Biomedical subjects

D J Shapiro

Publications and source records attributed to D J Shapiro.

At least 37 records · Page 2Linked to original sources

PubMed1996

A transcriptionally active estrogen receptor mutant is a novel type of dominant negative inhibitor of estrogen action.

We have characterized a human estrogen receptor (ER) mutant, V364E, which has a single amino acid substitution in its hormone-binding domain. This ER mutant is fully active or even superactive at saturating levels of estradiol (10(-8) M E2) yet has the capacity to act as a strong dominant negative inhibitor of the wild type ER. In transient transfection assays using ER-negative Chinese hamster ovary (CHO) cells and two different estrogen response element (ERE)-containing promoter reporter genes, V364E treated with 10(-8) M E2 exhibited approximately 250% and 100% of the activity of the wild type ER with these two promoter contexts, respectively. Despite the high activity of V364E when present alone in cells, coexpression of both V364E and wild type ER causes a significant decrease in overall ER-mediated transcriptional activity. On the TATA promoter, where V364E was more inhibitory, estrogen-stimulated activity was reduced by approximately 50% at a 1:1 ratio of mutant to wild type ER expression vector, and at a 10:1 ratio, 75% of ER activity was inhibited. V364E was expressed at lower levels than wild type ER and has a approximately 40-fold lower affinity for E2 compared with wild type ER. In promoter interference assays, V364E exhibited a strict dependence upon E2 for binding to an ERE. Surprisingly, even when V364E was unable to bind to ERE DNA (i.e. either at low E2 concentration or by mutation of its DNA-binding domain), this mutant retained full dominant negative activity. This highly active ER mutant is, thus, able to repress ER-mediated transcription when the mutant and wild type ER are present together in cells, even without DNA binding. Since competition for ERE binding and the formation of inactive heterodimers cannot fully account for the dominant negative activity of V364E, it is probable that altered interactions with proteins important in ER-mediated transcription play a key role in the repression of transcription by V364E. The properties and probable mechanism of action of V364E distinguish it from other previously described dominant negative inhibitors, in which competition for cis-acting DNA elements by transcriptionally inactive receptors played a large role in the resultant dominant negative phenotype.

Animals↗

PubMed1995

Analysis of mechanisms that determine dominant negative estrogen receptor effectiveness.

To analyze the mechanisms by which estrogen receptor (ER) activity is suppressed by dominant negative mutants, we examined the role of specific ER functions and domains in transcriptional repression. We previously described three transcriptionally inactive human ER mutants (the frameshift mutant S554fs, the point mutant L540Q, and the truncated receptor ER1-530), which act as effective dominant negative mutants, inhibiting the activity of wild type ER when they are coexpressed in mammalian cells. After additional mutational modifications, the ability of the ER mutants to suppress the activity of wild type ER was analyzed in cotransfection assays of the dominant negative mutants and wild type ER and an estrogen-responsive reporter gene (2ERE-TATA-CAT or 2ERE-pS2-CAT). Eliminating the ability of the three dominant negative mutants to bind to estrogen response element (ERE) DNA (by introducing three point mutations in their DNA binding domains) dramatically reduced, but did not completely abolish, the dominant negative activity of the ER mutants. The mutation G521R, which rendered the three mutants incapable of binding estradiol, also reduced, but did not abolish, their dominant negative activity. Immunoprecipitation with monoclonal or flag antibodies followed by Western blotting demonstrated that each of the original dominant negative ER mutants formed heterodimers with wild type ER. Rendering the dominant negative mutants dimerization deficient by the mutation L507R strongly reduced, but did not eliminate, their dominant negative activity. Deletion of the N-terminal A/B domain resulted in the nearly complete loss of inhibitory activity of the three dominant negative mutants. However, these double mutants retained their ability to heterodimerize with wild type ER, suggesting that dominant negative interference also occurs at an additional step beyond dimerization. Our data indicate that competition for ERE binding, formation of inactive heterodimers, and specific transcriptional silencing can all contribute to the dominant negative phenotype and that these receptors suppress the activity of wild type ER by acting at multiple steps in the ER-response pathway.

