8274405

pubmed:Citation

6

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.

eng

IM

MEDLINE

0960-0760

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NLM

713-30

pubmed-meshheading:8274405-Animals, pubmed-meshheading:8274405-Base Sequence, pubmed-meshheading:8274405-Binding, Competitive, pubmed-meshheading:8274405-Binding Sites, pubmed-meshheading:8274405-Cattle, pubmed-meshheading:8274405-DNA, pubmed-meshheading:8274405-DNA-Binding Proteins, pubmed-meshheading:8274405-Estradiol, pubmed-meshheading:8274405-Estrogen Antagonists, pubmed-meshheading:8274405-Female, pubmed-meshheading:8274405-Humans, pubmed-meshheading:8274405-Kinetics, pubmed-meshheading:8274405-Molecular Sequence Data, pubmed-meshheading:8274405-Oligodeoxyribonucleotides, pubmed-meshheading:8274405-Plasmids, pubmed-meshheading:8274405-Promoter Regions, Genetic, pubmed-meshheading:8274405-Receptors, Estrogen, pubmed-meshheading:8274405-Repetitive Sequences, Nucleic Acid, pubmed-meshheading:8274405-Sequence Homology, Nucleic Acid, pubmed-meshheading:8274405-Tamoxifen, pubmed-meshheading:8274405-Uterus

Differential impact of flanking sequences on estradiol- vs 4-hydroxytamoxifen-liganded estrogen receptor binding to estrogen responsive element DNA.

Journal Article, Comparative Study, Research Support, U.S. Gov't, P.H.S.