Transcription profiling by high throughput sequencing of Arabidopsis estradiol-inducible tor-es mutant
Ontology highlight
ABSTRACT: To validate that the transcriptional program rapidly activated by PP242, WYE354, KU63794 and Torin2 was specifically associated with the complete inhibition of TOR kinase, we performed genome-wide transcript profiling using the estradiol-inducible tor-es mutant after reaching complete TOR protein silencing in Arabidopsis seedlings grown in the full-nutrient medium. The estradiol inducible tor-es mutant was treated without/with estradiol for 72 hours to complete inhibition of TOR kinase. And RNA-seq was performed for comparison the transcriptomes changes (http://gbnci.abcc.ncifcrf.gov/geo/gse_view.php?ID=58096).
Project description:Using Western blot, we found the level of H3K27me3, but not H3K4me3, H3K9me2 and H3K36me3, was specifically reduced in the tor-es mutant. To gain a genome-wide view of the effects of TOR activity on H3K27me3 distribution, we performed quantitative chromatin immunoprecipitation with an exogenous reference genome (ChIP-Rx) followed by deep-sequencing. We find a global reduction of H3K27me3 occupancy in tor-es, whereas the H3K9me2 level was largely unaffected. These results suggest that TOR may be a specific and direct regulator of global deposition of H3K27me3. To investigate the function of TOR phosphorylation of FIE, we complemented the heterozygous fie/+ plants with GFP-FIE or the phosphorylation site mutant (SSTS/AAAA) under the control of the FIE promoter. To provide a parallel comparison with SSTS/AAAA/fie, we generated estradiol-inducible fie-amiR-es transgenic lines, that eliminated FIE protein. Quantitative ChIP-seq analyses revealed greatly reduced H3K27me3 levels across the genome in SSTS/AAAA and fie-amiR-es mutants. And transcriptome profiling by RNA-Seq was conducted to globally identify thousands of genes coordinately dysregulated in the shoots of SSTS/AAAA and fie-amiR-es plants. Furthermore, gene Ontology analysis of 986 TOR-FIE-PRC2 target genes revealed significant enrichment for transcription factors/regulators controlling a broad spectrum of developmental programs.
Project description:The aim was to identify genes associated to the down-regulation TARGET OF RAPAMYCIN (TOR) pathway in Arabidopsis. For this purpose, three independent amiR-tor lines (amiR-tor9, amiR-tor17 and amiR-tor 20) and EV lines 3 and 6 days after EST- or non-induction were used for expression profile analysis using Affymetrix microarray. Two weeks-old transgenic seedlings were transferred to MS plate with 20µM estradiol to induce the overexpression of amiR-tor (consequently, repression of AtTOR levels) under the control of an estradiol-(EST) inducible promoter. Identically treated wild-type Col-0 and pER8 empty-vector (EV) transformed seedlings were served as controls. Seedlings were harvested after 3 and 6 (amiR-tor lines) days.
Project description:The aim was to identify genes associated to the down-regulation TARGET OF RAPAMYCIN (TOR) pathway in Arabidopsis. For this purpose, three independent amiR-tor lines (amiR-tor9, amiR-tor17 and amiR-tor 20) and EV lines 3 and 6 days after EST- or non-induction were used for expression profile analysis using Affymetrix microarray. Two weeks-old transgenic seedlings were transferred to MS plate with 20µM estradiol to induce the overexpression of amiR-tor (consequently, repression of AtTOR levels) under the control of an estradiol-(EST) inducible promoter. Identically treated wild-type Col-0 and pER8 empty-vector (EV) transformed seedlings were served as controls. Seedlings were harvested after 3 and 6 (amiR-tor lines) days. genetic modification
Project description:The Tor kinase is one of the major regulatory nodes in a eukaryotic cell and until now, little is known about this kinase in filamentous fungi. Here, we analysed the Tor kinase in Aspergillus fumigatus, which is the most important airborne fungal pathogen of humans and the main causative agent of invasive aspergillosis. Because deletion of the single tor gene of A. fumigatus was apparently lethal, we generated a conditional lethal tor mutant. This was achieved by fusing the tor gene with the inducible xylose promoter and replacing the endogenous tor gene by the inducible xylp-tor gene cassette. The generated transgenic strain opened up the possibility to activate or silence the tor gene under controlled conditions. Since we did not observe any effect of rapamycin on Tor of A. fumigatus, the generation of the inducible Tor gene was even more valuable for an in-depth analysis of the Tor kinase. By LC-MS/MS analyses of mycelial proteins we identified the regulatory network of Tor. Tor controls a variety of genes and proteins involved in nutrient sensing, stress response, cell cycle progression, protein biosynthesis and degradation. Tor is a major kinase responsible for protein import into the mitochondria, and consequently responsible for the correct function of most processes occurring into these organelles, e.g. respiration and ornithine metabolism. Regulation of iron acquisition by Tor was found to be independent of the HapX transcription factor.
Project description:The goal of this experiment was to explore the molecular network of glucose-TOR signaling in Arabidopsis seedling autotrophic transition stage. We used the whole-genome microarrays to detail the global program of gene expression mediated by glucose and TOR. Arabidopsis WT and estradiol inducible RNAi-tor seedlings were germinated in low light condition for 3 days to deplete seed nutrient supply. Fifteen mM glucose was adding back to reactivate the quiescent seedlings and the samples were harvested for RNA isolation. Transcript profiles were analyzed and compared between with and without glucose treatment to identify early glucose-TOR target genes. Each experimental condition was repeated three times and total 12 samples were generated.
Project description:To screen miRNAs specifically regulated by mTORC1 or mTORC2, a global miRNA expression profile in MCF-7 cells treated with rapamycin or PP242 (mTORC1/2 kinase inhibitor) was developed using microarray. control, rapamycin or PP242 treated human MCF-7 cells were harvested 48h post-treatment and subjected to total RNA extraction.
Project description:gnp_blan06_torpten - rnai tor time course - The impact of the TOR pathway on growth and stress responses. Modification of translational and transcriptional profiles in Tor-inducible RNAi mutants and identification of TOR targets.
Project description:To screen miRNAs specifically regulated by mTORC1 or mTORC2, a global miRNA expression profile in MCF-7 cells treated with rapamycin or PP242 (mTORC1/2 kinase inhibitor) was developed using microarray.
Project description:The mammalian target of rapamycin (mTOR) is a central regulator of cell growth and proliferation in response to growth factor and nutrient signaling. Consequently, this kinase is implicated in metabolic diseases including cancer and diabetes so there is great interest in understanding mTOR regulatory networks. mTOR exists in two functionally distinct complexes, mTORC1 and mTORC2, and whereas the natural product rapamycin only inhibits a subset of mTORC1 functions, recently developed ATP-competitive mTOR inhibitors have revealed new roles for both complexes. To examine the complete spectrum of mTOR responsive cellular processes, we compared the transcriptional profiles of mammalian cells treated with rapamycin versus the ATP-competitive inhibitor PP242. Our analysis provides a genome-wide view of the transcriptional outputs of mTOR signaling that are insensitive to rapamycin. Gene expression in mouse NIH3T3 cells was measured after 18 hour treatment with DMSO (control), 50 nM rapamycin, or 2 uM PP242. Four independent experiments were performed for each condition.
Project description:gnp_blan06_torpten - rnai tor time course - The impact of the TOR pathway on growth and stress responses. Modification of translational and transcriptional profiles in Tor-inducible RNAi mutants and identification of TOR targets. 28 dye-swaps - normal vs. rnai mutant comparison.