Anticipatory estrogen activation of the unfolded protein response is linked to cell proliferation and poor survival in estrogen receptor α-positive breast cancer.

In response to cell stress, cancer cells often activate the endoplasmic reticulum (EnR) stress sensor, the unfolded protein response (UPR). Little was known about the potential role in cancer of a different mode of UPR activation, anticipatory activation of the UPR prior to accumulation of unfolded protein or cell stress. We show that estrogen, acting via estrogen receptor α (ERα), induces rapid anticipatory activation of the UPR, resulting in increased production of the antiapoptotic chaperone BiP/GRP78, preparing cancer cells for the increased protein production required for subsequent estrogen-ERα-induced cell proliferation. In ERα-containing cancer cells, the estrogen, 17ß-estradiol (E2) activates the UPR through a phospholipase C γ (PLCγ)-mediated opening of EnR IP3R calcium channels, enabling passage of calcium from the lumen of the EnR into the cytosol. siRNA knockdown of ERα blocked the estrogen-mediated increase in cytosol calcium and UPR activation. Knockdown or inhibition of PLCγ, or of IP3R, strongly inhibited the estrogen-mediated increases in cytosol calcium, UPR activation and cell proliferation. E2-ERα activates all three arms of the UPR in breast and ovarian cancer cells in culture and in a mouse xenograft. Knockdown of ATF6α, which regulates UPR chaperones, blocked estrogen induction of BiP and strongly inhibited E2-ERα-stimulated cell proliferation. Mild and transient UPR activation by estrogen promotes an adaptive UPR response that protects cells against subsequent UPR-mediated apoptosis. Analysis of data from ERα(+) breast cancers demonstrates elevated expression of a UPR gene signature that is a powerful new prognostic marker tightly correlated with subsequent resistance to tamoxifen therapy, reduced time to recurrence and poor survival. Thus, as an early component of the E2-ERα proliferation program, the mitogen estrogen, drives rapid anticipatory activation of the UPR. Anticipatory activation of the UPR is a new role for estrogens in cancer cell proliferation and resistance to therapy.

Interplay between the levels of estrogen and estrogen receptor controls the level of the granzyme inhibitor, proteinase inhibitor 9 and susceptibility to immune surveillance by natural killer cells.

Estrogens promote cell proliferation and metastases in several human cancers. Here, we describe a different action of estrogens likely to contribute to tumor development-blocking immunosurveillance. In breast cancer cells, increasing concentrations of estrogen induce increasing levels of the granzyme B inhibitor, SerpinB9/proteinase inhibitor 9 (PI-9) and progressively block cell death induced by NK92 natural killer (NK) cells, but do not block killing by a second NK cell line, NKL cells. RNA interference knockdown of PI-9 abolishes estrogen's ability to block NK92 cell-induced cytotoxicity. Expressing elevated levels of estrogen receptor alpha (ERalpha) increases the induced level of PI-9, and makes tamoxifen (TAM), but not raloxifene or ICI 182,780, a potent inducer of PI-9. At elevated levels of ERalpha, induction of PI-9 by estradiol or TAM blocks killing by both NK92 and NKL cells. When the Erk pathway is activated with epidermal growth factor, the concentration of estrogen required to induce a protective level of PI-9 is reduced to 10 pM. Elevated concentrations of estrogen and ER may provide a dual selective advantage to breast cancer cells by controlling PI-9 levels and thereby blocking immunosurveillance. Expressing elevated levels of ERalpha reveals a potentially important difference in the effects of TAM, raloxifene and ICI 182,780 on immunosurveillance in breast cancer.

A unique downstream estrogen responsive unit mediates estrogen induction of proteinase inhibitor-9, a cellular inhibitor of IL-1beta- converting enzyme (caspase 1).

