Biomedical subjects

C M Klinge

Publications and source records attributed to C M Klinge.

At least 37 records · Page 2Linked to original sources

PubMed1999

Role of estrogen receptor ligand and estrogen response element sequence on interaction with chicken ovalbumin upstream promoter transcription factor (COUP-TF).

Estrogen-responsive genes are regulated by altering the balance of estrogen receptor (ER) interaction with transcription activators and inhibitors. Here we examined the role of ER ligand on ER interaction with the Chicken Ovalbumin Upstream Promoter Transcription Factor (COUP-TF) orphan nuclear receptor. COUP-TF binding to half-site estrogen response elements (EREs) was increased by the addition of estradiol (E2) -liganded ER (E2-ER), but not by ER liganded with the antiestrogens 4-hydroxytamoxifen (4-OHT-ER) or tamoxifen aziridine (TAz-ER). ER did not bind to single half-sites. Conversely, COUP-TF enhanced the ERE binding of purified E2-ER, but did not affect TAz-ER-ERE binding. In contrast, only antiestrogens enhanced direct interaction between ER and COUP-TF as assessed by GST pull-down assays. Identical results were obtained using either purified bovine or recombinant human ERalpha. Co-immunoprecipitation assays showed that ER and COUP-TF interact in extracts from MCF-7 and ERalpha-transfected MDA-MB-231 cells. Here we document that ER ligand impacts COUP-TF-ER interaction. COUP-TF interaction is mediated by the DNA binding and ligand-binding domains of ER. We suggest that changes in ER conformation induced by DNA binding reduce ER-COUP-TF interaction. Transient transfection of human MCF-7 breast cancer cells with a COUP-TFI expression vector repressed E2-induced luciferase reporter gene expression from single or multiple tandem copies of a consensus ERE. COUP-TFI stimulated 4-OHT-induced luciferase activity from a minimal ERE. Alone, COUP-TFI increased transcription from ERE half-sites or a single ERE in a sequence-dependent manner. These data provide evidence that the ERE sequence and its immediate flanking regions influence whether COUP-TF enhances, inhibits, or has no effect on ER ligand-induced ERE reporter gene expression and that COUP-TFI activates gene transcription from ERE half-sites. We suggest that COUP-TFI plays a role in mitigating estrogen-responsive gene expression.

Animals↗

PubMed1999

Polyamine biosynthesis inhibitors alter protein-protein interactions involving estrogen receptor in MCF-7 breast cancer cells.

We investigated the effects of polyamine biosynthesis inhibition on the estrogenic signaling pathway of MCF-7 breast cancer cells using a protein-protein interaction system. Estrogen receptor (ER) linked to glutathione-S-transferase (GST) was used to examine the effects of two polyamine biosynthesis inhibitors, difluoromethylornithine (DFMO) and CGP 48664. ER was specifically associated with a 45 kDa protein in control cells. In cells treated with estradiol, nine proteins were associated with ER. Cells treated with polyamine biosynthesis inhibitors in the absence of estradiol retained the binding of their ER with a 45 kDa protein and the ER also showed low-affinity interactions with a number of cellular proteins; however, these associations were decreased by the presence of estradiol and the inhibitors. When samples from the estradiol+DFMO treatment group were incubated with spermidine prior to GST-ER pull down assay, an increased association of several proteins with ER was detected. The intensity of the ER-associated 45 kDa protein increased by 10-fold in the presence of 1000 microM spermidine. These results indicate a specific role for spermidine in ER association of proteins. Western blot analysis of samples eluted from GST-ER showed the presence of chicken ovalbumin upstream promoter-transcription factor, an orphan nuclear receptor, and the endogenous full-length ER. These results show that multiple proteins associate with ER and that the binding of some of these proteins is highly sensitive to intracellular polyamine concentrations. Overall, our results indicate the importance of the polyamine pathway in the gene regulatory function of estradiol in breast cancer cells.

Amidines↗

PubMed1998

Sequence requirements for estrogen receptor binding to estrogen response elements.