Animals↗

PubMed1995

An N-terminal deletion mutant of estrogen receptor exhibits increased synergism with upstream activators and enhanced binding to the estrogen response element.

To study the role of the N-terminal region of the estrogen receptor (ER) in transcription activation and in DNA binding, we constructed a mutant of the Xenopus laevis ER which lacks amino acids 1-159 (XER160/586). In transient transfections, XER160/586 exhibited < 10% of the activity of wild-type XER on a synthetic promoter containing two estrogen response elements (EREs). To examine transcriptional synergism by XER and by XER160/586, we determined the activity of promoters containing EREs and binding sites for either the vitellogenin activator, NF1, or AP1 upstream activator protein. For the three promoters transcription by XER was 2.8-fold greater than expected for additive activities, and transcription by XER160/586 was 6.2-fold greater. These data demonstrate that an upstream activator protein bound near the promoter can partially compensate for the loss of the internal N-terminal (AF1) transactivation domain in XER160/586. Using a promoter interference assay to study the intracellular interaction between ER and the estrogen response element, we found that XER160/586 exhibited a significant increase in affinity for the ERE. Its low basal activity and enhanced affinity for the ERE make XER160/586 an effective dominant negative mutant. When co-expressed with wild-type XER at 1:1 and 5:1 ratios, XER160/586 suppressed the activity of wild-type XER by 57% and > 80%, respectively.

Animals↗

PubMed1995

Intrinsically bent DNA in a eukaryotic transcription factor recognition sequence potentiates transcription activation.

Many eukaryotic transcription factors induce DNA bending on binding to their recognition sequences. DNA bending could play a structural role by altering contacts between the protein and DNA. Alternatively, DNA bending could play a more direct role in transcription activation. To distinguish between these possibilities, we inserted two to eight copies of the intrinsic bending sequence, AAAAAACGTG, into a minimal promoter containing only a TATA box. The intrinsic DNA bending sequence was a potent activator of transcription in both in vivo transfection experiments and in a cell-free transcription system. A protein binds to the intrinsic bending sequence with high specificity in gel mobility shift assays and was required for its transcription in cell-free extracts. The intercalator, distamycin, which eliminates the ability of the sequence to bend, specifically reduced its transcription by about 60%. Mutations in the sequence which abolished DNA bending reduced transcription by approximately 70% in vivo. Competition gel mobility shift assays showed that the transcription factor bound equally well to mutants in which DNA bending was abolished and to the intrinsic bending sequence. These data indicate that DNA bending can play a direct role in the activation of eukaryotic transcription.

Animals↗

PubMed1995

Tissue distribution, hormone regulation and evidence for a human homologue of the estrogen-inducible Xenopus laevis vitellogenin mRNA binding protein.

17 beta-estradiol induces the synthesis of massive amounts of the hepatic mRNA encoding the Xenopus laevis egg yolk precursor protein, vitellogenin. Vitellogenin mRNA exhibits a half life of approx. 500 h when 17 beta-estradiol is present, and 16 h after removal of 17 beta-estradiol from the culture medium. We recently reported that Xenopus liver contains a protein, which is induced by 17 beta-estradiol and binds with a high degree of specificity to a binding site in a segment of the 3'-untranslated region (3'-UTR) of vitellogenin mRNA implicated in 17 beta-estradiol stabilization of vitellogenin mRNA. To determine if this mRNA binding protein was specific to this system, or if it was present elsewhere, and regulated by other steroids, we examined the tissue distribution and androgen regulation of this protein. Substantial amounts of the vitellogenin 3'-UTR binding protein were found in several Xenopus tissues including testis, ovary and muscle. In the absence of hormone treatment, lung and intestine contained minimal levels of the mRNA binding protein. Testosterone administration induced the vitellogenin 3'-UTR RNA binding protein in several tissues. Additionally, we found a homologous mRNA binding protein in MCF-7, human breast cancer cells. Although the MCF-7 cell protein was not induced by 17 beta-estradiol, the MCF-7 cell mRNA binding protein appears to be closely related to the Xenopus protein since: (i) the human and Xenopus proteins elicit gel shifted bands with the same electrophoretic mobility using the vitellogenin mRNA 3'-UTR binding site; (ii) The human and Xenopus proteins exhibit similar binding specificity for the vitellogenin 3'-UTR RNA binding site; and (iii) RNA from MCF-7 cells is at least as effective as RNA from control male Xenopus liver in blocking the binding of the Xenopus and human proteins to the vitellogenin mRNA 3'-UTR binding site. Its broad tissue distribution and regulation by both 17 beta-estradiol and testosterone suggests that this mRNA binding protein may play a significant role in steroid hormone regulation of mRNA metabolism in many vertebrate cells.

