High affinity binding of the estrogen receptor to a DNA response element does not require homodimer formation or estrogen.
We have developed an antibody-based DNA binding assay to study the effects of the absence or presence of an estrogen receptor agonist and two antagonists on the thermodynamic binding parameters for estrogen receptor binding to a consensus estrogen response element in vitro. We first demonstrate that antibodies raised against a region of the estrogen receptor directly adjacent to the DNA-binding domain do not interfere with the receptor's ability to preferentially bind to the vitellogenin estrogen response element in plasmid DNA. Exploiting this property to develop a quantitative DNA binding assay, we demonstrate that estrogen is not required for high affinity binding of the receptor for an oligonucleotide containing this element, nor do antiestrogens alter this interaction. In addition, we find that 1 mol of estrogen receptor is complexed with high affinity to 1 mol of vitellogenin estrogen response element, instead of two estrogen receptors/response element as would be predicted if estrogen receptor homodimer formation was required for high affinity DNA binding. Our data best fit a model in which the active estrogen receptor form is a monomer or heterodimer, but not a homodimer, and the regulatory role of estrogen on the estrogen receptor is the induction of protein-protein, but not protein-DNA, interactions.
Department of Biochemistry, University of Wisconsin, Madison 53706-1569.
J D
JD
Furlow
F E
FE
Murdoch
J
J
Gorski
D000818:Animals; D000906:Antibodies; D001483:Base Sequence; D003600:Cytosol / Q000378:metabolism; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D004958:Estradiol / Q000031:analogs & derivatives / Q000378:metabolism* / Q000494:pharmacology; D004965:Estrogen Antagonists / Q000378:metabolism* / Q000494:pharmacology; D005260:Female; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis / Q000378:metabolism*; D053284:Polyunsaturated Alkamides; D051381:Rats; D017207:Rats, Sprague-Dawley; D011960:Receptors, Estrogen / Q000302:isolation & purification / Q000378:metabolism*; D015183:Restriction Mapping; D013629:Tamoxifen / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D013816:Thermodynamics; D014599:Uterus / Q000378:metabolism*
D000906:Antibodies; D004268:DNA-Binding Proteins; D004965:Estrogen Antagonists; D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; D053284:Polyunsaturated Alkamides; D011960:Receptors, Estrogen; D013629:Tamoxifen; D004958:Estradiol; C051781:ICI 164384; D004247:DNA