A Unique Downstream Estrogen Responsive Unit Mediates Estrogen Induction of Proteinase Inhibitor-9, a Cellular Inhibitor of IL-1ß- Converting Enzyme (Caspase 1)

Sacha A. Krieg, Adam J. Krieg and David J. Shapiro

Department of Biochemistry, University of Illinois, Urbana, Illinois 61801-3602

Address all correspondence and requests for reprints to: Dr. David Shapiro, Department of Biochemistry, 413 RAL, University of Illinois, 600 South Mathews Avenue, Urbana, Illinois 61801. E-mail: djshapir{at}uiuc.edu


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Recently, proteinase inhibitor 9 (PI-9) was identified as the first endogenous inhibitor of caspase 1 (IL-1ß-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 ER{alpha} 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.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
PROTEINASE INHIBITOR 9 (PI-9), a member of the ovalbumin serine proteinase inhibitor family, is of particular interest because of its unusual specificity for caspases 1 and 4 (1, 2). Previously, cytokine response modifier A, an intensively studied antiinflammatory and antiapoptotic cowpox virus protein, was the only serpin known to inhibit caspase 1 (3). Caspases 1 and 4 are atypical in that their primary function is not the induction of apoptosis but the maturation of inflammatory cytokines such as IL-1ß and IL-18. IL-1ß and IL-18 are synthesized as inactive precursors that are converted to their active forms by proteolytic cleavage catalyzed by IL-1ß-converting enzyme (caspase 1) and to a lesser extent by caspase 4 (4, 5). This subset of cytokines plays an important role in several chronic disease states, including atherosclerosis, osteoporosis, and cirrhosis of the liver (2, 4, 5). PI-9 expression is dysregulated in early-stage atherosclerotic plaques, suggesting that PI-9 has the potential to regulate inflammatory and immune responses in vivo (2). PI-9 is also a potent inhibitor of the protease granzyme B (6), which is found in granules produced by cytolytic lymphocytes (CTLs). Granzyme B plays a key role in the ability of CTLs to induce apoptosis of target cells (6, 7, 8). Thus, PI-9 inhibits both caspase 1, which is involved in the maturation of inflammatory cytokines, and granzyme B, which is used by CTLs to induce the death of target cells. Supporting the finding that PI-9 has an antiinflammatory role, recent work by Bladergroen and co-workers showed that PI-9 is expressed at high levels in immune-privileged sites, such as placenta and testis (8A ). PI-9 found in these tissues could abrogate CTL-induced apoptosis and caspase-1-mediated inflammation, possibly contributing to the ability of these organs to evade immune response.

Using a line of human hepatoblastoma cells (HepG2-ER7) stably transfected with ER, we used differential display to identify PI-9 as an estrogen-inducible mRNA. RT-PCR on a human liver biopsy sample confirmed that estrogen induces PI-9 mRNA in human liver cells (4). We showed that estrogen induction of PI-9 mRNA is a direct effect of estrogen and is blocked by the pure anti-estrogen ICI 182,780. That induction of PI-9 is a genomic action of ER was further confirmed by the strong estrogen induction of a construct containing the PI-9 promoter region linked to a luciferase reporter gene (4). The emergence of PI-9 as an endogenous caspase 1 inhibitor, linking estrogen action to crucial processes in inflammation and immune response, makes the regulation of PI-9 gene expression of great interest. Therefore, we began a more detailed analysis of the regulatory elements responsible for estrogen induction of PI-9 gene expression.

Transcriptional activation by ER involves either binding of the ER to specific DNA sequences, termed estrogen response elements (EREs), or tethering of the ER to the DNA through interaction of the ER with other transcription factors, such as AP1 or SP1 (9, 10, 11). Orphan receptors such as chicken ovalbumin upstream transcription factor (COUP-TF) can further modulate estrogen-dependent transcription by competing with ER for binding to DNA sequences and by interacting with ER-ERE complexes (12).

The consensus EREs (cERE) consists of two half-sites (aGGTCAnnnTGACCt) separated by a three-nucleotide spacer. Many estrogen-responsive genes contain imperfect EREs that differ from the cERE sequence by one to three nucleotides (13). Although the region of the PI-9 promoter upstream of the transcription start site contained several potential imperfect EREs, mutation and deletion of these elements separately or in combination suggested that they played no role in estrogen induction of PI-9 gene expression (4). Although the ER is typically thought to bind specifically to palindromes containing three-base spacers, synthetic constructs containing widely spaced direct repeats of the GGTCA half-site sequence interact with the ER in EMSAs and show 17ß estradiol (E2)-dependent transactivation in transient transfections (14, 15, 16). Although widely separated ERE half-sites had been proposed to explain estrogen regulation of a few genes, the weak transactivation potential of single copies of the putative elements and difficulty in demonstrating that ER binds directly to these half-sites has made several of these studies controversial (14, 17, 18). The osteopontin promoter is estrogen regulated through multiple ERE half-sites. Induction was dependent on the extended half-site sequence TCAAGGTCA (19). This extended half-site sequence is not present in the PI-9 estrogen-responsive unit (ERU).

To identify the DNA element(s) responsible for the strong estrogen induction of PI-9 mRNA, we prepared an extensive series of PI-9 deletions and mutations and analyzed their ability to activate transcription in HepG2 cells. Our studies identified an unusual downstream ERU containing an imperfect ERE immediately adjacent to a direct repeat of two consensus ERE half-sites separated by 13 nucleotides (DR13). To test whether the PI-9 promoter interacts directly with ER at the level of native chromatin, we performed chromatin immunoprecipitation (ChIP) assays. The ChIP assay showed that when the potent estrogen, moxestrol, was present, anti-ER antibodies immunoprecipitated the region of the PI-9 promoter encompassing the PI-9 ERU. This element was further demonstrated to be biologically active by its ability to interact with the orphan receptors COUP-TF I and II. To further define the roles of the imperfect ERE and the DR13 element, we carried out transfections, EMSA, and deoxyribonuclease I (DNase I) footprinting. Transfections demonstrated that all four of the half-sites in both the imperfect ERE and the DR13 are required for effective estrogen induction of reporter gene expression. EMSA analysis showed that two ER complexes are associated with the ERU and indicated that the imperfect PI-9 ERE is occupied by one ER dimer and that an isolated DR13 element also interacts with a single ER dimer. DNase I footprinting demonstrated that both ERE half-sites in the DR13 element are protected by purified human ER{alpha} (hER{alpha}). The critical role played by the DR13 element in the functioning of the downstream ERU in the PI-9 gene establishes direct repeats, without a TCA extension, as functional EREs and extends the spectrum of DNA elements that can bind and respond to ER in a native gene.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The PI-9 ERU Is Located Downstream of the Transcription Start Site
There are several potential imperfect EREs in the 5'-flanking region of the PI-9 gene. In previous work, we showed that mutating these potential EREs both individually and in combination did not alter estrogen inducibility of the PI-9 gene (4). To identify the regulatory elements that mediate estrogen induction of PI-9 expression, we prepared a series of constructs containing segments 5' and 3' of the PI-9 transcription initiation site cloned upstream of a luciferase reporter gene. The activity of the constructs was assayed by transient transfection of HepG2 cells. The full-length PI-9 promoter (-1,482 to +314) exhibited 10- to 15-fold induction in the presence of the estrogen moxestrol (Fig. 1Go). We used moxestrol in these studies because in liver cells it is metabolized more slowly than E2 (20). Progressive deletion of the region from -1,420 to -841 containing putative HNF-3 and HNF-1 sites did not affect estrogen inducibility (Fig. 1Go) and did not reduce the overall activity of the PI-9 gene (data not shown). In agreement with our earlier work, a construct containing the potential EREs (-901/+237) and constructs in which the entire region containing the potential EREs was deleted (-308/+237) showed similar estrogen inducibility. The -700/+237 and -700/+174 constructs both contain an AP1 site, but the -700/+237 construct retained full estrogen inducibility, whereas the -700/+174 construct completely lost estrogen inducibility (Fig. 1Go). Sequence analysis indicated a potential ERE in the downstream region between +174 and +237; therefore, we focused our attention on this region (+197/+237).