Animals↗

PubMed1995

Regulation of Xenopus laevis estrogen receptor gene expression is mediated by an estrogen response element in the protein coding region.

To investigate the 17 beta-estradiol induction of the mRNA coding for the Xenopus laevis estrogen receptor (XER), we cloned the promoter and the 5'-flanking region of the ER gene. Transcription initiation sites were identified by primer extension, and confirmed by the polymerase chain reaction. The promoter and 5'-flanking region contain an imperfect TATA box and a potential CAAT box at -51. Sequence analysis and transfections indicated that no functional estrogen response element (ERE) was present in approximately 3 kb of 5'-flanking region. An imperfect ERE, GGTCAGTTTGACG, which differs from the consensus ERE sequence by 1 nucleotide, was detected in the protein coding region of the gene, approximately 480 nucleotides downstream of the transcription initiation site. In transient transfections using a simple promoter containing two copies of this Xenopus estrogen receptor ERE (XERE), we observed an estrogen-dependent increase in CAT activity of four- to five-fold, to a level approximately 20-fold greater than the activity of the control plasmid lacking the XEREs. In competition gel mobility-shift assays, the XERE exhibited a weak, but clearly detectable, ability to compete for binding of human ER to a labeled consensus ERE. Because it exhibits sequence-specific binding to the ER in competition gel mobility-shift assays, and is able to confer estrogen-dependent transcription on a simple synthetic promoter, the novel XERE, located in the protein coding region of the XER gene appears to represent a weak, but functional, ERE.

Amino Acid Sequence↗

PubMed1995

Repression of endogenous estrogen receptor activity in MCF-7 human breast cancer cells by dominant negative estrogen receptors.

We have investigated the ability of several transcriptionally inactive estrogen receptor (ER) mutants to block endogenous ER-mediated transcription in MCF-7 human breast cancer cells. In transient transfections of MCF-7 cells, two of the mutants, a frame-shifted ER (S554fs) and a point-mutated ER (L540Q), strongly inhibit the ability of endogenous wild-type ER to activate transcription of estrogen-regulated reporter plasmids. A third mutant, ER1-530, which is missing 65 residues from its carboxy-terminus, is a weaker repressor of estradiol-stimulated transcription. When an estrogen response element (ERE)-thymidine kinase-chloramphenicol acetyltransferase reporter gene is used, S554fs, L540Q, and ER1-530 suppress the transcriptional activity of endogenous MCF-7 ER by 87%, 97%, and 62%, respectively. The magnitude of dominant negative repression is promoter specific; when an ERE-pS2-chloramphenicol acetyltransferase reporter is employed, inhibition of endogenous ER activity by equivalent amounts of S554fs, L540Q, and ER1-530 ranges from 85-97%. Dose-response studies show the S554fs mutant to be the most potent of the three ER mutants as a repressor of estrogen action in these cells. In addition, elevated levels of intracellular cAMP, achieved by the addition of 3-isobutyl-1-methylxanthine plus cholera toxin to cells, fail to compromise the effectiveness of these mutants as dominant negative ERs despite the cAMP-enhanced transcriptional activity of ER. The mutants are also powerful repressors of the agonist activity of trans-hydroxytamoxifen-stimulated ER transcription. The dominant negative activity of the three mutants is lost when the A/B domain of these receptors is deleted, implying an important role for this N-terminal region of the ER in the ability of these mutants to inhibit endogenous wild-type ER activity. All in all, the data suggest that S554fs in particular is a reasonable candidate for studies designed to use a dominant negative ER to inhibit the estrogen- and tamoxifen-stimulated growth of human breast cancer cells.

