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  • Defective p53 response and apoptosis associated with an ataxia-telangiectasia-like phenotype. 16540636

    Ataxia-telangiectasia mutated (ATM), the protein defective in ataxia-telangiectasia, plays a central role in DNA damage response and signaling to cell cycle checkpoints. We describe here a cell line from a patient with an ataxia-telangiectasia-like clinical phenotype defective in the p53 response to radiation but with normal ATM activation and efficient downstream phosphorylation of other ATM substrates. No mutations were detected in ATM cDNA. A normal level of interaction between p53 and peptidyl-prolyl-isomerase Pin1 suggests that posttranslational modification was intact in these cells but operating at reduced level. Defective p53 stabilization was accompanied by defective induction of p53 effector genes and failure to induce apoptosis in response to DNA-damaging agents. Continued association between p53 and murine double minute-2 (Mdm2) occurred in irradiated ATL2ABR cells in response to DNA damage, and incubation with Mdm2 antagonists, nutlins, increased the stabilization of p53 and its transcriptional activity but failed to induce apoptosis. These results suggest that ATM-dependent stabilization of p53 and induction of apoptosis by radiation involve an additional factor(s) that is defective in ATL2ABR cells.
    Document Type:
    Reference
    Product Catalog Number:
    07-164
    Product Catalog Name:
    Anti-phospho-H2A.X (Ser139) Antibody

  • COA 107164

    Document Type:
    Certificate of Analysis
    Product Catalog Number:
    107164

  • Clustering phenotype populations by genome-wide RNAi and multiparametric imaging. 20531400

    Genetic screens for phenotypic similarity have made key contributions to associating genes with biological processes. With RNA interference (RNAi), highly parallel phenotyping of loss-of-function effects in cells has become feasible. One of the current challenges however is the computational categorization of visual phenotypes and the prediction of biological function and processes. In this study, we describe a combined computational and experimental approach to discover novel gene functions and explore functional relationships. We performed a genome-wide RNAi screen in human cells and used quantitative descriptors derived from high-throughput imaging to generate multiparametric phenotypic profiles. We show that profiles predicted functions of genes by phenotypic similarity. Specifically, we examined several candidates including the largely uncharacterized gene DONSON, which shared phenotype similarity with known factors of DNA damage response (DDR) and genomic integrity. Experimental evidence supports that DONSON is a novel centrosomal protein required for DDR signalling and genomic integrity. Multiparametric phenotyping by automated imaging and computational annotation is a powerful method for functional discovery and mapping the landscape of phenotypic responses to cellular perturbations.
    Document Type:
    Reference
    Product Catalog Number:
    05-373
    Product Catalog Name:
    Anti-Cyclin B1 Antibody, clone GNS3 (8A5D12)

  • MeCP2 binds to nucleosome free (linker DNA) regions and to H3K9/H3K27 methylated nucleosomes in the brain. 22144686

    Methyl-CpG-binding protein 2 (MeCP2) is a chromatin-binding protein that mediates transcriptional regulation, and is highly abundant in brain. The nature of its binding to reconstituted templates has been well characterized in vitro. However, its interactions with native chromatin are less understood. Here we show that MeCP2 displays a distinct distribution within fractionated chromatin from various tissues and cell types. Artificially induced global changes in DNA methylation by 3-aminobenzamide or 5-aza-2'-deoxycytidine, do not significantly affect the distribution or amount of MeCP2 in HeLa S3 or 3T3 cells. Most MeCP2 in brain is chromatin-bound and localized within highly nuclease-accessible regions. We also show that, while in most tissues and cell lines, MeCP2 forms stable complexes with nucleosome, in brain, a fraction of it is loosely bound to chromatin, likely to nucleosome-depleted regions. Finally, we provide evidence for novel associations of MeCP2 with mononucleosomes containing histone H2A.X, H3K9me(2) and H3K27me(3) in different chromatin fractions from brain cortex and in vitro. We postulate that the functional compartmentalization and tissue-specific distribution of MeCP2 within different chromatin types may be directed by its association with nucleosomes containing specific histone variants, and post-translational modifications.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • Protein-Protein Interactions Occur Between p53 Phosphoforms and ATM and 53BP1 at Sites of Exogenous DNA Damage. 21361779

    Abstract We have previously shown that the Ser15-phosphorylated p53 phosphoform, p53(Ser15), can localize at sites of ionizing radiation-induced DNA damage. In this study, we hypothesized that the non-specific DNA binding domain (NSDBD) of the p53 carboxy-terminus (C-terminus) mediates chromatin anchoring at sites of DNA damage to interact with two key mediators of the DNA damage response (DDR): ATM and 53BP1. Exogenous YFP-p53 fusion constructs expressing C-terminus deletion mutants of p53 were transfected into p53-null H1299 cells and tracked by microscopy and biochemistry to determine relative chromatin-binding pre- and postirradiation. We observed that exogenous YFP-p53(WT) and YFP-p53(?367?393) associated with ATM(Ser1981) and 53BP1 in the nuclear, chromatin-bound fractions after DNA damage. Of interest, YFP-p53(?1?299) fusion proteins, which lack transcriptional trans-activation and the Ser15-residue, bound to ATM(Ser1981) but not to 53BP1. In support of these data, we used subnuclear UV-microbeam and immunoprecipitation analyses of irradiated normal human fibroblasts (HDFs) that confirmed an interaction between endogenous p53 and ATM or 53BP1. Based on these observations, we propose a model whereby a pre-existing pool of p53 responds immediately to radiation-induced DNA damage using the C-terminus to spatially facilitate protein-protein interactions and the DDR at sites of DNA damage.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • The ATM kinase signaling induced by the low-energy β-particles emitted by (33)P is essential for the suppression of chromosome aberrations and is greater than that induce ... 21315088

