 2007-01-01T00:00:00Z
REPOSITORIES: biostudies
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Project description:The histone variant H2AX is rapidly phosphorylated at the sites of DNA double-strand breaks (DSBs). This phosphorylated H2AX (gamma-H2AX) is involved in the retention of repair and signaling factor complexes at sites of DNA damage. The dependency of this phosphorylation on the various PI3K-related protein kinases (in mammals, ataxia telangiectasia mutated and Rad3-related [ATR], ataxia telangiectasia mutated [ATM], and DNA-PKCs) has been a subject of debate; it has been suggested that ATM is required for the induction of foci at DSBs, whereas ATR is involved in the recognition of stalled replication forks. In this study, using Arabidopsis as a model system, we investigated the ATR and ATM dependency of the formation of gamma-H2AX foci in M-phase cells exposed to ionizing radiation (IR). We find that although the majority of these foci are ATM-dependent, approximately 10% of IR-induced gamma-H2AX foci require, instead, functional ATR. This indicates that even in the absence of DNA replication, a distinct subset of IR-induced damage is recognized by ATR. In addition, we find that in plants, gamma-H2AX foci are induced at only one-third the rate observed in yeasts and mammals. This result may partly account for the relatively high radioresistance of plants versus yeast and mammals.
| S-EPMC1087258 | BioStudies
Project description:In response to DNA double-strand breaks (DSBs), eukaryotic cells rapidly phosphorylate histone H2A isoform H2AX at a C-terminal serine (to form gamma-H2AX) and accumulate repair proteins at or near DSBs. To date, these events have been defined primarily at the resolution of light microscopes, and the relationship between gamma-H2AX formation and repair protein recruitment remains to be defined.We report here the first molecular-level characterization of regional chromatin changes that accompany a DSB formed by the HO endonuclease in Saccharomyces cerevisiae. Break induction provoked rapid gamma-H2AX formation and equally rapid recruitment of the Mre11 repair protein. gamma-H2AX formation was efficiently promoted by both Tel1p and Mec1p, the yeast ATM and ATR homologs; in G1-arrested cells, most gamma-H2AX formation was dependent on Tel1 and Mre11. gamma-H2AX formed in a large (ca. 50 kb) region surrounding the DSB. Remarkably, very little gamma-H2AX could be detected in chromatin within 1-2 kb of the break. In contrast, this region contains almost all the Mre11p and other repair proteins that bind as a result of the break.Both Mec1p and Tel1p can respond to a DSB, with distinct roles for these checkpoint kinases at different phases of the cell cycle. Part of this response involves histone phosphorylation over large chromosomal domains; however, the distinct distributions of gamma-H2AX and repair proteins near DSBs indicate that localization of repair proteins to breaks is not likely to be the main function of this histone modification.
| S-EPMC4493763 | BioStudies
Project description:We show that DNA double-strand breaks (DSBs) induce complex subcompartmentalization of genome surveillance regulators. Chromatin marked by gamma-H2AX is occupied by ataxia telangiectasia-mutated (ATM) kinase, Mdc1, and 53BP1. In contrast, repair factors (Rad51, Rad52, BRCA2, and FANCD2), ATM and Rad-3-related (ATR) cascade (ATR, ATR interacting protein, and replication protein A), and the DNA clamp (Rad17 and -9) accumulate in subchromatin microcompartments delineated by single-stranded DNA (ssDNA). BRCA1 and the Mre11-Rad50-Nbs1 complex interact with both of these compartments. Importantly, some core DSB regulators do not form cytologically discernible foci. These are further subclassified to proteins that connect DSBs with the rest of the nucleus (Chk1 and -2), that assemble at unprocessed DSBs (DNA-PK/Ku70), and that exist on chromatin as preassembled complexes but become locally modified after DNA damage (Smc1/Smc3). Finally, checkpoint effectors such as p53 and Cdc25A do not accumulate at DSBs at all. We propose that subclassification of DSB regulators according to their residence sites provides a useful framework for understanding their involvement in diverse processes of genome surveillance.
