COOPERATIVE ESTROGEN-RECEPTOR INTERACTION WITH CONSENSUS OR VARIANT ESTROGEN RESPONSIVE ELEMENTS INVITRO

Specific binding of estradiol-liganded, partially purified calf uterine estrogen receptor (ER) to a 38-base pair estrogen responsive element (ERE) consensus sequence, containing the inverted repeat 5'-GGTCAnnnTGACC-3', was measured in vitro. The ERE sites were inserted as single or multiple tandem copies in a plasmid vector [pGEM-7Zf(+)]. Results showed that one dimeric ER can interact with one ERE, and steric constraints do not inhibit binding of ER to adjacent EREs. Molybdate-stabilized monomeric (4S) ER did not bind to EREs. ER bound to single and tandem double EREs with K(d) values of 0.24 and 0.23 nM, respectively. When the plasmid contained three or more tandem copies of the ERE, ER bound in a cooperative manner, as indicated by convex Scatchard plots and Hill coefficients greater than 1.5. To determine those characteristics of the consensus sequence that are important for maximal high-affinity ER binding, ten variant ERE oligomer sequences were synthesized and cloned into pGEM-7Zf(+) as single copies or as four copies in tandem. ER binding affinity was maximal for the consensus ERE and was reduced for variants containing one or two nucleotide changes in the inverted repent. The number of nucleotides separating the inverted repeat in the ERE was critical for high-affinity ER binding. Certain sequence-variant EREs when cloned as single copies bound less ER compared to the consensus ERE, yet when cloned as four tandem copies, ER binding displayed cooperativity by Scatchard and Hill analyses. Results demonstrate that cooperative interactions noted in vivo by others are present when measured in vitro. Results strongly imply that the number, spacing, and nucleotide sequence of EREs could precisely control the amount of ER binding to estrogen-responsive genes.