Project description:We compared the genome occupancy for FLAG-tagged versions of the ETS factors ERG and EHF in the normal prostate epithelial cell line RWPE1. Our in vitro binding studies support a model whereby oncogenic ETS factors like ERG bind cooperativly with AP1 factors at closly spaced ETS-AP1 sites, while certain non-oncogenic factors like EHF bind anti-cooperatively with AP1 at the same sites. ETS and AP1 binding motifs were enriched in both ChIP datasets, but the ERG-FLAG bound reginos contained a much higher percentage of ETS-AP1 sites spaced in close proximity, consistent with our in vitro binding data.
Project description:The goal of this project was to analyze the global gene expression profiles of RWPE1 and VCAP cells following transfection of GFP, GFP-ERG at 48 and 72hrs time points and stable ERG shRNA, scramble shRNA, respectively. RWPE1 cells were transfected with GFP or GFP-ERG. VCAP cells were transfected with ERG lenti-shRNA or scramble shRNA. Transfections were performed in duplicate. Total cellular RNA was isolated with Trizol and quality analysed by the bioanalyser kit.
Project description:The goal of this project was to analyze the global gene expression profiles of RWPE1 and VCAP cells following transfection of GFP, GFP-ERG at 48 and 72hrs time points and stable ERG shRNA, scramble shRNA, respectively.
Project description:OLFM4 gene expressing in RWPE1 stem/progenitor-like cells. To study OLFM4 gene functions and molecular mechanisms, we generated OLFM4-avtive and OLFM4-knock out-GFP reporter RWPE1 cells with CRISP-Cas 9 system. The cell transcriptome was studied with RNA sequencing.
Project description:E74-like factor 5 (ELF5) and ETS-homologous factor (EHF) are epithelial selective ETS family transcription factors (TFs) encoded by genes at chr11p13, a region associated with cystic fibrosis (CF) lung disease severity. EHF controls many key processes in lung epithelial function so its regulatory mechanisms are important. Using CRISPR/Cas9 technology, we removed three cis-regulatory elements (CREs) from the chr11p13 region in airway epithelial cells. Deletion of two enhancers and one CRE within a stretch enhancer at the EHF locus caused subtle changes in chromatin architecture, and though EHF expression did not change, ELF5 abundance increased. ELF5 is normally very low in airway cells so we next examined cell types that express more ELF5 (LNCaP and T47D). ATAC-seq experiments in these lines revealed novel peaks of open chromatin (potential CREs) at the 5’ end of chr11p13 that were associated with an expressed ELF5 gene. Furthermore, 4C-seq assays identified direct interactions between the active ELF5 promoter and sites within the EHF locus, suggesting coordinate regulation between these TFs. ChIP-seq for ELF5 in T47D cells revealed ELF5 occupancy within EHF introns 1 and 6 and siRNA-mediated depletion of ELF5 repressed EHF expression. These results define a new role for ELF5 in lung epithelial biology.
Project description:Our in vitro binding studies support a model whereby MED25 exhibits multivalent interactions with a subset of related ETS factors, ETV1/4/5. We hypothesize that the interaction would allow for coregulation of genes by ETV1/4/5 and MED25, acting perhaps to link the ETVs to the Mediator complex. To explore this possibility, we compared the genome occupancy for FLAG-tagged MED25 and ETV4 in the prostate cancer cell line PC3, which overexpresses ETV4. We also tested for relevance of MED25 and ETV4 binding to for gene expression in PC3s. We found a high degree of overlap in the FLAG-MED25 and ETV4 ChIPs datasets consistent with our model, and also identified a subset of target genes co-dependent on Med25 and ETV4.
Project description:Analysis of transcriptional changes during Myc or PI3K induced oncogenic transformation in RWPE1 cells (benign prostate epithelial cell line). The aim of the present study was to identify key epigenetic gene silencing events that occur during the oncogenic transformation events, hence emphasis was placed on downregulated genes. Results provide important information on which are the tumor suppressive pathways or genes that will be epigenetically silenced by during Myc or PI3K induced oncogenic transformation. Total RNA obtained from oncogenic transformed RWPE1 cells which were either overexpressing Myc or constitutive active mutant of PI3K (E545K) as compared to RWPE1 cells expressing the empty vector control PMN.