Breast Neoplasms↗

PubMed1995

Estrogen receptor mutants which do not bind 17 beta-estradiol dimerize and bind to the estrogen response element in vivo.

To investigate the stage in estrogen receptor (ER) action at which hormone functions, we prepared human ER mutants unable to bind 17 beta-estradiol. In transfected Chinese Hamster Ovary (CHO) cells, two of the ER mutants exhibited less than 5% of the ability to activate transcription shown by wild type ER. Immunoprecipitation followed by Western blotting with monoclonal antibodies was used to examine the ability of the ER mutants to form heterodimers with a truncated form of wild type ER. The non-hormone-binding mutants formed heterodimers with the truncated ER as efficiently as wild type ER. We used a promoter interference assay to measure the interaction of the ER with the estrogen response element (ERE) in vivo. Expression plasmids encoding the ER mutants and wild type ER were transfected into CHO cells across a range of concentrations, resulting in both high and low levels of promoter interference. The ER mutants and wild type ER elicited similar levels of promoter interference, indicating that although they were unable to bind ligand, the ER mutants bound to the ERE in vivo as effectively as wild type ER. Additional evidence that the non-hormone-binding ER mutants are not in a functionally inactive complex comes from their ability to suppress the activity of wild type ER, when they were coexpressed in the same cells. These data support a model for ER action in which the unliganded ER is free to dimerize and bind to the ERE. In this model, the primary role of 17 beta-estradiol in ER action is to induce a conformational change which activates the ligand-dependent transactivation domain.

Amino Acid Sequence↗

PubMed1994

Antiestrogens activate an estrogen receptor mutant exhibiting enhanced binding to the estrogen response element.

The appearance of constitutively active and antiestrogen resistant estrogen receptor variants has been proposed as one of several factors leading to the development of antiestrogen resistant breast cancers. We recently described the ER2, estrogen receptor mutant, which exhibits enhanced binding to the estrogen response element on DNA, and partial constitutive estrogen-independent) ability to activate transcription. In this work we used transient transfections to show that the antiestrogens trans hydroxytamoxifen and ICI 164,384 are unable to suppress the constitutive activity of the ER2 mutant. Instead, both antiestrogens were concentration-dependent activators of the ER2 mutant. The ER2 mutant appears to be the first estrogen receptor mutant to show activation of a simple estrogen response element-containing promoter using the "pure" antiestrogen ICI 164,384.

Animals↗

PubMed1994

Two functional forms of the Xenopus laevis estrogen receptor translated from a single mRNA species.

Steroid receptors are nuclear proteins that regulate gene transcription in a ligand-dependent manner. Over-expression of the Xenopus estrogen receptor in a vaccinia virus-derived expression system revealed that the receptor localized exclusively in the nucleus of the infected cells, irrespective of the presence or absence of the ligand. Furthermore, two forms of the receptor were produced, a full-length and a N-terminal truncated version, which are translated from a single mRNA species by the use of two AUG within the same reading frame. These 66- and 61-kDa receptors were also observed after in vitro translation of the mRNA as well as in primary Xenopus hepatocytes. Both forms are potent estrogen-dependent transcriptional activators in transient transfection experiments, as well as in in vitro transcription assays.

Animals↗

PubMed1994

An estrogen-inducible protein binds specifically to a sequence in the 3' untranslated region of estrogen-stabilized vitellogenin mRNA.