Recently, proteinase inhibitor 9 (PI-9) was identified as the first endogenous inhibitor of caspase 1 (IL-1beta-converting enzyme). The regulation of PI-9 expression, therefore, has great importance in the control of inflammatory processes. We reported that PI-9 mRNA and protein are rapidly and directly induced by estrogen in human liver cells. Using transient transfections to assay PI-9 promoter truncations and mutations, we demonstrate that this strong estrogen induction is mediated by a unique downstream estrogen responsive unit (ERU) approximately 200 nucleotides downstream of the transcription start site. Using primers flanking the ERU in chromatin immunoprecipitation assays, we demonstrate estrogen-dependent binding of ER to the cellular PI-9 promoter. The ERU consists of an imperfect estrogen response element (ERE) palindrome immediately adjacent to a direct repeat containing two consensus ERE half-sites separated by 13 nucleotides (DR13). In transient transfections, all four of the ERE half-sites in the imperfect ERE and in the DR13 were important for estrogen inducibility. Transfected chicken ovalbumin upstream transcription factor I and II down-regulated estrogen-mediated expression from the ERU. EMSAs using purified recombinant human ERalpha demonstrate high-affinity binding of two ER complexes to the ERU. Further EMSAs showed that one ER dimer binds to an isolated DR13, supporting the view that one ER dimer binds to the imperfect ERE and one ER dimer binds to DR13. Deoxyribonuclease I footprinting showed that purified ER protected all four of the half-sites in the ERU. Our finding that a direct repeat can function with an imperfect ERE palindrome to confer estrogen inducibility on a native gene extends the repertoire of DNA sequences able to function as EREs.

Vigilin binding selectively inhibits cleavage of the vitellogenin mRNA 3'-untranslated region by the mRNA endonuclease polysomal ribonuclease 1.

In Xenopus, estrogen induces the stabilization of vitellogenin mRNA and the destabilization of albumin mRNA. These processes correlate with increased polysomal activity of a sequence-selective mRNA endonuclease, PMR-1, and a hnRNP K homology-domain RNA-binding protein, vigilin. Vigilin binds to a region of the vitellogenin mRNA 3'-untranslated region (3'-UTR) implicated in estrogen-mediated stabilization. The vigilin-binding site in the vitellogenin B1 mRNA 3'-UTR contains two consensus PMR-1 cleavage sites. The availability of purified PMR-1 and recombinant vigilin made it possible to test the hypothesis that RNA-binding proteins interact with cis-acting elements to stabilize target mRNAs by blocking cleavage by site-specific mRNA endonucleases. Vigilin binds to the vitellogenin mRNA 3'-UTR site with at least 30-fold higher affinity than it exhibits for the albumin mRNA segment containing the mapped PMR-1 cleavage sites. This differential binding affinity correlates with differential in vitro susceptibility of the protein-RNA complexes to cleavage by PMR-1. Whereas recombinant vigilin has no detectable protective effect on PMR-1 cleavage of albumin mRNA, it retards in vitro cleavage of the vitellogenin mRNA 3'-UTR by purified PMR-1. The PMR-1 sites in the vitellogenin mRNA 3'-UTR are functional because they are readily cleaved in vitro by purified PMR-1. These results provide direct evidence for differential susceptibility to endonuclease-mediated mRNA decay resulting from the differential affinity of a RNA-binding protein for cis-acting stability determinants.

A histone deacetylase inhibitor potentiates estrogen receptor activation of a stably integrated vitellogenin promoter in HepG2 cells.

To compare the role of histone deactylation in estrogen activation of a transiently transfected vitellogenin (VIT) promoter and an integrated VIT promoter in the same cells, we produced three HepG2, human hepatoma, cell lines (HepG2ERV cells) stably expressing human estrogen receptor alpha (hERalpha) and containing an integrated VIT promoter-chloramphenicol acetyltransferase (VIT-CAT) reporter gene. The three ER-positive HepG2ERV cell lines and wild-type, ER-negative, HepG2 cells cotransfected with cytomegalovirus-hERalpha exhibited similar MOX-dependent inductions of 20- to 50-fold with a transiently transfected VIT-luciferase reporter and 15- to 50-fold with a transfected 4-estrogen response element-TATA-luciferase reporter gene. The histone deacetylase inhibitor, trichostatin A, did not enhance MOX induction of the transiently transfected VIT promoter in the HepG2ERV cells. In contrast, trichostatin A dramatically potentiated MOX induction of the stably integrated VIT-CAT reporter gene, resulting in MOX-ER-dependent increases in CAT activity of up to 600-fold. These data demonstrate that although liganded ER exhibits the capacity to fully activate a transiently transfected VIT promoter, under some circumstances the ability to reorganize a repressive chromatin structure may be limiting for steroid receptor action.

