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Endocrinology Vol. 142, No. 7 2879-2887
Copyright © 2001 by The Endocrine Society

ARTICLES

Cloning of the Ovine Estrogen Receptor-{alpha} Promoter and Functional Regulation by Ovine Interferon-{tau}1

Jo-Ann G. W. Fleming2, Youngsok Choi2, Greg A. Johnson, Thomas E. Spencer and Fuller W. Bazer

Center for Animal Biotechnology and Genomics and Department of Animal Science, Texas A&M University, College Station, Texas 77843-2471

Address all correspondence and requests for reprints to: Dr. Fuller W. Bazer, Center for Animal Biotechnology and Genomics, 2471 TAMU, Texas A&M University, College Station, Texas 77843-2471. E-mail: fbazer{at}cvm.tamu.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Interferon-{tau} (IFN{tau}), the ruminant pregnancy recognition signal, inhibits transcription of the estrogen receptor {alpha} (ER{alpha}) gene in the endometrial lumenal epithelium of the sheep uterus, thereby abrogating production of luteolytic PGF2{alpha} pulses. The effects of IFN{tau} are mediated in part by IFN-stimulated response elements (ISREs) and IFN regulatory factor elements (IRFEs). The promoter/enhancer region of the ovine ER{alpha} gene was cloned, sequenced, and predicted to contain four IRFEs and one ISRE. Electrophoretic mobility shift assays indicated that the -2110 IRFE bound only IRF-1, whereas the -1877 IRFE and the -1284 ISRE were functional in binding IRF-1 and IRF-2. IFN{tau} inhibited transcriptional activity of the 2.7-kb ovine ER{alpha} promoter in transfection assays using ovine lumenal epithelium cells. Analyses of sequential 5'-deletion mutants of the ovine ER{alpha} promoter indicated that the effects of IFN{tau} may be mediated by IRFEs as well as other elements. Overexpression of ovine IRF-2, but not IRF-1, inhibited transcriptional activity of several regions of the ovine ER{alpha} promoter containing an IRFE or an ISRE as well as some, but not all, regions lacking these elements.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
RUMINANTS, i.e. sheep, cattle, and goats, are spontaneous ovulators that undergo estrous cycles until the elongating conceptus (embryo and associated extraembryonic membranes) signals maternal recognition of pregnancy. In cyclic ewes, the endometrial luteolytic mechanism develops between days 11 and 15 postestrus (1, 2). In the endometrial lumenal epithelium (LE) and superficial glandular epithelium (sGE), loss of epithelial progesterone receptor (PR) expression between days 9 and 13 (3, 4) is accompanied by increases in epithelial estrogen receptor {alpha} (ER{alpha}) gene expression between days 11 and 13 (4, 5). The increase in ER{alpha} expression precedes the onset of oxytocin receptor (OTR) gene expression between days 13 and 14 of the cycle (6, 7, 8). Oxytocin, released in a pulsatile manner from the corpus luteum (CL) and posterior pituitary between days 8–16, binds to OTR on the LE and sGE to initiate pulsatile release of luteolytic PGF2{alpha} between days 14 and 16 (9). In the absence of a viable conceptus, the endometrial luteolytic mechanism develops to destroy the CL and allow the ewe to return to estrus for another opportunity to mate.

During pregnancy recognition, the conceptus secretes interferon-{tau} (IFN{tau}), a unique member of the type I IFN family, which acts in a paracrine fashion on the endometrium to prevent development of the endometrial luteolytic mechanism (1, 2). IFN{tau} are secreted in high amounts (2 x 107 antiviral units (AVU)/day) by the sheep conceptus between days 10 and 21 of early pregnancy and prevent increases in ER{alpha} and OTR gene expression in the LE and sGE (8, 10, 11, 12, 13). Intrauterine injections of recombinant ovine IFN{tau} into cyclic ewes were sufficient to prevent estrogen-induced increases in epithelial ER{alpha} and OTR expression (10, 12, 13). Available evidence in the ovine model supports the hypothesis that IFN{tau} inhibits transcription of the ER{alpha} gene, which precludes estrogen induction of OTR gene expression, formation of OTR on the endometrial LE and sGE, and the ability of oxytocin to elicit the release of luteolytic pulses of PGF2{alpha} from the endometrium (13). The cellular and molecular mechanisms mediating the inhibitory effects of IFN{tau} on endometrial gene transcription are not known.