The 3' untranslated region (3'-UTR) has been implicated in the estrogen stabilization of hepatic Xenopus laevis vitellogenin mRNA. We used RNA gel mobility shift assays to demonstrate that Xenopus liver contains a factor which binds with very high specificity to a segment of the 3'-UTR of vitellogenin B1 and B2 mRNAs. We detected a single high-affinity binding site in the vitellogenin mRNA 3'-UTR and localized the binding site to a 27-nucleotide region. Since binding was abolished by proteinase K digestion, at least a component of the factor is a protein. Following estrogen administration, binding was induced approximately four- to fivefold in extracts from liver polysomes. The hepatic vitellogenin mRNA-binding protein was found in both polysomes and cytosol. Since the protein was also estrogen inducible in cytosol, this represents a genuine induction, not simply recruitment of the cytosolic protein into polysomes. UV cross-linking studies with the 27-nucleotide recognition sequence revealed bands corresponding to bound proteins with apparent molecular weights of 71,000 and 141,000. This appears to be the first example of steroid hormone-inducible proteins binding to an mRNA 3'-UTR. Its induction by estrogen and its sequence-specific binding to a region of vitellogenin mRNA important in estrogen-mediated stabilization suggest that the protein may play a role in the regulation of mRNA stability.

Animals↗

PubMed1993

An estrogen receptor mutant exhibiting hormone-independent transactivation and enhanced affinity for the estrogen response element.

To study transactivation by the Xenopus laevis estrogen receptor (XER), we inserted one or two copies of a synthetic amphipathic helix at amino acid 276 of the XER. The XER mutants containing one or two copies of the amphipathic helix (XER/1AH and XER/2AH, respectively) and wild-type XER were expressed at similar levels. In transient transfection assays, XER/1AH exhibited only a modest, promoter-specific increase in transactivation. Constitutive (estrogen-independent) transcription of a synthetic promoter containing two estrogen response elements (EREs) was approximately 10-fold higher for the XER/2AH mutant than for wild-type XER. The XER/2AH mutant and wild-type XER exhibited similar 17 beta-estradiol dose-response curves for transactivation. In studies carried out over a broad range of DNA concentrations using the simple 2ERE-TATA promoter or a complex vitellogenin-derived promoter, the XER/2AH mutant exhibited an estrogen-dependent 2-3-fold increase in transactivation. A 2-3-fold increase in transactivation by XER/2AH was also observed using synthetic promoters in which the two EREs exhibit synergistic interactions with the NF1, AP1, or vitellogenin activator upstream activator sequences. Using a promoter interference assay to investigate intracellular interactions between the estrogen receptor and the ERE, we showed that binding of wild-type XER to the ERE was strongly estrogen-dependent. In the presence of 17 beta-estradiol, XER/2AH and wild-type XER exhibited similar promoter interference curves. In the absence of 17 beta-estradiol, the expression plasmid encoding the XER/2AH mutant achieved levels of promoter interference with 0.25-0.5 microgram of transfected DNA that were similar to those observed with 5-10 micrograms of the expression plasmid encoding wild-type XER. The ability of the XER/2AH mutant to activate transcription in the absence of estrogen therefore is likely to be related to the approximately 20-fold increase in its apparent ability to bind to the ERE. Since XER/2AH was unable to activate transcription from a glucocorticoid response element, enhanced binding of XER/2AH to the ERE did not result from a general increase in binding to DNA. The XER/2AH mutant appears to be the first nuclear receptor mutant to retain hormone-dependent transactivation and to exhibit enhanced hormone-independent binding to its hormone response element.

Animals↗

PubMed1993

Powerful dominant negative mutants of the human estrogen receptor.