View larger version (19K):
[in this window]
[in a new window]
 
Figure 1. The PI-9 Promoter Region Responsible for Estrogen Induction Is Located Downstream of the Transcriptional Start Site

HepG2 cells were transiently transfected with a PI-9 promoter-luciferase construct or with truncations of the PI-9 promoter. Truncations are named by the nucleotide in the promoter they contain, with the transcriptional start site denoted as +1. The imperfect EREs and the ERU are enclosed in quotation marks to signify that they contain nonconsensus sequences. Transfections were by calcium phosphate coprecipitation and were carried out in HepG2 cells as described in Materials and Methods using 25 ng of CMV-hER{alpha}, 15 ng of pRLSV40 as internal standard, 50 ng of PI-9 promoter-luciferase construct, and pTZ18U (as carrier DNA) to 2 µg of total DNA. Fold induction represents the increase in luciferase activity for the wild-type promoter and for each truncation in the presence of 10 nM moxestrol, with the non-hormone-treated sample equal to 1. The data represent the mean ± SEM for three separate transfections.

 
The Minimal PI-9 ERU Consists of an Imperfect ERE and a DR13
The failure of the -700/+174 construct to exhibit estrogen inducibility suggested that the consensus AP1 site was not responsible for estrogen induction. To further assess the role of the AP1 site and its flanking sequence in estrogen induction, we compared -308/+237, which retains the AP1 site, to -169/+237, in which the AP1 site has been deleted (Fig. 2Go). Deletion of the AP1 site did not abolish estrogen-dependent transactivation (Fig. 2Go) but did reduce the overall activity of the promoter by approximately 2-fold (data not shown). The region surrounding +200 contains several ERE half-sites. There is an everted ERE (5'-ACTGG-3'; region E) and an imperfect ERE (5'-GGGGAcccTGACC-3'; regions A and B), which contains two changes from the core consensus ERE. The imperfect ERE is followed immediately by two consensus ERE half-sites (5'-TGACC(N)13TGACC-3'; regions C and D; Fig. 2Go). We prepared a series of constructs in which the outer half-sites were deleted and the interior half-sites were mutated (to maintain the correct spacing of the remaining half-sites) in the context of the ERU (+197/+237) construct and assessed their estrogen inducibility in transient transfections (Fig. 2Go). The everted ERE (region E) did not contribute to estrogen inducibility. The construct encompassing the imperfect ERE and the DR13 (constructs A–D) was the functional, minimal ERU. A single copy of this element exhibited an approximately 6-fold increase in expression in response to moxestrol (Fig. 2Go). Analysis of deletions and/or mutations showed that all four of the ERE half-sites (A–D) in the imperfect ERE and in DR13 were required for strong estrogen-dependent transactivation. For example, deletion or mutation of either of the two half-sites in DR13 (constructs A,B,C and A,B,mutC,D) and deletion or mutation of either of the two ERE half-sites in the imperfect ERE (constructs B,C,D and A,mutB,C,D) greatly reduced, or virtually eliminated, estrogen inducibility (Fig. 2Go). Also notable was our finding that deletion of the highly imperfect half-site GGGGA (region A) in the imperfect ERE, which differs by two nucleotides from the GGTCA consensus, reduced estrogen inducibility to only about 2-fold (Fig. 2Go, construct B,C,D). These data indicate that the region necessary for efficient estrogen induction of PI-9 transcription is this unique downstream ERU consisting of an imperfect ERE and a DR13.



View larger version (34K):
[in this window]
[in a new window]
 
Figure 2. The Minimal ERU for PI-9 Consists of an Imperfect ERE and a DR13

HepG2 cells were transfected with fragments of the PI-9 promoter cloned into the luciferase reporter system pGL3 promoter (Promega Corp.). The -1482/+314 construct is referred to as the full-length promoter construct. Nonconsensus nucleotides are denoted with asterisks. Mutated residues were generated in the context of the +197/+237 ERU construct. Each transfection contained 25 ng of CMV-hER, 15 ng of pRLSV40 as internal standard, 50 ng of the PI-9 promoter-luciferase construct, and pTZ18U (as carrier DNA) to 2 µg of total DNA. Fold induction represents the increase in luciferase activity upon addition of moxestrol to 10 nM, with the nonhormone-treated sample equal to 1. The data represent the mean ± SEM for three separate transfections.

 
PI-9 Is Estrogen Inducible in Nonhepatic Cell Lines
To determine whether estrogen inducibility of the PI-9 promoter was limited to liver cells, we also carried out transient transfections in two widely used human cell lines, HeLa (human cervical cancer cells) and MCF-7 (human breast cancer cells). Transiently transfected PI-9 promoter-luciferase constructs exhibited estrogen-dependent expression both in ER-positive MCF-7 cells and in ER-negative HeLa cells cotransfected with a cytomegalovirus (CMV)-hER{alpha} expression plasmid (Fig. 3Go). When the full-length PI-9 promoter (-1,482/+314) was used, the fold induction by estrogen was similar in the HepG2, HeLa, and MCF-7 cells. The fold induction by a single ERU was highest in HeLa cells, intermediate in HepG2 cells, and lowest in MCF-7 cells (Fig. 3Go).



View larger version (21K):
[in this window]
[in a new window]
 
Figure 3. The PI-9 Promoter Is Estrogen Inducible in HeLa Cells and in MCF-7 Cells

HepG2, MCF-7, and HeLa cells were transfected with PI-9 promoter-luciferase constructs containing -1482/+314 or the minimal ERU (derived from +197/+237; Fig. 2Go, ABCD) and then assayed for luciferase activity. Fold induction represents the increase in luciferase activity in response to 10 nM E2 (HeLa and MCF-7 cells) or moxestrol (HepG2 cells), with activity in the presence of ethanol alone equal to 1. The data represent the mean ± SEM for three separate experiments.

