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PubMed1996

Estrogenic triarylethylene acetic acids: effect of structural variation on estrogen receptor affinity and estrogenic potency and efficacy in MCF-7 cells.

Triarylethylenecarboxylic acids exemplified by (E,Z)-2-{4-[1-(p-hydroxyphenyl)-2-phenyl]-1-butenyl}phenoxyacetic acid (8) are a new class of estrogen receptor (ER) ligands capable of tissue selective estrogen agonist and antagonist effects. We report the syntheses of 8 and of analogues incorporating structural features known or anticipated to facilitate ER affinity in triarylethylenes. These studies revealed that the p-hydroxyphenyl moiety, ethylenic bond, and ether oxygen of 8 were all critical for high ER affinity. Although a 1,1-bisphenolic analogue bearing the p-(oxyacetic acid) moiety on its 2-phenyl ring, 12, had low ER affinity, it exhibited estrogenic potency approaching that of 8 in MCF-7 cells. Unlike 8 which was a partial agonist with weak antagonist potency, 12 was a full agonist. A similar profile of potency/efficacy in MCF-7 cells was seen in 9, an ethylenic bond saturated analogue of 8. Growth-promoting effects of 8, 9, and 12 were fully antagonized by the antiestrogen tamoxifen, suggesting that such effects were mediated solely via ER. Thus, our studies in MCF-7 cells have confirmed the estrogenicity of 8 and have enabled identification of two analogues with favorable estrogenic potency and full estrogen efficacy. On this basis, these three (triarylethylene)acetic acids have been selected for more intensive animal studies of their extrareproductive tract estrogenic effects.

Animals↗
PubMed1988

Bromine-80m-labeled estrogens: Auger electron-emitting, estrogen receptor-directed ligands with potential for therapy of estrogen receptor-positive cancers.

To assess their possible use for estrogen receptor (ER)-directed radiotherapy of estrogen receptor-containing cancers, two estrogens were synthesized with the Auger electron-emitting nuclide bromine-80m and administered to immature female rats. Both the triphenylethylene-based estrogen, [80mBr]-2-bromo-1,1-bis(4-hydroxyphenyl)phenylethylene (Br-BHPE) and the steroidal estrogen [80mBr]17 alpha-bromovinylestradiol, showed substantial diethylstilbestrol-inhibitable localization only in the estrogen target tissues, the uterus, pituitary, ovaries, and vagina and, except for the liver and intestines, generally lower concentrations in all other tissues at both 0.5 and 2 h. The [80mBr]Br-BHPE (specific activity, 8700 Ci/mmol), was shown to bind specifically to the low salt extractable ER of the rat uterus. Comparing i.p., i.v., and s.c. administration of [80mBr]BHPE the i.p. route was found to be particularly advantageous to effect maximum, DES-inhibitable concentrations of radiobromine in the ER-rich target organs in the peritoneal cavity. When the tissue distribution of the [80mBr]Br-BHPE was compared with that of sodium bromide-80m, it was apparent that no substantial amounts of radiobromine were released from the bromoestrogen prior to its target tissue localization. The substantial concentration of these bromine-80m-labeled estrogens in ER-rich tissues, combined with previously reported evidence for the effective radiotoxicity of Auger electron-emitting nuclides within cell nuclei suggest a good potential for such ligands for therapy of ER positive cancers.

Animals↗
PubMed2003

Estrogenic diazenes: heterocyclic non-steroidal estrogens of unusual structure with selectivity for estrogen receptor subtypes.

Estrogens regulate many biological functions, often acting in a tissue-selective manner. Their tissue-selective action is believed to involve differential estrogen action through the two estrogen receptor (ER) subtypes, ERalpha and ERbeta, as well as differential interaction of the ligand-receptor complexes with promoters and coregulator proteins. In the latter case, selectivity is based on the induction of specific conformations of the ligand-ER complex, conformations that are influenced by the structure of the ligand. Estrogen pharmaceuticals having an ideal balance of tissue-selective activity are being sought for menopausal hormone replacement, breast cancer prevention and therapy, and other actions. To expand on the structural diversity of ER ligands that might show such tissue selectivity, we have prepared a series of diazenes (pyrazines, pyrimidines, and pyridazines) substituted with two to four aryl groups and various short-chain aliphatic substituents. All of the pyrazine and pyrimidines bind to ER, some with high affinity and with a considerable degree of preferential binding to either ERalpha or ERbeta. One pyrimidine and one pyrazine have ERalpha affinity preferences as high as 23 and 9, respectively, and one pyrimidine has an ERbeta affinity preference of 8. The pyridazines, by contrast, are quite polar and have only very low binding affinity for the ER. In cell-based transcription assays, several of the pyrimidines and a pyrazine were found to be considerably more agonistic on ERalpha than on ERbeta. Because these triaryl diazenes have the largest volumes among the ER ligands so far investigated, their high affinity demonstrates the flexibility of the ligand binding pocket of the ERs and its tolerance for large substituents. Thus, these novel heterocyclic ligands expand the repertoire of chemical structures that bind to the estrogen receptor, and they could prove to be useful in elucidating the biological behavior of the two ER subtypes and in forming the basis for new estrogen pharmaceuticals having desirable tissue selectivity.

