Figure 5

Estrogen stimulates the recruitment of Sp to a GC-rich region in the BRCA1 promoter. (a) Nuclear protein extracts (200 μg) were obtained from MCF-7 cells cultured for 1.5 hours in Dulbecco's modified Eagle's medium containing 5% charcoal dextran-stripped fetal bovine serum containing vehicle (DMEM) or DMEM plus 10 nmol/l 17β-estradiol (E2). DNA:protein complexes were generated by coincubation of nuclear extracts with the wild-type specificity protein (Sp)1/activator protein (AP)-1 oligonucleotide or the same oligonucleotide harboring a mutated Sp (Spmut) and separated by SDS-PAGE. The recruitment of Sp1, Sp3, or estrogen receptor (ER)-α was visualized by Western blotting with specific antibodies. Panels are representative of two independent experiments. Numbers represent molecular weight markers (kDa). (b) Nuclear extracts normalized to 5 μg of nuclear protein obtained from MCF-7 cells were incubated with ³²P-labeled BRCA1 promoter oligonucleotides containing the AP-1/CRE or the Sp plus AP-1/CRE sites. MCF-7 cells were cultured for 3 hours in control vehicle (DMEM) or DMEM plus 10 nmol/l E2. FP, free probe (lane 1). Arrows indicate DNA:protein complexes (A and B) visualized by electromobility binding assay, and cAMP response element (CRE)-binding protein(CREB)/DNA complexes supershifted with the CREB antibody. IgG, preimmune immunoglobulin control. (c) Basal and E2-induced recruitment of Sp1, Sp3, Sp4, and ER-α to the BRCA1 region containing the Sp, AP-1, and CRE sites was examined by chromatin immunoprecipitation assay followed by real-time PCR, as described in Materials and methods. The columns represent mean amplification products corrected for input ± standard error (bars) and normalized to vehicle control (DMEM). Asterisks indicate statistically significant (P < 0.05) induction of factor recruitment by E2 as compared with vehicle control DMEM.