The estrogen receptor binds tightly to its responsive element as a ligand-induced homodimer

Extracts containing wild-type or mutant human estrogen receptor (ER) have been used to study the binding of ER to its responsive element (ERE). Estradiol (E2) or the antiestrogen hydroxytamoxifen is required for ER binding as assayed by gel retardation. The DNA binding domain (DBD) encompasses the highly conserved region C. Both intact ER-E2 complexes and ER mutants truncated for the hormone binding domain (HBD) bind as dimers to an ERE. However, an HBD-truncated ER binds less tightly to an ERE than an intact ER-E2 complex. The DBD and HBD contain a constitutive and a stronger ER-induced dimerization function, respectively. Thus, in addition to inducing the activation function associated with the HBD, estrogen plays a crucial role in the formation of stable ER dimers that bind tightly to ERE.     

Stability of the ligand-estrogen receptor interaction depends on estrogen response element flanking sequences and cellular factors

To determine whether accessory proteins mediate the ligand- and DNA sequence-dependent specificity of estrogen receptor (ER) interaction with DNA, the binding of partly purified vs highly purified bovine ER to various estrogen response elements (EREs) was measured in the presence of different ER ligands. Partly purified estradiol-liganded ER (E₂-ER) binds cooperatively to stereoaligned tandem EREs flanked by naturally occurring AT-rich sequences, with a stoichiometry of one E₂-ER dimer per ERE. In contrast, highly purified E₂-ER binds with a 10-fold lower affinity and non-cooperatively to EREs flanked by the AT-rich region. Moreover, the binding stoichiometry of highly purified E₂-ER was 0.5 E₂-ER dimer, or one monomer per ERE, independent of the ERE flanking sequence. Interestingly, the binding of ER liganded with the antiestrogen 4-hydroxytamoxifen (4-OHT-ER) was non-cooperative with an apparent stoichiometry of 0.5 4-OHT-ER dimer per ERE, regardless of ER purity or ERE flanking sequence. We recently showed that when 4-OHT-ER binds DNA, one molecule of 4-OHT dissociates from the dimeric 4-OHT-ER-ERE complex, accounting for the reduced apparent binding stoichiometry. In contrast, ER covalently bound by tamoxifen aziridine (TAz) gave an ERE binding stoichiometry of one TAz-ER dimer per ERE, and TAz-ER binds cooperatively to multiple AT-rich EREs, regardless of the purity of the receptor. We have obtained evidence that purification of ER removes an accessory protein(s) that interacts with ER in a sequence- and/or DNA conformational-dependent manner, resulting in stabilization of E₂, but not 4-OHT, in the ligand binding domain when the receptor binds to DNA. We postulate that retention of ligand by ER maintains the receptor in a conformation necessary to achieve high-affinity, cooperative ERE binding.     

Differential DNA-binding abilities of estrogen receptor occupied with two classes of antiestrogens: studies using human estrogen receptor overexpressed in mammalian cells.

We have developed a transient transfection system using the Cytomegalovirus (CMV) promoter to express the human estrogen receptor (ER) at very high levels in COS-1 cells and have used it to study the interaction of agonist and antagonist receptor complexes with estrogen response element (ERE) DNA. ER can be expressed to levels of 20-40 pmol/mg or 0.2-0.3% of total soluble protein and all of the soluble receptor is capable of binding hormone. The ER binds estradiol with high affinity (Kd 0.2 nM), and is indistinguishable from native ER in that the receptor is capable of recognizing its cognate DNA response element with high affinity, and of transactivating a transgene in an estradiol-dependent manner. Gel mobility shift assays reveal interesting ligand-dependent differences in the binding of receptor complexes to ERE DNA. Receptors occupied by estradiol or the type I antiestrogen transhydroxytamoxifen bind to DNA response elements when exposed to the ligand in vitro or in vivo. Likewise, receptors exposed to the type II antiestrogen ICI 164,384 in vitro bind to ERE DNA. However, when receptor exposure to ICI 164,384 is carried out in vivo, the ER-ICI 164,384 complexes do not bind to ERE DNA, or do so only weakly. This effect is not reversed by subsequent incubation with estradiol in vitro, but is rapidly reversible by in vivo estradiol exposure of intact COS-1 cells. This suggests there may be some cellular process involved in the mechanism of antagonism by the pure antiestrogen ICI 164,384, which is not observed in cell-free extracts.     

Binding of type II nuclear receptors and estrogen receptor to full and half-site estrogen response elements in vitro.