 
The PI-9 ERU Activates Transcription from Its Native Downstream Position
The PI-9 ERU is approximately 200 nucleotides downstream of the transcription start site, a location that is unusual but not unprecedented (21, 22, 23, 24). The constructs tested in Figs. 1–3GoGoGo were cloned into the widely used reporter plasmid pGL3 promoter, which contains a simian virus 40 (SV40) TATA box downstream of the inserted promoter sequence. In these constructs, the ERU was positioned upstream of this TATA box. To determine whether the PI-9 ERU could activate transcription from its native downstream position, the full-length PI-9 promoter (-1,482/+314) and a smaller PI-9 promoter fragment containing the ERU (-308/+237) were cloned into the luciferase reporter plasmid pGL3 basic, which does not contain the SV40 TATA box. Transcription of these pGL3 basic PI-9 promoter constructs relies on the endogenous PI-9 start site, with the ERU in its native downstream position. The full-length PI-9 promoter cloned into the pGL3 basic plasmid retains full estrogen inducibility when transfected into HepG2 cells (Fig. 4Go). A smaller construct containing -308/+237 also shows strong estrogen inducibility (Fig. 4Go). These data show that a single downstream ERU is sufficient for estrogen inducibility.



View larger version (31K):
[in this window]
[in a new window]
 
Figure 4. The PI-9 ERU Is Functional in Its Native Downstream Position

To test the ability of the PI-9 ERU to activate transcription from its native downstream location, the PI-9 promoter construct was cloned upstream of the luciferase reporter gene in pGL3 basic, which lacks an SV40 TATA box, so that transcription is from the PI-9 start site. The activity of the promoter in the pGL3 basic and pGL3 promoter plasmid backbones was compared in transfected HepG2 cells as described in Materials and Methods. Activity is expressed as fold induction in the presence of 10 nM moxestrol relative to ethanol alone, which was set equal to 1. The data represent the mean ± SEM for three separate transfections.

 
The ERU Interacts with Two ER Complexes in EMSA
Transfection data (Fig. 2Go) showed that all four of the ERE half-sites in the imperfect ERE and in the DR13 were important for estrogen inducibility. Although these data suggest that more than one ER dimer may interact functionally with the ERU in vivo, the close proximity of the imperfect ERE and the DR13 (Fig. 2Go) made it important to explore the ER-ERU interaction in a more defined biochemical system. Therefore, we carried out EMSAs in which purified recombinant FLAG epitope-tagged hER{alpha} was incubated with labeled, equivalently sized probes containing the PI-9 ERU or a cERE. The cERE probe exhibits a strong up-shifted band (Fig. 5AGo, lane 3, top arrow). It is widely accepted that this represents occupancy of the ERE half-sites by one ER dimer. At low concentrations of added hER{alpha}, the ERU exhibited primarily a single up-shifted band with the same electrophoretic mobility seen with the cERE probe. As the amount of added ER increased, the intensity of this up-shifted band decreased (Fig. 5AGo, lanes 4–7, lower arrow) and a second, slower-migrating up-shifted band progressively increased in intensity (Fig. 5AGo, lanes 4–7, middle arrow). Thus, at higher ER concentrations, the ERU is presumably occupied by a second ER dimer. Although we used purified recombinant hER{alpha}, it was still necessary to show that the two up-shifted bands we observed were caused by specific ER-ERU interactions. Addition of increasing amounts of unlabeled competitor cERE caused a progressive decrease in the intensity of both up-shifted bands (Fig. 5AGo, lanes 8–11). To confirm that the up-shifted bands were cERE-ER and ERU-ER interactions, we performed an antibody supershift using monoclonal antibody against the FLAG epitope tag. As expected, the ER-ERE-antibody complex exhibited a single supershifted band (Fig. 5AGo, lane 12). The two ER-ERU complexes were both supershifted by the FLAG antibody (Fig. 5AGo, lane 13). Although it is generally accepted that the ER binds to the ERE as a dimer, we wanted to test whether these two supershifted bands did indeed correspond to two ER dimers. A DNA probe containing the PI-9 ERE and the directly adjacent half-site, but lacking the second half-site in DR13, resulted in a single up-shifted band that migrated to the same position as the up-shifted band seen with the equivalently sized cERE probe (Fig. 5BGo, lanes 3–5). A probe containing only the DR13 element also showed a single strong up-shifted band, with no evidence of a faster-migrating monomer band (Fig. 5BGo, lanes 6 and 7). The absence of a band corresponding to the ER monomer is consistent with the view that the ER dimer binds to DR13. These data do not support a model in which two ER dimers bind to DR13, with each dimer binding to one of the half-sites in DR13 through one of its two ER monomers. Because the mobility of the up-shifted band seen with DR13 is identical to the mobility of the up-shifted band seen with the cERE, a single ER dimer is bound to DR13. Neither the DR13 nor the probe containing the imperfect ERE and one half-site showed the slower-migrating up-shifted band seen with the complete ERU (compare Fig. 5BGo, lanes 4–7, with Fig. 5AGo, lanes 2–7 and Fig. 5BGo, lanes 1 and 2). The gel shift data demonstrate that the ERU effectively binds ER and support the view that two ER dimers can bind to the ERU.



View larger version (73K):
[in this window]
[in a new window]
 
Figure 5. Two ER Complexes Bind to the ERU in EMSAs

EMSAs were used to evaluate ER binding to this novel ERU. Equivalently sized DNAs containing either the cERE or the PI-9 ERU were radiolabeled and incubated with purified, FLAG epitope-tagged hER{alpha}. Probe bands are indicated with arrows. Complexes were resolved on a nondenaturing, low-ionic-strength polyacrylamide gel. A, Lane 3 illustrates the binding of 0.5 ng of purified hER{alpha} to the cERE probe showing a major up-shifted band (bottom arrow). In lanes 4–7, increasing amounts of purified hER{alpha} (0.5, 1.5, 2.5, and 10 ng of ER, respectively) were incubated with the PI-9 ERU probe. In lanes 8–11, increasing amounts (0, 10, 25, and 100-fold excess relative to ERU) of unlabeled cERE were added to a reaction containing labeled ERU and 1.5 ng of ER. In lanes 12 and 13, anti-FLAG (M2) monoclonal antibody was shown to supershift the FLAG-hER{alpha}-cERE and FLAG-hER{alpha}-ERU complexes. B, EMSA using mutated and truncated ERUs. In lanes 1 and 2, radiolabeled PI-9 ERU was incubated with 1.5 and 3 ng of ER. Lanes 3–7 represent the results from a separate experiment. In lanes 4 and 5, the PI-9 ERU was mutated to abolish the most distal half-site of the DR13 element, and the resulting sequence (PI9ERE + 1/2site) was incubated with 5 and 10 ng of ER, respectively. In lanes 6 and 7, the DR13 element alone was incubated with 10 and 20 ng of ER, respectively. In lane 3, 0.5 ng of ER was incubated with a cERE probe to illustrate the mobility of an hER{alpha} homodimer-cERE complex.

