Dimerizing the Estrogen Receptor DNA Binding Domain Enhances Binding to Estrogen Response Elements

Martin A Kuntz,David J Shapiro

doi:10.1074/jbc.272.44.27949

Abstract

In this work, we provide a rationale for the finding that the estrogen receptor (ER) binds to its DNA response element as a homodimer in vivo. Binding of the monomer estrogen receptor DNA binding domain (ER DBD) to a palindromic, consensus estrogen response element (ERE) is increased 5-6-fold when the ER DBD is dimerized either by a monoclonal antibody that recognizes an attached epitope tag or by expressing the ER DBD as a single molecule in which the two monomers are joined by a peptide linker. Most of the increase in binding is due to stabilization of the ER DBD.ERE complex. We observed only an approximately 2.5-fold reduction in binding when a consensus ERE was replaced with widely spaced ERE half-sites, suggesting that the interaction between ER DBDs on the ERE is relatively weak, and that in full-length ER the DBDs can move independently of each other. To test binding to an imperfect palindrome, typical of the imperfect EREs found in almost all natural estrogen receptor responsive genes, we used the pS2 ERE. Even at high concentrations of ER DBD, specific binding of the ER DBD to the imperfect pS2 ERE was undetectable. Both of the dimerized ER DBDs exhibited efficient binding to the imperfect pS2 ERE, with an affinity at least 25-fold greater than monomer ER DBD. These data support the view that steroid receptor dimerization provides an important mechanism facilitating the recognition of naturally occurring, imperfect hormone response elements.

Highlights

The intracellular actions of estrogens are mediated by the estrogen receptor
Expression and Purification of DBD Constructs—We constructed a core human estrogen receptor (ER) DBD containing a minimum sequence, which retains efficient binding to the estrogen response element (ERE)
The steroid receptors could interact with their half-sites by binding to them independently as monomers, by binding as monomers and forming interacting dimers on the DNA, or by forming dimers in solution and binding to their recognition sequences as dimeric units

Summary

Dimerizing the ER DBD Enhances ERE Binding

Labs (Beverly, MA) or Life Technologies, Inc. Radioisotopes were obtained from ICN (Costa Mesa, CA). To construct the linker-dimerized DBD plasmid pET-DDb, one copy of the DBD coding sequence was inserted into the NheI and BamHI sites of the pET-21b(ϩ) plasmid. This insert was prepared by PCR amplification from the human ER coding sequence using the primers 5Ј-TCAGGATCCACCATGGCTAGCGACTACAAGGACGACGATGACAAGATGTACCCTAGGGGCAAGGAGACTCGCTACTGT-3Ј and 5Ј-ATAGGATCCACCTCCACCTGAGCCACCCCCTCCTCTTCGGTCTTT-3Ј. This complex cloning procedure was performed to minimize the selection of mutants in the DNA binding domain with a reduced affinity for the ERE. All kinetics experiments were performed by incubation of the binding reaction at 4 °C for 1 h before the addition of probe (on-rate) or a 10-fold excess ERE containing competitor (off-rate), and loaded onto a gel running at 4 °C at the times indicated. Quantitation was performed by PhosphorImager analysis (Molecular Dynamics, Sunnyvale, CA)

RESULTS

DISCUSSION

Consensus ERE