Hierarchical regulation in a KRAS pathway-dependent transcriptional network revealed by a reverse-engineering approach (7TF and control)
Ontology highlight
ABSTRACT:
ORGANISM(S): Rattus norvegicus
SUBMITTER: Nils BlM-CM-<thgen
PROVIDER: E-GEOD-38584 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
Similar Datasets
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:This SuperSeries is composed of the following subset Series: GSE38584: Hierarchical regulation in a KRAS pathway-dependent transcriptional network revealed by a reverse-engineering approach (7TF and control) GSE38585: Hierarchical regulation in a KRAS pathway-dependent transcriptional network revealed by a reverse-engineering approach (RAS-ROSE and ROSE with siRNA) Refer to individual Series
2012-07-31 | E-GEOD-38614 | biostudies-arrayexpress
Project description:Cancer treatment decisions are increasingly guided by which specific genes are mutated within each patient’s tumor. For example, agents inhibiting the epidermal growth factor receptor (EGFR) benefit many colorectal cancer (CRC) patients, with the general exception of those whose tumor includes a KRAS mutation. However, among the various KRAS mutations, the G13D mutation behaves differently; for unknown reasons, CRC patients with the KRAS G13D mutation (also written KRASG13D) appear to benefit from the EGFR-blocking antibody cetuximab. Controversy surrounds this observation, because it appears to contradict the well-established mechanisms of EGFR signaling and of RAS mutations. Here, we identified a systems-level, mechanistic basis that explains why KRASG13D cancers respond to EGFR inhibition. We first investigated the problem with a computational model of RAS signaling, which unexpectedly revealed that the known biophysical differences between the three most common KRAS mutant proteins are sufficient to generate different sensitivities to inhibition. Computation and experimentation together revealed a non-intuitive, mutant-specific dependency of wild-type RAS activation by EGFR that is determined by the interaction strength between KRAS and the tumor suppressor neurofibromin (NF1). KRAS mutants that strongly interact with NF1 drive wild-type RAS activation in an EGFR independent manner through competitive inhibition of NF1, whereas KRAS G13D cells remain dependent upon EGFR for wild-type Ras activation because they cannot competitively inhibit NF1 due to a weak interaction between these two proteins. Overall, our work demonstrates how systems approaches enable mechanism-based inference in genomic medicine and can help identify patients for selective therapeutic strategies.
2020-05-27 | PXD014755 | Pride
Project description:The expansion of repressive epigenetic marks has been implicated in heterochromatin formation during embryonic development, but the general applicability of this mechanism is unclear. Here we show that nuclear rearrangement of repressive histone marks H3K9me3 and H3K27me3 into non-overlapping structural layers characterizes senescence-associated heterochromatic foci (SAHF) formation in human fibroblasts. However, the global landscape of these repressive marks remains unchanged upon SAHF formation, suggesting that in somatic cells heterochromatin can be formed through the spatial repositioning of pre-existing repressively marked histones. This model is reinforced by the correlation of pre-senescent replication timing with both the subsequent layered structure of SAHFs and the global landscape of the repressive marks, allowing us to integrate microscopic and genomic information. Furthermore, modulation of SAHF structure does not affect the occupancy of these repressive marks nor vice versa. These experiments reveal that high-order heterochromatin formation and epigenetic remodeling of the genome can be discrete events. ChIP-seq for different histone marks in both growing and Ras-induced senescent fibroblasts, in the presence or absence of