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Genome-wide occupancy of FLAG-ERG and FLAG-EHF in RWPE1 cells

ABSTRACT: Genome-wide occupancy of FLAG-ERG and FLAG-EHF in RWPE1 cells

PROVIDER: PRJNA470652 | ENA |

REPOSITORIES: ENA

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Genome-wide occupancy of FLAG-ERG and FLAG-EHF in RWPE1 cells

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.

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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.

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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.

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