We have identified and characterized three human estrogen receptor (ER) mutants, which, at low concentrations, are capable of blocking the intracellular activity of wild type ER. The mutants, a truncated ER (ER1-530), a point mutant (L540Q), and a frameshift (S554fs), were generated by random chemical mutagenesis of the ER hormone binding domain and screened first for low activity in a yeast selection system. In transient co-transfection assays using ER-deficient Chinese hamster ovary cells, all three mutants exhibited less than 10% of the transcription activation activity of wild type ER, and when co-expressed with wild type ER, each of the mutants effectively suppressed the ability of wild type ER to activate transcription of an estrogen-regulated reporter plasmid. When equal amounts of plasmid encoding the ER mutants and wild type ER were used, S554fs, ER1-530, and L540Q suppressed the activity of wild type ER by 80, 55, and 75%, respectively. At a ratio of 1 part S554fs to 10 parts wild type ER, transcription was still inhibited by 40%. Western blot analysis showed that all three mutants were expressed at approximately the same level as wild type ER. Suppression of transcription was specific for ER, since the mutants did not inhibit progesterone receptor-mediated transcription. Not all mutations leading to inactive ER confer the dominant negative phenotype, as five ER mutants rendered transcriptionally inactive by point mutations between residues 516 and 524 of the ER hormone binding domain were poor inhibitors of wild type ER activity. Binding studies showed that the L540Q and S554fs dominant negative mutants bound 17 beta-estradiol with wild type affinity (Kd = 0.3-0.5 nM), whereas ER1-530 exhibited a 15-fold reduction in affinity for estradiol. The three dominant negative ERs showed significant ability to interact with the estrogen response element (ERE) in promoter interference assays, but ER1-530 and S554fs displayed little or no binding to the ERE in gel mobility shift assays where higher affinity for the DNA may be required for the receptor-ERE complex to remain associated during the electrophoresis. These data support the idea that, in all three mutants, it is loss of function of the COOH-terminal transactivation domain which leads to the dominant negative phenotype. S554fs, a powerful dominant negative mutant, is a good candidate for further studies aimed at suppressing the estrogen-dependent growth of human breast cancer cells.

Amino Acid Sequence↗

PubMed1993

Human estrogen receptor bound to an estrogen response element bends DNA.

We have used gel mobility shift assays to examine changes in DNA bending induced by binding of human estrogen receptor (hER) to a series of estrogen response element (ERE) containing DNA fragments. Competition experiments with ERE-containing DNA fragments and antibody supershift experiments demonstrated that ER in crude extracts from MCF-7 human breast cancer cells exhibited specific interaction with the ERE. Using DNA bending standards, we found that binding of ER to a single ERE induced a reproducible DNA bend of 56 degrees. This was 1.65-fold greater than the 34 degrees bending angle we recently reported for binding of bacterially expressed ER DNA binding domain. The DNA bending angle induced was the same whether the salt-extracted receptor was unoccupied, occupied by 17 beta-estradiol, or occupied by trans-hydroxytamoxifen. To determine if proteins associated with ER in MCF-7 cells affect the degree of bending, we examined the ability of partially purified hER expressed in yeast to bend DNA. The degree of bending induced by the partially purified yeast ER was the same as the bending induced by crude MCF-7 cell ER. More highly purified ER from yeast extracts did not bind to an ERE-containing DNA fragment, suggesting that additional proteins may play an important role in the interaction of the ER with the ERE. When two EREs were present in the DNA fragment, a small but reproducible increase in bending was observed. Our demonstration that binding of hER to the ERE induces DNA bending suggests a possible role for DNA bending in ER-induced transcription activation.

Binding, Competitive↗

PubMed1993

DNA bending by nuclear receptors.

Although steroid hormone receptors constitute an intensively studied family of ligand-regulated transcription factors, the mechanism by which these receptors activate transcription has not been defined. Evidence has accumulated from prokaryotic and eukaryotic systems that many transcription factors are capable of binding to their cognate recognition sequences and causing DNA to bend. Therefore, it has been hypothesized that DNA bending and transcription activation may be functionally coupled. We have utilized circular permutation analysis to examine the ability of the estrogen receptor DNA binding domain and the intact estrogen receptor to bend DNA fragments containing estrogen response elements (EREs). The DNA binding domain, which is a less potent activator of transcription, bent ERE containing DNA fragments less (34 degrees) than the intact estrogen receptor (56 degrees), which is a more potent activator of transcription. In addition, when two EREs were present in a DNA fragment, the degree of DNA bending observed was greater than when one ERE was present. These data suggest that DNA bending may play a role in transcription activation of estrogen responsive genes.