The estrogen receptor (ER) is a transcription factor that binds to a specific DNA sequence found in the regulatory regions of estrogen-responsive genes, called the estrogen response element (ERE). Many genes that contain EREs have been identified, and most of these EREs contain one or more changes from the core consensus sequence, a 13-nucleotide segment with 10 nucleotides forming an inverted repeat. A number of genes have multiple copies of these imperfect EREs. In order to understand why natural EREs have developed in this manner, we have attempted to define the basic sequence requirements for ER binding. To this end, we measured the binding of homodimeric ER to a variety of nonconsensus EREs. We discovered that an ERE containing even a single change from the consensus may be unable to bind ER. However, an ERE with two changes from the consensus may be capable of binding avidly to ER in the context of certain flanking sequences. We found that changes in the sequences flanking a nonconsensus ERE can greatly alter ER-ERE affinity, either positively or negatively. Careful study of sequences flanking a series of EREs made it possible to develop rules that predict whether ER binds to a given natural ERE and also to predict the relative amounts of binding when comparing two EREs.

Animals↗

PubMed1998

Comparison of tamoxifen ligands on estrogen receptor interaction with estrogen response elements.

The estrogen receptor (ER) is a ligand-activated transcription factor that binds to specific DNA sequences, estrogen response elements (EREs). Estradiol-liganded ER (E2-ER) binds cooperatively to stereoaligned EREs that are surrounded by naturally-occurring AT-rich sequences with a stoichiometry of one E2-ER dimer per ERE. When ER is bound by 4-hydroxytamoxifen (4-OHT), the active metabolite of the widely used therapeutic antiestrogen tamoxifen (TAM), the receptor binds to EREs with high affinity. However, one molecule of 4-OHT ligand dissociates from the ER dimer apparently during the process of binding to DNA, yielding a stoichiometry of one [3H]4-OHT molecule per ERE. To determine whether DNA-binding induced ligand dissociation is a general property of type I antiestrogens that are not covalently attached to the ER, we examined the interaction of ER liganded by tamoxifen (TAM) with EREs. We demonstrate that TAM-ER binds EREs with lower affinity than E2-ER, 4-OHT-ER, or ER liganded by the covalent antiestrogen tamoxifen aziridine. Unlike E2-ER, both TAM and 4-OHT-ER bind EREs non-cooperatively. Like 4-OHT, TAM appears to dissociate from the liganded ER as the receptor binds EREs. Additionally, partial proteolysis of ERE-bound ER by trypsin revealed different cleavage patterns for E2 versus 4-OHT and TAM. These findings indicate that the behavior of the ER liganded by TAM is generally similar to that of the antiestrogen 4-OHT.

Animals↗

PubMed1997

Chicken ovalbumin upstream promoter-transcription factor interacts with estrogen receptor, binds to estrogen response elements and half-sites, and inhibits estrogen-induced gene expression.

Chicken ovalbumin upstream promoter-transcription factor (COUP-TF) was identified as a low abundance protein in bovine uterus that co-purified with estrogen receptor (ER) in a ligand-independent manner and was separated from the ER by its lower retention on estrogen response element (ERE)-Sepharose. In gel mobility shift assays, COUP-TF bound as an apparent dimer to ERE and ERE half-sites. COUP-TF bound to an ERE half-site with high affinity, Kd = 1.24 nM. In contrast, ER did not bind a single ERE half-site. None of the class II nuclear receptors analyzed, i.e. retinoic acid receptor, retinoid X receptor, thyroid receptor, peroxisome proliferator-activated receptor, or vitamin D receptor, were constituents of the COUP-TF.DNA binding complex detected in gel mobility shift assays. Direct interaction of COUP-TF with ER was indicated by GST "pull-down" and co-immunoprecipitation assays. The nature of the ER ligand influenced COUP-TF-ERE half-site binding. When ER was liganded by the antiestrogen 4-hydroxytamoxifen (4-OHT), COUP-TF-half-site interaction decreased. Conversely, COUP-TF transcribed and translated in vitro enhanced the ERE binding of purified estradiol (E2)-liganded ER but not 4-OHT-liganded ER. Co-transfection of ER-expressing MCF-7 human breast cancer cells with an expression vector for COUP-TFI resulted in a dose-dependent inhibition of E2-induced expression of a luciferase reporter gene under the control of three tandem copies of EREc38. The ability of COUP-TF to bind specifically to EREs and half-sites, to interact with ER, and to inhibit E2-induced gene expression suggests COUP-TF regulates ER action by both direct DNA binding competition and through protein-protein interactions.

Animals↗

PubMed1997

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 .