Type I IFNs bind to specific receptors and regulate transcription of target genes by stimulation of the signal transducers and activators of transcription (STAT) and IFN regulatory factor (IRF) pathways (14, 15, 16, 17). Activated STAT1 can homodimerize to form an {gamma}-activated factor that binds to {gamma} activation sequence (GAS) elements and activates transcription of genes such as IFN regulatory factor-1 (IRF-1) (18, 19, 20, 21). STAT1-STAT2 heterodimers may also associate with p48/ISGF3{gamma}/IRF-9 to form ISGF3, which binds to IFN-stimulated response elements (ISREs) and activates transcription of a number of IFN-stimulated genes (ISG), such as ubiquitin cross-reactive protein (UCRP/ISG17) and 2',5'-oligoadenylate synthetase (17, 22, 23). IFN{tau} binds endometrial epithelial receptors and up-regulates IRF-1 protein expression in vivo and in vitro (14, 15, 17). IRF-1 is a positive-acting transcription factor that binds to both ISREs and IRF elements (IRFEs) (24, 25, 26, 27, 28, 29, 30, 31, 32). In contrast, IRF-2 is a negative-acting transcriptional repressor of IRFE-containing target genes (24, 25, 26, 27, 28, 29, 30, 31, 32, 33). The mechanism of IRF-2 action has been shown to include direct transcriptional repression and repulsion of transcriptional coactivators (33).

In pregnant ewes, IRF-1 expression was detected transiently in LE and sGE on days 11 and 13, followed by IRF-2 expression in the same epithelia on days 13–20 (14). Infusion of recombinant IFN{tau} into the uterine horn of cyclic ewes transiently up-regulated the expression of IRF-1 and later IRF-2 proteins before the time that expression of ER{alpha} and OTR was detected in the contralateral uterine horn receiving control proteins (14). Our working hypothesis is that IFN{tau} inhibits expression of the ovine ER{alpha} gene in the endometrial epithelium by up-regulating IRF-2 in the endometrial epithelium, which negatively regulates transcription via IRFEs and/or ISREs in the 5'-flanking promoter/enhancer region of the gene (15, 16, 17). Therefore, the objectives of this study were to begin elucidating the cellular and molecular mechanisms mediating the inhibitory effects of IFN{tau} on ovine ER{alpha} (oER{alpha}) gene transcription by 1) cloning, sequencing, and analyzing the 5'-flanking promoter/enhancer region of the oER{alpha} gene; 2) identifying functional IRF-Es and ISREs by electrophoretic mobility shift analyses (EMSAs); and 3) determining the effects of oIFN{tau}, oIRF-1, and oIRF-2 on oER{alpha} promoter activity using transient transfection analyses of an immortalized ovine endometrial LE cell line.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cloning of the 5'-flanking promoter/enhancer region of the oER{alpha} gene
An NcoI-PstI fragment of exon 1 of the human ER{alpha} gene (nucleotides 1–367, where 1 is ATG; X62462) (34) was labeled with [32P]deoxy (d)-CTP by random primer labeling and used as a probe to screen an EMBL3 {lambda} sheep genomic library (CLONTECH Laboratories, Inc., Palo Alto, CA) under high stringency conditions. Plaque purification to homogeneity of several positive isolates, DNA isolation, and physical mapping by restriction endonuclease digestion and Southern blotting were performed using standard methods (35). A 4.7-kbp BamHI-SalI fragment was subcloned into pBluescript KS vector (Stratagene, La Jolla, CA) and sequenced with oER{alpha} exon 1-specific primers to confirm identity. The nucleotide sequence of the approximately 2.7-kbp region upstream of the ATG site was determined in both directions (GenBank AF159145).

5'-oER{alpha}-luciferase (LUC) constructs
A HindIII site that ablated the ATG of the oER{alpha} gene was introduced by cloning a NotI-HindIII adaptor oligonucleotide at the NotI site closest to the ATG in the oER{alpha} gene. This adapter maintained the oER{alpha} gene sequence between the NotI site and the introduced HindIII site. The primers for the adapter were: forward, 5'-GGCCGCTCACCA-3'; and reverse, 5'-AGCTTGGTGAGC-3'. The resultant BamHI-HindIII fragment was cloned into the BglII and HindIII sites upstream of the luciferase gene in the pGL3 Basic (Promega Corp., Madison, WI) reporter, which contains no endogenous promoter sequences. A series of 5'-upstream truncations was constructed by digesting reporter plasmid DNA with MluI and selected restriction endonucleases, blunt-ending with Klenow and/or T4 DNA polymerase, and religating. Plasmid DNA used for transfections and in vitro transcription-translation experiments was isolated by the alkaline lysis method and anion exchange chromatography (QIAGEN, Valencia, CA).

oER{alpha}-TK-LUC constructs
A TK-LUC reporter plasmid was constructed by obtaining the 164-bp thymidine kinase (TK) promoter from 3xGAS-TK-LUC (17) by digesting with BamHI and BglII and subcloning into the BglII site of pGL3 Basic. The -1877 IRFE was placed in front of the heterologous TK promoter by synthesizing specific oligonucleotides (forward, 5'-CGG GCC CAT GTT TGA AAA TTG AAA GTG TGA TTT G-3'; reverse, 5'-CTA GCA AAT CAC ACT TTC AAT TTT CAA ACA TGG GCC CGA GCT-3'), annealing them, and cloning them into the SacI and NheI sites upstream of the TK promoter in TK-LUC.

