Browse
Submit Data
Databases
API
Help

Dataset Information

8 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

BRCA1 Forms a Functional Complex with γ-H2AX as a Late Response to Genotoxic Stress.

ABSTRACT: Following genotoxic stress, the histone H2AX becomes phosphorylated at serine 139 by the ATM/ATR family of kinases. The tumor suppressor BRCA1, also phosphorylated by ATM/ATR kinases, is one of several proteins that colocalize with phospho-H2AX (γ-H2AX) at sites of active DNA repair. Both the precise mechanism and the purpose of BRCA1 recruitment to sites of DNA damage are unknown. Here we show that BRCA1 and γ-H2AX form an acid-stable biochemical complex on chromatin after DNA damage. Maximal association of BRCA1 with γ-H2AX correlates with reduced global γ-H2AX levels on chromatin late in the repair process. Since BRCA1 is known to have E3 ubiquitin ligase activity in vitro, we examined H2AX for evidence of ubiquitination. We found that H2AX is ubiquitinated at lysines 119 and 119 in vivo and that blockage of 26S proteasome function stabilizes γ-H2AX levels within cells. When BRCA1 levels were reduced, ubiquitination of H2AX was also reduced, and the cells retained higher levels of phosphorylated H2AX. These results indicate that BRCA1 is recruited into stable complexes with γ-H2AX and that the complex is involved in attenuation of the γ-H2AX repair signal after DNA damage.

SUBMITTER: Krum SA

PROVIDER: S-EPMC2948912 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

The WSTF-ISWI chromatin remodeling complex transiently associates with the human inactive X chromosome during late S-phase prior to BRCA1 and γ-H2AX.

Project description:Replicating the genome prior to each somatic cell division not only requires precise duplication of the genetic information, but also accurately reestablishing the epigenetic signatures that instruct how the genetic material is to be interpreted in the daughter cells. The mammalian inactive X chromosome (Xi), which is faithfully inherited in a silent state in each daughter cell, provides an excellent model of epigenetic regulation. While much is known about the early stages of X chromosome inactivation, much less is understood with regards to retaining the Xi chromatin through somatic cell division. Here we report that the WSTF-ISWI chromatin remodeling complex (WICH) associates with the Xi during late S-phase as the Xi DNA is replicated. Elevated levels of WICH at the Xi is restricted to late S-phase and appears before BRCA1 and γ-H2A.X. The sequential appearance of WICH and BRCA1/γ-H2A.X implicate each as performing important but distinct roles in the maturation and maintenance of heterochromatin at the Xi.

| S-EPMC3498190 | biostudies-literature

Intercellular communication of cellular stress monitored by gamma-H2AX induction.

Project description:When cells are exposed to ionizing radiation (IR), unexposed cells that share media with damaged cells exhibit similar effects to irradiated cells including increased levels of DNA double-strand breaks (DSBs). Hypothesizing that this effect, known as the radiation-induced bystander effect, may be a specific instance of communication between damaged and undamaged cells regardless of damage source, we demonstrated that exposure of target cells to non-IR induces bystander damage in non-targeted cells as measured by gamma-H2AX and 53BP1 focal formation. Initially, bystander damage was found primarily in S-phase cells, but at later times, non-S-phase cells were also affected. In addition, media from undamaged malignant and senescent cells also was found to induce DSBs in primary cultures. Media conditioned on cells targeted with either ionizing or non-IR as well as on undamaged malignant and senescent cells contained elevated levels of several cytokines. One of these, transforming growth factor beta (TGF-beta), and nitric oxide (NO) were found to elevate numbers of gamma-H2AX/53BP1 foci in normal cell cultures similar to levels found in bystander cells, and this elevation was abrogated by NO synthase inhibitors, TGF-beta blocking antibody and antioxidants. These findings support the hypothesis that damage in bystander cells results from their exposure to cytokines or reactive compounds released from stressed cells, regardless of damage source. These results have implications for oncogenesis in that they indicate that damaged normal cells or undamaged tumor cells may induce genomic instability, leading to an increased risk of oncogenic transformation in other cells with which they share media or contact directly.

| S-EPMC2757548 | biostudies-literature

A PP4-phosphatase complex dephosphorylates gamma-H2AX generated during DNA replication.

Project description:The histone H2A variant H2AX is rapidly phosphorylated in response to DNA double-stranded breaks to produce gamma-H2AX. gamma-H2AX stabilizes cell-cycle checkpoint proteins and DNA repair factors at the break site. We previously found that the protein phosphatase PP2A is required to resolve gamma-H2AX foci and complete DNA repair after exogenous DNA damage. Here we describe a three-protein PP4 phosphatase complex in mammalian cells, containing PP4C, PP4R2, and PP4R3beta, that specifically dephosphorylates ATR-mediated gamma-H2AX generated during DNA replication. PP4 efficiently dephosphorylates gamma-H2AX within mononucleosomes in vitro and does not directly alter ATR or checkpoint kinase activity, suggesting that PP4 acts directly on gamma-H2AX in cells. When the PP4 complex is silenced, repair of DNA replication-mediated breaks is inefficient, and cells are hypersensitive to DNA replication inhibitors, but not radiomimetic drugs. Therefore, gamma-H2AX elimination at DNA damage foci is required for DNA damage repair, but accomplishing this task involves distinct phosphatases with potentially overlapping roles.

| S-EPMC3242369 | biostudies-literature

Phosphoproteomic analysis reveals plant DNA damage signalling pathways with a functional role for histone H2AX phosphorylation in plant growth under genotoxic stress.