Endometrial Neoplasms↗
PubMed1997

Mouse estrogen receptor beta forms estrogen response element-binding heterodimers with estrogen receptor alpha.

The recent discovery that an additional estrogen receptor subtype is present in various rat tissues has advanced our understanding of the mechanisms underlying estrogen signaling. Here we report on the cloning of the cDNA encoding the mouse homolog of estrogen receptor-beta (ER beta) and the functional characterization of mouse ER beta protein. ER beta is shown to have overlapping DNA-binding specificity with that of the estrogen receptor-alpha (ER alpha) and activates transcription of reporter gene constructs containing estrogen-response elements in transient transfections in response to estradiol. Using a mammalian two-hybrid system, the formation of heterodimers of the ER beta and ER alpha subtypes was demonstrated. Furthermore, ER beta and ER alpha form heterodimeric complexes with retained DNA-binding ability and specificity in vitro. In addition, DNA binding by the ER beta/ER alpha heterodimer appears to be dependent on both subtype proteins. Taken together these results suggest the existence of two previously unrecognized pathways of estrogen signaling; I, via ER beta in cells exclusively expressing this subtype, and II, via the formation of heterodimers in cells expressing both receptor subtypes.

Amino Acid Sequence↗
PubMed2002

Relationship between estrogen receptor-binding and estrogenic activities of environmental estrogens and suppression by flavonoids.

In this study, we investigated the estrogenic activity of environmental estrogens by a competition binding assay using a human recombinant estrogens receptor (hERbeta) and by a proliferation assay using MCF-7 cells and a sulforhodamine-B assay. In the binding assay, pharmaceuticals had a stronger binding activity to hERbeta than that of some phytoestrogens (coumestrol, daidzein, genistein, luteolin, chrysin, flavone, and naringenin) or industrial chemicals, but phytoestrogens such as coumestrol had a binding activity as strong as pharmaceuticals such as 17alpha-ethynylestradiol (EE), tamoxifen (Tam), and mestranol. In the proliferation assay, pharmaceuticals such as diethylstilbestrol, EE, Tam, and clomiphene, and industrial chemicals such as 4-nonylphenol, bisphenol A, and 4-dihydroxybiphenyl had a proliferation-stimulating activity as strong as 17beta-estradiol (ES). In addition, we found that phytoestrogens such as coumestrol, daidzein, luteolin, and quercetin exerted a proliferation stimulating activity as strong as ES. Furthermore, we examined the suppression of proliferation-stimulating activity, induced by environmental estrogen, by flavonoids, such as daidzein, genistein, quercetin, and luteolin, and found that these flavonoids suppressed the induction of the proliferation-stimulating activity of environmental estrogens. The suppressive effect of flavonoids suggests that these compounds have anti-estrogenic and anti-cancer activities.

Binding, Competitive↗
PubMed2001

Regulation of prothymosin alpha gene expression by estrogen in estrogen receptor-containing breast cancer cells via upstream half-palindromic estrogen response element motifs.