The mechanism by which retinoids, thyroid hormone (T3) and estrogens modulate the growth of breast cancer cells is unclear. Since nuclear type II nuclear receptors, including retinoic acid receptor (RAR), retinoid X receptor (RXR) and thyroid hormone receptor (TR), bind direct repeats (DR) of the estrogen response elements (ERE) half-site (5'-AGGTCA-3'), we examined the ability of estrogen receptor (ER) versus type II nuclear receptors, i.e. RARalpha, beta and gamma, RXRbeta, TRalpha and TRbeta, to bind various EREs in vitro . ER bound a consensus ERE, containing a perfectly palindromic 17 bp inverted repeat (IR), as a homodimer. In contrast, ER did not bind to a single ERE half-site. Likewise, ER did not bind two tandem (38 bp apart) half-sites, but low ER binding was detected to three tandem copies of the same half-site. RARalpha,beta or gamma bound both ERE and half-site constructs as a homodimer. RXRbeta did not bind full or half-site EREs, nor did RXRbeta enhance RARalpha binding to a full ERE. However, RARalpha and RXRbeta bound a half-site ERE cooperatively forming a dimeric complex. The RARalpha-RXRbeta heterodimer bound the Xenopus vitellogenin B1 estrogen responsive unit, with two non-consensus EREs, with higher affinity than one or two copies of the full or half-site ERE. Both TRalpha and TRbeta bound the full and the half-site ERE as monomers and homodimers and cooperatively as heterodimers with RXRbeta. We suggest that the cellular concentrations of nuclear receptors and their ligands, and the nature of the ERE or half-site sequence and those of its flanking sequences determine the occupation of EREs in estrogen-regulated genes in vivo .     

The plasticity of estrogen receptor-DNA complexes: binding affinity and specificity of estrogen receptors to estrogen response element half-sites separated by variant spacers

The consensus estrogen response element (cERE) contains a palindromic sequence of two 6-base pair (bp) half-sites separated by a spacer size of 3bp. This study investigates the extent to which estrogen receptors, ERalpha and ERbeta can bind target sequences not considered as conventional EREs. We determined the effect of spacer size (n=0-4) on the binding affinity and conformation of ERalpha and ERbeta in these complexes and the effect of HMGB1 on the complexation. We find (1) both receptors bind similarly and with progressively reduced affinity to cEREn, as n differs from 3; (2) however, both receptors bind as strongly to the cERE with no spacer (cERE0) as to cERE3; (3) HMGB1 enhances ER binding affinity in all complexes, resulting in strong and comparable binding affinities in all complexes examined; (4) the full-length ER binding differs strikingly from similar binding studies for the ER DNA binding domain (ERDBD), with the full-length ER dimer exhibiting strong binding affinity, enormous plasticity and retaining binding cooperativity as the spacer size varies; (5) both protease digestion profiles and monoclonal antibody binding assays indicate the conformation of the receptor in the ER/ERE complex is sensitive to the spacer size; (6) the ER/cERE0 complex appears to be singularly different than the other ER/cEREn complexes in binding and conformation. This multifaceted approach reinforces the notion of the plasticity in ER binding and leads to the hypothesis that in most cases, the minimum requirement for estrogen receptor binding is the ERE half-site, in which one or more cofactors, such as HMGB1, can cooperate to decrease ER binding specificity, while increasing its binding affinity.     

A single d(GpG) cisplatin adduct on the estrogen response element decreases the binding of the estrogen receptor

Both cisplatin and the estrogen receptor (ER) are known to bend DNA. The influence of the bending of sequences by the d(GpG)cisPt adduct binding of ER to estrogen response element (ERE)-like sequences was examined. Three ERE-like oligonucleotides with different affinities for ER and which include a GG in the linker sequence were designed in order to form a single central d(GpG)cisPt adduct. Using electrophoretic mobility shift assay and Scatchard analysis, it was shown that the presence of a single d(GpG)cisPt adduct in the linker sequence decreases the ER affinity for DNA. These results do not support a critical role of a DNA bend in the initial recognition of ERE by ER. Then, the platination of DNA outside of the ERE half-sites decreases the interaction of ER with ERE.     

