Figure 4

Binding of recombinant, nuclear extract derived and endogenous GABP alpha/beta to the BRCA1 promoter. (a) A bandshift scanning assay of the BRCA1 promoter for GABP complex binding sites was preformed using recombinant GABP alpha/beta. Double-stranded DNA probes spanning putative protein binding sites were designed for the entire length of the BRCA1 proximal promoter and are indicated on the schematic (top). Binding reactions were performed with recombinant human GABP alpha and beta proteins and each of the ³²P-labelled probes as indicated by the vertical lines. Samples were run on a 6% nondenaturing acrylamide gel and visualized by autoradiography. The locations of the free and bound probe are indicated. (b) Supershift assays with the UPFR6 probe were performed with 5 μg of MCF-7 nuclear extract and an antibody directed against CREB, GABP alpha or Ets-2. Complexes are as indicated. (c) ChIP assays were preformed using chromatin isolated from MCF-7 cells with antibodies against acetylated histone H3, GABP beta, GABP alpha and haemagglutinin (HA). PCR products obtained using BRCA1-specific primers and the immunoprecipitation products are shown.