Estrogen receptor-KRAB chimeras are potent ligand-dependent repressors of estrogen-regulated gene expression.

As an approach to targeted repression of genes of interest, we describe the development of human estrogen receptor (ER) alpha-KRAB repressor domain chimeras that are potent ligand-dependent repressors of the transcription of estrogen response element (ERE)-containing promoters and analyze their mechanisms of action. Repression by the KRAB domain was dominant over transactivation mediated by ER AF1 and AF2. An ERE and an ER ligand (estrogen or antiestrogen) were required for repression. Studies with several promoters and cell lines demonstrated that the presence of EREs, rather than the capacity for estrogen induction, determines the potential for repression of a gene by the KRAB-ERalpha-KRAB (KERK) chimera. A single consensus ERE was sufficient for repression, but the KERK chimera was unable to suppress transcription from the imperfect ERE in the native pS2 promoter. We recently reported mutations that enhance binding of a steroid receptor DNA-binding domain to the ERE. Introducing these mutations into wild-type ER enhanced transactivation from the pS2 ERE. Insertion of these mutations into KERK created the novel repressor KERK-3M, which is a potent repressor of both ER-induced and basal transcription on a promoter containing the pS2 ERE. These modified ER-KRAB chimeras should prove useful as new tools for the functional analysis and repression of ER-regulated genes.

Proteinase inhibitor 9, an inhibitor of granzyme B-mediated apoptosis, is a primary estrogen-inducible gene in human liver cells.

Although liver is an estrogen target tissue, the number of hepatic genes known to be directly induced by estrogen is very small. We identified proteinase inhibitor 9, or PI-9, as being rapidly and strongly induced by estrogen in an estrogen receptor-positive human liver cell line (HepG2-ER7). Since PI-9 mRNA was also induced by estrogen in a human liver biopsy sample, PI-9 is a genuine estrogen-regulated human gene. PI-9 is a potent inhibitor of granzyme B and of granzyme B-mediated apoptosis. Estrogens induced PI-9 mRNA within 2 h, PI-9 mRNA levels reached a plateau of 30-40-fold induction in 4 h, and induction was not blocked by cycloheximide, indicating that induction of PI-9 mRNA is a primary response. The antiestrogen trans-hydroxytamoxifen was a partial agonist for PI-9 mRNA induction, whereas the antiestrogen ICI 182, 780 was a pure antagonist. Western blot analysis showed that estrogen strongly increases PI-9 protein levels. Inhibition of transcription with actinomycin D resulted in identical rates of PI-9 mRNA decay in the presence and absence of estrogen. We isolated genomic clones containing the PI-9 promoter region, identified a putative transcription start site, and carried out transient transfections of PI-9-luciferase reporter gene constructs. The estrogen, moxestrol, elicited a robust induction from the PI-9-luciferase reporter. Mutational inactivation of three potential imperfect estrogen response elements in the PI-9 5'-flanking region had no effect on moxestrol estrogen receptor induction.

Activation of the p38 mitogen-activated protein kinase pathway by estrogen or by 4-hydroxytamoxifen is coupled to estrogen receptor-induced apoptosis.

