Figure 4.

Luciferase reporter assays of cloned genomic loci bound by ERα and displaying high enhancer activity in MCF7 cells. Genomic loci (average length 776 bp) containing one or more ERE sequences were cloned into a minimal promoter reporter construct driving luciferase gene expression. Specific wild-type ERE sequences and mutated (i.e. E1M) ERE sequences are indicated above each clone being tested (repetitive element EREs are indicated by red font, mismatches from the classical ERE sequence are indicated by lower case lettering, and mutated sequences were changed to tttttt where indicated). Basal and E2-stimulated luciferase values are shown normalized to co-transfected β-galactosidase-expressing plasmid. Clone D54 contains three ERE sequences and the third is in a repetitive element (see Table 2) which contributes to overall enhancer function (A). B68 contains two ERE sequences both of which reside within repetitive DNA elements and both contribute to enhancer function (B). D70 also contains two ERE sequences both of which reside within repetitive DNA elements (C). Values are the average of three experiments, performed in triplicate, with SEM indicated. *P < 0.01 comparing E2-treated wild type with E2-treated mutated reporter. ^§P < 0.01 comparing E2-treated empty vector with E2-treated mutated reporter. ^†P < 0.01 comparing vehicle control-treated with E2-treated reporter.