2006-01-01 | S-EPMC2063811 | BioStudies
Project description:The DNA damage response (DDR) encompasses the cellular response to DNA double-stranded breaks (DSBs), and includes recognition of the DSB, recruitment of numerous factors to the DNA damage site, initiation of signaling cascades, chromatin remodeling, cell-cycle checkpoint activation, and repair of the DSB. Key drivers of the DDR are multiple members of the phosphatidylinositol 3-kinase-related kinase family, including ataxia telangiectasia mutated (ATM), ataxia telangiectasia and Rad3-related (ATR), and the DNA-dependent protein kinase catalytic subunit (DNA-PKcs). ATM and ATR modulate multiple portions of the DDR, but DNA-PKcs is believed to primarily function in the DSB repair pathway, non-homologous end joining. Utilizing a human cell line in which the kinase domain of DNA-PKcs is inactivated, we show here that DNA-PKcs kinase activity is required for the cellular response to DSBs immediately after their induction. Specifically, DNA-PKcs kinase activity initiates phosphorylation of the chromatin factors H2AX and KAP1 following ionizing radiation exposure and drives local chromatin decondensation near the DSB site. Furthermore, loss of DNA-PKcs kinase activity results in a marked decrease in the recruitment of numerous members of the DDR machinery to DSBs. Collectively, these results provide clear evidence that DNA-PKcs activity is pivotal for the initiation of the DDR.
| S-EPMC6765147 | BioStudies
Project description:In Saccharomyces cerevisiae, a single double-strand break (DSB) triggers extensive phosphorylation of histone H2A (known as gammaH2AX) over 50 kb on either side of the DSB. This modification is carried out by either of yeastM-^Rs checkpoint kinases, the ATM homolog, Tel1, or the ATR homolog, Mec1. In G1-arrested cells, where there is very little 5M-^R to 3M-^R processing of DSB ends, only Tel1 promotes this modification. We have recently described a second modification gammaH2B - the phosphorylation of the C terminal T129 locus of histone H2B which is also carried out by both Mec1 and Tel1 kinases. To understand in detail how gamma-H2AX and gamma-H2B spread along the chromosome from a DSB we have undertaken a high-density analysis of their occupancy where there is a DSB on three different chromosomes. gamma-H2AX and gamma-H2B modifications are similar, but there is a marked absence of gamma-H2B near telomeres. We find that there is reduced gamma-H2AX and gamma-H2B modification over strongly transcribed regions, even taking into account the reduced histone occupancy of these genes. When transcription of the galactose-regulated genes GAL1, GAL10, GAL7 are turned off by the addition of glucose, gamma-H2AX is restored within 5 min; when these genes are again induced, gamma-H2AX is rapidly lost. Regions more distal to the GAL genes have markedly reduced gamma-H2AX levels that rise rapidly when transcription is repressed, suggesting that transcription acts as a barrier to the propagation of gamma-H2AX away from the DSB. The restoration of gamma-H2AX in transcribed regions can be carried out by either Mec1 or Tel1, even 7 h after break induction, suggesting that Tel1 remains associated with damaged chromosomes for an extended time. In addition, we show that gamma-H2AX can be transferred in trans, to regions unlinked to the DSB that lie in close proximity the DSB. Specifically, if a DSB is generated 14 kb from CEN2, gamma-H2AX is transferred to regions around all the other centromeres, in keeping with observed close proximity of all centromere-adjacent chromosome arms. This transfer can be observed even in the absence of formaldehyde crosslinking of the samples.