The -1949/-1492 oER{alpha}-TK-LUC and -1492/-958-oER{alpha}-TK-LUC constructs were created by digesting the oER{alpha} promoter with EcoRI and subcloning the fragments into pBluescript. The oER{alpha} regions in the pBluescript clones were excised with XbaI and XhoI and then subcloned into the NheI and XhoI sites upstream of the TK promoter in TK-LUC. The -958/-736 oER{alpha}-TK-LUC construct was prepared by digesting oER{alpha} with EcoRI and PstI and subcloning into pCRII (Invitrogen, Carlsbad, CA). The -958/-736 region was excised from the pCRII subclone using SacI and XbaI and subcloned into the SacI and NheI sites of TK-LUC. The -2677/-1949 and -2677/-2163 fragments were cloned by digesting the full-length pBluescript oER{alpha} clone with HindIII and either EcoRI or NcoI, respectively, end-filling with Klenow, and religating. The deleted clones were digested with SacI and XhoI and cloned directionally (5' to 3') into the SacI-XhoI sites upstream of the TK promoter of TK-LUC. The -732/-266 PstI-SacI fragment was subcloned into the PstI-SacI sites of pBluescript and then excised with SacI and XhoI. This fragment was cloned similarly in the SacI and XhoI sites of TK-LUC, but is in reverse orientation (3' to 5'). The -2163/-1949 construct was made by first digesting the full-length BamHI-HindIII oER{alpha} clone with EcoRI and religating to create the internally deleted clone poERdEcoRI (poERd [-1949/-958]). This clone was digested with BamHI and NcoI, blunted with Klenow, and religated. The -2163/-1949 fragment was excised by digesting the BamHI/NcoI fusion clone with EcoRI, end-filling with Klenow, and then digesting with SacI. The 214-bp fragment was inserted 5' to 3' upstream of the TK promoter in TK-LUC digested with SacI and SmaI.

Cloning of ovine IRF-1 and IRF-2 messenger RNAs and construction of mammalian overexpression vectors
A partial complementary DNA (cDNA) for oIRF-1 was amplified by RT-PCR from day 15 pregnant ovine endometrial messenger RNA using primers based on the human IRF-1 sequence (GenBank NM002198; forward, 5'-TCC ACC TCT CAC CAA GAA CC-3'; reverse, 5'-TTC TGG CTC CTC CTT ACA GC-3'). PCR conditions were denaturation at 95 C for 2 min, followed by 30 cycles of 30 sec at 95 C, 30 sec at 57 C, and 30 sec at 72 C with a final extension at 72 C for 10 min. The 502-bp product was cloned into pCRII vector, and its identity was confirmed by sequencing. A random primer-labeled ovine IRF-1 partial cDNA and a full-length murine IRF-2 cDNA were used as probes for screening a {lambda} ZAP II cDNA library of day 15 pregnant ovine endometrium (Stratagene) at low stringency. Clones containing the entire oIRF-1 and oIRF-2 coding sequences were isolated and sequenced in both directions (GenBank AF331970 and AF228445). oIRF-1 and oIRF-2 cDNAs were subcloned into pEF1-Myc/His lacZ mammalian expression vector (Invitrogen, Carlsbad, CA). BamHI-NotI-digested vector was ligated to a BamHI-NotI oIRF-2 fragment or blunt-ended at the BamHI site with Klenow before ligation to an EcoRV-NotI oIRF-1 fragment.

EMSAs
Oligonucleotides containing a consensus IRFE or the putative ovine ER{alpha} IRFEs and the immediately adjacent flanking sequences and their complements were synthesized as follows: consensus IRFE: forward, 5'-GGA AGC GAA AAT GAA ATT GAC T-3'; reverse, 5'-AGT CAA TTT CAT TTT CGC TTC C-3'; -2110 IRFE: forward, 5'-GTA GGA CTA TTT TGA TTT CTC TTC GCA GCT TTA-3'; reverse, 5'-ATG CTA AAG CTG CGA AGA GAA ATC AAA ATA GTC-3'; -1877 IRFE: forward, 5'-TTA AGA TGT TTG AAA ATT GAA AGT GTG ATT T-3'; reverse, 5'-CCG TAA ATC ACA CTT TCA ATT TTC AAA CAT-3'; -1803 IRFE: forward, 5'-TGG TTG CTG GCC ATA AAG AGA AAT ATC TGT GAT TCG-3'; reverse, 5'-GGC GAA TCA CAG ATA TTT CTC TTT ATG GCC AGC-3'; -1317 IRFE: forward, 5'-TAA TGT ATT TGA GCA GGT TTA ACT TTT AAC TCA G-3'; reverse, 5'-GGT ACT GAG TTA AAA GTT AAA CCT GCT CAA ATA C-3'; and -1284 IRFE: forward, 5'-TCA GTA CCA AAC TTT TTC CTG TTT CTT TTT CAA TCT GG-3'; reverse, 5'-ATA TCC AGA TTG AAA AAG AAA CAG GAA AAA GTT TGG T-3'. Probes (SA, >=108 cpm/µg) were prepared by end-labeling with [32P]ATP and T4 polynucleotide kinase or end-filling annealed primers with [32P]dATP and [32P]dCTP using Klenow.

