Figure 2

Analysis of the effect of mutation of the UP site on BRCA1 proximal promoter activity. (a) Schematic representation of the BRCA1 renilla-luciferase reporter plasmids: mutations were introduced in the context of both the full-length proximal promoter (L6-pRL) and a promoter construct from which the RIBS site had been deleted (L6DR-pRL). Point mutations were made in the UP site (L6-mUP-pRL, L6DR-mUP-pRL) and the downstream E2F binding site (L6-mE2F-pRL, L6DR-mE2F-pRL), or both sites in combination (L6-mUP-mE2F-pRL, L6DR-mUP-mE2F). See (b) for sequences. (b) Transfection assays for the effects of the various mutants on promoter activity. MCF-7 and T-47D cells were cotransfected with 225 ng of one of the BRCA1 promoter constructs described above and 25 ng of the internal control vector (CMV-Luc). Both renilla and firefly luciferase values were measured using a dual luciferase assay. The data presented is a representative experiment of mean values of triplicates ± the standard deviation of the relative light units of the pRL reporter constructs normalized to the luciferase activity of the internal control vector. Independent experiments were performed a minimum of three times. Statistical significance was determined using a t-test and significant results (p = 0.05) are indicated by asterisks and are in relation to the L6 vector.