certain sh-RNAs K9me3Grow2.bed (growing) Chip Seq Analysis of H3K9me3 in ER:Ras expressing IMR90 human diploid fibroblasts d6 4OHT K9me3Sen2.bed (senescent) Chip Seq Analysis of H3K9me3 in ER:Ras expressing IMR90 human diploid fibroblasts with no treatment K9me2Grow3.bed (growing) Chip Seq Analysis of H3K9me2 in ER:Ras expressing IMR90 human diploid fibroblasts with no treatment K9me2Sen3.bed (senescent) Chip Seq Analysis of H3K9me2 in ER:Ras expressing IMR90 human diploid fibroblasts d6 4OHT K27me3Sen3.bed (senescent) Chip Seq Analysis of H3K27me3 in ER:Ras expressing IMR90 human diploid fibroblasts d6 4OHT K27me3Grow2.bed (growing) Chip Seq Analysis of H3K27me3 in ER:Ras expressing IMR90 human diploid fibroblasts with no treatment K36me3Grow2.bed (growing) Chip Seq Analysis of H3K36me3 in ER:Ras expressing IMR90 human diploid fibroblasts with no treatment K36me3Sen2.bed (senescent) Chip Seq Analysis of H3K36me3 in ER:Ras expressing IMR90 human diploid fibroblasts d6 4OHT K4me3Grow2.bed (growing) Chip Seq Analysis of H3K4me3 in ER:Ras expressing IMR90 human diploid fibroblasts with no treatment K4me3Sen3.bed (senescent) Chip Seq Analysis of H3K4me3 in ER:Ras expressing IMR90 human diploid fibroblasts d6 4OHT
2012-07-09 | E-GEOD-38442 | 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.
2010-05-06 | E-GEOD-14658 | biostudies-arrayexpress
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.
2013-11-27 | E-GEOD-45222 | biostudies-arrayexpress
Project description:Serum-dependent transcriptional networks identify distinct functional roles for H-ras and N-ras during initial stages of the cell cycle; Using oligonucleotide microarrays, we compared gene expression transcriptional profiles corresponding to the initial cell cycle stages of mouse fibroblasts lacking H-Ras and/or NRas with those of matching, WT controls. The similarity of transcription profiles among serum-starved fibroblasts of all different WT and ras knockout genotypes tested, indicated that H-Ras and N-Ras do not play significant roles in control of transcriptional responses to serum deprivation stress. In contrast, genomic disruption of H-ras or N-ras, individually or in combination, determined highly specific, differential gene expression profiles in response to post-starvation stimulation with serum for 1 hour (G0/G1 transition) or for 8 hours (mid-G1 progression). The absence of N-Ras caused significantly higher changes than the absence of H-Ras on the wave of transcriptional activation linked to G0/G1 transition. In contrast, the absence of H-Ras affected more potently the profile of the transcriptional wave detected during mid-G1 progression. Functional analysis demonstrated a predominant functional association of H-Ras with growth and proliferation, whereas N-Ras exhibited a closer functional link to development or cell cycle regulation as well as immunomodulation and apoptosis. Mechanistic analysis indicated that ERK-dependent activation of Stat1 mediates the regulatory effect of N-Ras on defense and immunity, whereas the pro-apoptotic effects of N-Ras are mediated through ERK and p38 signaling. Our observations support previous reports of an absolute requirement for different peaks of Ras activity during the initial stages of the cell cycle and confirm the notion of functional specificity for the H-Ras and N-Ras isoforms. Experiment Overall Design: Mus musculus cell lines from the appropriate ras genotype were harvested on Dulbeccoâs modified Eagleâs medium (DMEM) supplemented with fetal bovine serum (10% FBS), glutamine (2mM), penicillin (100 U/ml) and streptomycin (100 mg/ml). Cultures were grown in a humidified CO2 (5%) atmosphere at 37°C and when subconfluent cells were starved for 24 hours. After starvation cells were either used for RNA isolation, or induced for 1 hour or 8 hours with 20% fetal bovine serum and then RNA extraction and isolation was carried out for hybridization on Affymetrix microarrays.