    Ataxia-telangiectasia mutated (ATM) encodes a nuclear serine/threonine protein kinase whose activity is increased in cells exposed to low doses of ionizing radiation (IR). Here we examine ATM kinase activation in cells exposed to either (32)P- or (33)P-orthophosphate under conditions typically employed in metabolic labelling experiments. We calculate that the absorbed dose of IR delivered to a 5cm×5cm monolayer of cells incubated in 2ml media containing 1mCi of the high-energy (1.70MeV) β-particle emitter (32)P-orthophosphate for 30min is ∼1Gy IR. The absorbed dose of IR following an otherwise identical exposure to the low-energy (0.24MeV) β-particle emitter (33)P-orthophosphate is ∼0.18Gy IR. We show that low-energy β-particles emitted by (33)P induce a greater number of ionizing radiation-induced foci (IRIF) and greater ATM kinase signaling than energetic β-particles emitted by (32)P. Hence, we demonstrate that it is inappropriate to use (33)P-orthophosphate as a negative control for (32)P-orthophosphate in experiments investigating DNA damage responses to DNA double-strand breaks (DSBs). Significantly, we show that ATM accumulates in the chromatin fraction when ATM kinase activity is inhibited during exposure to either radionuclide. Finally, we also show that chromosome aberrations accumulate in cells when ATM kinase activity is inhibited during exposure to ∼0.36Gy β-particles emitted by (33)P. We therefore propose that direct cellular exposure to (33)P-orthophosphate is an excellent means to induce and label the IR-induced, ATM kinase-dependent phosphoproteome.Copyright © 2011 Elsevier B.V. All rights reserved.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • Estrogen receptor α-mediated transcription induces cell cycle-dependent DNA double-strand breaks. 21112959

    Prolonged exposure to estrogen increases breast cancer risk. Estrogen is known to induce chromosomal aberrations, yet the mechanisms by which estrogen promotes genomic instability are not fully understood. Here, we show that exposure of MCF-7 cells to 17β-estradiol (E2) induces DNA double-strand breaks (DSBs), as determined by the formation of γH2AX foci. Foci formation was dependent upon estrogen receptor-α (ERα) and the catalytic activity of the type II topoisomerase, topoisomerase IIβ (topoIIβ). Moreover, we show by chromatin immunoprecipitation that topoIIβ-dependent E2-induced γH2AX localizes to the promoter of the estrogen-inducible gene, trefoil factor 1. E2-induced foci were associated with cyclin A expression and inhibited by pre-incubation with the DNA polymerase inhibitor aphidicolin suggesting that E2-induced DSBs are mediated by progression through S phase. Furthermore, E2-induced γH2AX foci colocalized with Rad51, suggesting that E2-induced DSBs are repaired by homologous recombination. We propose that DNA DSBs formed by the strand-cleaving activity of the topoIIβ-DNA cleavage complex at estrogen-inducible genes can present a barrier to DNA replication, leading to persistent DNA DSBs in ERα-positive breast cancer cells.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • Meiotic cohesin complexes are essential for the formation of the axial element in mice. 22711701

    Cohesin is a conserved multisubunit protein complex that participates in chromosome segregation, DNA damage repair, chromatin regulation, and synaptonemal complex (SC) formation. Yeast, but not mice, depleted of the cohesin subunit Rec8 are defective in the formation of the axial elements (AEs) of the SC, suggesting that, in mammals, this function is not conserved. In this paper, we show that spermatocytes from mice lacking the two meiosis-specific cohesin subunits RAD21L and REC8 were unable to initiate RAD51- but not DMC1-mediated double-strand break repair, were not able to assemble their AEs, and arrested as early as the leptotene stage of prophase I, demonstrating that cohesin plays an essential role in AE assembly that is conserved from yeast to mammals.
    Document Type:
    Reference
    Product Catalog Number:
    07-164
    Product Catalog Name:
    Anti-phospho-H2A.X (Ser139) Antibody

  • Constructive Technology Assessment (CTA) as a tool in coverage with evidence development: the case of the 70-gene prognosis signature for breast cancer diagnostics. 19126254

    Constructive Technology Assessment (CTA) is a means to guide early implementation of new developments in society, and can be used as an evaluation tool for Coverage with Evidence Development (CED). We used CTA for the introduction of a new diagnostic test in the Netherlands, the 70-gene prognosis signature (MammaPrint) for node-negative breast cancer patients.
    Document Type:
    Reference
    Product Catalog Number:
    07-164
    Product Catalog Name:
    Anti-phospho-H2A.X (Ser139) Antibody