Animals↗

PubMed1997

Effects of multiple estrogen responsive elements, their spacing, and location on estrogen response of reporter genes.

Most highly estrogen-responsive genes possess multiple estrogen-responsive elements (EREs) that act synergistically to activate expression. Synergism between EREs appears to depend on structural features of the EREs and the promoter. To examine the activation process, we cloned single or multiple tandem copies of the consensus ERE into reporter plasmids. These plasmids contained either a chloramphenicol acetyl transferase reporter gene driven by a minimal promoter or a luciferase reporter gene driven by the Simian virus 40 (SV40) promoter. Using MCF-7 human breast cancer cells, we demonstrate that synergism among EREs depends on the number of EREs, their spacing, and the distance of the EREs from the promoter. The induction capacity of EREs falls off slowly with distance from the promoter. Remarkably, multiple EREs can induce effectively and synergize even when they are located more than 2000 nucleotides from the promoter. For EREs located immediately upstream of the promoter, both the distance separating the EREs and the distance to the promoter have to be optimal for synergy. Altering either distance changes the response from synergistic to additive. For distant EREs, presumed to interact by a looping mechanism at the promoter, the length of DNA between the EREs and the promoter is not critical. Synergy among closely spaced EREs that are far from the promoter only requires an optimal distance separating the ERE centers of symmetry. Interestingly, very widely separated EREs can also synergize, presumably also because of their ability to interact by looping. The estrogen response from single or multiple tandem copies of ERE half-palindromes near the SV40 promoter was also tested. The negligible induction capacity of a single half-site was not significantly increased in multiple sites. The biological role of half-EREs is not apparent in the system employed here.

Animals↗

PubMed1996

Phosphorylation of purified estradiol-liganded estrogen receptor by casein kinase II increases estrogen response element binding but does not alter ligand stability.

The estrogen receptor is a ligand-activated transcription factor that binds to specific DNA sequences, estrogen response elements. Recent studies have characterized the location of tyrosine and serine residues in the estrogen receptor that are phosphorylated either by purified protein kinases in vitro or in response to ligand and DNA binding in vivo. Here we examined how phosphorylation of purified bovine uterine estrogen receptor in vitro by casein kinase II impacts estrogen receptor-estrogen response element binding and 17 beta-estradiol ligand binding stability. Our results show that phosphorylation doubles estrogen receptor-estrogen response element binding, but does not affect estradiol binding stability. These finding suggest that phosphorylation by casein kinase II on serine residues within the A/B domain results in intramolecular interactions affecting the DNA binding domain but not the ligand binding domain of the estrogen receptor.

Animals↗

PubMed1996

Site-directed estrogen receptor antibodies stabilize 4-hydroxytamoxifen ligand, but not estradiol, and indicate ligand-specific differences in the recognition of estrogen response element DNA in vitro.

Conformational differences between type I antiestrogen-liganded estrogen receptor and estradiol (E2)-liganded estrogen receptor (ER) are thought to be responsible for differentiating agonist versus antagonist ER activity at individual genes. To examine the impact of ER ligand on estrogen-response element (ERE) binding kinetics and receptor conformation, we quantitated the effect of site-directed, ER-specific antibodies raised against synthetic peptides corresponding to the DNA-binding domain of human ER on ER-ERE binding in vitro. Although 4-hydroxytamoxifen-liganded-ER (4-OHT-ER) and E2-ER bind a consensus ERE with equal high affinity, the stoichiometry of 4-OHT-ER-ERE binding at saturation is approximately 50% lower than that of E2-ER binding to all ERE sequences tested. In contrast, the ERE binding stoichiometry of tamoxifen aziridine-liganded ER (TAz-ER) is identical to that of E2-ER: one receptor dimer bound per ERE. The difference in binding stoichiometry is caused by dissociation of one molecule of 4-OHT from the ER as the dimeric receptor binds DNA. Addition of low concentrations of ER-specific polyclonal antibodies AT3A or AT3B prevented 4-OHT ligand dissociation, yielding an increase in specific 4-OHT-ER-ERE binding to a level equal to that of E2-ER- or TAz-ER-ERE binding. However, higher amounts of AT3A or AT3B inhibited specific ERE binding of both 4-OHT- and E2-ER. We conclude that differences in ER conformation when liganded with 4-OHT versus E2 are revealed by these antibodies and that such differences in receptor conformation may influence subsequent interaction of the receptor with other proteins necessary for transactivation.