 
Both Regions of the PI-9 ERU Are Protected by ER in DNase I Footprinting
The mobility shift data indicated that two ER complexes could bind to the ERU and suggested that all four half-sites were required for occupancy by two ER dimers. To further elucidate the ER-ERU interaction, we carried out DNase I footprinting. Increasing amounts of purified recombinant FLAG-hER{alpha} were added to a 140-bp end-labeled PI-9 ERU probe before digestion with DNase I. The footprinting experiment clearly showed two protected regions (Fig. 6Go). One protected region corresponds to the imperfect ERE and the directly adjacent consensus half-site of the DR13 element (regions E, A, B, and C), and the second protected region corresponds to the distal half-site in the DR13 (region D) (Fig. 6Go). Interestingly, the ER did not protect the entire 13-nucleotide spacer region between the two half-sites in DR13 (Fig. 6Go, N13). ER binding to the adjacent half-sites enhanced the susceptibility of part of the spacer region to cleavage by DNase I, suggesting that after ER binding at least some of the spacer DNA may be in an exposed bend or loop between the two direct repeat half-sites.



View larger version (78K):
[in this window]
[in a new window]
 
Figure 6. DNase I Footprinting of the ER Bound to the PI-9 ERU Shows Two Distinct Protected Regions

A 140-bp DNA containing the PI-9 ERU probe was end labeled and incubated with the indicated amounts of purified FLAG-hER{alpha}. The mixture was digested with DNase I as described in Materials and Methods. Both the PI-9 ERE and the half-sites found in the DR13 were protected from DNase I digestion.

 
The ER Interacts with the Cellular PI-9 Promoter
To demonstrate that the ER interacts with the PI-9 gene in intact cells, ChIPs were performed. HepG2-ER cells were treated with hormone for 1 h and then cross-linked, harvested, and sonicated. Using monoclonal antibodies specific for the ER (Fig. 7CGo), promoter regions were immunoprecipitated. After immunoprecipitation, cross-linking was reversed and the DNA was amplified by PCR using primers corresponding to the region -169/+237, within which the ERU is located. The moxestrol-treated cells showed an appropriately sized PCR product (Fig. 7BGo) that was not seen with the ethanol-treated samples. Control experiments in which no antibody was used showed no immunoprecipitation or amplification of the -169/+237 promoter region (Fig. 7BGo). Additionally, primers that anneal approximately 1.5 kb from the ERU did not amplify any product (Fig. 7Go, A and B). These data demonstrate that moxestrol-liganded ER interacts with the PI-9 promoter in intact cells. Although the ER epitope recognized by one of the anti-ER monoclonal antibodies used in the immunoprecipitations is not known, the antibody was specific for ER. Using this antibody, a Western blot of a crude HepG2-ER cell extract showed only the expected 66-kDa ER band (Fig. 7CGo).



View larger version (25K):
[in this window]
[in a new window]
 
Figure 7. ChIP Shows ER Interaction with the PI-9 ERU

A, PI-9 promoter diagram illustrating the location of primers used in ChIP assays. B, HepG2 ER cells were treated with hormone for 1 h and processed as described in Materials and Methods. Immunoprecipitations gave the appropriately sized ~400-bp PCR product for the -169/+237 primer set and ~250 PCR product for the -1,482/-1,255 primer set. Input DNA corresponds to 5% of the DNA that was originally added to the immunoprecipitation reaction to show differences in loading. C, Western blot of HepG2 ER cell extract illustrating that the Novacastra monoclonal antibody used in the ChIP interacts specifically with the ER. This specific immunoprecipitation is representative of several independent experiments.

 
Coexpression of the Orphan Receptor COUP-TF Reduces Estrogen Inducibility of PI-9
The orphan receptors COUP-TF I and COUP-TF II interact with ERE half-sites as either homodimers or heterodimers, and their expression reduces the estrogen inducibility of several genes (12, 17, 25, 26, 27). To evaluate the effect of coexpression of COUP-TF on estrogen inducibility of the PI-9 promoter region, we cotransfected increasing amounts of COUP-TF I or COUP-TF II expression plasmids into HepG2 cells and determined the expression of a luciferase reporter gene driven by the PI-9 promoter region in the presence and absence of moxestrol (Fig. 8AGo). Neither COUP-TF I nor COUP-TF II repressed basal (moxestrol-independent) expression of the PI-9 promoter region. With increasing amounts of cotransfected COUP-TF I or COUP-TF II expression plasmids, there was a reduction in the estrogen-dependent (Fig. 8AGo, +MOX) expression of the PI-9 gene. To determine whether COUP-TF repression was exerted through the ERU, we tested the ability of COUP-TF to repress expression of a reporter gene containing the ERU. COUP-TF I and COUP-TF II each repressed estrogen induction of the reporter gene containing a single copy of the ERU (Fig. 8BGo, +MOX). Again, COUP-TF I and II had no effect on basal transcription of this reporter. These data demonstrate that COUP-TF is capable of negatively regulating the expression of PI-9 through the ERU.



View larger version (15K):
[in this window]
[in a new window]
 
Figure 8. COUP-TF I and II Interaction with the PI-9 ERU Represses estrogen Inducibility

Transient transfection of HepG2 cells was carried out as described in Materials and Methods and the legend to Fig. 1Go. A, Increasing amounts of COUP-TF I or COUP-TF II expression plasmids were cotransfected into HepG2 cells along with CMV-hER and the full-length (-1,482/+314) PI-9 promoter-luciferase reporter gene. Luciferase activity in the presence or absence of moxestrol (MOX) was assayed and reported as relative luciferase units (RLU). B, The ability of increasing amounts of COUP-TF I or COUP-TF II to repress ER-dependent transcription from a reporter gene containing only the PI-9 ERU (Fig. 2Go, A, B, C, and D) was evaluated. The data represent the mean ± SEM for three separate experiments.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Estrogen has long been known to play a protective role in several chronic diseases with inflammatory components in their pathogenesis (28). Identification of the antiinflammatory protein PI-9 as an estrogen-inducible gene product is of great interest. Our earlier indirect data suggested that induction of PI-9 mRNA was a direct effect of estrogen (4). Our ChIP assay data provide clear evidence that ER directly regulates PI-9 gene expression. Anti-ER monoclonal antibody immunoprecipitated the PI-9 promoter from liver cells treated with moxestrol but did not immunoprecipitate the promoter from untreated cells (Fig. 7Go).

