MULTIPLE PIT-1-BINDING SITES FACILITATE ESTROGEN RESPONSIVENESS OF THE PROLACTIN GENE

Previous studies have shown that estrogen responsiveness of the rat PR L gene requires the presence of both the estrogen receptor and the tis sue-specific transcription factor, Pit-1. To examine the contribution of individual Pit-1-binding sites in permitting an estrogen response, we mutated specific sites in both the proximal and distal regions of t he rat PRL gene. The studies reveal that mutation of Pit-1-binding sit es in either the proximal or the distal region can have an effect on e strogen responsiveness. The most important Pit-1-binding site appears to be the site in the distal enhancer, which is adjacent to the estrog en receptor-binding site. However, mutation of combinations of other P it-1-binding sites reveals that these sites also contribute to the est rogen response of the PRL gene. The binding sequences for another tran scription factor cannot substitute for Pit-1 sites in bringing about a wild-type estrogen response, as shown by replacement of Pit-1-binding sites with a consensus cAMP-responsive element. Conversion of the imp erfect palindromic estrogen response element of the PRL gene to a perf ect palindrome eliminated the positive effects of an intact 1D Pit-1-b inding site. To examine potential physical interactions between the es trogen receptor and Pit-1, a protein interaction assay was performed. The results demonstrate that labeled estrogen receptor can bind to Pit -1 immobilized on glutathione agarose beads. However, most of the inte raction between Pit-1 and the estrogen receptor appears to be DNA depe ndent. Overall, the results demonstrate a distributed role for multipl e Pit-1 sites in permitting an estrogen response of the rat PRL gene.