Unoccupied Estrogen Receptor Research Articles

Hypoxia-inducible factor 1α activates and is inhibited by unoccupied estrogen receptor β

Previously, we showed that hypoxia induces ligand-independent estrogen receptor (ER)α activation. In this study, we found that hypoxia activated the ERβ-mediated transcriptional response in HEK293 cells in the absence of estrogen. ERβ transactivation was induced by the expression of the hypoxia-inducible factor 1α (HIF-1α) under normoxia. ERβ interacted with HIF-1α, and SRC1 and CBP potentiated the effect of HIF-1α on ERβ-mediated transcription. We then examined the effect of ERβ on HIF1-α transactivation. Surprisingly, ERβ attenuated the transcriptional activity of HIF-1α, as measured by HRE-driven reporter gene expression and hypoxic induction of VEGF mRNA in HEK293 cells. Taken together, these data show that HIF-1α activates ERβ-mediated transcription in the absence of a ligand, and ERβ inhibits HIF-1α-mediated transcription.

Effects of estrogen receptor and estrogen on the chromatin structure in estrogen receptor stable transfectants.

The estrogen receptor (ER), a member of the nuclear hormone receptor superfamily recruits coactivators that modify local chromatin structure. Here we investigated the effect of the estrogen receptor and estrogen on the global chromatin structure and the local chromatin structure of the progesterone receptor gene during the process of transcriptional activation using Rat1+ER cells stably expressing the estrogen receptor. The total chromatin was more accessible to DNasel in Rat1+ER cells than in the parental estrogen receptor-negative Rat1 cells. After 18 h of estrogen treatment, total chromatin was more dispersed in Rat1+ER cells than in Rat1 cells. The chromatin structure of the progesterone receptor gene was more sensitive to DNasel in Rat1+ER cells than in Rat1 cells. However, the chromatin structure of the progesterone receptor gene did not change further on estrogen treatment. Our results suggest that under certain circumstances unoccupied estrogen receptors may play some role in reorganizing the repressive chromatin structure to induce gene activation.

Identification of a unique liganded estrogen receptor complex released from the nucleus by decavanadate.

Unoccupied estrogen receptor (ER) can be extracted from tissues by homogenization with a hypotonic buffer, whereas hormone-occupied ER becomes tightly bound to the nuclear pellet and must be extracted with high-salt-containing buffers. The molecular basis for estrogen-induced tight nuclear binding of ER remains an important puzzle. The different subcellular fractionation behaviors of the occupied and unoccupied ER are presumed to be due to a difference in their ability to interact with nuclear components, such as DNA and proteins. The proteins that are the targets for interaction with the hormone-occupied ER may be important for transcriptional regulation. However, the salt-extracted ER is recovered as a homodimer, and associated proteins are presumably lost due to the high-salt conditions. We have discovered an alternate method of releasing the occupied ER from the nucleus. Inclusion of 2 mM orthovanadate, polymerized primarily to decavanadate, in a hypotonic buffer efficiently releases over 90% of estrogen-bound ER from the nuclear pellet. The recovered ER complex is fully functional in terms of estrogen and DNA binding and is full-length by western blot analysis. Our data suggest that the mechanism of ER release is by decavanadate competition with nuclear DNA, rather than by inhibition of a phosphotyrosine phosphatase. Of particular interest, the decavanadate released occupied ER complex shows distinct behavior by sucrose density gradient sedimentation analysis. It is larger than the salt-extracted transformed ER, suggesting that an occupied ER in complex with nuclear proteins may be released from the nucleus by decavanadate.