Prothymosin alpha (PTalpha), a protein associated with cell proliferation and chromatin remodeling, and found to selectively enhance ER transcriptional activity by interacting with a repressor of ER activity, is shown to be a primary response gene to estrogen. Prothymosin alpha mRNA was rapidly increased by estrogen, followed by a 6-fold increase in prothymosin alpha protein content in ER-containing breast cancer cells. Analysis of the prothymosin alpha promoter and 5'-flanking region, and electrophoretic gel mobility shift studies showed the strong inducibility by the estradiol-ER complex to be mediated by two consensus half-palindromic estrogen response elements at -750 and -1051, which directly bind the ER. Estrogenic stimulation of prothymosin alpha required a DNA binding form of ER with a functional activation function-2 domain. The prothymosin alpha 5'-regulatory region also contains multiple Sp1 sites. Although addition of Sp1 did not further enhance estradiol-ER stimulated prothymosin alpha transcriptional activity in breast cancer cells, transfection and response element mutagenesis studies using Drosophila cells, which are deficient in Sp1, revealed that Sp1 and the estradiol occupied-ER can each activate the prothymosin alpha gene independently of the other and act in an additive manner. These observations, documenting robust prothymosin alpha up-regulation by the estradiol-ER complex via widely spaced half-palindromic estrogen response element motifs, are reminiscent of those shown previously for the ovalbumin gene and suggest that the use of multiple half response elements may be a more common mode for regulation of gene expression by the ER than previously appreciated. In addition, these observations suggest interrelationships between cell proliferation and gene transcriptional activities and indicate a positive mechanism by which PTalpha, which increases ER transcriptional effectiveness, is itself up-regulated by the estrogen-ER complex.

Amino Acid Motifs↗
PubMed1984

Differences between estrogen- and antiestrogen-estrogen receptor complexes from human breast tumors identified with an antibody raised against the estrogen receptor.

Radiolabeled estrogens 17 beta-[3H]estradiol and diethylstilbestrol ( [3H]DES) and the antiestrogen [3H]monohydroxytamoxifen ( [3H]MHT) all bind with high affinity to the extranuclear estrogen receptor (ER) from the MCF-7 human breast tumor cell line (Kd = 3 X 10(-10), 2 X 10(-10), and 0.63 X 10(-10) M, respectively). A polyclonal antibody raised in a goat to the calf nuclear ER selectively decreased the binding affinity and number of binding sites for 17 beta-[3H]estradiol, but did not appear to affect these binding parameters for [3H]MHT. In the presence of goat antibody, the binding of the nonsteroidal estrogen DES was so perturbed that it was not possible to quantitate the decreased number of binding sites or affinity of this compound as assessed by Scatchard saturation analysis. These results were confirmed in human breast tumor cytosols by sucrose density gradient analysis. The binding of 17 beta-[3H]-estradiol and [3H]DES to the ER was significantly reduced by preincubation with the polyclonal antibody, whereas the binding of [3H]MHT was reduced only when the tumor cytosol was preincubated with a very high concentration of antibody. At these concentrations of antibody, the binding of 17 beta-[3H]estradiol and [3H]DES to the receptor was prevented completely. In contrast, when the antibody was added to the tumor cytosol after the 3H-ligand had bound to the receptor, the binding properties of all 3H-ligands were unaffected. The [3H]MHT-ER antibody complex consistently sedimented as a higher-molecular-weight complex on sucrose density gradients than did the corresponding estrogenic complexes. The decrease in the affinity of estrogenic ligands can be explained in part by an increase in the dissociation rate at 4 degrees of these compounds from the ER. The dissociation rate of MHT was unaffected by the goat antibody. These results imply that there are important differences in the binding of antiestrogen and estrogens to the tumor cytosol ER. A ligand-binding model is proposed that may aid in the understanding of antiestrogen action.

Antibodies↗
PubMed1997

The novel estrogen receptor-beta subtype: potential role in the cell- and promoter-specific actions of estrogens and anti-estrogens.

The recent discovery that an additional estrogen receptor (ER) subtype is present in various rat, mouse and human tissues has advanced our understanding of the mechanisms underlying estrogen signalling. The discovery of a second ER subtype (ERbeta) suggests the existence of two previously unrecognised pathways of estrogen signalling: via the ERbeta subtype in tissues exclusively expressing this subtype and via the formation of heterodimers in tissues expressing both ER subtypes. Various models have been suggested as explanations for the striking cell- and promoter-specific effects of estrogens and anti-estrogens, all on the basis of the assumption that only a single ER gene exists. This minireview describes several of these models and focuses on the potential role which the novel ERbeta subtype might have in this regard.

Animals↗
PubMed1985

Estrogen and antiestrogen binding to rat uterine and pituitary estrogen receptor: evidence for at least two physicochemical forms of the estrogen receptor.