For initial EMSAs, in vitro translated proteins were synthesized from plasmids containing murine IRF-1 in pRcCVM vector or oIRF-2 using a TNT T7 Quick Coupled Transcription/Translation System (Promega Corp.). Binding reactions were conducted by incubating probes (30,000 cpm/reaction) with in vitro translated proteins as described by Harada et al. (25). Reactions were electrophoresed in 5% acrylamide gels in 0.5 or 1 x TBE buffer (0.09 M Tris, 0.09 M borate, and 2 mM EDTA, pH 8.0). Autoradiography of dried gels was performed using Kodak XAR-5 film (Eastman Kodak Co., Rochester, NY).

Cell culture
The immortalized ovine endometrial LE cell line was developed in our laboratory and described by Johnson et al. (15). Cells were maintained in DMEM (DMEM/F-12) supplemented with 5% FCS (Life Technologies, Inc., Gaithersburg, MD). Recombinant oIFN{tau} (roIFN{tau}) was prepared and assayed for biological (antiviral) activity as described previously (36). The specific activity of roIFN{tau} was 1.8 x 108 AVU/mg protein.

Nuclear extract preparation and EMSAs
Ovine LE cells were treated with roIFN{tau} (104 AVU/ml) for 0, 3, 6, 12, or 24 h. Washed cells were resuspended in HEGD buffer (25 mM HEPES, 1.5 mM EDTA, 1 mM dithiothreitol, and 10% glycerol, pH 7.6), incubated on ice for 15 min, and pelleted at 12,000 x g for 5 min at 4 C. The pellet was resuspended in HED (HEGD buffer without glycerol), incubated for 5 min on ice, and centrifuged as described above. After resuspension in HEGD, cells were disrupted with a Dounce homogenizer (Kontes Co., Vineland, NJ) and centrifuged (16,000 x g, 10 min, 4 C). Nuclear pellets were resuspended in HEGDK (HEGD plus 0.5 mM KCl), incubated for 30 min on ice, and centrifuged (16,000 x g, 15 min, 4 C). The supernatant nuclear extracts were collected and assayed for protein concentration with a Bradford assay (Bio-Rad Laboratories, Inc., Hercules, CA), using BSA as the standard.

Nuclear extracts (9 µg) were incubated in 25 µl of 1 x binding buffer [0.2 M NaCl, 0.1 M HEPES (pH 7.6), 5 mM MgCl2, 0.5 mM EGTA, 0.002% Ficoll, and 2.5 mM dithiothreitol] for 10 min. For competition reactions, a 100-fold molar excess of specific or nonspecific (consensus GC-rich motif that binds Sp1/Sp3 proteins) oligonucleotide was added to binding reactions and incubated for 5 min. Radiolabeled oligonucleotide (40,000 cpm) was then added in the presence of 0.5 µg poly d(A-T) and 0.5 µg salmon sperm DNA and incubated for 15 min at room temperature. For supershift analyses, anti-IRF-1 or anti-IRF-2 antibodies or normal mouse or rabbit IgG (1 µg) was added to binding reactions and incubated for an additional 15 min at room temperature. Binding reactions were analyzed by electrophoresis in native 5% polyacrylamide gels with 1 x TBE buffer. Gels were dried, and radiographic images were captured using a Storm A60 PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA).

