Figure 4

Sp site enhances the binding of nuclear proteins to the BRCA1 promoter. (a) Nuclear extracts normalized to 5 μg of nuclear protein obtained from MCF-7 cells were incubated with ³²P-labeled BRCA1 promoter oligonucleotides containing the activator protein (AP)-1/cAMP response element (CRE) or the Sp plus AP-1/CRE sites. MCF-7 cells were cultured for 3 hour in control vehicle (DMEM) or DMEM plus 10 nmol/l 17β-estradiol (E2). FP, free probe (lane 1). (b, c) In competition studies, nuclear extracts from MCF-7 cells cultured in the presence of E2 were incubated with ³²P-labeled BRCA1 oligonucleotides containing the wild-type specificity protein (Sp)/AP-1/CRE sequences in the absence (lane 2) or presence (100-fold) of competing cold wild-type Sp/AP-1/CRE oligonucleotide (lane 3), or 100-fold of competing cold oligonucleotides mutated for Sp (lane 4), AP-1 (lanes 5 and 6), intervening sequences between AP-1 and CRE (lanes 7 and 8), and CRE (lanes 9 and 10). The arrows indicate the DNA:protein complexes visualized by electromobility binding assay.