17beta-Estradiol (E(2)) or the antiestrogen, 4-hydroxytamoxifen (OHT), induce apoptosis in stably transfected estrogen receptor (ER)-positive HeLa-ER5 cells. p38 mitogen-activated protein kinase is implicated in cellular processes involving apoptosis. The p38 kinase inhibitor, SB203580, partially protects HeLa-ER5 cells against apoptosis induced by E(2) or by OHT. E(2) induces the p38 pathway 12-36-fold in ER-positive cell lines, while OHT induces p38 activity 2-5-fold. In an ER-positive cell line selected for resistance to E(2)-induced apoptosis, E(2) no longer induced p38, and the ER no longer bound to the estrogen response element, while OHT induced both p38 and apoptosis. In cells selected for resistance to OHT-induced apoptosis, OHT no longer induced p38, while E(2) induced p38 and apoptosis, and transactivated an estrogen response element-containing reporter gene. In MCF-7 cells, whose growth is stimulated by estrogen, E(2) did not induce p38 or apoptosis, while OHT induced both p38 and apoptosis, and SB203580 protected against OHT-induced apoptosis. This work shows that E(2) and OHT activate the p38 pathway, suggests that they use different pathways for p38 activation, and links activation of the p38 pathway to apoptosis induced by E(2) and by OHT.

High level expression of full-length estrogen receptor in Escherichia coli is facilitated by the uncoupler of oxidative phosphorylation, CCCP.

The expression of high levels of full-length human estrogen receptor alpha (hERalpha) in Escherichia coli has proven difficult. We found that expression of the ER DNA binding domain is highly toxic to E. coli, resulting in rapid loss of the expression plasmid. Using a tightly regulated arabinose expression system and the antibiotic Timentin, we were able to overcome ER toxicity and express substantial levels of ER. The expressed ER exhibited protease cleavage at a single site near the N-terminus of the hinge region. Of the many measures we tested to eliminate ER cleavage, only addition of carbonyl cyanide m-chlorophenyl-hydrazone (CCCP), an uncoupler of oxidative phosphorylation, completely blocked intracellular proteolysis of the ER. Using CCCP and our expression methods, full-length FLAG epitope-tagged hERalpha (fER) was expressed in E. coli at approximately 1 mg/l. The fER was purified to homogeneity in a single step by immunoaffinity chromatography with anti-FLAG monoclonal antibody. Purified full-length bacterial fER binds 17beta-estradiol with the same affinity as hER expressed in human cells (K(D) approximately 0.5 nM). At high concentrations of fER (20 nM), a bell-shaped estrogen binding curve with a Hill coefficient of 1.7 was seen. Bacterially-expressed fER exhibits a reduced affinity for the estrogen response element (ERE). Anti-FLAG antibody restores high affinity binding of the fER to the ERE, suggesting that impaired dimerization may be responsible for the reduced affinity of bacterially-expressed fER for the ERE. The use of Timentin and CCCP may provide a general method for high level bacterial expression of steroid/nuclear receptors and other proteins important in hormone action.

Analysis of estrogen response element binding by genetically selected steroid receptor DNA binding domain mutants exhibiting altered specificity and enhanced affinity.

To analyze the role of amino acids in the steroid receptor DNA binding domain (DBD) recognition helix in binding of the receptor to the estrogen response element (ERE), we adapted the powerful P22 challenge phage selection system for use with a vertebrate protein. We used the progesterone receptor DNA binding domain and selected for mutants that gained the ability to bind to the ERE. We used a mutagenesis protocol based on degenerate oligonucleotides to create a large and diverse pool of mutants in which 10 nonconsensus amino acids in the DNA recognition helix of the progesterone receptor DNA binding domain were randomly mutated. After a single cycle of modified P22 challenge phage selection, 37 mutant proteins were identified, all of which lost the ability to bind to the progesterone response element. In gel mobility shift assays, approximately 70% of the genetically selected mutants bound to the consensus ERE with a >4-fold higher affinity than the naturally occurring estrogen receptor DBD. In the P-box region of the DNA recognition helix, the selected mutants contained the amino acids found in the wild-type estrogen receptor DBD, as well as other amino acid combinations seen in naturally occurring steroid/nuclear receptors that bind the aGGTCA half-site. We also obtained high affinity DBDs with Trp(585) as the first amino acid of the P-box, although this is not found in the known steroid/nuclear receptors. In the linker region between the two zinc fingers, G597R was by far the most common mutation. In transient transfections in mammalian cells using promoter interference assays, the mutants displayed enhanced affinity for the ERE. When linked to an activation domain, the transfected mutants activated transcription from ERE-containing reporter genes. We conclude that the P-box amino acids can display considerable variation and that the little studied linker amino acids play an important role in determining affinity for the ERE. This work also demonstrates that the P22 challenge phage genetic selection system, modified for use with a mammalian protein, provides a novel, single cycle selection for steroid/nuclear receptor DBDs with altered specificity and greatly enhanced affinity for their response elements.