Modulation of Estrogen Receptor Levels in Mouse Uterus by Protein Kinase C Isoenzymes

We have recently shown that protein kinase C (PKC) modifies estrogen receptor (ER) binding and modulates the responsiveness to estrogens in a clonal osteoblast-like cell line stably transfected with the ER. The purpose of the present study was to determine whether the interaction observed between the ER and PKC signaling in these cells occurs in additional estrogen target organs, such as the uterus. When uteri were incubated for 2 h with increasing concentrations of a kinase inhibitor (H7), ER binding was enhanced in a dose-dependent manner. Stimulation of PKC with phorbol ester reduced PKC activity levels, but increased ER binding. Interestingly, the changes in binding appeared to be due primarily to alterations in cytosolic ER levels, as binding in the nuclear fraction was minimally enhanced. When levels of ER messenger RNA were evaluated by Northern blot analysis, no differences were observed among the H7- or 12-O-tetradecanoylphorbol-13-acetate (TPA)-treated and untreated groups. Western blot analysis, however, demonstrated that levels of ER cytosolic protein in the H7-, TPA-, and staurosporine-treated groups were increased relative to those in the untreated controls. When uteri were incubated with diethylstilbestrol in the presence of either H7 or TPA, no change in cytosolic ER levels was found, suggesting that only unoccupied ERs are responsive to modulation by PKC. Western blotting of the various PKC isoforms indicated that although PKCα, -βI, -βII, -δ, and -ζ are expressed in the uterus, only PKCα and -βI are translocated from the soluble to the particulate fraction and then degraded after phorbol ester stimulation. Hence, one or both of these latter PKC isoforms may regulate cytosolic ER levels. Collectively, these data indicate that PKC may play an important role in the modulation of uterine ER levels and that PKC may exert its effect on the ER at some posttranscriptional or posttranslational step. Finally, our results show that an ER-PKC interaction occurs in a whole organ such as the uterus and that this interaction may be important in the regulation of the ER activity in a variety of estrogen-responsive tissues.

Modulation of estrogen receptor levels in mouse uterus by protein kinase C isoenzymes.

We have recently shown that protein kinase C (PKC) modifies estrogen receptor (ER) binding and modulates the responsiveness to estrogens in a clonal osteoblast-like cell line stably transfected with the ER. The purpose of the present study was to determine whether the interaction observed between the ER and PKC signaling in these cells occurs in additional estrogen target organs, such as the uterus. When uteri were incubated for 2 h with increasing concentrations of a kinase inhibitor (H7), ER binding was enhanced in a dose-dependent manner. Stimulation of PKC with phorbol ester reduced PKC activity levels, but increased ER binding. Interestingly, the changes in binding appeared to be due primarily to alterations in cytosolic ER levels, as binding in the nuclear fraction was minimally enhanced. When levels of ER messenger RNA were evaluated by Northern blot analysis, no differences were observed among the H7- or 12-O-tetradecanoylphorbol-13-acetate (TPA)-treated and untreated groups. Western blot analysis, however, demonstrated that levels of ER cytosolic protein in the H7-, TPA-, and staurosporine-treated groups were increased relative to those in the untreated controls. When uteri were incubated with diethylstilbestrol in the presence of either H7 or TPA, no change in cytosolic ER levels was found, suggesting that only unoccupied ERs are responsive to modulation by PKC. Western blotting of the various PKC isoforms indicated that although PKC alpha, -beta1, -betaII, -delta, and -zeta are expressed in the uterus, only PKC alpha and -beta1 are translocated from the soluble to the particulate fraction and then degraded after phorbol ester stimulation. Hence, one or both of these latter PKC isoforms may regulate cytosolic ER levels. Collectively, these data indicate that PKC may play an important role in the modulation of uterine ER levels and that PKC may exert its effect on the ER at some posttranscriptional or posttranslational step. Finally, our results show that an ER-PKC interaction occurs in a whole organ such as the uterus and that this interaction may be important in the regulation of the ER activity in a variety of estrogen-responsive tissues.

Altered ligand binding properties and enhanced stability of a constitutively active estrogen receptor: evidence that an open pocket conformation is required for ligand interaction.

