Figure 3.

Treatment of MCF-7 cells with estrogen stimulates BRCA-1 promoter occupancy by ERα and p300 to an AP-1 site. (A) MCF-7 cells were precultured for 4 days in DMEM with 5% FBS and then treated with 10 nM E₂ for various periods of time. Cells were processed for ChIP assay using an antibody against ERα (Neomarkers, Fremont, CA). Inputs are control bands generated by PCR from cross-linked chromatin. (B) At 24 hours, estrogen stimulates the recruitment of ERα and p300 (antibody from Affinity Bioreagents), whereas 1 µM TMX (Sigma) antagonizes E₂-dependent recruitment of ERα and p300. (C) E₂-induced BRCA-1 transcription in transiently transfected MCF-7 cells is antagonized by cotreatment with TMX. (D) E₂ stimulates the recruitment of c-Jun and FosB. (E) Coincubation with IgG followed by PCR amplification does not produce a band comprising the AP-1 segment. (F) The recruitment of ERα to a region of exon 7 in the BRCA-1 gene (negative control) is not stimulated by E₂, which stimulates (G) the recruitment of ERα to an ERE in the pS2 gene (positive control). The size of the amplicon was 237 bp for BRCA-1 (-98 to +139 bp from +1 on exon 1B), 289 bp for the pS2 ERE, and 140 bp for exon 7 of the BRCA-1 gene. (H) EMSA for ERα at the BRCA-1 promoter. MCF-7 cells were precultured for 4 days in DMEM with 5% FBS and then treated for 24 hours with E₂. Nuclear extracts were coincubated with a ³²P-labeled BRCA-1 oligonucleotide (-40/-19 bp) plus various amounts of mouse IgG or an antibody for ERα. The ERα antibody supershifted a complex (band A) in a dose-dependent manner, thus confirming the presence of ERα at this region; FP, free probe.