Transient transfection assays, luciferase assays, and ß-galactosidase assays
Immortalized ovine LE cells were subcultured into 12-well plates (70–80% confluent) and transiently cotransfected (n = 4 wells/construct and treatment) with the indicated LUC reporter construct (0.5 µg/well) and pEF1-Myc/His-lacZ (0.05 µg/well) using the GenePORTER transfection reagent (Gene Therapy Systems, San Diego, CA) according to the manufacturer’s recommendations. The transfected cells were placed in DMEM/F-12 medium with 5% FCS and then treated with roIFN{tau} or left untreated (control). Cell lysates were prepared in Cell Culture Lysis buffer (Promega Corp.). Luciferase and ß-galactosidase expression assays were performed according to the manufacturer’s instructions with a Luciferase Assay System (Promega Corp.) and a Galacto-Light Plus Kit (Tropix, Bedford, MA), respectively, and measured with a luminometer. Each transfection experiment contained four replicates and was repeated in at least four independent experiments. To compare data between experiments, luciferase data were normalized with the ß-galactosidase data. The ß-galactosidase values corrected for differences in transfection efficiency between wells and plates within an individual transfection experiment. Normalized luciferase data were then used to calculate the effect of roIFN{tau} treatment or, in some cases, the effect of IRF-1 or IRF-2 overexpression.

Western blot analyses
Monolayer cultures of immortalized ovine LE cells were grown in culture medium to 80% confluence on 100-mm diameter dishes. Cells were then left untreated as a control or were treated with roIFN{tau} (104 AVU/ml) for 0, 0.5, 1, 6, 12, 24, or 48 h in each of three independent experiments. Total cellular protein was harvested for Western blot analyses as described previously (17) and assayed for protein concentration as above. Whole cell extracts (20 µg/sample) were separated by 10% SDS-PAGE, transferred to nitrocellulose, and probed with rabbit antihuman IRF-2 polyclonal antibodies (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) or normal rabbit IgG (Sigma, St. Louis, MO) according to the manufacturer’s recommendations using methods described previously (17). Immunoreactive proteins were detected by chemiluminescence (SuperSignal West Pico, Pierce Chemical Co., Rockford, IL) according to the manufacturer’s recommendations using X-OMAT AR x-ray film (Kodak). Multiple exposures of each Western blot were performed to ensure linearity of chemiluminescent signals.

Statistical analyses
The effects of dose of roIFN{tau} and time of IFN{tau} treatment on activity of the full-length oER{alpha} promoter luciferase reporter construct were analyzed by least squares ANOVAs using the general linear models procedures of the Statistical Analysis System (SAS Institute, Inc., Cary, NC). If an effect of IFN{tau} was detected, least squares regression analyses were conducted using the ß-galactosidase data as a covariate to correct for differences in transfection efficiency. The relative effects of roIFN{tau} on oIRF-1 and oIRF-2 overexpression on activity of oER{alpha} promoter-reporter constructs were analyzed using a one-way ANOVA. P <= 0.05 was considered statistically significant. Data are reported as either the least squares mean relative light units with SE or the mean percent suppression with SE.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Cloning and sequence analysis of the 5'-flanking promoter/enhancer region of the oER{alpha} gene
Southern blot analysis of sheep genomic DNA using a cDNA probe derived from exon 1 of the human ER{alpha} gene detected single hybridizing bands of 6 and 3 kb in EcoRI-digested ovine and human genomic DNA, respectively (data not shown). These results are consistent with a single copy of the ER{alpha} gene being present in both human and sheep genomes. An exon 1-specific fragment of the human ER{alpha} cDNA was used to screen an EMBL3 sheep genomic DNA library under high stringency conditions. Several positive plaques were purified to homogeneity and analyzed by restriction mapping and Southern blot analyses. A 4.7-kb BamHI-SalI fragment was subcloned, and initial sequencing with gene-specific primers indicated that the clone contained exon 1 and approximately 2.8 kb of genomic sequence upstream of the translational start site (+1), as predicted from the human and pig ER{alpha} genes. The 2.7 kb of 5'-flanking promoter/enhancer region upstream of the translational start site were sequenced in both directions (GenBank AF159145). Sequence alignments indicated that this region of the oER{alpha} gene displayed high overall sequence similarity (>85%) to the 5'-flanking region of the ER{alpha} gene of the pig (AF034972), human (AF082876, X068051, X63118, X62462, X74439), mouse (M38652), horse (AF124093), and rat (X98236).

Computer-assisted prediction of promoter regions in the 5'-flanking region of the oER{alpha} gene was performed using the PromoterInspector Web-based program (Genomatix Software GmbH, Munich, Germany). A single promoter region was found beginning at position 2409. Two coding regions were predicted in the 5'-flanking promoter/enhancer region of the oER{alpha} gene using the Open Reading Frame Finder tool available through the National Center for Biotechnology Information (Bethesda, MD). The first predicted coding region began with a methionine at position 116 and ended with a stop codon at position 235. The predicted protein sequence contained no significant homology to any known protein using the NCBI Basic Alignment and Search Tool. The second predicted coding region began at position 2678 and continued to the end of the clone. The predicted protein sequence is highly homologous to the N-terminus of ER{alpha} from a number of species, including sheep, pig, human, mouse, horse, and rat.