Project description:DNA damage responses are crucial for plant growth under genotoxic stress. Accumulating evidence indicates that DNA damage responses differ between plant cell types. Here, quantitative shotgun phosphoproteomics provided high-throughput analysis of the DNA damage response network in callus cells. MS analysis revealed a wide network of highly dynamic changes in the phosphoprotein profile of genotoxin-treated cells, largely mediated by the ATAXIA TELANGIECTASIA MUTATED (ATM) protein kinase, representing candidate factors that modulate plant growth, development and DNA repair. A C-terminal dual serine target motif unique to H2AX in the plant lineage showed 171-fold phosphorylation that was absent in atm mutant lines. The physiological significance of post-translational DNA damage signalling to plant growth and survival was demonstrated using reverse genetics and complementation studies of h2ax mutants, establishing the functional role of ATM-mediated histone modification in plant growth under genotoxic stress. Our findings demonstrate the complexity and functional significance of post-translational DNA damage signalling responses in plants and establish the requirement of H2AX phosphorylation for plant survival under genotoxic stress.

| S-EPMC6900162 | biostudies-literature

An optimized method for detecting gamma-H2AX in blood cells reveals a significant interindividual variation in the gamma-H2AX response among humans.

Project description:Phosphorylation of histone H2AX on serine 139 (gamma-H2AX, gammaH2AX) occurs at sites flanking DNA double-strand breaks (DSBs) and can provide a measure of the number of DSBs within a cell. Here we describe a rapid and simple flow-cytometry-based method, optimized to measure gamma-H2AX in non-fixed peripheral blood cells. No DSB induced signal was observed in H2AX-/- cells indicating that our FACS method specifically recognized gamma-H2AX accumulation. The gamma-H2AX assay was capable of detecting DNA damage at levels 100-fold below the detection limit of the alkaline comet assay. The gamma-H2AX signal was quantitative with a linear increase of the gamma-H2AX signal over two orders of magnitude. We found that all nucleated blood cell types examined, including the short-lived neutrophils induce gamma-H2AX in response to DSBs. Interindividual difference in the gamma-H2AX signal in response to ionizing radiation and the DSB-inducing drug calicheamicin was almost 2-fold in blood cells from patients, indicating that the amount of gamma-H2AX produced in response to a given dose of radiation varies significantly in the human population. This simple method could be used to monitor response to radiation or DNA-damaging drugs.

| S-EPMC1865071 | biostudies-literature

Wip1 directly dephosphorylates gamma-H2AX and attenuates the DNA damage response.

Project description:The integrity of DNA is constantly challenged throughout the life of a cell by both endogenous and exogenous stresses. A well-organized rapid damage response and proficient DNA repair, therefore, become critically important for maintaining genomic stability and cell survival. When DNA is damaged, the DNA damage response (DDR) can be initiated by alterations in chromosomal structure and histone modifications, such as the phosphorylation of the histone H2AX (the phosphorylated form is referred to as gamma-H2AX). gamma-H2AX plays a crucial role in recruiting DDR factors to damage sites for accurate DNA repair. On repair completion, gamma-H2AX must then be reverted to H2AX by dephosphorylation for attenuation of the DDR. Here, we report that the wild-type p53-induced phosphatase 1 (Wip1) phosphatase, which is often overexpressed in a variety of tumors, effectively dephosphorylates gamma-H2AX in vitro and in vivo. Ectopic expression of Wip1 significantly reduces the level of gamma-H2AX after ionizing as well as UV radiation. Forced premature dephosphorylation of gamma-H2AX by Wip1 disrupts recruitment of important DNA repair factors to damaged sites and delays DNA damage repair. Additionally, deletion of Wip1 enhances gamma-H2AX levels in cells undergoing constitutive oncogenic stress. Taken together, our studies show that Wip1 is an important mammalian phosphatase for gamma-H2AX and shows an additional mechanism for Wip1 in the tumor surveillance network.

| S-EPMC2904079 | biostudies-literature

Genome-wide characterization reveals complex interplay between TP53 and TP63 in response to genotoxic stress.