HMG-1 stimulates estrogen response element binding by estrogen receptor from stably transfected HeLa cells.

Estrogen receptor (ER) toxicity has hampered the development of vertebrate cell lines stably expressing substantial levels of recombinant wild-type ER. To isolate clonal lines of HeLa cells stably expressing epitope-tagged ER, we used a construction encoding a single bicistronic mRNA, in which FLAG-epitope-tagged human ER alpha (fER) was translated from a 5'-translation initiation site and fused to the neomycin resistance gene, which was translated from an internal ribosome entry site. One stable HeLa-ER-positive cell line (HeLa-ER1) produces 1,300,000 molecules of fER/cell (approximately 20-fold more ER than MCF-7 cells). The HeLa fER is biologically active in vivo, as judged by rapid death of the cells in the presence of either 17 beta-estradiol or trans-hydroxytamoxifen and the ability of the cell line to activate a transfected estrogen response element (ERE)-containing reporter gene. The FLAG-tagged ER was purified to near homogeneity in a single step by immunoaffinity chromatography with anti-FLAG monoclonal antibody. Purified fER exhibited a distribution constant (KD) for 17 beta-estradiol of 0.45 nM. Purified HeLa fER and HeLa fER in crude nuclear extracts exhibit similar KD values for the ERE (0.8 nM and 1 nM, respectively), which are approximately 10 times lower than the KD of 10 nM we determined for purified ER expressed using the baculovirus system. HMG-1 strongly stimulated binding of both crude and purified HeLa fER to the ERE (KD of 0.25 nM). In transfected HeLa cells, HMG-1 exhibited a dose-dependent stimulation of 17 beta-estradiol-dependent transactivation. At high levels of transfected HMG-1 expression plasmid, transactivation by ER became partially ligand-independent, and transactivation by trans-hydroxytamoxifen was increased by more than 25-fold. These data describe a system in which ER, stably expressed in HeLa cells and easily purified, exhibits extremely high affinity for the ERE, and suggest that intracellular levels of HMG-1 may be limiting for ER action.

In vitro genetic analysis of the RNA binding site of vigilin, a multi-KH-domain protein.

The function(s) and RNA binding properties of vigilin, a ubiquitous protein with 14 KH domains, remain largely obscure. We recently showed that vigilin is the estrogen-inducible protein in polysome extracts which binds specifically to a segment of the 3' untranslated region (UTR) of estrogen-stabilized vitellogenin mRNA. In order to identify consensus mRNA sequences and structures important in binding of vigilin to RNA, before vigilin was purified, we developed a modified in vitro genetic selection protocol. We subsequently validated our selection procedure, which employed crude polysome extracts, by testing natural and in vitro-selected RNAs with purified recombinant vigilin. Most of the selected up-binding mutants exhibited hypermutation of G residues leading to a largely unstructured, single-stranded region containing multiple conserved (A)nCU and UC(A)n motifs. All eight of the selected down-binding mutants contained a mutation in the sequence (A)nCU. Deletion analysis indicated that approximately 75 nucleotides are required for maximal binding. Using this information, we predicted and subsequently identified a strong vigilin binding site near the 3' end of human dystrophin mRNA. RNA sequences from the 3' UTRs of transferrin receptor and estrogen receptor, which lack strong homology to the selected sequences, did not bind vigilin. These studies describe an aproach to identifying long RNA binding sites and describe sequence and structural requirements for interaction of vigilin with RNAs.