To elucidate the ligand binding properties of the estrogen receptor (ER) and how ligand access to and release from the ligand binding pocket is affected by the conformational state of the receptor, we have measured the rates of estradiol association and dissociation, the equilibrium binding, and the stability of estradiol binding to denaturants, comparing wild-type human ER and a point mutant (Y537S ER) that shows full constitutive activity, i.e., the same full transcriptional activity in the absence or presence of estrogen. Ligand binding kinetics and affinity were measured with the full-length (1-595) ERs and with truncated forms of both receptors containing domains C through F (including the DNA binding, hinge, and ligand binding domains, amino acids 175-595) or domains E and F (the ligand binding domain; amino acids 304-595). With all ERs, the rates of ligand association and dissociation were considerably slower with the Y537S mutant ER than with wild-type ER (6-fold and 3-4-fold, respectively). These marked differences in ligand on and off rates for the wild-type and Y537S receptors result in a predicted (k-1/k+1) and measured Kd that is 2-fold lower for Y537S ER compared to wild-type ER. The binding of estradiol by wild-type ER was disrupted by high concentrations of urea (above 2 M), whereas the Y537S ER was distinctly more resistant to this disruption. These results are consistent with a model in which wild-type ER in the absence of ligand adopts a transcriptionally inactive collapsed pocket conformation, stabilized by specific interactions of Y537 with nearby regions of ER. When estradiol is bound, the wild-type ER adopts a transcriptionally active, closed pocket (ligand occupied) conformation. By contrast, the Y537S mutant ER favors the transcriptionally active closed pocket conformation, whether occupied by ligand or not, the latter state (closed pocket but unoccupied) accounting for its constitutive activity. Our findings suggest that the entry or exit of ligand from the binding pocket requires that ER adopt an open pocket conformation. The reduced rates of ligand association and dissociation in the constitutively active form of the ER, as well as its greater resistance to disruption of ligand binding by urea, support the supposition that the rate at which this open pocket conformation can be accessed from the unoccupied or ligand-occupied Y537S ER is slower than from the unoccupied or occupied forms of wild-type ER. Thus, the binding and release of ligand by ER require that the receptor access an open pocket state, and the ease with which this state can be accessed is affected by mutations that alter receptor conformation.

Equilibrium Binding of Estrogen Receptor with DNA Using Fluorescence Anisotropy

Interaction of estrogen receptor (ER) with DNA sequences known as estrogen response elements (ERE) is required for estrogen regulation of the expression of target genes. To characterize the affinity and specificity of ER interaction with ERE sequences in vitro under equilibrium conditions, fluorescence anisotropy assays were performed using recombinant, purified ER and a fluorescein-labeled 35-base pair oligonucleotide bearing an idealized palindromic ERE. In buffer containing 100 mM KCl, the baculovirus-expressed, purified human ER bound with similar affinity to the consensus ERE and a mutant ERE with a single base pair change per half-site. Above 225 mM KCl, ER exhibited discrimination between the consensus and mutated ERE targets. Between 225 and 275 mM KCl, binding to the consensus ERE was independent of salt concentration and occurred with an equilibrium dissociation constant (Kd) of 1.8 +/- 0.6 nM, whereas binding to the mutant ERE was not detected at ER concentrations below 100 nM under the same conditions. At 300 mM KCl, the Kd for the consensus ERE increased approximately 25-fold, suggesting complex salt concentration dependence. Both estrogen-occupied and unoccupied ER bound to the consensus ERE sequence with similar affinity, indicating that estrogen affects ER activity at a step other than DNA binding. Unlike the full-length ER, the recombinant DNA binding domain of ER did not discriminate between the consensus and mutated ERE sequences even at buffer salt concentrations greater than 200 mM NaCl, suggesting that ER sequences outside the DNA binding domain may be important in promoting specific binding.

Two populations of the estrogen receptor separated and characterized using aqueous two-phase partitioning.

Two populations of the rat uterine estrogen receptor (ER) were separated and characterized using aqueous two-phase partitioning. Countercurrent distribution of rat uterine cytosolic ER allowed rapid and efficient separation of two populations, one population partitioned preferentially into the top phase (T, K(obs) = 3-6) and the other into the bottom phase (B, K(obs) = 0.01-0.03). The majority of unoccupied cytosolic ER is in the T population. Upon estrogen binding and/or heating to 30 degrees C in vitro the T population is converted to the B population. The transition from T to B does not exclusively involve loss of heat shock protein 90 and does not alter the ligand binding ability of the steroid binding domain. Using the human ER steroid binding domain overproduced in Escherichia coli and the steroid binding domain generated by partial trypsinization of the rat uterine ER, we demonstrate that the characteristic distinguishing T and B populations is not localized to this domain alone but may be associated with the amino terminal half of the ER (the A/B and DNA binding domains). The T to B transition of the ER also occurs in human MCF-7 breast cancer cells upon treatment with estrogen at 37 degrees C.