Antibody Specificity↗

PubMed1996

Dissociation of 4-hydroxytamoxifen, but not estradiol or tamoxifen aziridine, from the estrogen receptor as the receptor binds estrogen response element DNA.

Estradiol-liganded estrogen receptor (E2-ER) binds EREs with a stoichiometry of one E2-ER dimer per estrogen response element (ERE). In contrast, although 4-hydroxytamoxifen (4-OHT)-liganded ER (4-OHT-ER) binds EREs with high affinity, its saturation ERE binding capacity is consistently half that of E2-ER, giving an apparent stoichiometry of one 4-OHT-ER monomer per ERE. Here we show that one molecule of 4-OHT ligand dissociates from the ER dimer apparently during the process of binding to DNA. Under equilibrium conditions, the type I antiestrogen tamoxifen aziridine (TAz), covalently attached to ER (TAz-ER), binds a single ERE with high affinity (Kd = 0.27 nM), comparable to that of E2-ER and 4-OHT-ER. In contrast to 4-OHT-ER, the ERE binding stoichiometry of TAz-ER was identical to that of E2-ER: one dimeric receptor per ERE. By measuring [3H]ligand that was initially bound to ER, a significant loss of [3H]4-OHT from ER was detected after ERE binding, whereas all [3H]E2 or [3H]TAz remained ER-bound. These results confirm that one molecule of 4-OHT ligand dissociates from the ER dimer as a consequence of ERE binding. Binding of 4-OHT and TAz are likely to induce a conformation in ER dimers that alters their capacity for gene activation. Upon ER binding to DNA, this conformation reveals itself by allowing 4-OHT dissociation, and predictably would allow TAz dissociation were it not bound covalently.

Base Sequence↗

PubMed1996

Footprint analysis of estrogen receptor binding to adjacent estrogen response elements.

Quantitative DNase I footprinting assays were employed to simultaneously measure the amount of estrogen receptor (ER) bound to each site in constructs containing multiple estrogen response elements (EREs). These assays revealed identical, high affinity ER-ERE binding, Kd of approximately 0.25 nM, for estradiol-liganded ER (E2-ER), 4-hydroxytamoxifen liganded ER (4-OHT-ER), tamoxifen aziridine liganded ER (TAz-ER), and unliganded dimeric ER, for each ERE in constructs containing up to four tandem EREs. Increasing concentrations of ER resulted in the same pattern of occupancy for each ERE, whether or not the site was located near other EREs. Similarly, the presence or absence of E2, 4-OHT, or TAz ligand did not change ER-ERE interaction. Since activated ER-ERE binding affinity is identical, whether ER is liganded or unliganded, ligand cannot regulate ER-ERE binding affinity. These results support the hypothesis that ligand-dependent conformational changes primarily determine how ER interacts with components of the transcription initiation complex that mediate gene transactivation. In addition, footprint assays revealed that, following ER binding, an AT-rich site adjacent to the ERE becomes hypersensitive to DNase I digestion. This sequence may be easily or intrinsically bent, assisting in recruiting ER to ERE sites.

Animals↗

PubMed1996

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.

Animals↗

PubMed1995

A test of the hypothesis that a 60-Hz magnetic field affects ornithine decarboxylase activity in mouse L929 cells in vitro.

Four replicate experiments were performed to test the hypothesis that a 4-hr exposure of L929 cells in vitro to a 60 Hz, 10 microT magnetic field results in a large increase in ornithine decarboxylase (ODC) enzyme activity (1-2). A positive control yielded a highly statistically significant increase in ODC activity. However, magnetic field exposure had no statistically significant effect on extractable ODC activity of L929 cells relative to that of sham-exposed cells.

Analysis of Variance↗

PubMed1995

Cooperative binding of estrogen receptor to DNA depends on spacing of binding sites, flanking sequence, and ligand.

