DIFFERENTIAL IMPACT OF FLANKING SEQUENCES ON ESTRADIOL- VS 4-HYDROXYTAMOXIFEN-LIGANDED ESTROGEN-RECEPTOR BINDING TO ESTROGEN-RESPONSIVE ELEMENT DNA

The mechanism by which antiestrogens antagonize the ability of estroge n receptor (ER) to induce the transcription of estrogen-regulated gene s is only partially understood. To examine the effect of estrogen resp onsive element (ERE) stereoalignment and flanking sequences on estradi ol-liganded ER (E(2)-ER)-ERE and antiestrogen-liganded ER (4-hydroxyta moxifen-liganded ER or 4-OHT-ER)-ERE binding, several dimeric EREs, co ntaining a perfect inverted repeat (5'-GGTCAgagTGACC-3') but lacking t he AT-rich flanking sequences typical of highly estrogen-responsive pr omoters, were cloned into a plasmid vector. The ERE centers of symmetr y were spaced 1.5, 2.0, 3.0, 6.4 and 6.7 helical turns apart. E(2)-ER and 4-OHT-ER binding to these constructs was specific and saturable, b ut orientation-independent and, in contrast to our earlier work with E (2)-ER binding to AT-rich EREs, not cooperative. The affinity of E(2)- ER binding decreased as the distance between adjacent EREs was increas ed, suggesting that E(2)-ER binding to closely spaced EREs is more sta ble (K-d = 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 increas ed with increased ERE spacing (K-d= 2.90, 4.79, 1.39, 1.77, and 0.92 n M, respectively). The presence of AT-rich sequences flanking the ERE i ncreased the binding affinity of E(2)-ER and 4OHT-ER, an increase refl ected in slower dissociation rates of ER from these EREs. The AT-rich sequence also enhanced the binding capacity of E(2)-ER but not 4OHT-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 importa nt in stabilizing E(2)-ER binding and may be critical for cooperative binding to stereoaligned EREs.