Hierarchical regulation in a KRAS pathway-dependent transcriptional network revealed by a reverse-engineering approach
Ontology highlight
ABSTRACT:
ORGANISM(S): Rattus norvegicus
SUBMITTER: Nils BlM-CM-<thgen
PROVIDER: E-GEOD-38614 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:RAS mutations are highly relevant for progression and therapy response of human tumours, but the genetic network that ultimately executes the oncogenic effects is poorly understood. Here we used a reverse-engineering approach in an ovarian cancer model to reconstruct KRAS oncogene-dependent cytoplasmic and transcriptional networks from perturbation experiments based on gene silencing and pathway inhibitor treatments. We measured mRNA and protein levels in manipulated cells by microarray, RT-PCR and Western Blot analysis, respectively. The reconstructed model revealed complex interactions among the transcriptional and cytoplasmic components, some of which were confirmed by double pertubation experiments. Interestingly, the transcription factors decomposed into two hierarchically arranged groups. To validate the model predictions we analysed growth parameters and transcriptional deregulation in the KRAS-transformed epithelial cells. As predicted by the model, we found two functional groups among the selected transcription factors. The experiments thus confirmed the predicted hierarchical transcription factor regulation and showed that the hierarchy manifests itself in downstream gene expression patterns and phenotype. RAS-ROSE cells and ROSE cells treated with Scrambled siRNA
2012-07-31 | E-GEOD-38585 | biostudies-arrayexpress
Project description:RAS mutations are highly relevant for progression and therapy response of human tumours, but the genetic network that ultimately executes the oncogenic effects is poorly understood. Here we used a reverse-engineering approach in an ovarian cancer model to reconstruct KRAS oncogene-dependent cytoplasmic and transcriptional networks from perturbation experiments based on gene silencing and pathway inhibitor treatments. We measured mRNA and protein levels in manipulated cells by microarray, RT-PCR and Western Blot analysis, respectively. The reconstructed model revealed complex interactions among the transcriptional and cytoplasmic components, some of which were confirmed by double pertubation experiments. Interestingly, the transcription factors decomposed into two hierarchically arranged groups. To validate the model predictions we analysed growth parameters and transcriptional deregulation in the KRAS-transformed epithelial cells. As predicted by the model, we found two functional groups among the selected transcription factors. The experiments thus confirmed the predicted hierarchical transcription factor regulation and showed that the hierarchy manifests itself in downstream gene expression patterns and phenotype. RAS-ROSE cells were treated with siRNA against 7 transcription factors or controls,
2012-07-31 | E-GEOD-38584 | biostudies-arrayexpress
Project description:A genome-wide survey of transcriptional targets responding to chronic KRAS/MAPK (mitogen-activated protein kinase) pathway activation in rat ovarian surface epithelial cells (ROSE 199) has identified multiple differentially expressed transcriptional regulators including the architectural transcription factor high mobility group AT hook 2 (HMGA2). To elucidate the role of HMGA2 in the transcriptional network affected by oncogenic signaling, we used an integrated approach combining RNAi-mediated silencing, microarray-based expression profiling, computational prediction of transcription factor binding sites in target gene promoters and phenotypic analysis. Knocking-down HMGA2 resulted in the reversion of epithelial-mesenchymal transition, loss of anchorage independence and in a substantial restoration of the gene expression pattern characteristic of non-transformed ROSE cells. Computational prediction of transcription factor binding sites in the promoters of HMGA2-regulated genes and expression profiling revealed a preferential role of activator complex-1 (AP-1) components Fra-1 and JunB in target gene regulation. Forced expression of HMGA2 did not transform normal ovarian epithelial cells, suggesting that HMGA2 up-regulation is not sufficient for inducing the transformed phenotype, but mediates cancer-specific phenotypic traits in cells expressing mutated KRAS and, hence, oncogene addiction. The Ras signaling target array was designed and produced as described (Tchernitsa et al., 2005). Briefly, we selected 121 genes recovered from subtracted cDNA libraries representing sequences differentially expressed in parental and KRAStransformed ROSE 199 cells (Tchernitsa et al., 2004). In addition, we selected genes from subtracted libraries representing sequences differentially expressed in normal 208F fibroblasts and the HRAS-transformed derivative FE-8 (Zuber et al., 2000). Previously described Ras pathway targets and 13 different genes described as housekeeping genes in many different tissues and cell lines were included to permit normalization of hybridization intensities on microarrays. The total number of gene probes is 325 on the array. Unmodified 70-mer DNA oligonucleotides were chosen as probe sets for the array. The oligonucleotide design was performed with software available on the web (http://oligo.lnatools.com/expression/) using the following parameters: option for LNA frequency=0, melting temperature between 74°C and 76°C, GC content between 45- 55%, absence of strong secondary structures and proximity to 3â end of coding sequence. Oligonucleotides were synthesized at Illumina, Inc (San-Diego, USA) at the 50 nmol scale. Each of the 325 oligonucleotides was spotted four times at a different location on the slides. Thus, the array contains 1,300 features. Overall the study contains six samples. We studied for conditions: First condition is influence of empty plasmid on ROSE199 cells. No additional hybridization replica. Sample 199_199emptyvector Second condition is influence of siEGFP (used as a scramble duplex) on ROSEA25 cell line. As there was no influence of siEGFP on A25cell line and genes differentially expressed between ROSE199 and ROSEA25 were previously described in our several publication here we also used only one hybridization replica. Sample ROSE199_ROSEA25siEGFP. Third condition is influence of HMGA2 expression vector on ROSE199 cells. One dye swap replica was done. Samples: ROSE199_ROSE199HMGA2 and 199HMGA2_199. Forth condition is influence of siHMGA2 duplex on ROSEA25 cell line compared to ROSE199 cell line. One replica with dye swap. Samples ROSE199_ROSEA25siGMGA2 and ROSEA25siHMGA2_ROSE199.