It has been suggested that cooperative binding of estrogen receptor (ER) may, in part, be responsible for the synergistic activation of transcription of estrogen-responsive genes that contain multiple estrogen-response elements (EREs). Experiments described here show that estradiol-liganded ER (E2-ER) binds cooperatively to stereoaligned EREs that are surrounded by naturally occurring flanking sequences, such as an AT-rich region. In contrast, EREs lacking these sequences do not bind E2-ER cooperatively, regardless of ERE spacing or stereoalignment. Moreover, binding is of lower affinity and capacity in the absence of these critical flanking sequences. By varying the sequence of nucleotides adjacent to the ERE, features important for the flanking sequence effect were characterized. Interestingly, when ER was liganded with 4-hydroxytamoxifen (4-OHT), the active metabolite of the widely used therapeutic antiestrogen tamoxifen, the antiestrogen-liganded ER complex (4-OHT-ER) did not bind cooperatively to multiple EREs, regardless of spacing or flanking sequence. We postulate that ERE flanking sequences bestow upon E2-ER enhanced ERE binding capacity and cooperativity, but do not affect 4-OHT-ER-ERE binding.

Animals↗

PubMed1995

Polyamine-mediated conformational perturbations in DNA alter the binding of estrogen receptor to poly(dG-m5dC).poly(dG-m5dC) and a plasmid containing the estrogen response element.

The binding estrogen receptor (ER) to the upstream regions of estrogen-responsive genes, the estrogen response elements (ERE), is of fundamental importance in the regulation of gene expression by estradiol. Multiple cell-specific factors affect ER-ERE binding and modulate the responses of estradiol. We studied the role of polyamines in the recognition of ER, a ligand-activated transcription factor, with a left-handed Z-DNA forming polynucleotide as well as with a plasmid containing ERE. Polyamines are cellular organic cations with multiple functions in cell growth and differentiation. Polyamines induce Z-DNA conformation in alternating purine-pyrimidine sequences. To understand the role of polyamine-induced DNA conformational transition in ER-DNA interaction, we studied the binding of partially purified rabbit uterine ER to poly(dG-m5dC).poly(dG-m5dC). The induction of Z-DNA in the polynucleotide was monitored by circular dichroism and ultraviolet spectroscopic measurements. Binding of ER to poly(dG-m5dC).poly(dG-m5dC) increased from 15% to approx. 50-60% in the presence of 7.5 mM putrescine, 0.5 mM spermidine or 0.25 mM spermine. Maximal binding of ER to the polynucleotide was observed near the midpoint of the B-DNA to Z-DNA transition of the polynucleotide. N1-acetyl spermidine and N1-acetyl spermine facilitated the B-DNA to Z-DNA transition and the binding of ER although they were less effective than the unacetylated analog. Co(NH3)6(3+), a trivalent inorganic cation, also provoked the B-DNA to Z-DNA transition of the polynucleotide and increased its binding to ER. At higher polyamine concentrations, there was an inhibition of ER binding to the polynucleotide. In the presence of polyamines, the binding of ER to a plasmid containing ERE was 2-3-fold higher than that to a control plasmid devoid of ERE. Polyamine-induced facilitation of ER-ERE binding was also confirmed by gel mobility shift assay. Our data indicate that conformational perturbations, similar to that of the early stages of B-DNA to Z-DNA transition, are important in the recognition of ER and ERE.

Animals↗

PubMed1993

Differential impact of flanking sequences on estradiol- vs 4-hydroxytamoxifen-liganded estrogen receptor binding to estrogen responsive element DNA.

The mechanism by which antiestrogens antagonize the ability of estrogen receptor (ER) to induce the transcription of estrogen-regulated genes is only partially understood. To examine the effect of estrogen responsive element (ERE) stereoalignment and flanking sequences on estradiol-liganded ER (E2-ER)-ERE and antiestrogen-liganded ER (4-hydroxytamoxifen-liganded ER or 4-OHT-ER)-ERE binding, several dimeric EREs, containing a perfect inverted repeat (5'-GGTCAgagTGACC-3') but lacking the AT-rich flanking sequences typical of highly estrogen-responsive promoters, were cloned into a plasmid vector. The ERE centers of symmetry were spaced 1.5, 2.0, 3.0, 6.4 and 6.7 helical turns apart. E2-ER and 4-OHT-ER binding to these constructs was specific and saturable, but orientation-independent and, in contrast to our earlier work with E2-ER binding to AT-rich EREs, not cooperative. The affinity of E2-ER binding decreased as the distance between adjacent EREs was increased, suggesting that E2-ER binding to closely spaced EREs is more stable (Kd = 0.38, 0.58, 0.83, 1.23, and 0.96 nM, respectively, for the above spacings). In contrast, the affinity of 4-OHT-ER binding increased with increased ERE spacing (Kd = 2.90, 4.79, 1.39, 1.77, and 0.92 nM, respectively). The presence of AT-rich sequences flanking the ERE increased the binding affinity of E2-ER and 4-OHT-ER, an increase reflected in slower dissociation rates of ER from these EREs. The AT-rich sequence also enhanced the binding capacity of E2-ER but not 4-OHT-ER. Since the binding capacity of 4-OHT-ER is identical with or without an AT-rich region, we suggest that flanking sequences are more important in stabilizing E2-ER binding and may be critical for cooperative binding to stereoaligned EREs.

