Equilibrium Binding of Estrogen Receptor with DNA Using Fluorescence Anisotropy

Mary Szatkowski Ozers,John J Hill,Kerry Ervin,Jennifer R Wood,Ann M Nardulli,Catherine A Royer,Jack Gorski

doi:10.1074/jbc.272.48.30405

Abstract

Interaction of estrogen receptor (ER) with DNA sequences known as estrogen response elements (ERE) is required for estrogen regulation of the expression of target genes. To characterize the affinity and specificity of ER interaction with ERE sequences in vitro under equilibrium conditions, fluorescence anisotropy assays were performed using recombinant, purified ER and a fluorescein-labeled 35-base pair oligonucleotide bearing an idealized palindromic ERE. In buffer containing 100 mM KCl, the baculovirus-expressed, purified human ER bound with similar affinity to the consensus ERE and a mutant ERE with a single base pair change per half-site. Above 225 mM KCl, ER exhibited discrimination between the consensus and mutated ERE targets. Between 225 and 275 mM KCl, binding to the consensus ERE was independent of salt concentration and occurred with an equilibrium dissociation constant (Kd) of 1.8 +/- 0.6 nM, whereas binding to the mutant ERE was not detected at ER concentrations below 100 nM under the same conditions. At 300 mM KCl, the Kd for the consensus ERE increased approximately 25-fold, suggesting complex salt concentration dependence. Both estrogen-occupied and unoccupied ER bound to the consensus ERE sequence with similar affinity, indicating that estrogen affects ER activity at a step other than DNA binding. Unlike the full-length ER, the recombinant DNA binding domain of ER did not discriminate between the consensus and mutated ERE sequences even at buffer salt concentrations greater than 200 mM NaCl, suggesting that ER sequences outside the DNA binding domain may be important in promoting specific binding.

Highlights

Interaction of estrogen receptor (ER) with DNA sequences known as estrogen response elements (ERE) is required for estrogen regulation of the expression of target genes
The abundance of the single human ER complex with F-vitellogenin ERE (vitERE) was not reduced when mutated form of the ERE (mutERE) was included as competitor but its formation was diminished when vitERE was used as competitor, indicating that the purified human ER binds to the vitERE sequence
These results indicated that the purified mouse ER and human ER exhibit non-negligible affinity for the mutERE sequence under these conditions

Summary

Equilibrium Binding of Estrogen Receptor with DNA Using Fluorescence Anisotropy*

(Received for publication, July 17, 1997, and in revised form, September 12, 1997). Mary Szatkowski Ozers‡, John J. The ability of unpurified ER from rat uterine cytosolic extracts to discriminate the consensus ERE from other DNA sequences was examined by using an avidin-biotin complexed with DNA assay to quantify the binding interaction [17] These assays indicated that ER recognized the vitellogenin ERE (vitERE) with an affinity that was three orders of magnitude greater than that for a mutated form of the ERE (mutERE) bearing 1 base change per half-site in a salt-dependent manner. It has been shown that human ER expressed from a recombinant baculovirus and produced by in vitro transcription/translation requires E2 for DNA binding [28] In these studies, we used fluorescence anisotropy to examine the specificity and affinity of the ER-ERE interaction under equilibrium conditions. The role of E2 in ER binding to the ERE was examined using this equilibrium technique

EXPERIMENTAL PROCEDURES

RESULTS

Thermodynamic constants for ER binding to the ERE

Fit App Kd

Unoccupied ER c ND

DISCUSSION