Our laboratory has previously reported that calf uterine cytosol prepared in buffer containing 10 mM molybdate and chromatographed on DEAE-Sephadex contains two forms of the unactivated estrogen receptor, Peak I and Peak II; however, cytosol receptor bound to the high-affinity antiestrogen, H1285 (4-(N,N-diethylaminoethoxy)-4'-methoxy-alpha- (p-hydroxyphenyl)-alpha'-ethylstilbene), eluted only as Peak I. We have extended these studies to the rat uterus and pituitary in order to determine the organ and species specificity of this phenomenon. Cytosol prepared in Tris-molybdate buffer from immature and adult rat uteri or pituitaries was labelled with 10 nM [3H]estradiol or [3H]H1285 and chromatographed on QAE-Sephadex. Uterine estrogen receptors bound to either [3H]estradiol or [3H]H1285 eluted from QAE-Sephadex as a large Peak I (approximately 0.21 M KCl) and a smaller Peak II (approximately 0.25 M KCl). Analyses of these partially purified estrogen receptor fractions using high-salt sucrose density gradients showed that Peak I [3H]estradiol-receptor complexes sedimented predominantly as a lighter form (4.0S). In contrast, Peak I [3H]H1285-receptor complexes sedimented primarily as a heavier form (5.5S) often accompanied by a smaller lighter form (4.0S). Peak II [3H]estradiol- and [3H]H1285-receptor complexes sedimented as the heavier form (5.3-5.5S). These data suggest a monomer-dimer relationship between estrogen receptor forms with antiestrogen binding favoring the formation of the dimeric form. Further analysis of these Peak I receptor complexes by gel filtration chromatography yielded molecular forms of approx 70 KDaltons for [3H]estradiol-receptor complexes and 73 KDaltons and 165 KDaltons for [3H]H1285-receptor complexes, supporting the monomer-dimer concept. Data from experiments with the pituitary also suggest that H1285 causes the formation of the dimeric receptor form whereas estradiol interaction with the receptor results only in the monomeric form. These differences in estrogen receptor forms when bound by estrogen versus antiestrogen may be related to the different biological responses induced by these ligands.

Animals↗
PubMed1998

Estrogenic and anti-estrogenic regulation of estrogen receptor in MCF-7 breast-cancer cells: comparison of immunocytochemical data with biochemical measurements.

Data from immunocytochemical assessment of estrogen receptor (ER) regulation in MCF-7 cells under estrogenic and anti-estrogenic stimulation were compared with those obtained by enzyme immunoassay (Abbott ER-EIA). Similar trends were observed, although ER level variations were less marked when assessed immunocytochemically. We confirmed reports of ER disappearance in the presence of estrogens (Es; E2 and DES) and pure anti-estrogens (AEs; RU 58,668 and ICI 164,384) as well as its increase with partial AEs (4-OH-TAM and RU 39,119). E2-induced ER down-regulation was partly blocked by actinomycin D (AMD), okadaic acid (OK) and cycloheximide (CHX) when assessed by these 2 methods. Down-regulation by pure AEs was not impeded by CHX, indicating that they operate differently from Es (i.e., transformation of ER to a form sensitive to constitutive degradation activity). In situ pre-labeling of the cells with [3H]TAZ indicated that all investigated ligands eliminate pre-existing ER through binding to newly synthetized receptors, since [3H]TAZ co-valently associates with ER; E2 and RU 58,668 were more effective than 4-OH-TAM in this regard. CHX blocked ER disappearance even in the presence of pure AEs, which is in contrast to the data established with cells not pre-exposed to [3H]TAZ. Nuclear location of [3H]TAZ-ER complexes may explain this discrepancy, since pure AE-ER complexes were reported to be incapable of nuclear translocation.

Breast Neoplasms↗
PubMed2006

The modulation of aromatase and estrogen receptor alpha in cultured human dermal papilla cells by dexamethasone: a novel mechanism for selective action of estrogen via estrogen receptor beta?