Computer-assisted transcription factor-binding site analyses were conducted with the Genomatix MatInspector Professional Web-based program (37). As summarized in Table 1Go, the 2.7-kb oER{alpha} promoter contained one putative ISRE (position -1284) and four putative IRFEs (positions -2110, -1877, -1803, and -1317) relative to the translational start site (+1) with high sequence similarity to the consensus elements found in other genes. In addition, three putative estrogen response elements (EREs) were found at positions -1061, -837, and -9. The putative -9 ERE contained two inverted repeats (GCCGCTCACCATGACCATG) with high similarity to a consensus ERE (AGGTCANNNTGACCT), whereas the -1061 and -837 sites were half-sites (TGACC) positioned in front of putative activator protein-1 (AP-1) sites. A typical TATA sequence (-1187) and a potential CAAT box sequence (-1236 and -1293) were also present.


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  		Table 1. Comparison of ISRE and IRFEs in the oER{alpha} gene with consensus IRFE, ICS, and ISRE found in other genes responsive to type I IFNs

 
EMSAs
To determine whether the putative IRFEs identified in the oER{alpha} promoter were functional, we examined the ability of each IRFE to bind mouse IRF-1 or ovine IRF-2 in vitro transcribed-translated proteins in EMSAs (Fig. 1Go). Specific binding of IRF-1 was detected on radiolabeled oligonucleotides containing a consensus IRFE, the oER{alpha} IRFEs at positions -2110 and -1877, and the oER{alpha} ISRE at -1284 (Fig. 1AGo), whereas IRF-2 bound only the -1877 and -1284 oER{alpha} elements and the control consensus oligonucleotide (Fig. 1BGo). The binding was specific, because it was effectively competed by a 100-fold molar excess of cold oligonucleotide, but not by a nonspecific oligonucleotide containing an Sp1/Sp3 response element.



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  		Figure 1. Characterization of putative IRFEs in the ovine ER{alpha} promoter using in vitro EMSAs. A, Oligonucleotides containing a consensus IRFE or the predicted oER{alpha} IRFE were radiolabeled and incubated with in vitro transcribed-translated IRF-1 as described in Materials and Methods. The arrows (*) indicate mIRF-1-DNA complexes. The other arrow indicates free probe (FP). B, Oligonucleotides containing a consensus IRFE or the predicted oER{alpha} IRFE were radiolabeled and incubated with in vitro transcribed-translated IRF-2. The arrows indicate oIRF-2-DNA complex (*) or free probe (FP).

 
Nuclear extracts were then assayed in an EMSA with -2110 IRFE (Fig. 2Go, A and B), -1877 IRFE (Fig. 2Go, C and D), or -1284 ISRE (Fig. 2Go, E and F) oligonucleotide probes. Three specific protein-DNA complexes were formed with either the -2110 or -1877 IRFE probes, and five complexes were formed with the -1284 probe. The complexes were competed away by a 100-fold molar excess of nonradioactive ISRE or IRFE DNA, but not by a 100-fold molar excess of an oligonucleotide containing a consensus GC-rich motif that binds Sp1/Sp3 proteins. One complex on the -1877 and -1284 probes supershifted in reactions containing an rabbit anti-IRF-2 antibody (Fig. 2Go, C and E), but not in reactions containing nonspecific rabbit IgG. Another complex on the -2110, -1877, and -1284 probes was found to contain IRF-1 by supershift analyses using rabbit anti-IRF-1 antibody (Fig. 2Go, B, D, and F). The IRF-1-containing complex increased in relative abundance between 0 and 3 h and declined thereafter, similar to the reported pattern of IRF-1 protein expression in IFN{tau}-treated ovine LE cells (15, 17). In contrast, the IRF-2-containing complex did not change in abundance between 0 and 24 h post-IFN{tau} treatment. The mobility of the remaining complexes was unaffected by antibodies against either IRF-1 or IRF-2, and their relative abundance was unchanged between 0 and 24 h of IFN{tau} treatment. Similar to results from EMSAs using IRF-1 and IRF-2 proteins, no specific binding of IRF-1 and IRF-2 in nuclear extracts was detected using the -1803 and -1317 oligonucleotide probes (data not shown).



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  		Figure 2. Characterization of IRFEs in the oER{alpha} promoter using EMSAs and supershift identification of IRF-1 and IRF-2 binding. Nuclear extracts were prepared from ovine endometrial LE cells treated with roIFN{tau} (104 AVU/ml) for 0, 1, 3, 6, 12, or 24 h. Oligonucleotides containing the oER{alpha} IRFEs at position -2110 (A and B), -1877 (C and D), or -1284 (E and F) were radiolabeled and incubated with nuclear extracts as described in Materials and Methods. Rabbit antibodies to human IRF-2 (A, C, and E) and human IRF-1 (B, D, and F) were used to identify the nature of specific protein-DNA complexes. FP, Free probe; SS, supershift.