Previous differential display experiments demonstrated that estrogen induces PI-9 in liver cells (4). Our observations that a reporter gene containing the PI-9 promoter region is strongly estrogen inducible in MCF-7 cells and in HeLa cells (Fig. 3Go) suggest that estrogen may regulate PI-9 levels in a variety of cell types. The idea that PI-9 may be regulated by estrogen in nonhepatic cells is supported by our finding that deletion of the putative liver-specific HNF-1 and HNF-3 sites (-1,482 to -841) did not influence PI-9 expression (Fig. 1Go). The contribution of COUP-TF to PI-9 gene expression was also explored. In several estrogen-regulated promoters, binding of COUP-TF homodimers or heterodimers to ERE half-sites prevented binding of ER (26, 29) and subsequent ER-mediated transactivation (12, 17, 25, 26, 30). Expression of COUP-TF I or COUP-TF II reduced estrogen inducibility of the intact PI-9 promoter region and the ERU, providing further evidence that the ERE half-sites in the ERU are functional. COUP binding to ERE half-sites in the native PI-9 promoter provides a potential mechanism by which COUP might attenuate ER binding and provide an additional level of PI-9 regulation.

Our identification of a downstream sequence as critical for estrogen induction of PI-9 is unusual but not unique. EREs have been identified downstream of the transcription start site in several other estrogen-responsive genes (21, 22, 23, 24). Typically, a downstream ERE is identified by cloning the promoter region under study into the 5'-flanking region of a reporter plasmid, often after multimerization of the putative element. To test the effect of ERU position on estrogen inducibility, we cloned the PI-9 promoter region containing the downstream ERU into a reporter plasmid lacking a transcription start site, so that initiation relies on the endogenous PI-9 start site. Moxestrol strongly activated expression of both the full-length PI-9 promoter and the ERU-containing fragment, demonstrating that a single copy of the ERU is functional in its native downstream position.

The downstream PI-9 ERU consists of an imperfect ERE and an immediately adjacent DR13 element. Our observation that deletion or mutation of either of the half-sites in the imperfect ERE abolished estrogen inducibility (Fig. 2Go) suggests that an isolated direct repeat of the ERE half-site lacks the capacity to act as a strong independent activator of estrogen-dependent transcription. Although neither the imperfect ERE nor the DR13 component has much capacity for estrogen-dependent transactivation alone, together they form a powerful ERU. Consistent with our hypothesis, the levels of ER required for two ER complexes to bind to the ERU are lower than the levels of ER calculated to occur in cell nuclei (18).

The possibility that a direct repeat of an ERE half-site could function as an estrogen-inducible regulatory element was initially suggested by studies examining the transactivation potential of a large pool of DNA fragments in yeast (31) and studies examining ER binding and transactivation of synthetic sequences containing direct repeats of ERE half-sites separated by 5 to more than 100 nucleotides (14). Maximum estrogen transactivation was achieved with synthetic DR10 and DR15 elements (14). This spacing is very similar to the spacing in the DR13 element we identified. To confirm that ER was binding to the direct repeat of ERE half-sites and not to separated half-sites, Gronemeyer and co-workers (14) also showed that mutation of one half-site in a DR element abolished binding by ER. Although there is currently no detailed structural information on how ER can bind to a direct repeat, there are several types of comparative and experimental data suggesting that the ER is well suited to bind to such a sequence. Orphan receptors, which are related more closely to the ER than to other steroid receptors, are able to bind direct repeats of an extended ERE half-site (32). Structural studies of orphan receptors show that members of the nuclear receptor superfamily that bind to extended direct repeats of the GGTCA half-site contain an additional element known as the GRIP box in the C-terminal extension region of the DNA-binding domain. These studies show that the GRIP box contributes to a DNA-dependent dimerization event on direct repeats (32). Of interest, amino acids 260–263 of hER{alpha} constitute a GRIP box. The presence of a GRIP box and the ability of ER to bind direct repeats suggest that the DNA-binding properties of the ER may lie somewhere between those of thyroid/retinoid receptors and other steroid receptors such as GR or PR.

We initially considered models for the interaction of ER with the DR13 in the ERU in which ER monomers bind to one or both half-sites in DR13 or an ER dimer binds specifically to each half-site and nonspecifically to nearby DNA. However, our in vivo and in vitro data, and the work of others, is most consistent with a model in which one ER dimer binds to the imperfect ERE and a second ER dimer binds to the DR13. 1) EMSA data demonstrate that binding of ER to DR13 leads to the same up-shifted band as binding of ER to the cERE (Fig. 5BGo). The absence of a faster-migrating monomer band (Fig. 5BGo, lanes 6 and 7), and earlier studies by Kato et al. (14) showing that both half-sites in a direct repeat element are required for ER binding do not support models in which an ER monomer binds to a half-site or one monomer of an ER dimer binds to each half-site in DR13. 2) A second, slower-migrating band is seen on ER binding to the ERU. This slower-migrating band disappears when one half-site in DR13 is mutated (Fig. 5Go, A and B). A diffuse, slower-migrating third band is also visible in EMSAs with the ERU probe (Fig. 5AGo, lanes 6 and 7). Because this band appears at concentrations of ER 5–10 times higher than those required to protect all four ERE half-sites in a DNAse I footprint, it seems likely that the two ER dimers bound to the ERU stabilize weak, low-affinity binding of ER to other sequences in the probe. It is possible, but less likely, that the third band results from interaction of an additional ER complex with two ER dimers bound to the ERU. Also, all four ERE half-sites in the ERU are protected in DNase I footprinting assays at the concentration of ER showing only two up-shifted bands in the EMSA experiments (Fig. 6Go, 150-fmol ER). These footprinting data demonstrate increased susceptibility to digestion in the DR13 spacer, suggesting that specific dimer binding to this element causes the spacer region to bend or loop out to accommodate ER binding. 3) The ERE half-sites in the DR13 (and in the imperfect ERE) do not contain the TCAAGGTCA sequence suggested to enable ER binding to an extended ERE. Competition experiments led Laudet and co-workers (19) to conclude that the 5' extension was essential for ER to bind to an ERE half-site. Binding of orphan receptors to isolated ERE half-sites involves an AT-rich region flanking the half-site. However, the sequence around the ERU is very GC rich. This supports the view that DR13 functions as a discrete element, not as two independent half-sites. 4) Lastly, the transient transfection data show that deletion or mutation of any of the four ERE half-sites greatly reduces or abolishes estrogen inducibility. A model in which one ER dimer binds to the imperfect ERE and one ER dimer binds to the DR13 is most consistent with these data.

