Journal
JOURNAL OF BIOLOGICAL CHEMISTRY
Volume 279, Issue 6, Pages 5025-5034Publisher
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M307076200
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- NICHD NIH HHS [HD-16720] Funding Source: Medline
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To examine the role of the estrogen response element (ERE) sequence in binding of liganded estrogen receptor (ER) to promoters, we analyzed in vivo interaction of liganded ER with the imperfect ERE in the pS2 gene and the composite estrogen-responsive unit (ERU) in the proteinase inhibitor 9 (PI-9) gene. In transient transfections of ER-positive HepG2-ER7 cells, PI-9 was strongly induced by estrogen, moxestrol (MOX), and 4-hydroxytamoxifen (OHT). PI-9 was not induced by raloxifene or ICI 182,780. Quantitative reverse transcriptase-PCR showed that moxestrol strongly induced cellular PI-9 and pS2 mRNAs, whereas OHT moderately induced PI-9 mRNA and weakly induced pS2 mRNA. Chromatin immunoprecipitation experiments demonstrated strong and similar association of 17beta-estradiol-hERalpha and MOX-hERalpha with the PI-9 ERU and with the pS2 ERE. Binding of MOX-hERalpha to the PI-9 ERU and the pS2 ERE was rapid and continuous. Although MOX-hERalpha bound strongly to the PI-9 ERU and less well to the pS2 ERE in chromatin immunoprecipitation, gel shift assays showed that estrogen-hERalpha binds with higher affinity to the deproteinized pS2 ERE than to the PI-9 ERU. Across a broad range of OHT concentrations, OHT-hERalpha associated strongly with the pS2 ERE and weakly with the PI-9 ERU. ICI-hERalpha bound poorly to the PI-9 ERU and effectively to the pS2 ERE. Raloxifene-hERalpha and MOX-hERalpha exhibited similar binding to the PI-9 ERU and the pS2 ERE. These studies demonstrate that ER ligand and ERE sequence work together to regulate in vivo binding of ER to estrogen-responsive promoters.
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