The DNA binding domain of estrogen receptor alpha is required for high-affinity nuclear interaction induced by estradiol.
Estrogen receptor alpha (ER) is a member of the nuclear hormone receptor family, which upon binding estrogen shows increased apparent affinity for nuclear components (tight nuclear binding). The nuclear components that mediate this tight nuclear binding have been proposed to include both ER-DNA interactions and ER-protein interactions. In this paper, we demonstrate that tight nuclear binding of ER upon estrogen occupation requires ER-DNA interactions. Hormone-bound ER can be extracted from the nucleus in low-salt buffer using various polyanions, which mimic the phosphate backbone of DNA. The importance of specific ER-DNA interactions in mediating tight nuclear binding is also supported by the 380-fold lower concentration of the ERE oligonucleotide necessary to extract estrogen-occupied ER from the nucleus compared to the polyanions. We also demonstrate that estrogen-induced tight nuclear binding requires both the nuclear localization domain and the DNA binding domain of ER. Finally, enzymatic degradation of nuclear DNA allows us to recover 45% of tight nuclear-bound ER. We further demonstrate that ER-AIB1 interaction is not required for estrogen-induced tight nuclear binding. Taken together, we propose a model in which tight nuclear binding of the estrogen-occupied ER is predominantly mediated by ER-DNA interactions. The effects of estrogen binding on altering DNA binding in whole cells are proposed to occur through estrogen-induced changes in ER-chaperone protein interactions, which alter the DNA accessibility of ER but do not directly change the affinity of the ER for DNA, which is similar for both unoccupied and occupied ER.
Weinberg
Amy L
AL
Department of Pathology and Laboratory Medicine, University of Wisconsin, Madison, Wisconsin 53706, USA.
Carter
Damien
D
Ahonen
Minna
M
Alarid
Elaine T
ET
Murdoch
Fern E
FE
Fritsch
Michael K
MK
D019943:Amino Acid Substitution; D001667:Binding, Competitive / Q000187:drug effects; D002460:Cell Line; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004720:Endonucleases / Q000378:metabolism; D004958:Estradiol / Q000378:metabolism* / Q000494:pharmacology; D047628:Estrogen Receptor alpha / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017353:Gene Deletion; D006367:HeLa Cells; D051548:Histone Acetyltransferases / Q000235:genetics / Q000378:metabolism; D006801:Humans; D019913:Nuclear Localization Signals / Q000235:genetics; D056921:Nuclear Receptor Coactivator 3; D009841:Oligonucleotides / Q000235:genetics / Q000378:metabolism; D011122:Polyphosphates / Q000737:chemistry / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects; D020218:Response Elements / Q000235:genetics; D012995:Solubility; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism; D014638:Vanadates / Q000737:chemistry / Q000494:pharmacology
D004268:DNA-Binding Proteins; D047628:Estrogen Receptor alpha; D019913:Nuclear Localization Signals; D009841:Oligonucleotides; D011122:Polyphosphates; D015534:Trans-Activators; D014638:Vanadates; D004958:Estradiol; D004247:DNA; D051548:Histone Acetyltransferases; C538866:NCOA3 protein, human; D056921:Nuclear Receptor Coactivator 3; D004720:Endonucleases