Diverse stresses dramatically alter genome-wide p53 binding and transactivation landscape in human cancer cells
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
SUBMITTER: NIEHS Microarray Core
PROVIDER: E-GEOD-46642 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
Publications
Diverse stresses dramatically alter genome-wide p53 binding and transactivation landscape in human cancer cells.
Menendez Daniel D Nguyen Thuy-Ai TA Freudenberg Johannes M JM Mathew Viju J VJ Anderson Carl W CW Jothi Raja R Resnick Michael A MA
Nucleic acids research 20130617 15
The effects of diverse stresses on promoter selectivity and transcription regulation by the tumor suppressor p53 are poorly understood. We have taken a comprehensive approach to characterizing the human p53 network that includes p53 levels, binding, expression and chromatin changes under diverse stresses. Human osteosarcoma U2OS cells treated with anti-cancer drugs Doxorubicin (DXR) or Nutlin-3 (Nutlin) led to strikingly different p53 gene binding patterns based on chromatin immunoprecipitation ...[more]
Similar Datasets
Project description:The effects of diverse stresses on promoter selectivity and transcription regulation by the tumor suppressor p53 are poorly understood. We have taken a comprehensive approach to characterizing the human p53 network that includes p53 levels, binding, expression and chromatin changes under diverse stresses. Human osteosarcoma U2OS cells treated with anti-cancer drugs Doxorubicin or Nutlin-3 led to strikingly different p53 gene binding patterns based on ChIP-seq experiments. While two contiguous RRRCWWGYYY decamers is the consensus binding motif, p53 can bind a single decamer and function in vivo. Although the number of sites bound by p53 was 6-times greater for Nutlin-3 than Doxorubicin, expression changes induced by Nutlin-3 were much less dramatic compared to Doxorubicin. Unexpectedly, the solvent DMSO alone induced p53 binding to many sites common to Doxorubicin; however, this binding had no effect on target gene expression. Together, these data imply a two-stage mechanism for p53 transactivation where p53 binding only constitutes the first stage. Furthermore, both p53 binding and transactivation were associated with increased active histone modification H3K4me3. We discovered 149 putative new p53 target genes including several that are relevant to tumor suppression, revealing potential new targets for cancer therapy and expanding our understanding of the p53 regulatory network. Gene expression analysis (using Affymetrix Human Genome U133 Plus 2.0 GeneChip® arrays) of the p53 response in U2OS cells treated with either Doxorubicin or Nutlin-3, relative to their controls No Treatment and DMSO, respectively.
2013-07-01 | E-GEOD-46493 | biostudies-arrayexpress
Project description:The tumor suppressor p53 plays a crucial role in cellular growth control inducing a plethora of cellular response pathways. The molecular mechanisms that discriminate between the distinct p53-responses towards different stress treatments have remained largely elusive. Here, we have analyzed the p53-regulated pathways induced by two chemotherapeutical treatments, Actinomycin D inducing growth arrest and Etoposide resulting in apoptosis. We found that the genome-wide p53-binding patterns are almost identical upon both treatments notwithstanding transcriptional differences that we observed in genome-wide transcriptome analysis. To assess the role of post-translational modifications in target gene choice and activation we investigated the extent of phosphorylation of Serine 46 of p53 bound to DNA (p53-pS46), a modification that has been linked to apoptosis-pathways, and the extent of phosphorylation of Serine 15 (p53-pS15), a general p53-activation mark. Interestingly, the overall extent of S46 phosphorylation of p53 bound to DNA is considerably higher in cells directed towards apoptosis while the degree of phosphorylation at S15 of DNA bound p53 remains highly similar upon both treatments. Moreover, our data suggest that, following different chemotherapeutical treatments, the extent of chromatin-associated p53 phosphorylated at S46 but not at pS15 is higher on certain apoptosis related target genes, including the BAX and PUMA genes. These data provide evidence that cell fate decisions are not made primarily on the level of general p53 DNA-binding, but possibly through post-translational modifications of chromatin bound p53. ChIP-seq profiles of p53, p53phosphorylated at Serine 15 (p53-pS15) and p53 phosphorylated at Serine 46 (p53-pS46) in U2OS cells treated with either Actinomycin D or Etoposide.
