Interplay Between Estrogen Response Element Sequences and Ligands Controls Binding of Estrogen Receptor to Regulated Genes
Krieg, Adam Jeremy
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Permalink
https://hdl.handle.net/2142/84798
Description
Title
Interplay Between Estrogen Response Element Sequences and Ligands Controls Binding of Estrogen Receptor to Regulated Genes
Author(s)
Krieg, Adam Jeremy
Issue Date
2003
Doctoral Committee Chair(s)
Shapiro, David J.
Department of Study
Biochemistry
Discipline
Biochemistry
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Chemistry, Biochemistry
Language
eng
Abstract
Dissecting the mechanism of estrogen receptor-mediated transcription is essential for understanding diseases ranging from breast cancer to osteoporosis. Central to estrogen receptor (ER)-dependent gene activation is the interaction of ER with its DNA recognition sequence, the estrogen response element (ERE). Although the ER preferentially interacts with the consensus ERE (PuGGTCAnnnTGACCPy), natural genes use diverse sequences to regulate estrogen action. To investigate the influence of ERE sequence on estrogen-dependent gene expression, I established both in vitro and cell-based systems. To study estrogen regulation of transcription in vitro, I developed the CHO-S system for expression of ER. Appropriate concentrations of CHO-S-expressed ER activated transcription in cell-free systems and bound DNA in electrophoretic mobility shift assays, thereby providing a useful new source of ER. To assess the interplay between ligand and ERE sequence in estrogen-dependent gene expression, I used chromatin immunoprecipitation assays (ChIP) to compare the interaction of the ER with the promoters of the Proteinase Inhibitor-9 (PI-9) and pS2 genes. PI-9 is an anti-inflammatory protein regulated by a unique estrogen response unit (ERU) containing an imperfect ERE directly adjacent to an element containing two ERE half-sites. pS2 is a breast cancer prognostic gene regulated through a near-consensus ERE palindrome. When bound to the potent estrogen Moxestrol, ERalpha was strongly recruited to the PI-9 ERU, and to the pS2 ERE. The breast cancer drug 4-hydroxytamoxifen (OHT) weakly activates PI-9 and barely activates pS2. In contrast, OHT-liganded ER bound well to the pS2 ERE, but poorly to the PI-9 ERU. Neither the pure antiestrogen ICI 182,780 nor the SERM Raloxifene activates either PI-9 or pS2. Surprisingly, ICI-liganded ER behaves more like OHT-liganded ER on PI-9 and pS2, while Raloxifene-liganded ER behaves more like Moxestrol-liganded ER. Based on these combined data, I propose that the conformations assumed by the ER in the presence of different ligands and EREs work together to determine the ability of liganded ER to bind to specific ERE sequences, thereby providing one mechanism by which SERMs elicit their promoter-specific effects.
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