Transcriptome and translatome profiling of MCF7 vector cells after p53 activation
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE50650 | GEO | 2014/04/17
SECONDARY ACCESSION(S): PRJNA218203
REPOSITORIES: GEO
Similar Datasets
Project description:Several genome-wide transcriptome analyses that focused on p53-induced cellular responses in many cellular contexts have continued to expand the already vast p53-regulated transcriptional networks. To investigate post-transcriptional controls as an additional dimension of p53-directed gene expression responses we performed translatome analysis by polysomal profiling on MCF7 cells treated with Doxorubicin and Nutlin-3a. A comparison between the transcriptome and the translatome revealed a large number of uncoupled genes, whose transcription changes did not correlate with translation changes. Overall, we establish p53 as a master regulator of translational control and identify many p53 target genes affecting translation that can contribute to p53-dependent cellular responses. Keywords: p53, transcriptome, translatome, polysomal RNA, subpolysomal RNA, uncoupling, Doxorubicin, Nutlin-3a Total RNA (tot) was extracted from MCF7 vector cells after 16h of treatment with Doxorubicin (1.5uM) and Nutlin-3a (10uM) or DMSO (solvent, as control treatment). Polysomal profiling was performed after the same conditions. We collected all subpolysomal mRNA fractions (sub) and the polysomal ones (pol) after sucrose gradient fractionation of cytoplasmic lysates to analyze separately mRNAs that are not actively translated from those that are considered in active translation, respectively. Experiments were performed in three biological replicates.
2014-04-17 | E-GEOD-50650 | biostudies-arrayexpress
Project description:Translatome analysis by sucrose gradient centrifugation of cell lysates followed by microarray profiling of the polysomal and subpolysomal RNA fractions represents a way of both studying translational control networks and better approximating the proteomic representation of cells. It is an established notion that translational control takes place essentially at the translation initiation level, therefore the variation in abundance of a given mRNA species on polysomes can be directly related to the variation in abundance of the corresponding protein. Comparison of translatome profile changes with corresponding transcriptome profile changes can provide a measure of the degree of concordance between cellular controls affecting mRNA abundance and cellular controls affecting mRNA availability to translation. To provide a direct experimental evaluation of the phenomenon, we decided to study a classical example of transcriptional reprogramming of gene expression: Epidermal Growth Factor (EGF) treatment. This stimulus triggers a well known chain of intracellular transduction events, ultimately resulting in a multifaceted phenotypic spectrum of changes with prevalent induction of cell growth and proliferation. We subjected HeLa cells to serum starvation for 12h and then we added EGF at final concentration of 1 μg/ml, profiling before and after 40 minutes of treatment the transcriptome, the translatome, coming from the polysomal pool of mRNAs after sucrose gradient separation, and also the mRNA content of the subpolysomal pool, expected not to be actively translated. Keywords: translatome profiling, polysomal profiling, polysomal RNA, translational control, translational profiling, polysome profiling, post-transcriptional regulation, EGF starvation release. The comparison between transcriptional and polysomal profiling was used for the discovery of general and mRNA-specific changes in the translation state of the serum starved HeLa cells transcriptome in response to EGF stimulus. To identify translationally regulated mRNA molecules, gene expression signals derived from the polysomal and subpolysomal RNA populations were compared by microarrays analysis to those obtained from unfractionated total RNAs. Polysomal RNA, subpolysomal RNA and total RNA were isolated from HeLa cells serum starved and treated with EGF. Cells lysates were collected before (t = 0 min) and after (t = 40 min) EGF treatment. All experiments were run in triplicates.
2010-05-27 | E-GEOD-20277 | biostudies-arrayexpress
Project description:Translatome analysis by sucrose gradient centrifugation of cell lysates followed by microarray profiling of the polysomal and subpolysomal RNA fractions represents a way of both studying translational control networks and better approximating the proteomic representation of cells. It is an established notion that translational control takes place essentially at the translation initiation level, therefore the variation in abundance of a given mRNA species on polysomes can be directly related to the variation in abundance of the corresponding protein. Comparison of translatome profile changes with corresponding transcriptome profile changes can provide a measure of the degree of concordance between cellular controls affecting mRNA abundance and cellular controls affecting mRNA availability to translation. To provide a direct experimental evaluation of the phenomenon, we decided to study a classical example of transcriptional reprogramming of gene expression: Epidermal Growth Factor (EGF) treatment. This stimulus triggers a well known chain of intracellular transduction events, ultimately resulting in a multifaceted phenotypic spectrum of changes with prevalent induction of cell growth and proliferation. We subjected HeLa cells to serum starvation for 12h and then we added EGF at final concentration of 1 μg/ml, profiling before and after 40 minutes of treatment the transcriptome, the translatome, coming from the polysomal pool of mRNAs after sucrose gradient separation, and also the mRNA content of the subpolysomal pool, expected not to be actively translated. Keywords: translatome profiling, polysomal profiling, polysomal RNA, translational control, translational profiling, polysome profiling, post-transcriptional regulation, EGF starvation release.