Steroid hormones have important modulatory effects on the hair follicle, but the mechanisms by which they regulate human hair growth are still poorly understood. It is now clear that there are two distinct estrogen receptors (estrogen receptor alpha (ERalpha) and estrogen receptor beta (ERbeta)) that bind 17beta-estradiol. Since the follicular dermal papilla is known to control hair growth, and steroid hormones regulate receptor and aromatase expression in other tissues, we tested the hypothesis that steroid hormones would similarly modulate estrogen receptor and/or aromatase expression in cultured dermal papilla cells derived from human hair follicles. Primary cultures of non-balding occipital and frontal scalp and beard dermal papilla cells (n = 10) were established. Immunocytochemical studies showed the expression of ERalpha in both the cytoplasm and nucleus, whereas ERbeta was confined to the nuclei. The cells derived from occipital scalp were also incubated for 24 hours with 10 nM of either 17beta-estradiol, estrone, testosterone, 5alpha-dihydrotestosterone, 5alpha-androstane-3alpha, 17beta-diol, 5alpha-androstane-3beta, 17beta-diol, or 100 nM tamoxifen or dexamethasone in phenol red-free, serum-free medium to measure the steady-state levels of ERalpha, ERbeta, and aromatase mRNA by semiquantitative reverse transcriptase-PCR. Although androgens and estrogens did not alter ERalpha mRNA levels, treatment with dexamethasone significantly reduced ERalpha levels to 38% of the untreated control. By contrast, ERbeta mRNA levels were unaffected by any steroid treatment. Furthermore, dexamethasone significantly stimulated the expression of aromatase mRNA approximately 9-fold. Aromatase activity, assayed by the tritiated water method, was stimulated in both frontal scalp and beard dermal papilla cell cultures by dexamethasone. These observations provide evidence for a glucocorticoid-dependent mechanism whereby the selective action of estradiol via ERbeta may be promoted. Additionally, upregulation of aromatase combined with downregulation of ERalpha provides a basis for selective action of estradiol produced locally by autocrine or paracrine mechanisms.

Androgens↗
PubMed1999

Underdeveloped uterus and reduced estrogen responsiveness in mice with disruption of the estrogen-responsive finger protein gene, which is a direct target of estrogen receptor alpha.

The biological roles of estrogen-responsive finger protein (efp) in vivo were evaluated in mice carrying a loss-of-function mutation in efp by gene-targeted mutagenesis. Although efp homozygous mice were viable and fertile in both sexes, the uterus that expressed abundant estrogen receptor alpha exhibited significant underdevelopment. When the ovariectomized homozygotes were subjected to 17beta-estradiol treatment, they showed remarkably attenuated responses to estrogen, as exemplified by decreased interstitial water imbibition and retarded endometrial cell increase, at least, attributable to the lower ratio of G1 to S-phase progression in epithelial cells. These results suggest that efp is essential for the normal estrogen-induced cell proliferation and uterine swelling as one of the direct targets of estrogen receptor alpha.

Animals↗
PubMed2006

Estrogen-induced proliferation of uterine epithelial cells is independent of estrogen receptor alpha binding to classical estrogen response elements.

Acting via the estrogen receptor (ER), estradiol exerts pleomorphic effects on the uterus, producing cyclical waves of cellular proliferation and differentiation in preparation for embryo implantation. In the classical pathway, the ER binds directly to an estrogen response element to activate or repress gene expression. However, emerging evidence supports the existence of nonclassical pathways in which the activated ER alters gene expression through protein-protein tethering with transcription factors such as c-Fos/c-Jun B (AP-1) and Sp1. In this report, we examined the relative roles of classical and nonclassical ER signaling in vivo by comparing the estrogen-dependent uterine response in mice that express wild-type ERalpha, a mutant ERalpha (E207A/G208A) that selectively lacks ERE binding, or ERalpha null. In the compound heterozygote (AA/-) female, the nonclassical allele (AA) was insufficient to mediate an acute uterotrophic response to 17beta-estradiol (E2). The uterine epithelial proliferative response to E2 and 4-hydroxytamoxifen was retained in the AA/-females, and uterine luminal epithelial height increased commensurate with the extent of ERalpha signaling. This proliferative response was confirmed by 5-bromo-2'-deoxyuridine incorporation. Microarray experiments identified cyclin-dependent kinase inhibitor 1A as a nonclassical pathway-responsive gene, and transient expression experiments using the cyclin-dependent kinase inhibitor 1A promoter confirmed transcriptional responses to the ERalpha (E207A/G208A) mutant. These results indicate that nonclassical ERalpha signaling is sufficient to restore luminal epithelial proliferation but not other estrogen-responsive events, such as fluid accumulation and hyperemia. We conclude that nonclassical pathway signaling via ERalpha plays a critical physiologic role in the uterine response to estrogen.

Animals↗
PubMed1996

Human estrogen receptor ligand activity inversion mutants: receptors that interpret antiestrogens as estrogens and estrogens as antiestrogens and discriminate among different antiestrogens.