DNA↗

PubMed1992

Binding of the estrogen receptor DNA-binding domain to the estrogen response element induces DNA bending.

We have used circular permutation analysis to determine whether binding of purified Xenopus laevis estrogen receptor DNA-binding domain (DBD) to a DNA fragment containing an estrogen response element (ERE) causes the DNA to bend. Gel mobility shift assays showed that DBD-DNA complexes formed with fragments containing more centrally located EREs migrated more slowly than complexes formed with fragments containing EREs near the ends of the DNA. DNA bending standards were used to determine that the degree of bending induced by binding of the DBD to an ERE was approximately 34 degrees. A 1.55-fold increase in the degree of bending was observed when two EREs were present in the DNA fragment. These in vitro studies suggest that interaction of nuclear receptors with their hormone response elements in vivo may result in an altered DNA conformation.

Animals↗

PubMed1992

The role of estrogen response elements in expression of the Xenopus laevis vitellogenin B1 gene.

We have used site-directed mutagenesis and a homologous transient transfection system to investigate the role of the two imperfect estrogen response elements (EREs) located at -302/-334 in the 5'-flanking region of the estrogen-regulated Xenopus laevis vitellogenin B1 gene. Deletion of either ERE effectively abolishes estrogen-dependent transcription of the vitellogenin promoter. Neither replacement of the two imperfect EREs with a single consensus ERE at -334, nor insertion of one or two consensus EREs at -359, restores full estrogen responsiveness to the mutant promoter. In competition gel mobility shift assays using the DNA binding domain of the Xenopus estrogen receptor, the consensus ERE was a severalfold more effective competitor than the two imperfect B1 EREs. These data suggest that flanking DNA sequences may exert a significant effect on the activity of EREs as hormone-dependent transcription activators. When the imperfect EREs at -302/-334 were present, an additional consensus ERE at -359 exhibited synergistic activation of transcription. However, two consensus EREs located close to the TATA box showed additive, not synergistic, activation of transcription. In contrast, synergistic activation of transcription was observed in synthetic promoters containing two EREs and either the vitellogenin activator element or the NF1 or AP1 upstream activator elements.

Animals↗

PubMed1991

Purified estrogen receptor DNA binding domain expressed in Escherichia coli activates transcription of an estrogen-responsive promoter in cultured cells.

The region of the Xenopus laevis estrogen receptor responsible for interaction with DNA, the DNA binding domain (DBD), has been cloned and overexpressed in Escherichia coli using a T7 RNA polymerase expression system. Extracts from cells transformed with the DBD expression vector contain a single protein which reacts with polyclonal antibodies to estrogen receptor and exhibits sequence-specific binding to a DNA fragment containing a consensus estrogen response element. The DBD protein has been purified to near homogeneity. Determination of the rotational relaxation time of the dansylated DBD by fluorescence polarization and size fractionation by Superdex column chromatography indicate that the DBD is a monomer in solution. The DBD forms a single protein-estrogen response element complex in gel mobility shift assays at DBD concentrations of 18-3,600 nM, suggesting that the DBD is bound to both halves of the palindromic estrogen response element. To investigate the ability of the DBD expressed in bacteria to activate gene expression, we have developed a simple liposome-based system for delivery of protein into cultured cells. Transfected DBD protein elicited large, concentration-dependent increases in transcription of an estrogen receptor regulated reporter gene. These data demonstrate that the bacterially expressed DNA binding domain, which represents a small portion of the Xenopus laevis estrogen receptor, retains significant ability to activate transcription of an estrogen-responsive promoter in vertebrate cells.

Animals↗