2010-05-09 | GSE20277 | GEO
Project description:In most lymphomas, p53 signaling pathway is inactivated by various mechanisms independent to p53 gene mutations or deletions. In many cases, p53 function is largely regulated by alterations in the protein abundance levels by the action of E3 ubiquitin-protein ligase MDM2, targeting p53 to proteasome-mediated degradation. In the present study, an integrating transcriptomics and pro-teomics analysis was employed to investigate the effect of p53 activation by a small-molecule MDM2-antagonist, nutlin 3a, on three lymphoma cell models following p53 activation. Our analy-sis revealed a system-wide nutlin 3a-associated effect in all examined lymphoma types, identifying in total of 4037 differentially affected proteins involved in a plethora of pathways, with significant heterogeneity among lymphomas. Our findings include known p53-targets and novel p53 activa-tion effects, involving transcription, translation, or degradation of protein components of path-ways, such as a decrease in key members of PI3K/mTOR pathway, heat-shock response, and gly-colysis, and an increase in key members of oxidative phoshosphorylation, autophagy and mito-chondrial translation. Combined inhibition of HSP90 or PI3K/mTOR pathway with nutlin 3a-mediated p53-activation enhanced the apoptotic effects suggesting a promising strategy against human lymphomas. Integrated omic profiling after p53 activation offered novel insights on the regulatory role specific proteins and pathways may have in lymphomagenesis.
2023-07-30 | PXD009290 | Pride
Project description:We treated Jurkat cells for 48 hr with a sublethal dose of FK866 (5 nM) and DMSO (Mock, control treatment). RNA samples for microarrays derived from fractionated samples by sucrose gradient (sub-polysomes, polysomes), giving us the chance to perform an analysis among polysomal/subpolysomal distribution in treated or untreated cells and the possibility to identify the multi-level gene expression regulation effects of FK866. We are interested to find differentially expressed genes, in the early phase of cell response to FK866, and genes that account for a specific post-transcriptional regulation exerted by the cell in response to the drug. Keywords: polysomal profiling, translatome profiling, polysomal RNA, subpolysomal RNA, translational profiling, polysome profiling, post-transcriptional regulation, FK866, translational efficiency. Gene expression signals derived from the polysomal and subpolysomal RNA populations were compared by microarrays analysis to those obtained from total RNAs (derived from the sum of all the fractions in the polysomal gradient). Polysomal RNA, subpolysomal RNA and total RNA were isolated from Jurkat cells treated with FK866 5 nM or DMSO (mock, control treatment) for 48 hr. Cells lysates were collected from control cells (mock) and from treated cells (FK866). All experiments were run in biological quadruplicates.
2015-04-01 | E-GEOD-53772 | biostudies-arrayexpress
Project description:RNA-seq and ChIP-seq on MCF-7 breast cancer cell line upon activation of p53 by the non-genotoxic small molecule Nutlin-3a ChIP-seq on p53 in MCF7 with Nutlin-3a stimulation (S) in triplicate, and the control (input), in stimulated and non stimulated, using illumina HiSeq 2000
2014-05-27 | E-GEOD-47041 | biostudies-arrayexpress
Project description:This study has examined the molecular mechanisms underlying sensitivity of sarcomas to Nutlin-3a, a non-genotoxic activator of the p53 pathway. Human patient material was collected immediately following surgical resection, dissected into small pieces and ex planted onto gelatin sponges immersed in media containing either vehicle control or Nutlin-3a (10uM and/or 50uM) for 48 hours. Nutlin-3a represents a novel targeted therapy for the treatment of sarcomas. We have examined expression profiles of genes upregulated in sarcoma patient derived tissues following nutlin-3a treatment ex vivo 16 samples ((Vehicle control and Nutlin-3a (10uM and 50uM) treated samples)) from 6 sarcoma patients
2014-03-10 | E-GEOD-48296 | biostudies-arrayexpress
Project description:We treated Jurkat cells for 48 hr with a sublethal dose of FK866 (5 nM) and DMSO (Mock, control treatment). RNA samples for microarrays derived from fractionated samples by sucrose gradient (sub-polysomes, polysomes), giving us the chance to perform an analysis among polysomal/subpolysomal distribution in treated or untreated cells and the possibility to identify the multi-level gene expression regulation effects of FK866. We are interested to find differentially expressed genes, in the early phase of cell response to FK866, and genes that account for a specific post-transcriptional regulation exerted by the cell in response to the drug. Keywords: polysomal profiling, translatome profiling, polysomal RNA, subpolysomal RNA, translational profiling, polysome profiling, post-transcriptional regulation, FK866, translational efficiency.
2015-04-01 | GSE53772 | GEO
Project description:We sequenced mRNA from HCT116 p21-/- cells treated with Nutlin-3a, doxorubicin, or DMSO for 24 h.
2016-06-09 | GSE77841 | GEO
Project description:Telomere erosion contributes to age-associated tissue dysfunction and senescence, and p53 plays a crucial role in this response. We undertook a genome-wide screen to identify gene deletions that sensitized p53-positive human cells to loss of telomere integrity, and uncovered a previously unannotated gene, C16ORF72, which we term Telomere Attrition and p53 Response 1: TAPR1. CRISPR-Cas9 mediated deletion of TAPR1 led to elevated p53 and induction of p53 transcriptional targets. TAPR1-disrupted cells exhibited a synthetic-sick relationship with the loss of telomerase, or treatment with the topoisomerase II inhibitor doxorubicin. Stabilization of p53 with nutlin-3a further decreased cell fitness in cells lacking TAPR1 or telomerase, whereas deletion of TP53 rescued the decreased fitness of TAPR1-deleted cells. We propose that TAPR1 regulates p53 turnover, thereby tapering the p53-dependent response to telomere erosion. We discuss the possible implications of such a mechanism in the preservation of genome integrity during senescence or aging.
2021-03-04 | PXD022128 | Pride