In vitro binding of the purified hormone-binding subunit of the estrogen receptor to oligonucleotides containing natural or modified sequences of an estrogen-responsive element.
Estrogen receptor (ER) was purified from calf uterus by immunoaffinity chromatography in the absence of the ligand. The purified ER consists of a mixture of monomer and homodimer forms of 67-kDa hormone-binding subunit (no 90-kDa heat shock protein is present). The purified ER was incubated with a 32P-labeled 61-basepair oligonucleotide containing the sequence of the estrogen response element (ERE) of the Xenopus laevis A2 vitellogenin gene. DNA mobility shift assays showed formation of specific complexes of the ERE containing oligonucleotide with ER, formation which did not require and was not affected by estradiol or antiestrogenic molecules. Both the monomer and the dimer were equally able to interact with the ERE-containing oligonucleotide. Sucrose gradient experiments showed that only the ER monomer is able to interact with an oligonucleotide in which a single mutation destroyed the dyad symmetry of ERE. Multiple symmetric mutations which did not alter the dyad symmetry of ERE nevertheless totally destroyed the ability of the oligonucleotide to form complexes with either the monomeric or dimeric form of ER. These results suggest that ER is able to bind to ERE independently of the presence of estradiol or other proteins and, therefore, that estradiol does not act by modulating the ability of ER to bind to ERE on DNA.
Istituto di Patologia Generale ed Oncologia, I Facoltà di Medicina e Chirurgia, Università di Napoli, Italy.
N
N
Medici
V
V
Nigro
C
C
Abbondanza
B
B
Moncharmont
A M
AM
Molinari
G A
GA
Puca
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002417:Cattle; D002845:Chromatography; D004591:Electrophoresis, Polyacrylamide Gel; D004958:Estradiol / Q000378:metabolism; D005260:Female; D005786:Gene Expression Regulation; D066298:In Vitro Techniques; D011960:Receptors, Estrogen / Q000378:metabolism*
D011960:Receptors, Estrogen; D004958:Estradiol