EFFECTS OF MULTIPLE ESTROGEN-RESPONSIVE ELEMENTS, THEIR SPACING, AND LOCATION ON ESTROGEN RESPONSE OF REPORTER GENES

Most highly estrogen-responsive genes possess multiple estrogen-respon sive elements (EREs) that act synergistically to activate expression. Synergism between EREs appears to depend on structural features of the EREs and the promoter. To examine the activation process, we cloned s ingle or multiple tandem copies of the consensus ERE into reporter pla smids. These plasmids contained either a chloramphenicol acetyl transf erase reporter gene driven by a minimal promoter or a luciferase repor ter gene driven by the Simian virus 40 (SV40) promoter. Using MCF-7 hu man breast cancer cells, we demonstrate that synergism among EREs depe nds on the number of EREs, their spacing, and the distance of the EREs from the promoter. The induction capacity of EREs falls off slowly wi th distance from the promoter. Remarkably, multiple EREs can induce ef fectively and synergize even when they are located more than 2000 nucl eotides from the promoter. For EREs located immediately upstream of th e promoter, both the distance separating the EREs and the distance to the promoter have to be optimal for synergy. Altering either distance changes the response from synergistic to additive. For distant EREs, p resumed to interact by a looping mechanism at the promoter, the length of DNA between the EREs and the promoter is not critical. Synergy amo ng closely spaced EREs that are far from the promoter only requires an optimal distance separating the ERE centers of symmetry. Interestingl y, very widely separated EREs can also synergize, presumably also beca use of their ability to interact by looping. The estrogen response fro m single or multiple tandem copies of ERE half-palindromes near the SV 40 promoter was also tested. The negligible induction capacity of a si ngle half-site was not significantly increased in multiple sites. The biological role of half-EREs is not apparent in the system employed he re.