Project description:In response to genotoxic stress the TP53 tumour suppressor activates target gene expression to induce cell cycle arrest or apoptosis depending on the extent of DNA damage. These canonical activities can be repressed by TP63 in normal stratifying epithelia to maintain proliferative capacity or drive proliferation of squamous cell carcinomas, where TP63 is frequently overexpressed/amplified. Here we use ChIP-sequencing, integrated with microarray analysis, to define the genome-wide interplay between TP53 and TP63 in response to genotoxic stress in normal cells. We reveal that TP53 and TP63 bind to overlapping, but distinct cistromes of sites through utilization of distinctive consensus motifs and that TP53 is constitutively bound to a number of sites. We demonstrate that cisplatin and adriamycin elicit distinct effects on TP53 and TP63 binding events, through which TP53 can induce or repress transcription of an extensive network of genes by direct binding and/or modulation of TP63 activity. Collectively, this results in a global TP53-dependent repression of cell cycle progression, mitosis and DNA damage repair concomitant with activation of anti-proliferative and pro-apoptotic canonical target genes. Further analyses reveal that in the absence of genotoxic stress TP63 plays an important role in maintaining expression of DNA repair genes, loss of which results in defective repair.

| S-EPMC4041465 | biostudies-literature

RNF168 forms a functional complex with RAD6 during the DNA damage response.

Project description:Protein ubiquitination plays an important role in initiating the DNA damage response. Following DNA damage, E2 ubiquitin conjugating enzymes are crucial for catalyzing substrate ubiquitination that recruits downstream DNA repair factors to DNA lesions. To identify novel E2 conjugating enzymes important for initiating the DNA-damage-induced ubiquitination cascade, we screened most of the known E2 enzymes and found that RAD6A and RAD6B function together with RNF168 in the ionizing radiation (IR)-induced DNA damage response. Similarly to RNF168-deficient cells, RAD6A- or RAD6B-deficient cells exhibit a reduction in DNA-damage-induced protein ubiquitination. Correspondingly, DNA-damage-induced foci formation of DNA damage repair proteins, such as BRCA1 and 53BP1, is impaired in the absence of RAD6A or RAD6B. Moreover, the RNF168-RAD6 complex targeted histone H1.2 for ubiquitination in vitro and regulated DNA-damage-induced histone H1.2 ubiquitination in vivo. Collectively, these data demonstrate that RNF168, in complex with RAD6A or RAD6B, is activated in the DNA-damage-induced protein ubiquitination cascade.

| S-EPMC3666256 | biostudies-literature

Whole-genome landscape of histone H2AX and γ-H2AX

Project description:Phosphorylation of the histone variant H2AX forms γ-H2AX that marks DNA double-strand break (DSB). Here we generated the sequencing-based maps of H2AX and γ-H2AX positioning in resting and proliferating cells before and after ionizing irradiation. Genome-wide locations of possible endogenous and exogenous DSBs were identified based on γ-H2AX distribution in dividing cancer cells without irradiation and that in resting cells upon irradiation, respectively. γ-H2AX-enriched regions of endogenous origin in replicating cells included telomeres and active transcription start sites, apparently reflecting replication- and transcription-mediated stress during rapid cell division. Surprisingly, H2AX itself, prior to phosphorylation, was specifically located at these endogenous hotspots. This phenomenon was only observed in dividing cancer cells but not in resting cells. Endogenous H2AX was concentrated on the transcription start site of actively transcribed genes but was irrelevant to pausing of RNA polymerase II (pol II), which precisely coincided with γ-H2AX of endogenous origin. γ-H2AX enrichment upon irradiation also coincided with actively transcribed regions, but unlike endogenous γ-H2AX, it extended into the gene body and was not specifically concentrated on the pausing site of pol II. Subtelomeres were not responsive to external DNA damage. Our findings provide insight into DNA repair programs of cancer and may have implications for cancer therapy.

2012-04-04 | GSE25577 | GEO

Cotranscriptional exon skipping in the genotoxic stress response

Project description:Pre-mRNA splicing is functionally coupled to transcription, and genotoxic stresses can enhance alternative exon inclusion by affecting elongating RNA polymerase II. We report here that various genotoxic stress inducers, including camptothecin, inhibit the interaction between EWS, an RNA polymerase II-associated factor, and YB-1, a spliceosome-associated factor. This results in the cotranscriptional skipping of several exons of the MDM2 gene encoding the main p53 ubiquitin-ligase. This reversible exon skipping participates in the timely regulation of MDM2 expression, and may contribute to the accumulation of p53 during stress exposure and its rapid shut off when stress is removed. Finally, a splicing-sensitive microarray identified numerous exons that are skipped in response to camptothecin and EWS/YB-1 depletion. These data demonstrate genotoxic stress-induced alteration of the communication between the transcriptional and splicing machineries, resulting in widespread exon skipping and playing a central role in the genotoxic stress response. 6 samples of MCF7 cells exposed to different treatments were analyzed: 3 x control_6 hours; 3 x camptothecin_6 hours.

| S-ECPF-GEOD-21840 | biostudies-other

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data