Animals↗

PubMed1992

Cooperative estrogen receptor interaction with consensus or variant estrogen responsive elements in vitro.

Specific binding of estradiol-liganded, partially purified calf uterine estrogen receptor (ER) to a 38-base pair estrogen responsive element (ERE) consensus sequence, containing the inverted repeat 5'-GGTCAnnnTGACC-3', was measured in vitro. The ERE sites were inserted as single or multiple tandem copies in a plasmid vector [p GEM-7Zf(+)]. Results showed that one dimeric ER can interact with one ERE, and steric constraints do not inhibit binding of ER to adjacent EREs. Molybdate-stabilized monomeric (4S) ER did not bind to EREs. ER bound to single and tandem double EREs with Kd values of 0.24 and 0.23 nM, respectively. When the plasmid contained three or more tandem copies of the ERE, ER bound in a cooperative manner, as indicated by convex Scatchard plots and Hill coefficients greater than 1.5. To determine those characteristics of the consensus sequence that are important for maximal high-affinity ER binding, ten variant ERE oligomer sequences were synthesized and cloned into pGEM-7Zf(+) as single copies or as four copies in tandem. ER binding affinity was maximal for the consensus ERE and was reduced for variants containing one or two nucleotide changes in the inverted repeat. The number of nucleotides separating the inverted repeat in the ERE was critical for high-affinity ER binding. Certain sequence-variant EREs when cloned as single copies bound less ER compared to the consensus ERE, yet when cloned as four tandem copies, ER binding displayed cooperativity by Scatchard and Hill analyses. Results demonstrate that cooperative interactions noted in vivo by others are present when measured in vitro. Results strongly imply that the number, spacing, and nucleotide sequence of EREs could precisely control the amount of ER binding to estrogen-responsive genes.

Base Sequence↗

PubMed1992

Antiestrogen-liganded estrogen receptor interaction with estrogen responsive element DNA in vitro.

The mechanism whereby antiestrogens alter the ability of the estrogen receptor (ER) to enhance transcription of estrogen-regulated genes is largely unknown. The effect that selected estrogenic and antiestrogenic ligands have on binding of ER to specific DNA sequences, estrogen responsive elements (EREs) has been quantitated. No differences in purification properties of calf uterine ER liganded with 4-hydroxytamoxifen (4-OHT-ER), ICI 164,384 (ICI 164,384-ER) or estradiol (E2-ER) were detected. A microtiter well plate assay was employed in which liganded ER bound to plasmid DNA is preferentially retained compared to free liganded ER. Binding of E2-ER, 4-OHT-ER, or ICI 164,384-ER was measured to plasmids containing or lacking a 38bp consensus ERE in vitro. The EREs tested contain an inverted repeat (5'-CAGGTCAGAGTGACCTG-3'). Both E2-ER and 4-OHT-ER showed similar high affinity specific binding (Kd = 0.24 and 0.16 nM, respectively) to one copy of the ERE. ICI 164,384-ER did not bind to plasmids containing one ERE. At saturation, however, 4-OHT-ER binding was about 50% of that observed for E2-ER. When the plasmid contained 3 or 4 tandem copies of the ERE, binding of E2-ER, 4-OHT-ER, and ICI 164,384-ER binding was measurable. E2-ER bound in a cooperative manner as suggested by convex Scatchard plots and Hill coefficients > 1.5. In contrast, 4-OHT-ER binding displayed much reduced cooperativity, and ICI 164,384-ER did not display cooperative binding. From these results, we propose that the conformation of ER induced by 4-OHT reduces its binding capacity to this consensus ERE without altering its affinity of binding. Furthermore, higher order protein-protein interactions between antiestrogen-liganded ER bound to DNA differ from those of E2-ER bound to ERE.

Animals↗