Sex differences in estrogen receptor and progestin receptor induction in the guinea pig hypothalamus and preoptic area

Quantitative in vitro autoradiography was used to determine if regional sex differences in estrogen receptor (ER) content and/or estrogen responsiveness, as indicated by an increase in progestin receptor (PR), are present in the adult guinea pig brain. Adult male and female guinea pigs were gonadectomized 1 week before subcutaneous injection of 25 μg estradiol benzoate (EB)/kg body wt or the sesame oil vehicle. Animals were killed by decapitation 44 h after injection. Unoccupied PRs, and unoccupied and occupied ERs, were measured in discrete brain regions by quantitative in vitro autoradiography using [ 3H]R5020 and [ 3H]estradiol as ligands, respectively. In vehicle-injected controls, a higher level of ER was found in the arcuate nucleus (ARC), dorsal medial nucleus (DMN) and ventrolateral nucleus (VLN) of females as compared to males. At 44 h after EB injection, 32–55% of the ERs were occupied; however, EB treatment caused a marked down-regulation of total receptor (calculated as occupied + unoccupied receptor) in most of the brain regions examined, including the periventricular preoptic area (PVP), medial preoptic area (WO), bed nucleus of the stria terminalis, paraventricular nucleus, ARC, ventrolateral hypothalamus (VLH), VLN, and DMN. In EB-treated animals, PR binding was detectable in the PVP, MPO, ARC, VLH, and VLN, with higher levels of binding observed in the PVP, MPO, and VLN of the female as compared to the male. No PR binding was observed in oil-injected control animals. These results demonstrate region-specific sex differences in ER as well as estrogen-induced regulation of progestin and ERs in the guinea pig brain. The discordance between the regional distributions of sex differences in ER and estrogen-induced PR implies that sex differences in ER and estrogen-induced PR implies that sex differences in estrogen response may not be clearly linked to a sex difference in receptor number. Instead, sex differences in response may involve differences in receptor number within specific subpopulations of estrogen target cells or may involve differences in ER dynamics.

In vitro labeling of gonadal steroid hormone receptors in brain tissue sections

Autoradiographic methods have been developed for measurement of gonadal steroid receptors in situ in brain tissue sections. Based on principles established previously for estrogen receptors in the rat brain using a 125I-labeled ligand, procedures have been developed for in vitro labeling of estrogen, androgen, and progestin receptors with commercially available tritiated ligands. Addition of protamine sulfate to the incubation buffer precipitates the receptors in situ in the tissue sections, allowing them to be detected autoradiographically after incubation with labeled steroid and subsequent washing to remove unbound and nonspecifically bound ligand. Occupied and unoccupied estrogen receptors can be measured selectively using appropriately modified incubation conditions. In the case of androgen and progestin receptors, unoccupied receptors are readily detected by in vitro labeling of tissue sections, but occupied receptors do not appear to label efficiently. Preliminary data suggest that these methods should be equally applicable to a variety of laboratory animals, including the rat, mouse, guinea pig, and monkey.

Estrogen receptor-induced DNA bending: orientation of the bend and replacement of an estrogen response element with an intrinsic DNA bending sequence.

The estrogen receptor (ER) binds to DNA fragments containing estrogen response elements (EREs) and causes them to bend. To characterize this ER-induced DNA bend and determine if it is involved in transcription activation, three different lines of investigation were used. Using MCF-7 human breast cancer cell extracts and circular permutation analysis, it was determined that molybdate-stabilized, unoccupied cytosolic ER was unable to bind to ERE-containing DNA fragments when maintained at 4 C, but that thermal activation enabled the cytosolic receptor to bind and bend ERE-containing DNA fragments to the same extent as ER present in whole cell extracts. DNA phasing analysis was utilized to determine that ER binding induced DNA fragments containing EREs to bend toward the major groove of the DNA helix. The orientation of this bend was the same with thermally activated, unoccupied cytosolic ER and with unoccupied ER, 17 beta-estradiol-occupied ER, and 4-hydroxytamoxifen-occupied ER present in whole cell extracts. Using transient cotransfection assays, the ability of an intrinsically bent DNA sequence to replace an ERE was tested. When a single consensus ERE, which is induced to bend 56 degrees on ER binding, was replaced with a 54 degrees intrinsic DNA bending sequence, transcription was effectively activated. Similar levels of transcription were also observed when promoters contained either a 108 degrees intrinsic DNA bending sequence or two consensus EREs. However, the 54 degrees DNA bending sequence and a single ERE were unable to cooperatively activate transcription. Because the magnitude and orientation of ER-induced DNA bends are the same with the unoccupied and occupied receptor, DNA bending alone probably does not function as a transcriptional switch to turn on gene transcription. However, DNA bending may be required to provide the architecture needed for modulation of target genes.