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2019-01-16 | PXD011943 | Pride
Project description:Plants have evolved sophisticated systems to respond to environmental changes, and growth arrest is a common strategy to enhance stress tolerance. Despite the growth-survival tradeoffs is essential for shaping plant productivity, the mechanisms balancing growth and survival remain largely unknown. Aquaporins play a crucial role in growth and stress responses by controlling water transport across membranes. Here, we present that RhPIP2;1, an aquaporin from rose (Rosa sp.), interacts with a membrane-tethered MYB protein, RhPTM. Water deficiency triggers nuclear translocation of RhPTM C-terminus. Silencing of RhPTM causes continuous growth under drought stress, and a consequent decrease in survival rate. RNA-seq indicates that RhPTM influences expression of genes related to carbohydrate metabolism. Water deficiency induces phosphorylation of RhPIP2;1 at Ser273, which is sufficient to promote nuclear translocation of RhPTM C-terminus. These results indicate that RhPIP2;1-RhPTM module serves as a critical player to orchestrate the tradeoff between growth and stress survival in rose.
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Project description:Analysis of the transcriptional profiles of mRNA and microRNA in Rasless fibroblasts. 4-Hydroxy-tamoxifen (4-OHT) treatment triggers removal of K-Ras expression in [H-Ras-/-;N-Ras-/-;K-Raslox/lox;RERTert/ert] mouse fibroblasts (named K-Raslox) generating M-bM-^@M-^\RaslessM-bM-^@M-^] MEFs which are unable to proliferate, but recover proliferative ability after ectopic expression of constitutively active downstream kinases such as BRAF and MEK1. Using Affymetrix microarrays, we compared the transcriptional profiles of Rasless fibroblasts to those of K-Raslox (MEFs lacking only H-Ras and N-Ras). We also compared the transcriptional profile of Rasless cells with those of BRAF and MEK1-transfected Rasless cells which recovered proliferative ability. Re-entry of the Rasless cells into cell cycle progression through ectopic expression of activated BRAF or MEK1 resulted in full reversion of most transcriptional alterations identified in cells lacking the three canonical Ras proteins. Pre-confluent cell cultures corresponding to the different Ras genotypes were harvested for extraction of total RNA and miRNA samples, and subsequent hybridization on Affymetrix microarrays. The different sets of experimental mRNA samples analyzed here included (i) 8 samples K-Raslox (proliferating cells expressing only K-Ras, considered as Control): 4 biological replicates, technical duplicates each; (ii) 7 samples Rasless: the same cells after treatment with 4-OHT for 12 days (to become non-proliferating fibroblasts), including 4 biological replicates and technical duplicates from 3 of them; (iii) 3 samples BRAF-rescued MEFs (Rasless cells harboring activated BRAF constructs, with regained proliferative ability): 3 biological replicates; (iv) 3 samples MEK1-rescued MEFs (Rasless cells harboring activated MEK1 constructs, with regained proliferative ability): 3 biological replicates; and their respective controls harbouring the vempty vectors (v) 2 samples Hygromycin-resistant (control for BRAF-transfected Rasless cells) including 2 biological replicates, and (vi) 4 samples Puromycin-resistant (control for MEK1-transfected Rasless cells) including 2 biological replicates, technical duplicates each. The gene expression study contains a total of 27 samples. Regarding the miRNA expression study, the experimental samples analyzed included (i) 4 samples samples K-Raslox (used as Control): 2 biological replicates, technical duplicates each; (ii) 8 samples Rasless: the same cells after treatment with 4-OHT for 6 days (2 biological replicates, technical duplicates each) and 12 days (2 biological replicates, technical duplicates each); (iii) 4 samples BRAF-rescued, including 2 biological replicates, technical duplicates each; (iv) 4 samples MEK1-rescued,including 2 biological replicates, technical duplicates each, and (v) 4 samples Puromycin-resistant empty vector, containing 2 biological replicates, technical duplicates each. The miRNA expression study contains a total of 24 samples.
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