The estrogen receptor (ER) is a transcription factor whose activity is normally activated by the hormone estradiol and inhibited by antiestrogen. It has been found that certain mutational changes in the activation function-2 region in the hormone-binding domain of the human ER result in ligand activity inversion mutants, i.e. receptors that are now activated by antiestrogen and inhibited by estrogen. The ER point mutant L540Q is activated by several antiestrogens (the more pure antiestrogens ICI 164,384 and RU 54,876 or the partial antiestrogen trans-hydroxytamoxifen) but not by estradiol. The presence of the F domain and an intact activation function-i in the A/B domain are required for this activity, as is the DNA-binding ability of the receptor. This inverted ligand activity is observed with several estrogen-responsive promoters, both simple and complex; however, the activating ability of antiestrogens is observed only in some cells, highlighting the important role of cell-specific factors in ligand interpretation. The introduction of two additional amino acid changes close to 540 results in receptors that are still not activated by estradiol but are now able to distinguish between partial antiestrogens (which remain agonistic) and pure antiestrogens (which show a greatly reduced stimulatory activity). These ligand activity inversion mutants remain stable in cells in the presence of the antiestrogen ICI 164,384, as does a related ER mutant receptor that shows the normal, wild type ER ligand activity profile in which ICI 164,384 is transcriptionally inactive. Thus, the presence of adequate levels of mutant ER may be necessary but not sufficient for ICI 164,384 to elicit transcriptional activity. These findings highlight the means by which the carboxyl-terminal region in domain E functions to interpret the activity of a ligand, and they demonstrate that rather minimal changes in the ER can result in receptors with inverted response to antiestrogen and estrogen. Such point mutations, if present in estrogen target cells, would result in antiestrogens being seen as growth stimulators, rather than suppressors, with potentially detrimental consequences in terms of breast cancer treatment with antiestrogens.

3T3 Cells↗
PubMed2002

Comparison of the effects of transdermal estrogen, oral estrogen, and oral estrogen-progestogen therapy on bone mineral density in postmenopausal women.

It is now clear that estrogen intervention reduces bone loss in postmenopausal osteoporosis. The aim of this study was to investigate whether the route of estrogen administration or addition of progesterone changes this positive effect. Transdermal estrogen (T-E), oral estrogen (E), and oral estrogen plus progestogen (E-P) were administered to 15, 18, and 17 postmenopausal women, respectively, who all had normal bone mineral density (BMD) before hormone replacement therapy (HRT). Calcium (500 mg/day) was routinely added to all HRT regimens. The BMD of the lumbar spine (L2-L4) was measured initially and at the end of the first and second years of treatment. The paired-sample t test, independent-sample t test, and Pearson correlation analysis were used for the statistical evaluation. The initial BMD measurements and the values at the end of the first and second years of the therapy were not significantly different either within or among the groups (P > 0.05). These results indicate a similar therapeutic value of each HRT regimen in the prevention of bone loss in postmenopausal women.

Administration, Cutaneous↗
PubMed1999

Synergism between a half-site and an imperfect estrogen-responsive element, and cooperation with COUP-TFI are required for estrogen receptor (ER) to achieve a maximal estrogen-stimulation of rainbow trout ER gene.

In all oviparous, liver represents one of the main E2-target tissues where estrogen receptor (ER) constitutes the key mediator of estrogen action. The rainbow trout estrogen receptor (rtER) gene expression is markedly up-regulated by estrogens and the sequences responsible for this autoregulation have been located in a 0.2 kb upstream transcription start site within - 40/- 248 enhancer region. Absence of interference with steroid hormone receptors and tissue-specific factors and a conserved basal transcriptional machinery between yeast and higher eukaryotes, make yeast a simple assay system that will enable determination of important cis-acting regulatory sequences within rtER gene promoter and identification of transcription factors implicated in the regulation of this gene. Deletion analysis allowed to show a synergistic effect between an imperfect estrogen-responsive element (ERE) and a consensus half-ERE to achieve a high hormone-dependent transcriptional activation of the rtER gene promoter in the presence of stably expressed rtER. As in mammalian cells, here we observed a positive regulation of the rtER gene promoter by the chicken ovalbumin upstream promoter-transcription factor I (COUP-TFI) through enhancing autoregulation. Using a point mutation COUP-TFI mutant unable to bind DNA demonstrates that enhancement of rtER gene autoregulation requires the interaction of COUP-TFI to the DNA. Moreover, this enhancement of transcriptional activation by COUP-TFI requires specifically the AF-1 transactivation function of ER and can be observed in the presence of E2 or 4-hydroxytamoxifen but not ICI 164384. Thus, this paper describes the reconstitution of a hormone-responsive transcription unit in yeast in which the regulation of rtER gene promoter could be enhanced by the participation of cis-elements and/or trans-acting factors, such as ER itself or COUP-TF.

Animals↗