| E-MTAB-1409 | BioStudies
Project description:Numerous viruses manipulate host factors for viral production. We demonstrated that human enterovirus A71 (EVA71), a primary causative agent for hand, foot, and mouth disease (HFMD), increased the level of the DNA damage response (DDR) marker γ-H2AX. DDR is primarily mediated by the ataxia telangiectasia mutated (ATM), ATM and Rad3-related (ATR), or DNA-dependent protein kinase (DNA-PK) pathways. Upregulation of γ-H2AX by EVA71 was dependent on the ATR but not the ATM or DNA-PK pathway. As a nuclear factor, there is no previous evidence of cytoplasmic distribution of γ-H2AX. However, the present findings demonstrated that EVA71 encouraged the localization of γ-H2AX to the cytoplasm. Of note, γ-H2AX formed a complex with structural protein VP3, non-structural protein 3D, and the viral genome. Treatment with an inhibitor or CRISPR/Cas9 technology to decrease or silence the expression of γ-H2AX decreased viral genome replication in host cells; this effect was accompanied by decreased viral protein expression and virions. In animal experiments, caffeine was used to inhibit DDR; the results revealed that caffeine protected neonatal mice from death after infection with EVA71, laying the foundation for new therapeutic applications of caffeine. More importantly, in children with HFMD, γ-H2AX was upregulated in peripheral blood lymphocytes. The consistent <i>in vitro</i> and <i>in vivo</i> data on γ-H2AX from this study suggested that caffeine or other inhibitors of DDR might be novel therapeutic agents for HFMD.
| S-EPMC8786350 | BioStudies
Project description:One of the hallmarks of DNA damage is the rapid spreading of phosphorylated histone H2A (?-H2AX) around a DNA double-strand break (DSB). In the budding yeast Saccharomyces cerevisiae, nearly all H2A isoforms can be phosphorylated, either by Mec1ATR or Tel1ATM checkpoint kinases. We induced a site-specific DSB with HO endonuclease at the MAT locus on chromosome III and monitored the formation of ?-H2AX by chromatin immunoprecipitation (ChIP)-qPCR in order to uncover the mechanisms by which Mec1ATR and Tel1ATM propagate histone modifications across chromatin. With either kinase, ?-H2AX spreads as far as ?50 kb on both sides of the lesion within 1 h; but the kinetics and distribution of modification around the DSB are significantly different. The total accumulation of phosphorylation is reduced by about half when either of the two H2A genes is mutated to the nonphosphorylatable S129A allele. Mec1 activity is limited by the abundance of its ATRIP partner, Ddc2. Moreover, Mec1 is more efficient than Tel1 at phosphorylating chromatin in trans-at distant undamaged sites that are brought into physical proximity to the DSB. We compared experimental data to mathematical models of spreading mechanisms to determine whether the kinases search for target nucleosomes by primarily moving in three dimensions through the nucleoplasm or in one dimension along the chromatin. Bayesian model selection indicates that Mec1 primarily uses a three-dimensional diffusive mechanism, whereas Tel1 undergoes directed motion along the chromatin.
| S-EPMC7474660 | BioStudies
Project description:Eukaryotic cells employ a suite of replication and mitotic checkpoints to ensure the accurate transmission of their DNA. In budding yeast, both the DNA damage checkpoint and the spindle assembly checkpoint (SAC) block cells prior to anaphase. The presence of a single unrepaired double-strand break (DSB) activates ATR and ATM protein kinase homologs Mec1 and Tel1, which then activate downstream effectors to trigger G2/M arrest and also phosphorylate histone H2A (creating gamma-H2AX) in chromatin surrounding the DSB. The SAC monitors proper attachment of spindle microtubules to the kinetochore formed at each centromere and the biorientation of sister centromeres toward opposite spindle pole bodies. Although these two checkpoints sense quite different perturbations, recent evidence has demonstrated both synergistic interactions and cross-talk between them. Here we report that Mad2 and other SAC proteins play an unexpected role in prolonging G2/M arrest after induction of a single DSB. This function of the SAC depends not only on Mec1 and other components of the DNA damage checkpoint but also on the presence of the centromere located > or = 90 kb from the DNA damage. DNA damage induces epigenetic changes at the centromere, including the gamma-H2AX modification, that appear to alter kinetochore function, thus triggering the canonical SAC. Thus, a single DSB triggers a response by both checkpoints to prevent the segregation of a damaged chromosome.
