FIG. 3.

In vitro binding of transcription factors to the estrogen-responsive region of the CCND1 gene. (A) EMSAs with nuclear extracts from hormone-responsive hBC cells treated or not treated with 50 nM E2 for 2 h and challenged with ³²P-labeled AP/dE double-stranded oligonucleotide; in one case (+ICI), a 100-fold molar excess of ICI 182,780 was also added to the culture media as described in Materials and Methods. In some experiments, a 100-fold molar excess of unlabeled oligonucleotide carrying consensus binding sites for the indicated factors was used as a competitor (comp.), or the nuclear extracts were pretreated with the indicated antibodies (Ab). NIS, pool of preimmune sera; ss, supershifted complexes observed with anti-ER, anti-c-Fos, and anti-Oct-1 antibodies (antibodies against c-Jun and YY1 hamper the interactions of these factors with DNA). The protein-DNA complexes indicated as c1, c2, and c3 were consistently observed with different nuclear extracts from either MCF-7 or ZR-75.1 cells and are specific, while the smaller ones represent nonspecific complexes, which were erratic or resulted from sample degradation during preparation or handling. Data displayed are representative of results obtained in multiple tests carried out with different nuclear extracts. (B) Results of EMSAs performed with equal amounts of nuclear proteins extracted from hormone-deprived (−E2) or hormone-stimulated (+E2) hBC cells and increasing amounts of AP/dE-labeled probe. Following electrophoresis and autoradiography, the intensity of the signal corresponding to complexes c1 (Oct-1), c2 (AP-1), or c3 (YY1) in each lane was measured by densitometry. Bars represent standard deviations. (C) Representative immunoblot of nuclear extracts from hBC cells treated with E2 and analyzed by Western blotting with antibodies against the represented proteins. Only the area of the filters where each protein of interest was detected is displayed. (D) Mutant analysis of in vitro transcription factor binding to the AP/dE composite element by EMSAs with nuclear extracts from hormone-treated hBC cells. In the top panel are shown the sequences tested by direct binding, with mutant residues in evidence; numbers to the right mark the corresponding lane in the autoradiogram below. Data displayed are from representative experiments carried out with MCF-7 cells; identical results were obtained with ZR-75.1 cells.