 
Effects of IFN{tau} on IRF-2 protein expression in immortalized ovine LE cells
To confirm the effects of IFN{tau} on IRF-2 protein expression in ovine LE cells, Western blot analyses were conducted. An immunoreactive protein of approximately 55 kDa was detected in extracts of ovine LE cells (Fig. 3Go). No cross-reactive bands were detected on replicate Western blots of LE cells extracts probed with normal rabbit IgG (data not shown). Consistent with EMSA results, immunoreactive IRF-2 protein expression in LE cells was unaffected by roIFN{tau} treatment.



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  		Figure 3. Effects of IFN{tau} on IRF-2 protein expression in ovine endometrial LE cells. Ovine endometrial LE cells were treated with roIFN{tau} (104 AVU/ml) for 0, 0.5, 1, 6, 12, 24, or 48 h. Cell lysate protein was separated by SDS-PAGE and analyzed by Western blotting using a rabbit antihuman IRF-2 antibody. The amount of IRF-2 protein did not change in response to IFN{tau} treatment.

 
Effects of IFN{tau} on transcriptional activity of the oER{alpha} promoter
To determine the effects of oIFN{tau} on transcriptional activity, immortalized, IFN{tau}-responsive ovine endometrial LE cells were transiently transfected with a construct containing the entire 2.7-kb oER{alpha} promoter upstream of a luciferase (LUC) reporter gene and treated for 48 h with increasing doses of roIFN{tau} (102–106 AVU/ml) selected to mimic the very high amounts of IFN{tau} (~2 x 107 AVU/conceptus) present in the ovine uterus between days 13 and 17 of pregnancy. As illustrated in Fig. 4AGo, IFN{tau} inhibited basal activity of promoter in a dose-dependent fashion (P < 0.01, quadratic effect of dose). IFN{tau} did not affect basal activity of the pGL3 luciferase vector (data not shown).



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  		Figure 4. IFN{tau} inhibits transcriptional activity of the oER{alpha} promoter. A, Dose response. Immortalized ovine endometrial LE cells were transiently cotransfected with 2.7 kb oER{alpha}-LUC and pEF1{alpha}-lacZ-Myc/His, left untreated or treated with increasing amounts of roIFN{tau} (102–106 AVU/ml), and assayed for luciferase and ß-galactosidase expression as described in Materials and Methods. B, Time course. Ovine endometrial LE cells were transiently cotransfected with 2.7 kb oER{alpha}-LUC and pEF1{alpha}-lacZ-Myc/His, treated with roIFN{tau} (105 AVU/ml) or left untreated as a control for the indicated time, and assayed for luciferase and ß-galactosidase expression as described in Materials and Methods. Luciferase expression is presented as the percent inhibition from four replicates of the experiment.

 
Ovine LE cells were then transfected with the 2.7-kb oER{alpha}-LUC construct, treated with roIFN{tau} (104 AVU/ml), or left untreated as a control and harvested at 0, 6, 12, 24, or 48 h posttreatment (Fig. 4BGo). The basal activity of the 2.7-kb oER{alpha}-LUC construct increased 3.7-fold between 0 and 48 h in untreated control LE cells (P < 0.01, quadratic effect of time). IFN{tau} inhibition of oER{alpha} promoter activity was evident by 6 h and continued to 48 h after IFN{tau} treatment compared with that in the untreated control (P < 0.01, treatment x time interaction). Activity of the pGL3 vector was not affected by IFN{tau} at any time point (data not shown).

Effects of IFN{tau} on sequential 5' deletion mutants of the oER{alpha} promoter
A series of 5' oER{alpha} promoter deletions was generated using convenient restriction enzyme sites (Fig. 5Go). Ovine LE cells were transiently transfected with the 2.7-kb oER{alpha}-LUC or each 5'-deletion construct and treated for 24 h with roIFN{tau} (104 AVU/ml). Transcriptional activity of the oER{alpha} promoter varied in untreated controls as the promoter was sequentially truncated, but IFN{tau} decreased expression from all except the shortest promoter construct. IFN{tau} inhibited (P < 0.0001) the activity of the full-length 2.7-kb oER{alpha}-LUC construct. Deletion to -2163 or -1620 increased promoter activity, but this activity was still inhibited by roIFN{tau} (P = 0.003 and P = 0.006, respectively). Further 5'-truncation in the -958 deletion mutant decreased the activity of the remaining oER{alpha} promoter, although the level of activity was much greater than that for the pGL3 vector. Nevertheless, IFN{tau} inhibited (P = 0.008) the activity of this promoter deletion mutant. The -735 mutant had higher overall activity and was inhibited (P < 0.05) by IFN{tau}. The -268 deletion had high basal activity that was not affected (P = 0.08) by IFN{tau}. The activity of the pGL3 backbone vector was not affected (P = 0.39) by IFN{tau} treatment.