A microtiter well assay for quantitative measurement of estrogen receptor binding to estrogen-responsive elements

Reproducible, rapid measurement of estrogen receptor (ER) binding to DNA was accomplished in microtiter wells treated so that ER-DNA complexes or DNA bound in preference to free ER. Mixtures of 35S-labeled DNA and [3H]estrogen-charged ER ([3H]ER), incubated to equilibrium in microfuge tubes, were transferred to microtiter wells previously treated with histone followed by gelatin. After binding of the DNA or ER-DNA complex to the treated wells, free ER was removed by washing. Radioactivity retained in each well was measured by placing individual wells from snap-apart microtiter plates directly in scintillation fluid. Binding of DNA was saturable, and ER-DNA complex binding was complete within 2 h at 4 C. The use of 35S-labeled DNA and [3H]ER allowed stoichiometric determination of ER bound to DNA. The amount of ER specifically bound to a consensus estrogen-responsive element (ERE) containing the inverted repeat GGTCAgagTGACC was determined by comparing ER bound to plasmid containing or lacking the ERE. At saturating concentrations of ER, plasmids bearing one, two, and four EREs in tandem bound approximately one, two, and four dimeric ER molecules, respectively. Scatchard analysis of saturation binding data revealed a Kd of 0.15 nM for specific ER binding to a single ERE site. Thus, the assay detects ER retaining both DNA-binding and estrogen-binding functions. ER complexed with DNA in the well was also detected using a monoclonal antibody specific for the receptor. Simple modifications of this method would allow study of other DNA-protein interactions.     

Ligands differentially modulate the protein interactions of the human estrogen receptors alpha and beta

The interactions of human estrogen receptor subtypes ERalpha and ERbeta with DNA and a 210 amino acid residue fragment of the coactivator protein SRC-1 bearing three nuclear receptor interaction motifs were investigated quantitatively using fluorescence anisotropy in the presence of agonist and antagonist ligands. ERalpha and ERbeta were found to bind in a similar manner to DNA, and both salt and temperature affected the affinity and/or stoichiometry of these interactions. The agonist ligands estradiol, estrone and estriol did not modify the binding of ERalpha to the fluorescein-labeled target estrogen response element. However, in the case of ERbeta, these ligands led to the formation of some higher-order protein-DNA complexes and a small decrease in affinity. The partial agonist 4-hydroxytamoxifen had little effect on either ER subtype, whereas the pure antagonist ICI 182,780 led to the cooperative formation of protein-DNA complexes of higher order than dimer, as further demonstrated by competition experiments and gel mobility-shift assays. In addition to DNA binding, the interaction of both ER subtypes with the Alexa488-labeled SRC-1 coactivator fragment was investigated by fluorescence anisotropy. The agonist ligands estrone, estradiol, estriol, genistein and ethynyl estradiol exhibited distinct capacities for inducing the recruitment of SRC-1 that were not correlated with their affinity for the receptor. Moreover, estrone and genistein exhibited subtype specificity in that they induced SRC-1 recruitment to ERbeta with much higher efficiency than in the case of ERalpha. The differential coactivator recruitment capacities of the ER agonists and their receptor subtype coactivator recruitment specificity may be linked to the molecular structure of the agonists with respect to their interactions with a specific histidine residue located at the back of the ligand-binding pocket. Altogether, these quantitative in vitro studies of ER interactions reveal the complex energetic and stoichiometric consequences of changes in the chemical structures of these proteins and their ligands.     

hsp70 is not required for high affinity binding of purified calf uterine estrogen receptor to estrogen response element DNA in Vitro

Bovine estrogen receptor (ER) was purified to near homogeneity by estrogen response element (ERE) affinity chromatography, and its ERE binding ability was measured in vitro. Highly purified ER bound EREs with reduced affinity compared to partially purified ER. Partially purified ER contained hsp70, but highly purified ER did not. We examined whether addition of purified recombinant human hsp70 or purified bovine hsp70 would restore the higher ERE binding affinity, stoichiometry, and ligand retention detected with partially purified receptor and how hsp70 affected the rate of ER-ERE association and dissociation. ER-ERE binding was not affected by antibodies to either constitutive or induced forms of hsp70, regardless of ER purity. Addition of purified hsp70, with or without ATP and Mg²⁺, did not affect the association or dissociation rates of highly purified liganded ER binding to ERE. hsp70 Did not alter the total amount of ER-ERE complex formed. Similarly, hsp70 did not affect the rate of [³H]estradiol (E₂) or [³H]4-hydroxytamoxifen (4-OHT) ligand dissociation from ER in the presence or absence of EREs. These data contrast with a report showing that maximal ERE binding by highly purified recombinant human ER required hsp70. We conclude that ER, purified from a physiological source, i.e., calf uterus, does not require hsp70 for maximal ER-ERE binding in vitro. Additionally, once ER is activated and bound by ligand, the receptor assumes its proper tertiary structure, and hsp70 does not impact ER ligand binding domain conformation.