| S-EPMC2811853 | BioStudies
Project description:In Saccharomyces cerevisiae, a single double-strand break (DSB) triggers extensive phosphorylation of histone H2A (known as gammaH2AX) over 50 kb on either side of the DSB. This modification is carried out by either of yeasts checkpoint kinases, the ATM homolog, Tel1, or the ATR homolog, Mec1. In G1-arrested cells, where there is very little 5 to 3 processing of DSB ends, only Tel1 promotes this modification. We have recently described a second modification gammaH2B - the phosphorylation of the C terminal T129 locus of histone H2B which is also carried out by both Mec1 and Tel1 kinases. To understand in detail how gamma-H2AX and gamma-H2B spread along the chromosome from a DSB we have undertaken a high-density analysis of their occupancy where there is a DSB on three different chromosomes. gamma-H2AX and gamma-H2B modifications are similar, but there is a marked absence of gamma-H2B near telomeres. We find that there is reduced gamma-H2AX and gamma-H2B modification over strongly transcribed regions, even taking into account the reduced histone occupancy of these genes. When transcription of the galactose-regulated genes GAL1, GAL10, GAL7 are turned off by the addition of glucose, gamma-H2AX is restored within 5 min; when these genes are again induced, gamma-H2AX is rapidly lost. Regions more distal to the GAL genes have markedly reduced gamma-H2AX levels that rise rapidly when transcription is repressed, suggesting that transcription acts as a barrier to the propagation of gamma-H2AX away from the DSB. The restoration of gamma-H2AX in transcribed regions can be carried out by either Mec1 or Tel1, even 7 h after break induction, suggesting that Tel1 remains associated with damaged chromosomes for an extended time. In addition, we show that gamma-H2AX can be transferred in trans, to regions unlinked to the DSB that lie in close proximity the DSB. Specifically, if a DSB is generated 14 kb from CEN2, gamma-H2AX is transferred to regions around all the other centromeres, in keeping with observed close proximity of all centromere-adjacent chromosome arms. This transfer can be observed even in the absence of formaldehyde crosslinking of the samples.
2013-12-15 | E-MTAB-1409 | ArrayExpress
Project description:There were studies investigating the effects of broadband infrared radiation (IR) on cancer cell, while the influences of middle-infrared radiation (MIR) are still unknown. In this study, a MIR emitter with emission wavelength band in the 3-5 µm region was developed to irradiate A549 lung adenocarcinoma cells. It was found that MIR exposure inhibited cell proliferation and induced morphological changes by altering the cellular distribution of cytoskeletal components. Using quantitative PCR, we found that MIR promoted the expression levels of ATM (ataxia telangiectasia mutated), ATR (ataxia-telangiectasia and Rad3-related and Rad3-related), TP53 (tumor protein p53), p21 (CDKN1A, cyclin-dependent kinase inhibitor 1A) and GADD45 (growth arrest and DNA-damage inducible), but decreased the expression levels of cyclin B coding genes, CCNB1 and CCNB2, as well as CDK1 (Cyclin-dependent kinase 1). The reduction of protein expression levels of CDC25C, cyclin B1 and the phosphorylation of CDK1 at Thr-161 altogether suggest G(2)/M arrest occurred in A549 cells by MIR. DNA repair foci formation of DNA double-strand breaks (DSB) marker ?-H2AX and sensor 53BP1 was induced by MIR treatment, it implies the MIR induced G(2)/M cell cycle arrest resulted from DSB. This study illustrates a potential role for the use of MIR in lung cancer therapy by initiating DSB and blocking cell cycle progression.
| S-EPMC3546001 | BioStudies