Dimerizing the estrogen receptor DNA binding domain enhances binding to estrogen response elements.

In this work, we provide a rationale for the finding that the estrogen receptor (ER) binds to its DNA response element as a homodimer in vivo. Binding of the monomer estrogen receptor DNA binding domain (ER DBD) to a palindromic, consensus estrogen response element (ERE) is increased 5-6-fold when the ER DBD is dimerized either by a monoclonal antibody that recognizes an attached epitope tag or by expressing the ER DBD as a single molecule in which the two monomers are joined by a peptide linker. Most of the increase in binding is due to stabilization of the ER DBD.ERE complex. We observed only an approximately 2.5-fold reduction in binding when a consensus ERE was replaced with widely spaced ERE half-sites, suggesting that the interaction between ER DBDs on the ERE is relatively weak, and that in full-length ER the DBDs can move independently of each other. To test binding to an imperfect palindrome, typical of the imperfect EREs found in almost all natural estrogen receptor responsive genes, we used the pS2 ERE. Even at high concentrations of ER DBD, specific binding of the ER DBD to the imperfect pS2 ERE was undetectable. Both of the dimerized ER DBDs exhibited efficient binding to the imperfect pS2 ERE, with an affinity at least 25-fold greater than monomer ER DBD. These data support the view that steroid receptor dimerization provides an important mechanism facilitating the recognition of naturally occurring, imperfect hormone response elements.

Prebending the estrogen response element destabilizes binding of the estrogen receptor DNA binding domain.

Binding of many eukaryotic transcription regulatory proteins to their DNA recognition sequences results in conformational changes in DNA. To test the effect of altering DNA topology by prebending a transcription factor binding site, we examined the interaction of the estrogen receptor (ER) DNA binding domain (DBD) with prebent estrogen response elements (EREs). When the ERE in minicircle DNA was prebent toward the major groove, which is in the same direction as the ER-induced DNA bend, there was no significant effect on ER DBD binding relative to the linear counterparts. However, when the ERE was bent toward the minor groove, in a direction that opposes the ER-induced DNA bend, there was a four- to eightfold reduction in ER DBD binding. Since reduced binding was also observed with the ERE in nicked circles, the reduction in binding was not due to torsional force induced by binding of ER DBD to the prebent ERE in covalently closed minicircles. To determine the mechanism responsible for reduced binding to the prebent ERE, we examined the effect of prebending the ERE on the association and dissociation of the ER DBD. Binding of the ER DBD to ERE-containing minicircles was rapid when the EREs were prebent toward either the major or minor groove of the DNA (k(on) of 9.9 x 10(6) to 1.7 x 10(7) M(-1) s(-1)). Prebending the ERE toward the minor groove resulted in an increase in k(off) of four- to fivefold. Increased dissociation of the ER DBD from the ERE is, therefore, the major factor responsible for reduced binding of the ER DBD to an ERE prebent toward the minor groove. These data provide the first direct demonstration that the interaction of a eukaryotic transcription factor with its recognition sequence can be strongly influenced by altering DNA topology through prebending the DNA.

Vigilin, a ubiquitous protein with 14 K homology domains, is the estrogen-inducible vitellogenin mRNA 3'-untranslated region-binding protein.

RNA-binding proteins containing KH domains are widely distributed. One KH domain protein of unknown function, vigilin (also known as the high density lipoprotein-binding protein), contains 14 KH domains and is ubiquitous in vertebrate cells. We previously used RNA gel mobility shift assays to describe an estrogen-inducible protein which binds specifically to a segment of the 3'-untranslated region (3'-UTR) of vitellogenin mRNA, an area which has been implicated in the estrogen-mediated stabilization of vitellogenin mRNA. Here we show that the vitellogenin mRNA-binding protein (VitRNABP) is vigilin. The VitRNABP was isolated as a 150-155-kDa protein on a vitellogenin mRNA 3'-UTR affinity column. Peptide microsequencing revealed that the purified protein was vigilin, a conclusion confirmed in Western blot analysis with antibodies to vigilin. Direct confirmation that vigilin is the VitRNABP was obtained from RNA gel mobility shift assays which demonstrated that antibodies to chicken vigilin supershifted the Xenopus VitRNABP band. Xenopus liver vigilin mRNA and the VitRNABP exhibited similar induction by estrogen, providing additional confirmation that vigilin is the estrogen-inducible protein which binds to the 3'-UTR of estrogen-stabilized vitellogenin mRNA. These data support a role for vigilin in the hormonal control of mRNA metabolism.