2009-09-15 | E-GEOD-14829 | biostudies-arrayexpress
Project description:It has been recently shown that N-ras plays a preferential role in immune cell development and function; specifically: N-ras, but not H-ras or K-ras, could be activated at and signal from the Golgi membrane of immune cells following a low level TCR stimulus. The goal of our studies was to test the hypothesis that N-ras and H-ras played distinct roles in immune cells at the level of the transcriptome. First, we showed via mRNA expression profiling that there were over four hundred genes that were uniquely differentially regulated either by N-ras or H-ras, which provided strong evidence in favor of the hypothesis that N-ras and H-ras have distinct functions in immune cells. We next characterized the genes that were differentially regulated by N-ras in T cells following a low-level TCR stimulus. Of the large pool of candidate genes that were differentially regulated by N-ras downstream of TCR ligation, four genes were verified in qRT-PCR-based validation experiments as being differentially regulated by N-ras (Dntt, Slc9a6, Chst1, and Lars2). Finally, although there was little overlap between individual genes that were regulated by N-ras in unstimulated thymocytes and stimulated CD4+ T-cells, there was a nearly complete correspondence between the signaling pathways that were regulated by N-ras in these two immune cell types. Since we were interested primarily in genes that were differentially regulated by N-ras following a low-level TCR stimulus, our microarray data comparison was between data from TCR-stimulated, WT CD4+ T-cells and from TCR-stimulated, N-ras KO CD4+ T-cells. Genes that were differentially regulated in the comparison between stimulated N-ras KO CD4+ T-cells and unstimulated N-ras KO CD4+ T-cells, as well as those genes that were differentially regulated in the comparison between stimulated WT CD4+ T-cells and unstimulated WT CD4+ T-cells were excluded from this analysis. To determine if N-ras and H-ras regulate different sets of genes in thymocytes, a comparison was made between the set of genes that were differentially regulated by N-ras in the [WT] vs. [N-ras KO] comparison and the set of genes that were differentially regulated by H-ras in the [WT] vs. [H-ras KO] comparison. RNA was extracted from CD4+ T cell splenocytes isolated from 6-20 week old N-Ras KO and WT mice following growth in T cell growth media either with or without 1 microgram/milliliter ant-CD3 and anti-CD28 antibodies. RNA was extracted from thymocytes isolated directly from 6-20 week old N-Ras KO, H-Ras KO and WT mice.
2013-04-04 | E-GEOD-45739 | biostudies-arrayexpress
Project description:p65-/-Ras cells show delayed tumor formation in SCID mice. However, after prolonged latency, tumor formation was observed from these mice. To understand the changes of NF-kB regulated genes before and after tumor formation, RNA from p65+/+Ras, p65+/+RasTumor, p65-/-Ras, p65-/-RasTumor cells were isolated and microarray were performed. p65+/+Ras and p65-/-Ras cells were injected into SCID mice. Tumors were removed and put back into tissue culture. RNA was isolated from these cells and microarrays were performed.
2014-08-01 | E-GEOD-59545 | biostudies-arrayexpress
Project description:The expansion of repressive epigenetic marks has been implicated in heterochromatin formation during embryonic development, but the general applicability of this mechanism is unclear. Here we show that nuclear rearrangement of repressive histone marks H3K9me3 and H3K27me3 into non-overlapping structural layers characterizes senescence-associated heterochromatic foci (SAHF) formation in human fibroblasts. However, the global landscape of these repressive marks remains unchanged upon SAHF formation, suggesting that in somatic cells heterochromatin can be formed through the spatial repositioning of pre-existing repressively marked histones. This model is reinforced by the correlation of pre-senescent replication timing with both the subsequent layered structure of SAHFs and the global landscape of the repressive marks, allowing us to integrate microscopic and genomic information. Furthermore, modulation of SAHF structure does not affect the occupancy of these repressive marks nor vice versa. These experiments reveal that high-order heterochromatin formation and epigenetic remodeling of the genome can be discrete events. mRNA expression profiles were compared between normal growing (control) and Ras-induced senescent IMR90 cells. Each includes 5 biological replicates.
2012-07-08 | E-GEOD-38410 | biostudies-arrayexpress