title: Differential impact of flanking sequences on estradiol- vs 4-hydroxytamoxifen-liganded estrogen receptor binding to estrogen responsive element DNA.
abstract: The mechanism by which antiestrogens antagonize the ability of estrogen receptor (ER) to induce the transcription of estrogen-regulated genes is only partially understood. To examine the effect of estrogen responsive element (ERE) stereoalignment and flanking sequences on estradiol-liganded ER (E2-ER)-ERE and antiestrogen-liganded ER (4-hydroxytamoxifen-liganded ER or 4-OHT-ER)-ERE binding, several dimeric EREs, containing a perfect inverted repeat (5'-GGTCAgagTGACC-3') but lacking the AT-rich flanking sequences typical of highly estrogen-responsive promoters, were cloned into a plasmid vector. The ERE centers of symmetry were spaced 1.5, 2.0, 3.0, 6.4 and 6.7 helical turns apart. E2-ER and 4-OHT-ER binding to these constructs was specific and saturable, but orientation-independent and, in contrast to our earlier work with E2-ER binding to AT-rich EREs, not cooperative. The affinity of E2-ER binding decreased as the distance between adjacent EREs was increased, suggesting that E2-ER binding to closely spaced EREs is more stable (Kd = 0.38, 0.58, 0.83, 1.23, and 0.96 nM, respectively, for the above spacings). In contrast, the affinity of 4-OHT-ER binding increased with increased ERE spacing (Kd = 2.90, 4.79, 1.39, 1.77, and 0.92 nM, respectively). The presence of AT-rich sequences flanking the ERE increased the binding affinity of E2-ER and 4-OHT-ER, an increase reflected in slower dissociation rates of ER from these EREs. The AT-rich sequence also enhanced the binding capacity of E2-ER but not 4-OHT-ER. Since the binding capacity of 4-OHT-ER is identical with or without an AT-rich region, we suggest that flanking sequences are more important in stabilizing E2-ER binding and may be critical for cooperative binding to stereoaligned EREs.
authors.affiliation: Department of Biochemistry, University of Rochester School of Medicine and Dentistry, NY 14642.
authors.forename: J H
authors.initials: JH
authors.lastname: Anolik
authors.forename: C M
authors.initials: CM
authors.lastname: Klinge
authors.forename: R A
authors.initials: RA
authors.lastname: Bambara
authors.forename: R
authors.initials: R
authors.lastname: Hilf
mesh_terms: D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D002417:Cattle; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D004958:Estradiol / Q000378:metabolism*; D004965:Estrogen Antagonists / Q000378:metabolism*; D005260:Female; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis / Q000378:metabolism; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011960:Receptors, Estrogen / Q000302:isolation & purification / Q000378:metabolism*; D012091:Repetitive Sequences, Nucleic Acid; D012689:Sequence Homology, Nucleic Acid; D013629:Tamoxifen / Q000031:analogs & derivatives* / Q000378:metabolism; D014599:Uterus / Q000378:metabolism*
chemical_list: D004268:DNA-Binding Proteins; D004965:Estrogen Antagonists; D009838:Oligodeoxyribonucleotides; D011960:Receptors, Estrogen; D013629:Tamoxifen; C016601:afimoxifene; D004958:Estradiol; D004247:DNA