Intranuclear Localization of Hormone-Occupied and -Unoccupied Estrogen Receptors in the Mouse Uterus: Application of 1 nm Immunogold-Silver Enhancement Procedure to Ultrathin Frozen Sections

Employing immunoelectron microscopy with ultrathin frozen sections, intranuclear localization of hormone-occupied and -unoccupied estrogen receptors (ER) was examined in the uterine epithelium; after 1 h of 17 beta-estradiol (E2, 20 micrograms/kg b.w.) or saline intraperitoneal injection, adult ovariectomized mice were sacrificed. ER were localized with both anti-ER monoclonal antibody and polyclonal antibody (AS 409) and 1 nm immunogold followed by silver enhancement. No significant differences in ultrastructure were recognized in either the nuclei or the cytoplasm of the uterine epithelium in E2-treated and control animals. E2-occupied and -unoccupied ER showed the same distribution pattern in the nuclei of both luminal and glandular epithelium. ER were exclusively localized in the dispersed and slightly condensed chromatin; the margins of the condensed chromatin also showed strong immunoreaction. Little immunolabeling was observed in the highly condensed chromatin associated with nuclear membranes. The reaction was negative in the nucleoli. Both antibodies provided the same ER intranuclear localization. It is suggested that marked change in the distribution of steroid receptors, i.e. rapid translocation from condensed chromatin to dispersed chromatin or the margins of condensed chromatin as described in some previous reports, probably takes place as a consequence of conformational changes in the chromatin-binding receptors.

Distribution of occupied and unoccupied estrogen receptors in the rat brain: effects of physiological gonadal steroid exposure.

In vitro autoradiographic methods have been developed for selective measurement of occupied and unoccupied estrogen receptors (ERs) in brain tissue sections. Addition of protamine sulfate traps unoccupied ERs in the tissue sections, allowing them to be detected after a short period of incubation with labeled estrogen. Occupied ERs are assessed, after washing in buffer without protamine to eliminate unoccupied receptor, by incubating the sections for 2 h at 37 C to exchange isotopically labeled steroid for the endogenous unlabeled ligand. Total ER binding capacity is estimated by summing the values for occupied and unoccupied ER. In all brain regions of normal females, ER occupation is low at estrus, reflecting the very low levels of circulating estradiol present at this stage of the estrous cycle, rising to approximately 50% of binding capacity at proestrus. By contrast, in intact males ER occupation varies considerably between brain regions, from a high of 55% of binding capacity in the bed nucleus of the stria terminalis to a low of 21% in the hypothalamic arcuate nucleus. Gonadectomy or treatment of intact males with the aromatase inhibitor 4-hydroxy androstenedione greatly reduces or eliminates ER occupation, depending on the brain region. In both sexes, changes in levels of endogenous gonadal steroids have little effect on total (occupied plus unoccupied) ER concentrations, with the exception of the hypothalamic ventromedial nucleus of the female, in which total ER concentration declines at estrus. These results are consistent with the hypothesis that local aromatization may be the primary determinant of regional ER occupation in the brain of the male rat, in contrast to the female, in which high levels of ER occupation are found only during the preovulatory estrogen surge. Although physiological changes in circulating estradiol and aromatizable androgen concentrations induce large changes in ER occupation, they have little effect on total ER content in most regions of the brain, suggesting that previous reports of changes in ER messenger RNA levels under different conditions of gonadal steroid exposure may not be directly reflected in steady state levels of the cognate receptor site.