The strong estrogen induction of PI-9 provides a novel link between estrogen action and hormone modulation of inflammatory processes. We have shown that the promoter element mediating estrogen control of PI-9 expression is an unusual downstream response element combining an imperfect ERE and a direct repeat. Our finding that a direct repeat of ERE half-sites can function as an ERE in a composite element with an imperfect ERE palindrome broadens the range of DNA sequences that can function as EREs in native genes.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Generation of Promoter Truncations
Promoter truncations were generated by PCR with primers containing either NheI or BglII restriction sites from a PI-9 promoter plasmid (4). Because of the high GC content of the PI-9 promoter region, standard Pfu turbo (Stratagene, La Jolla, CA) reactions were used with a GC melt (CLONTECH Laboratories, Inc. Palo Alto, CA) concentration of 1 M. Promoter fragments were cloned into the NheI and BglII sites of pGL3 promoter plasmid (Promega Corp., Madison, WI). Smaller promoter fragments were made by annealing primers corresponding to the region of interest containing NheI and BglII restriction sites. The annealed promoter fragments were then cloned into the pGL3 promoter. For experiments using the pGL3 basic vector (Promega Corp.), promoter fragments were cloned in a similar manner. All constructs were verified using the Big Dye terminator cycle sequencing kit (PE Applied Biosystems, Foster City, CA). All primer sequences were based on a promoter sequence in GenBank (accession no. AF200209).

Transient Transfection and Luciferase Assays
HepG2 cells were maintained in DMEM (Life Technologies, Inc., Gaithersburg, MD), 10% charcoal dextran-treated FBS, and penicillin-streptomycin (1,000 U/ml each). Transient transfections in HepG2 cells were carried out using calcium phosphate coprecipitation (33). Transfections were performed in 12-well plates using the following DNA quantities per well (unless stated otherwise in the figure legends): 25 ng of CMV-hER, 50 ng of PI-9-luciferase construct, 15 ng of pRLSV40 as internal standard, and 1.9 µg of pTZ18U as carrier. Immediately after a 20% glycerol shock, the cells were placed in medium containing 10 nM moxestrol or an equal volume of ethanol in the minus-hormone samples.

MCF-7 cells were maintained in MEM (Life Technologies, Inc.) without phenol red, 20 mM HEPES, 8 mM NaHCO3, 10% charcoal dextran-treated bovine calf serum, and penicillin-streptomycin. Transient transfections in MCF-7 cells were carried out using TFX-20 (Promega Corp.) according to the manufacturer’s instructions using serum-free DME:F12. The following concentrations of DNA were used: 5 ng of ER, 10 ng of pRLSV40, and 540 ng of PI-9 promoter-luciferase construct. After liposomes were added, hormone was added to 10 nM.

HeLa cells were cultured under the same conditions as HepG2 cells. Transfections were done using lipofectin (Life Technologies, Inc.) according to the manufacturer’s instructions. Transfections were carried out in 12-well plates with 30 ng of CMV-hER, 50 ng of PI-9 promoter-luciferase construct, 20 ng of pRLSV40 (Promega Corp.) as internal standard, and 0.3 µg of pTZ18U as carrier.

EMSAs
EMSAs were carried out essentially as described previously (34). For these studies, FLAG epitope-tagged hER{alpha} was overexpressed and purified from transiently transfected CHO-S cells (Life Technologies, Inc.). FLAG-hER{alpha} was purified by immunoaffinity chromatography as described (35) and quantitated by Western blot analysis with ER of known concentration as a standard. For supershift experiments, the indicated amount of ER was added to the following reaction mix with BSA to bring the total protein concentration to 5 µg. Reactions contained 3 ng of poly(dI:dC) as nonspecific competitor, 10% glycerol, 50 mM KCl, 15 mM Tris-HCl, pH 7.9, 0.2 mM EDTA, and 0.4 mM dithiothreitol in a total volume of 10 µL and were incubated on ice for 10 min. After incubation, the 60-nucleotide end-labeled PI-9 ERU probe, or a 60-nucleotide cERE probe (10,000 cpm), was added and the reaction mix was incubated at room temperature for 15 min. In the antibody supershift experiments 0.5 µg of FLAG M2 antibody (Sigma, St. Louis, MO) was added with the probe. After incubation with probe, the products were resolved on a low-ionic-strength 6% polyacrylamide gel with buffer recirculation using a water jacket to maintain the gel at 4 C. Gels were dried before autoradiography.

DNase I Footprinting
To create the probe containing the PI-9 ERU, oligonucleotides containing the PI-9 ERU used for EMSA were annealed and A-tailed using Taq polymerase (Life Technologies, Inc.) and then ligated into the pGEM-T vector (Promega Corp.). This PI-9 probe construct in pGEM was then prepared as follows to give a 140-bp probe. The PI-9 ERU fragment was cut once with SacI and treated with calf intestine alkaline phosphatase (Life Technologies, Inc.) and then cut with ApaI, and the fragment was gel purified. The gel-purified probe was then end labeled with T4 kinase, digested with SphI, heat inactivated, and desalted over a Centrisep column (Princeton Separations, Adelphia, NJ) to generate the 140-bp ERU-containing probe. DNase I footprinting assays were carried out using the same reaction mix as for EMSA assays with the exception that KCl was at 20 mM. Purified ER was used in a total volume of 10 µL. ER was preincubated in the absence of probe for 10 min on ice, and the 140-bp PI-9 ERU-containing probe was added and incubated at room temperature for 20 min. One unit of RQ1 DNase (Promega Corp.) was then added for 45 sec. The reaction was extracted with phenol-chloroform and ethanol precipitated two times before loading on an 8% sequencing gel. The gel was dried before autoradiography. Digestion products were compared with a Maxam and Gilbert chemically degraded PI-9 probe (36).

Chromatin Coimmunoprecipitations
The ChIP protocol is a modification of the method published by Meluh and Broach (37) with some minor modifications. HepG2-ER cells were grown in hormone-free medium for at least 1 wk before harvest, as previously described (4). Cells were grown to 95% confluence before use on 15-cm plates and treated with 10 nM moxestrol for 1 h before harvest. After 1 h of hormone treatment, cells were fixed for 10 min by the addition of formalin to a final concentration of 1%. After fixation, the cells were washed three times with PBS and harvested in PBS plus 1 mM EDTA. Cells were resuspended in 500 µl of lysis buffer (25 mM Tris, pH 8.1, 1% Triton X-100, 1% SDS, 3 mM EDTA, and 1x complete protease inhibitor cocktail Roche Molecular Biochemicals, Indianapolis, IN). Cells were sonicated to yield DNA fragments 200–1,000 bp in size by sonicating four times for 10 sec with 1 min of cooling between pulses (Heat Systems Ultrasonics sonic dismembrator with one eighth inch ultramicrotip set at 15% output). One tenth of sonicated lysate was added to each immunoprecipitation. All samples were diluted 1:10 in dilution buffer (lysis buffer without SDS). One-tenth of the diluted sonicate (500 µl) was used per immunoprecipitation. Samples were precleared with 40 µl of BSA-blocked protein A-Sepharose before antibody addition. Dilutions (1:150) of the following ER{alpha} antibodies were used in each immunoprecipitation: ER-NCL6F11 (Novacastra Labs, Newcastle-On-Tyne, UK) and Neomarkers ERAb-1 and ERAb-10 (Lab Vision, Freemont, CA). The specificity of the Neomarkers antibodies in ChiP studies was demonstrated recently (38). We demonstrated the specificity of the Novacastra antibody by Western blotting (Fig. 7C).

