Unknown,Transcriptomics,Genomics,Proteomics

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Hierarchical regulation in a KRAS pathway-dependent transcriptional network revealed by a reverse-engineering approach (RAS-ROSE and ROSE with siRNA)

ABSTRACT: 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

ORGANISM(S): Rattus norvegicus

SUBMITTER: Nils BlM-CM-<thgen

PROVIDER: E-GEOD-38585 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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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 74Â°C and 76Â°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.

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

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Hierarchical regulation in a KRAS pathway-dependent transcriptional network revealed by a reverse-engineering approach (RAS-ROSE and ROSE with siRNA)

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