Distribution of occupied and unoccupied estrogen receptors in the rat brain: effects of physiological gonadal steroid exposure

In vitro autoradiographic methods have been developed for selective measurement of occupied and unoccupied estrogen receptors (ERs) in brain tissue sections. Addition of protamine sulfate traps unoccupied ERs in the tissue sections, allowing them to be detected after a short period of incubation with labeled estrogen. Occupied ERs are assessed, after washing in buffer without protamine to eliminate unoccupied receptor, by incubating the sections for 2 h at 37 C to exchange isotopically labeled steroid for the endogenous unlabeled ligand. Total ER binding capacity is estimated by summing the values for occupied and unoccupied ER. In all brain regions of normal females, ER occupation is low at estrus, reflecting the very low levels of circulating estradiol present at this stage of the estrous cycle, rising to approximately 50% of binding capacity at proestrus. By contrast, in intact males ER occupation varies considerably between brain regions, from a high of 55% of binding capacity in the bed nucleus of the stria terminalis to a low of 21% in the hypothalamic arcuate nucleus. Gonadectomy or treatment of intact males with the aromatase inhibitor 4-hydroxy androstenedione greatly reduces or eliminates ER occupation, depending on the brain region. In both sexes, changes in levels of endogenous gonadal steroids have little effect on total (occupied plus unoccupied) ER concentrations, with the exception of the hypothalamic ventromedial nucleus of the female, in which total ER concentration declines at estrus. These results are consistent with the hypothesis that local aromatization may be the primary determinant of regional ER occupation in the brain of the male rat, in contrast to the female, in which high levels of ER occupation are found only during the preovulatory estrogen surge. Although physiological changes in circulating estradiol and aromatizable androgen concentrations induce large changes in ER occupation, they have little effect on total ER content in most regions of the brain, suggesting that previous reports of changes in ER messenger RNA levels under different conditions of gonadal steroid exposure may not be directly reflected in steady state levels of the cognate receptor site.

Down-regulation of estrogen receptor immunoreactivity by 17 beta-estradiol in the guinea pig forebrain.

Low amplitude pulses of estradiol-17 beta (E2-17 beta) are more effective than large single bolus injections or constant exposure to E2-17 beta in inducing progesterone-facilitated sex behavior in female rats and guinea pigs. The present study examined whether the increased responsiveness to E2-17 beta is due to an increase in the number of estrogen receptors in the estrogen receptor rich areas of the hypothalamus and amygdala. Initial studies examined the rapid effects (20 min) of a high dose of E2-17 beta (50 micrograms) on estrogen receptor immunostaining using either the H222 antibody or the ER 21 antiserum. ER 21 immunostaining was not affected by the E2-17 beta treatment suggesting that it binds to both occupied and unoccupied estrogen receptors. Therefore the ER 21 antiserum was used to characterize the regulation of estrogen receptor immunoreactivity (ER-IR) by E2-17 beta. ER-IR was examined for 48 h and serum E2-17 beta for 24 h following a 2 micrograms s.c. injection of E2-17 beta (a dose similar to that used in multiple pulse paradigms). Serum E2-17 beta peaked 15 to 30 min following the injection and returned to baseline values by 1 h. In all but one area maximal suppression of ER-IR occurred at 12 h. In summary, 1) decreases in estrogen receptor immunoreactivity following E2-17 beta are consistent with studies in which estrogen receptors were assayed by binding assays and estrogen receptor mRNA was determined by in situ hybridization; 2) the ER 21 antiserum is able to detect both occupied and unoccupied estrogen receptors and 3) H222 immunoreactivity is influenced by the presence of E2-17 beta, so that the level of H222-IR is a reflection of ligand/receptor binding dynamics. The data suggest that up-regulation of estrogen receptors does not account for the increase in behavioral sensitivity which is observed following multiple pulses of E2-17 beta.

Structural characterization of the trypsinized estrogen receptor.

