Mapping ERb Genomic Binding Sites Reveals Unique Genomic Features and Identifies EBF1 as an ERb

Considerable effort by numerous laboratories has resulted in an improved understanding of estrogen and SERM action mediated by the two estrogen receptors, ERa and ERb. However, many of the targets for ERb in cell physiology remain elusive. Here, the C4-12/Flag.ERb cell line which stably expressed Flag.ERb is used to study ERb genomic functions without ERa interference. Mapping ERb binding sites in these cells reveals ERb 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 ERb target genes. Gene ontology analysis reveals that ERb targets are enriched in differentiation, development and apoptosis. Concurrently, E2 treatment suppresses proliferation in these cells. Within ERb binding sites, while the most prevalent binding motif is the canonical ERE, motifs of known ER interactors are also enriched in ERb binding sites. Moreover, among enriched binding motifs are those of GFI, REST and EBF1, which are unique to ERb 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 ERb genomic functions in our cell model, we confirm the anti-proliferative role of ERb and discover the novel cross talk of ERb with EBF1 which has various implications in normal physiology