The antibody mixture was added to each tube and incubated overnight at 4 C. Fifty percent protein A-Sepharose (60 µl) was then added to each tube, and samples were incubated at room temperature for 1 h and sedimented by centrifugation. Beads were washed sequentially with 1 ml of dilution buffer + 150 mM NaCl + 0.1% SDS, 1 ml of dilution buffer + 500 mM NaCl + 0.1% SDS, 1 ml of buffer III (10 mM Tris, pH 8.1, 0.25 M LiCl, 1% Nonidet P-40, 1% sodium deoxycholate, and 1 mM EDTA), and two 1-ml washes of Tris-Cl, 10 mM, pH 8.0 EDTA, 1 mM. Two hundred fifty microliters of elution buffer (0.1 M NaHCO3 and 1% SDS) was added to the beads, and the samples were incubated at 65 C for 10 min. Elution was repeated, and subsequent steps were performed as described by Meluh and Broach (37). The DNA was resuspended in 20 µl of TE, and 7 µl of this reaction was used for PCR. The following PCR primers were used: -169, 5'-GATATCTAGCTAGCCACCTGTGGCTAGAGAAGGGC-3'; +237, 5'-GTTAGGAAGATCTGAGGTCAGAGCTGGGACAG-3'; +1255, 5'-GGCTTCAAATCAATGACCTCAGTTTC-3'; and +1482, 5'-GATATCTAGCTAGCGTTCTCCAGCCTAGGCGATAGAG-3'. PCR was carried out using Taq polymerase (Life Technologies, Inc.) according to the manufacturer’s directions with the exception that 1 M betaine (Sigma, St. Louis) was added. Thermocycling conditions used a 55 C annealing temperature and 29 cycles. PCR products were separated on a 2.5% agarose gel.


    ACKNOWLEDGMENTS
 
We are grateful to Dr. M. J. Tsai for providing the COUP-TF I and II expression plasmids. We thank Dr. A. Nardulli and M. Loven for helpful advice on DNase I footprinting. We are grateful to Drs. D. Edwards and A. Cooney for helpful discussions concerning the GRIP box and orphan receptors and to Dr. R. Dodson for helpful comments on the manuscript.


    FOOTNOTES
 
This work was supported by Grant HD-16720 from the National Institutes of Child Health and Human Development. S.A.K. was supported by predoctoral fellowship DAMD 17-98-1-8197 from the U.S. Army Medical Research and Materiel Command Breast Cancer Research Program.

Abbreviations: cERE, Consensus estrogen response element; ChIP, chromatin immunoprecipitation; CMV, cytomegalovirus; COUP-TF, chicken ovalbumin upstream trans-cription factor; CTL, cytolytic lymphocytes; DNase, deoxyribonuclease; DR13, direct repeat containing two consensus ERE half-sites separated by 13 nucleotides; ERU, estrogen-responsive unit; hER{alpha}, human ER{alpha}; PI-9, proteinase inhibitor 9; SV40, simian virus 40.