2011-03-24 | E-GEOD-21939 | biostudies-arrayexpress
Project description:The tumor suppressor p53 plays a crucial role in cellular growth control inducing a plethora of cellular response pathways. The molecular mechanisms that discriminate between the distinct p53-responses towards different stress treatments have remained largely elusive. Here, we have analyzed the p53-regulated pathways induced by two chemotherapeutical treatments, Actinomycin D inducing growth arrest and Etoposide resulting in apoptosis. We found that the genome-wide p53-binding patterns are almost identical upon both treatments notwithstanding transcriptional differences that we observed in genome-wide transcriptome analysis. To assess the role of post-translational modifications in target gene choice and activation we investigated the extent of phosphorylation of Serine 46 of p53 bound to DNA (p53-pS46), a modification that has been linked to apoptosis-pathways, and the extent of phosphorylation of Serine 15 (p53-pS15), a general p53-activation mark. Interestingly, the overall extent of S46 phosphorylation of p53 bound to DNA is considerably higher in cells directed towards apoptosis while the degree of phosphorylation at S15 of DNA bound p53 remains highly similar upon both treatments. Moreover, our data suggest that, following different chemotherapeutical treatments, the extent of chromatin-associated p53 phosphorylated at S46 but not at pS15 is higher on certain apoptosis related target genes, including the BAX and PUMA genes. These data provide evidence that cell fate decisions are not made primarily on the level of general p53 DNA-binding, but possibly through post-translational modifications of chromatin bound p53. Microarray analysis (Affymetrix Human Exon array) of the p53 response in U2OS cells treated with either Etoposide or Actinomycin D
2011-03-23 | E-GEOD-22184 | biostudies-arrayexpress
Project description:HCT116 microarray done 12 hours after treatment with DMSO (control) or Nutlin Total RNA from HCT116 cells was harvested with an RNeasy kit (Qiagen) and analyzed on Affymetrix HuGene 1.0 ST arrays following the manufacturer’s instructions. Microarray data were processed using Partek Genomics Suite 6.6. Anova was used to call differentially expressed genes for which any isoform showed a fold change > |+/-1.5| with FDR <0.05. There were 362 genes called as upregulated and 367 genes as downregulated.
2014-05-15 | E-GEOD-53965 | biostudies-arrayexpress
Project description:To evaluate the effect on gene expression by p53 family members and AKR1B10, we overexpressed p53 family members in H1299 cells or knocked down AKR1B10 in HCT116 cells and evaluated the gene expression by microarray analysis. Gene expression was measured in H1299 cells infected with adenovirus expressing p53, p63g and p73b, and in HCT116 cells transfected with siRNAs targeting AKR1B10 and then treated with adriamycin.
2013-10-31 | E-GEOD-47096 | biostudies-arrayexpress
Project description:This SuperSeries is composed of the SubSeries listed below. Refer to individual Series
2011-03-24 | E-GEOD-22186 | biostudies-arrayexpress
Project description:This SuperSeries is composed of the following subset Series: GSE27900: Gene expression analysis of mesenchymal stem cells (MSC), osteoblasts and the U2OS (osteosarcoma) cell line GSE35573: ChIP-seq analysis of H3K4Me3- and H3K27Me3-marked chromatin in mesenchymal stem cells (MSCs), osteoblasts derived from MSCs and the osteosarcoma cell line U2OS Refer to individual Series
2012-03-02 | E-GEOD-35576 | biostudies-arrayexpress
Project description:Many DNA-hypermethylated cancer genes are occupied by the polycomb (PcG) repressor complex in embryonic stem cells (ESCs). Their prevalence in the full spectrum of cancers, the exact context of chromatin involved, and their status in adult cell renewal systems are unknown. Using a genome-wide analysis, we demonstrate that approximately 75% of hypermethylated genes are marked by PcG in the context of bivalent chromatin in both ESC and adult stem/progenitor cells. A large number of these genes are key developmental regulators and a subset, which we call the "DNA hypermethylation module", comprise a portion of the PcG target genes that are downregulated in cancer. Genes with bivalent chromatin have a low, poised gene transcription state that has been shown to maintain stemness and self-renewal in normal stem cells. However, when DNA-hypermethylated in tumors, we find these genes are further repressed. We also show that the methylation status of these genes can cluster important subtypes of colon and breast cancers. By evaluating the subsets of genes that are methylated in different cancers with consideration of their chromatin