Structural differences between the unoccupied and ligand-occupied rat uterine estrogen receptors (ERs) were investigated using partial proteolysis followed by immunoblotting, affinity labeling, and gel filtration chromatography. Trypsin digestion of the unoccupied ER at 4 degrees C resulted in retention of 70-80% of high-affinity [3H]estradiol binding. Only two fragments of the rat ER were detected after prolonged trypsin treatment of the unoccupied ER followed by affinity labeling with [3H]tamoxifen aziridine. One fragment represents the intact steroid binding domain (28 kDa), and the other fragment is about 10 kDa. The small 10-kDa fragment of the ER detected by denaturing gel electrophoresis is shown to be held in a large oligomeric complex in solution using gel filtration chromatography. This oligomeric complex probably represents the steroid binding domain, which has its tertiary structure maintained predominantly by noncovalent interactions between the trypsin-generated fragments. The estrogen, anti-estrogen, and unoccupied trypsinized ERs all result in similar patterns of fragments after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and detection by immunoblotting. Although no new trypsin cleavage sites were exposed, the sensitivity of the available trypsin sites was altered by heating the ER and, to a lesser extent, by hormone treatment. Gel filtration chromatography of the trypsinized estradiol- and 4-hydroxytamoxifen-occupied ERs demonstrates similar, diffuse peaks centered at about the correct size for the intact steroid binding domain (28 kDa), whereas the trypsinized unoccupied ER results in a sharp, discrete peak centered at about 80 kDa.(ABSTRACT TRUNCATED AT 250 WORDS)

Examination of the DNA-binding ability of estrogen receptor in whole cells: implications for hormone-independent transactivation and the actions of antiestrogens.

We describe an assay employing the competitive binding of estrogen receptor (ER) with basal transcription factors on a constitutive promoter (cytomegalovirus-hormone response element[s]-chloramphenicol acetyltransferase [CMV-(HRE)n-CAT, containing a hormone response element(s) between the TATA box and the start site of transcription]) to examine the DNA-binding ability of the human ER in whole cells. We used this promoter interference assay to examine the DNA binding of ER in cell lines containing high and low levels of endogenous ER, as well as in CHO cells expressing wild-type and mutant ERs from cotransfected expression vectors. The ER is capable of binding to the promoter interference constructs in the absence of added ligand, and estrogen (estradiol) or antiestrogen (trans-hydroxytamoxifen or ICI 164,384) enhances or stabilizes this interaction. The binding of unoccupied ER to reporter gene activation plasmids results in ligand-independent transactivation, presumably due to the TAF-1 function of the receptor. DNA binding of ER in the absence of ligand is observed in cells containing endogenous ER, or expressed ER, and occurs in cells with high or low receptor contents. Although estrogen- and antiestrogen-occupied ER complexes bind to DNA and reduce the template promoter activity, the extent of suppression achieved by ICI-bound ERs is consistently less than that achieved with the other ligands, presumably caused by the fact that ICI rapidly reduces the level of ER in most of the cells examined. However, the ICI-ER complexes that remain are in sufficient quantity to bind to gene activation reporter constructs, and in these cells, ICI still behaves as a pure antagonist of gene transcription and does not activate reporter genes. Hence, obstruction of ER DNA binding or reduction of ER in target cells may contribute to, but cannot fully explain, the pure antagonist character of the antiestrogen ICI 164,384. In addition, DNA binding by the ER alone is clearly not sufficient for ensuring full activation of transcription and argues for an intermediate in the receptor activation of promoters.

Examination of the DNA-binding ability of estrogen receptor in whole cells: implications for hormone-independent transactivation and the actions of antiestrogens.

