Mapping ERß genomic binding sites reveals unique genomic features and identifies EBF1 as an ERß interactor [Gro-Seq]
The C4-12/Flag.ERß cell line which stably expressed Flag.ERß is used to study ERß genomic functions without ERa interference. Mapping ERß binding sites in these cells reveals ERß unique distribution and motif enrichment patterns. Accompanying our mapping results, nascent RNA profiling is performed on cells at the same treatment time. The combined results allow the identification of ERß target genes. Gene ontology analysis reveals that ERß targets are enriched in differentiation, development and apoptosis. Concurrently, E2 treatment suppresses proliferation in these cells. Within ERß binding sites, while the most prevalent binding motif is the canonical ERE, motifs of known ER interactors are also enriched in ERß binding sites. Moreover, among enriched binding motifs are those of GFI, REST and EBF1, which are unique to ERß binding sites in these cells. Further characterization confirms the association between EBF1 and the estrogen receptors, which favors the N-terminal region of the receptor. Furthermore, EBF1 negatively regulates ERs at the protein level. In summary, by studying ERß genomic functions in our cell model, we confirm the anti-proliferative role of ERß and discover the novel cross talk of ERß with EBF1 which has various implications in normal physiology. Overall design: C4-12/Flag.ERß cells were treated with 10nM E2 (or ethanol as vehicle control) for 1 hour. Nuclei were extracted and processed with run-on assay. The resultant run-on RNA was reverse-transcribed to generate cDNA library which was subsequently sequenced by Illumina Genome Analyzer II or HiSeq2000. Two samples for each treatment were included in this experiment.