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Molecular Endocrinology 11 (13): 1994-2003
Copyright © 1997 by The Endocrine Society

Effects of Multiple Estrogen Responsive Elements, Their Spacing, and Location on Estrogen Response of Reporter Genes

G. Sathya, Wenzhuo Li, Carolyn M. Klinge, Jennifer H. Anolik, Russell Hilf and Robert A. Bambara

Department of Biochemistry and Biophysics and the Cancer Center (G.S., W.L., J.H.A., R.H., R.A.B.) University of Rochester School of Medicine and Dentistry Rochester, New York 14642
Department of Biochemistry (C.M.K.) University of Louisville School of Medicine Louisville, Kentucky 40292

Most highly estrogen-responsive genes possess multiple estrogen-responsive 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 single or multiple tandem copies of the consensus ERE into reporter plasmids. These plasmids contained either a chloramphenicol acetyl transferase reporter gene driven by a minimal promoter or a luciferase reporter gene driven by the Simian virus 40 (SV40) promoter. Using MCF-7 human breast cancer cells, we demonstrate that synergism among EREs depends on the number of EREs, their spacing, and the distance of the EREs from the promoter. The induction capacity of EREs falls off slowly with distance from the promoter. Remarkably, multiple EREs can induce effectively and synergize even when they are located more than 2000 nucleotides from the promoter. For EREs located immediately upstream of the 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, presumed to interact by a looping mechanism at the promoter, the length of DNA between the EREs and the promoter is not critical. Synergy among closely spaced EREs that are far from the promoter only requires an optimal distance separating the ERE centers of symmetry. Interestingly, very widely separated EREs can also synergize, presumably also because of their ability to interact by looping.

The estrogen response from single or multiple tandem copies of ERE half-palindromes near the SV40 promoter was also tested. The negligible induction capacity of a single half-site was not significantly increased in multiple sites. The biological role of half-EREs is not apparent in the system employed here.




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