Analysis of ligand dependence and hormone response element synergy in transcription by estrogen receptor.

In this work we examined two questions: (1) Is the low, but readily detectable, ability of estrogen receptor (ER) to activate transcription in the absence of added 17beta-estradiol caused by traces of estrogen in the growth medium, or by a weak ligand-independent ability of ER to activate transcription? (2) Does the ER exhibit synergistic activation of transcription on reporter genes containing multiple estrogen response elements (EREs)? To study these questions we developed a powerful new reporter gene, containing four EREs, which achieves inductions of up to 330-fold in the presence of liganded ER. We provided several types of evidence indicating that under standard cell culture conditions unliganded ER is unable to activate transcription. We demonstrated that when cells are grown in serum-free medium, estrogenic compounds may be in the base tissue culture medium. We demonstrated a strong cell and ER-dependence in transcriptional synergy, and suggest that cooperative binding of ER to multiple EREs can be responsible for transcriptional synergy in vivo.

DNA bending is induced by binding of the glucocorticoid receptor DNA binding domain and progesterone receptors to their response element.

Circular permutation analysis was used to determine the degree of DNA bending induced by binding of the glucocorticoid receptor (GR) DNA binding domain (DBD), the human progesterone receptor (PR) DBD, PR-A:A and PR-B:B homodimers, and PR-A:B heterodimers to the glucocorticoid response element/progesterone response element (GRE/PRE). The bending angles induced by the GR DBD and the PR DBD were approximately 28 degrees and 25 degrees, respectively. The PR-B:B and PR-A:A homodimers and the PR-A:B heterodimers all induced similar DNA bending angles of 72-77 degrees. The substantially greater DNA bend induced by full-length PR compared to the PR DBD indicates that sequences outside the classic zinc finger DNA binding domain may play an important role in the interaction of PR with the GRE/PRE. Because PR-A:A and PR-B:B homodimers and the PR-A:B heterodimers induce similar DNA bends, the different abilities of the PR-A and PR-B isoforms to activate transcription are not due to differences in their abilities to distort DNA structure.

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.

In simple synthetic promoters YY1-induced DNA bending is important in transcription activation and repression.

Depending on promoter context, YY1 can activate or repress transcription, or provide a site for transcription initiation. To investigate whether the ability of YY1 to induce DNA bending influenced its ability to activate and repress transcription, simple synthetic promoters were constructed in which the YY1 binding site was inserted between the TATA box and either the NF1 or AP1 recognition sequences. In transient transfections of COS cells, the NF1YY1TATA and NF1RYY1TATA promoters exhibited a dramatic 15-20-fold increase in correctly initiated transcription. These promoters exhibited even larger 60-80-fold increases in transcription in HeLa cells. Neither multiple copies of the YY1 binding site alone, nor placement of a YY1 site upstream of the NF1 site activated transcription. Deletion of 4 bp between the NF1 and YY1 sites, which changes the phase of the DNA bends, abolished the 16-fold activation of transcription by NF1YY1TATA. Insertion of the YY1 site between the AP1 site and the TATA box decreased transcription approximately 3-fold. Replacing the YY1 binding site with an intrinsic DNA bending sequence mimicked this transcription repression. Sequences of similar length which do not bend DNA fail to repress AP1-mediated transcription. Gel mobility shift assays were used to show that binding of YY1 to its recognition sequence did not repress binding of AP1 to its recognition sequences. Our data indicate that YY1-induced DNA bending may activate and repress transcription by changing the spatial relationships between transcription activators and components of the basal transcription apparatus.