Received for publication January 3, 2001. Accepted for publication July 13, 2001.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Annand RR, Dahlen JR, Sprecher CA, De Dreu P, Foster DC, Mankovich JA, Talanian RV, Kisiel W, Giegel DA 1999 Caspase-1 (interleukin-1ß-converting enzyme) is inhibited by the human serpin analogue proteinase inhibitor 9. Biochem J 342:655–665[CrossRef][Medline]
  2. Young JL, Sukhova GK, Foster D, Kisiel W, Libby P, Schonbeck U 2000 The serpin proteinase inhibitor 9 is an endogenous inhibitor of interleukin 1ß-converting enzyme (caspase-1) activity in human vascular smooth muscle cells. J Exp Med 191:1535–1544[Abstract/Free Full Text]
  3. Ray CA, Black RA, Kronheim SR, Greenstreet TA, Sleath PR, Salvesen GS, Pickup DJ 1992 Viral inhibition of inflammation: cowpox virus encodes an inhibitor of the interleukin-1ß converting enzyme. Cell 69:597–604[Medline]
  4. Kanamori H, Krieg S, Mao C, Di Pippo VA, Wang S, Zajchowski DA, Shapiro DJ 2000 Proteinase inhibitor 9, an inhibitor of granzyme B-mediated apoptosis, is a primary estrogen-inducible gene in human liver cells. J Biol Chem 275:5867–5873[Abstract/Free Full Text]
  5. Kumar V, Green S, Stack G, Berry M, Jin JR, Chambon P 1987 Functional domains of the human estrogen receptor. Cell 51:941–951[Medline]
  6. Sun J, Bird CH, Sutton V, McDonald L, Coughlin PB, De Jong TA, Trapani JA, Bird PI 1996 A cytosolic granzyme B inhibitor related to the viral apoptotic regulator cytokine response modifier A is present in cytotoxic lymphocytes. J Biol Chem 271:27802–27809[Abstract/Free Full Text]
  7. Heusel JW, Wesselschmidt RL, Shresta S, Russell JH, Ley TJ 1994 Cytotoxic lymphocytes require granzyme B for the rapid induction of DNA fragmentation and apoptosis in allogeneic target cells. Cell 76:977–987[Medline]
  8. Bird CH, Sutton VR, Sun J, Hirst CE, Novak A, Kumar S, Trapani JA, Bird PI 1998 Selective regulation of apoptosis: the cytotoxic lymphocyte serpin proteinase inhibitor 9 protects against granzyme B-mediated apoptosis without perturbing the Fas cell death pathway. Mol Cell Biol 18:6387–6398[Abstract/Free Full Text]
  9. Bladergroen BA, Strik MC, Bovenshen N, Van Berkum O, Scheffer GR, Mejer CJ, Hak CE, Kummer J 2001 The granzyme B inhibitor, proteinase inhibitor 9, is mainly expressed by dendritic cells and at immune privileged sites. J Immunol 166:3218–3225[Abstract/Free Full Text]
  10. Wang F, Porter W, Xing W, Archer TK, Safe S 1997 Identification of a functional imperfect estrogen-responsive element in the 5'-promoter region of the human cathepsin D gene. Biochemistry 36:7793–7801[CrossRef][Medline]
  11. Qin C, Singh P, Safe S 1999 Transcriptional activation of insulin-like growth factor binding protein-4 by 17ß estradiol in MCF-7 cells: role of estrogen receptor-sp1 complexes. Endocrinology 140:2501–2508[Abstract/Free Full Text]
  12. Webb P, Nguyen P, Valentine C, Lopez GN, Kwok GR, McInerney E, Katzenellenbogen BS, Enmark E, Gustafsson JA, Nilsson S, Kushner PJ 1999 The estrogen receptor enhances AP-1 activity by two distinct mechanisms with different requirements for receptor transactivation functions. Mol Endocrinol 13:1672–1685[Abstract/Free Full Text]
  13. Chu K, Boutin JM, Breton C, Zingg HH 1998 Nuclear orphan receptors COUP-TFII and Ear 2: presence in oxytocin-producing uterine cells and functional interaction with the oxytocin gene promoter. Mol Cell Endocrinol 137:145–154[CrossRef][Medline]
  14. Driscoll MD, Sathya G, Muyan M, Klinge CM, Hilf R, Bambara RA 1998 Sequence requirements for estrogen receptor binding to estrogen response elements. J Biol Chem 273:29321–29330[Abstract/Free Full Text]
  15. Kato S, Sasaki H, Suzawa M, Masushige S, Tora L, Chambon P, Gronemeyer H 1995 Widely spaced, directly repeated PuGGTCA elements act as promiscuous enhancers for different classes of nuclear receptors. Mol Cell Biol 15:5858–5867[Abstract]
  16. Klinge CM, Bodenner DL, Desai D, Niles RM, Traish AM 1997 Binding of type II nuclear receptors and estrogen receptor to full and half-site estrogen response elements in vitro. Nucleic Acids Res 25:1903–1912[Abstract/Free Full Text]
  17. Aumais JP, Lee HS, DeGannes C, Horsford J, White JH 1996 Function of directly repeated half-sites as response elements for steroid hormone receptors. J Biol Chem 271:12568–12577[Abstract/Free Full Text]
  18. Park HM, Haecker SE, Hagen SG, Sanders MM 2000 COUP-TF plays a dual role in the regulation of the ovalbumin gene. Biochemistry 39:8537–8545[CrossRef][Medline]
  19. Anderson I, Gorski J 2000 Estrogen receptor {alpha} interaction with estrogen response element half-sites from the rat prolactin gene. Biochemistry 39:3842–3847[CrossRef][Medline]
  20. Vanacker JM, Pettersson K, Gustafsson JA, Laudet V 1999 Transcriptional targets shared by estrogen receptor-related receptors (ERRs) and estrogen receptor (ER) {alpha} but not ERß. EMBO J 18:4270–4279[Abstract/Free Full Text]
  21. Barkhem T, Andersson-Ross C, Hoglund M, Nilsson S 1997 Characterization of the "estrogenicity" of tamoxifen and raloxifene in HepG2 cells: regulation of gene expression from an ERE controlled reporter vector vs. regulation of the endogenous SHBG and PS2 genes. J Steroid Biochem Mol Biol 62:53–64[CrossRef][Medline]
  22. Choi I, Gudas LJ, Katzenellenbogen BS 2000 Regulation of keratin 19 gene expression by estrogen in human breast cancer cells and identification of the estrogen responsive gene region. Mol Cell Endocrinol 164:225–237[CrossRef][Medline]
  23. Lee JH, Kim J, Shapiro DJ 1995 Regulation of Xenopus laevis estrogen receptor gene expression is mediated by an estrogen response element in the protein coding region. DNA Cell Biol 14:419–430[Medline]
  24. Hyder SM, Stancel GM, Nawaz Z, McDonnell DP, Loose-Mitchell DS 1992 Identification of an estrogen response element in the 3'-flanking region of the murine c-fos protooncogene. J Biol Chem 267:18047–18054[Abstract/Free Full Text]
  25. Gill RK, Christakos S 1995 Regulation by estrogen through the 5'-flanking region of the mouse calbindin-D28k gene. Mol Endocrinol 9:319–326[Abstract]
  26. Zhang Y, Dufau ML 2000 Nuclear orphan receptors regulate transcription of the gene for the human luteinizing hormone receptor. J Biol Chem 275:2763–2770[Abstract/Free Full Text]
  27. Narayanan CS, Cui Y, Zhao YY, Zhou J, Kumar A 1999 Orphan receptor Arp-1 binds to the nucleotide sequence located between TATA box and transcriptional initiation site of the human angiotensinogen gene and reduces estrogen induced promoter activity. Mol Cell Endocrinol 148:79–86[CrossRef][Medline]
  28. Leng X, Cooney AJ, Tsai SY, Tsai MJ 1996 Molecular mechanisms of COUP-TF-mediated transcriptional repression: evidence for transrepression and active repression. Mol Cell Biol 16:2332–2340[Abstract]
  29. Kumar V, Cotran RS, Robbins SL 1997 Basic pathology. Philadelphia, PA: W.B. Saunders Co.
  30. Chu K, Boutin JM, Breton C, Zingg HH 1998 Nuclear orphan receptors COUP-TFII and Ear-2: presence in oxytocin-producing uterine cells and functional interaction with the oxytocin gene promoter. Mol Cell Endocrinol 137:145–154[CrossRef][Medline]
  31. Klinge CM, Silver BF, Driscoll MD, Sathya G, Bambara RA, Hilf R 1997 Chicken ovalbumin upstream promoter-transcription factor interacts with estrogen receptor, binds to estrogen response elements and half-sites, and inhibits estrogen-induced gene expression. J Biol Chem 272:31465–31474[Abstract/Free Full Text]
  32. Dana SL, Hoener PA, Wheeler DA, Lawrence CB, McDonnell DP 1994 Novel estrogen response elements identified by genetic selection in yeast are differentially responsive to estrogens and antiestrogens in mammalian cells. Mol Endocrinol 8:1193–1207[Abstract]
  33. Zhao Q, Khorasanizadeh S, Miyoshi Y, Lazar MA, Rastinejad F 1998 Structural elements of an orphan nuclear receptor-DNA complex. Mol Cell 1:849–861[Medline]
  34. Mattick S, Glenn K, de Haan G, Shapiro DJ 1997 Analysis of ligand dependence and hormone response element synergy in transcription by estrogen receptor. J Steroid Biochem Mol Biol 60:285–294[CrossRef][Medline]
  35. Kim J, de Haan G, Nardulli AM, Shapiro DJ 1997 Prebending the estrogen response element destabilizes binding of the estrogen receptor DNA binding domain. Mol Cell Biol 17:3173–3180[Abstract]
  36. Zhang CC, Krieg S, Shapiro DJ 1999 HMG-1 stimulates estrogen response element binding by estrogen receptor from stably transfected HeLa cells. Mol Endocrinol 13:632–643[Abstract/Free Full Text]
  37. Sambrook J, Fritsch EF, Maniatis T 1989 Molecular cloning: a laboratory manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press
  38. Meluh PB, Broach JR 1999 Immunological analysis of yeast chromatin. Methods Enzymol 304:414–430[Medline]
  39. Shang Y, Hu X, DiRenzo J, Lazar MA, Brown M 2000 Cofactor dynamics and sufficiency in estrogen receptor-regulated transcription Cell 103:843–852[Medline]