We describe an assay employing the competitive binding of estrogen receptor (ER) with basal transcription factors on a constitutive promoter (cytomegalovirus-hormone response element[s]-chloramphenicol acetyltransferase [CMV-(HRE)n-CAT, containing a hormone response element(s) between the TATA box and the start site of transcription]) to examine the DNA-binding ability of the human ER in whole cells. We used this promoter interference assay to examine the DNA binding of ER in cell lines containing high and low levels of endogenous ER, as well as in CHO cells expressing wild-type and mutant ERs from cotransfected expression vectors. The ER is capable of binding to the promoter interference constructs in the absence of added ligand, and estrogen (estradiol) or antiestrogen (trans-hydroxytamoxifen or ICI 164,384) enhances or stabilizes this interaction. The binding of unoccupied ER to reporter gene activation plasmids results in ligand-independent transactivation, presumably due to the TAF-1 function of the receptor. DNA binding of ER in the absence of ligand is observed in cells containing endogenous ER, or expressed ER, and occurs in cells with high or low receptor contents. Although estrogen- and antiestrogen-occupied ER complexes bind to DNA and reduce the template promoter activity, the extent of suppression achieved by ICI-bound ERs is consistently less than that achieved with the other ligands, presumably caused by the fact that ICI rapidly reduces the level of ER in most of the cells examined. However, the ICI-ER complexes that remain are in sufficient quantity to bind to gene activation reporter constructs, and in these cells, ICI still behaves as a pure antagonist of gene transcription and does not activate reporter genes. Hence, obstruction of ER DNA binding or reduction of ER in target cells may contribute to, but cannot fully explain, the pure antagonist character of the antiestrogen ICI 164,384. In addition, DNA binding by the ER alone is clearly not sufficient for ensuring full activation of transcription and argues for an intermediate in the receptor activation of promoters.

Cross-Linking of Estrogen Receptor to Chromatin in Intact MCF-7 Human Breast Cancer Cells: Optimization and Effect of Ligand

To investigate the effect of ligand (be it hormone, antihormone, or no hormone) on the interaction between estrogen receptor (ER) and chromatin, we have used formaldehyde as a cross-linking agent in intact MCF-7 human breast cancer cells. After a 1- to 2-h hormone treatment, the cells are exposed for 8 min to formaldehyde, which is added directly to their culture medium to minimize environmental perturbation. Nuclei are prepared from formaldehyde-treated cells and their contents are fractionated on CsCl density gradients to separate DNA-protein complexes from free protein. Peak gradient fractions are assayed for the presence of specific proteins by immunoblot of sodium dodecyl sulfate-polyacrylamide gel patterns. Using this approach, we find that 0.15% formaldehyde is optimal for cross-linking ER to chromatin. We detect ER and the large subunit of RNA polymerase II with DNA from formaldehyde-treated, but not from untreated cells. On the other hand, actin (a cytoplasmic protein) and small nuclear ribonucleoprotein particle proteins (nuclear RNA binding proteins) are not cross-linked to DNA. Therefore, cross-linking appears to be selective and fractionation is efficient. Interestingly, we detect similar levels of ER (as well as RNA polymerase II) with DNA from formaldehyde-treated cells, regardless of whether the cells are preexposed to estrogen (17 beta-estradiol at 10(-8) M), antiestrogen (ICI 164,384 at 10(-7) or 10(-6) M), or no hormone. These results, using covalent cross-linking in intact cells, indicate that both ligand-occupied and unoccupied ER are associated with chromatin.

Identification of unoccupied but transformed nuclear estrogen receptor in cultured mouse leydig cell

The molecular forms of estrogen receptor (ER) in estrogen-responsive mouse Leydig cell line (B-l) have been examined in relation to their biological activity. ER was predominantly recovered in the nuclear fraction upon homogenization even after cells were precultured in the absence of E 2 and Phenol Red. This unoccupied nuclear ER (ERn) whose hormone binding ability was extremely thermostable could be extracted with 0.4 M KC1. This stability enabled us to determine hydrodynamic parameters in the ligand-free condition. The Stokes radius and sedimentation constant of this ERn in high salt condition were 5.5 nm and 6.0S, respectively, resulting in its molecular weight of 140,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of ER labeled with [ 3H]tamoxifen aziridine gave a single band of 65,000 Da, indicating that this ERn had a oligomer structure similar to that of transformed nuclear ER complexed with estrogen in the putative target cells. Therefore, we further examined the possibility that this ERn in B-l cells can activate estrogen-responsive genes without any aid from estrogen. Estrogen responsive element-thymidine kinase promoter-chloramphenicol acetyltransferase fusion gene (ERE-tk-CAT) was transfected into B-1 cells. CAT activity was enhanced only in cells stimulated with estrogen. It may be concluded from these results that transformed ERn can be formed in the absence of estrogen but that binding to estrogen may be required in order to exert its biological activity.