status in ESCs, we provide evidence that DNA-hypermethylation preferentially targets the subset of PcG genes that are developmental regulators, and this may contribute to the stem-like state of cancer. Additionally, the capacity for global methylation profiling to cluster tumors by phenotype may have important implications for further refining tumor behavior patterns that may ultimately aid therapeutic interventions. The goal was to analyze the chromatin in MSCs, osteoblasts and U2OS cells of genes that are methylated in the osteosarcoma cell line U2OS. Cell culture: MSCs derived from adult bone marrow were cultured and differentiated to osteoblasts as described by Jaiswal et al., 1997 (J Cell Biochem 64, 295-312). U2OS cells were cultured in McCoy's 5A medium supplemented with 10% FBS. ChIP-seq: Cells were crosslinked in 3.7% formaldehyde, lysed and sonicated to obtain chromatin fragments in the range of 200 to 600 base pairs. Sonicated chromatin was incubated with primary antibody, H3K4Me3 (Millipore, 07-473) or H3K27Me3 (kind gift from Thomas Jenuwein/ Nicholas Shukeir), overnight at 4°C, followed by capturing primary antibodies by adding ProteinA/G magnetic beads (DynaBeads) and incubating at room temperature for 3-4 hrs. Captured chromatin was washed in low- and high-salt buffers, as well as TE. Chromatin fragments were subjected to simultaneous elution, de-crosslinking and Proteinase-K treatment at 65°C in elution buffer followed by phenol-chloroform-isoamyl alcohol extraction and ethanol precipitation. Sequencing and alignment: Purified DNA was used to prepare the sequencing library and sequenced on Applied Biosystems SOLiD (V3). Sequencing reads were aligned to hg18 (NCBI 36) using Bioscope 1.2.1. The software guarantees finding all alignments between the first 25 base pairs of the read (seed) and the reference sequence with up to 2 mismatches. Each match is extended to the full length of the read, scoring 1 point for matching and -2 points for mismatching bases. The read is trimmed to the length with the highest score. If there is only one alignment or if an alignment scores significantly higher than the others for the same read, it is considered unique and reported. Peak identification: Aligned reads were analyzed using Model-based Analysis of ChIP-Seq (MACS) to detect regions enriched for the histone marks (called peaks) with default settings. Input was used as background for peak identification.
2012-03-02 | E-GEOD-35573 | biostudies-arrayexpress
Project description:Recently, more than a thousand large intergenic non-coding RNAs (lincRNAs) have been reported. These RNAs are evolutionarily conserved in mammalian genomes and thus presumably function in diverse biological processes. Here, we report the identification of lincRNAs that are regulated by p53. One of these lincRNAs (lincRNA-p21) serves as a repressor in p53-dependent transcriptional responses. Inhibition of lincRNA-p21 affects the expression of hundreds of gene targets enriched for genes normally repressed by p53. The observed transcriptional repression by lincRNA-p21 is mediated through the physical association with hnRNP-K. This interaction is required for proper genomic localization of hnRNP-K at repressed genes and regulation of p53 mediated apoptosis. We propose a model whereby transcription factors activate lincRNAs that serve as key repressors by physically associating with repressive complexes and modulating their localization to sets of previously active genes. Gene expression profiles of different p53 inducible mouse embryonic fibroblasts (p53LSL/LSL MEFs) across different time points after DNA damage. A large intergenic noncoding RNA induced by p53 mediates global gene repression in the p53 transcriptional response. [1] MEF profiling: p53 LSL/LSL MEFs were treated with adenoCRE for p53 restoration or adenoGFP as control for 24h and treated with 500nM doxorubicin for the indicated times. [2] hnRNPKKD profiling: p53 LSL/LSL MEFs were treated with adenoCRE for p53 restoration for 24h, transfected with siRNA oligos targeting different transcripts for 24h and then treated with 100nM doxorubicin for 13h. [3] lincP21KD profiling: p53 LSL/LSL MEFs were treated with adenoCRE for p53 restoration for 24h, transfected with siRNA oligos targeting different transcripts for 24h and then treated with 100nM doxorubicin for 13h. [4] RAS profiling: For p53 restoration, cultured tumor cell lines were incubated with 500nM 4-hydroxytamoxifen for the indicated times.
2010-08-09 | E-GEOD-21761 | biostudies-arrayexpress
Project description:Human colorectal carcinoma-derived cell lines, p53 -/-, +/-, +/+ at 0 and 12 hrs after growth arrest.
2007-09-28 | E-GEOD-90 | biostudies-arrayexpress