ER binds promoter regions encoding both conserved and non-conserved predicted response elements in an estrogen-dependent manner. (a) EREs (underlined) found upstream of NRIP1 and GREB1 coding regions are conserved in human, chimpanzee, mouse, and rat genomes. (b) PCR primers flanking the predicted conserved (NRIP1 and GREB1) and non-conserved (ABCA3 and TFF1/pS2) EREs were designed to detect ER binding following ChIP assays. The relative positions of the primers and ERE, relative to the TSS, are indicated. (c) Interactions between ER and predicted EREs were enhanced by estrogen treatment. MCF-7 cells were either mock-treated with the carrier dimethyl sulfoxide (-E2, gray bars) or treated with estradiol (+E2, black bars), followed by ChIP experiments. Black and gray bars indicate the enrichment of the binding site in anti-ER ChIP experiments over anti-GST ChIP experiments. Enrichment of all EREs was observed in hormone-treated cells whereas the mock-treated cells displayed less or very little enrichment. There was no enrichment of actin exon 3 control region or any of the input controls (open bars).