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  		Figure 5. Effects of IFN{tau} on transcriptional activity of 5'-promoter deletion oER{alpha}-LUC constructs. A series of 5'-oER{alpha} promoter deletions was generated using convenient restriction enzyme digestion sites as described in Materials and Methods. Immortalized ovine LE cells were transiently cotransfected with the indicated oER{alpha}-LUC constructs and pEF1{alpha}-lacZ-Myc/His and treated with roIFN{tau} (104 AVU/ml) or left untreated as a control for 24 h, and luciferase and ß-galactosidase expressions were measured as described in Materials and Methods. Luciferase expression is presented as the least squares mean ± SE relative light units (RLU). The percent inhibition by IFN{tau} treatment for each oER{alpha}-LUC construct is shown based on four replicate experiments. All constructs maintained basal levels of LUC activity above the pGL3 vector backbone. Constructs containing IRFEs and/or ISREs (depicted at the top) were inhibited by IFN{tau}. The positions of the identified IRFEs and ISRE are indicated. The asterisk denotes a significant effect of IFN{tau} treatment (P < 0.05).

 
Effects of IFN{tau} on heterologous fusions of the oER{alpha} gene and TK promoter
To determine whether oER{alpha} promoter fragments containing an ISRE and/or IRFEs were able to confer IFN{tau} regulation of a heterologous promoter, selected regions of the oER{alpha} promoter were cloned upstream of the TK promoter in a luciferase reporter construct (TK-LUC; Fig. 6Go). Ovine LE cells were transiently transfected with these oER{alpha}-TK-LUC constructs or the TK-LUC backbone vector. Transfected cells were left untreated (control) or were treated with roIFN{tau} (104 AVU/ml) and harvested at 24 h posttreatment.



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  		Figure 6. IFN{tau} suppresses transcriptional activity of oER{alpha} TK (tk) promoter constructs. The indicated regions of the oER{alpha} promoter were cloned upstream of the heterologous TK promoter (TK-LUC) as described in Materials and Methods. Immortalized ovine LE cells were transiently cotransfected with the indicated ER{alpha} promoter constructs, 3xGAS-TK-LUC or TK-LUC and pEF1-Myc/His-lacZ. Transfected cells were treated with roIFN{tau} (104 AVU/ml) or left untreated as a control. Cells were harvested at 24 h posttreatment, and luciferase and ß-galactosidase expressions were measured as described in Materials and Methods. The asterisk denotes a significant effect of IFN{tau} treatment (P < 0.05).

 
IFN{tau} inhibited the activity of all oER{alpha} promoter TK-LUC constructs containing an IRFE or an ISRE (-1877, -1284) that bound IRF-2 in EMSAs using nuclear extracts. The activities of the -2677 to -2163 oER{alpha}-TK-LUC and -958 to -736 oER{alpha} promoter TK-LUC construct were also inhibited by IFN{tau}. IFN{tau} treatment did not affect the activity of the -732 to -266 oER{alpha}-TK-LUC or the basal activity of the TK-LUC backbone vector construct. The inhibitory actions of IFN{tau} on promoter-reporter constructs are specific, because the activity of a 5xISRE-TK-LUC construct was increased 3.1-fold by IFN{tau} in ovine LE cells (data not shown), as reported previously (17).

Effects of oIRF-1 and oIRF-2 overexpression on heterologous fusions of the oER{alpha} gene and TK promoter
Although IRF-2 gene expression increases in the endometrial LE during early pregnancy (14), the results of the present study indicated that IFN{tau} does not increase IRF-2 expression in the immortalized ovine LE cells. Therefore, the ovine LE cells were transiently cotransfected with overexpression vectors for oIRF-1 or oIRF-2 and TK-LUC (Fig. 7Go). Transfected cells were left untreated and harvested at 24 h posttransfection.



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  		Figure 7. oIRF-2, but not IRF-1, suppresses transcriptional activity of oER{alpha} TK (tk) promoter constructs. The indicated regions of the oER{alpha} promoter were cloned in front of the heterologous TK promoter (TK-LUC) as described in Materials and Methods. Immortalized ovine LE cells were transiently cotransfected with the indicated reporter constructs, pEF1-oIRF-1 and/or pEF1-oIRF-2, and pEF1-Myc/His-lacZ. Transfected cells were treated with roIFN{tau} (104 AVU/ml) or left untreated as a control. Cells were harvested at 24 h posttreatment, and luciferase and ß-galactosidase expressions were measured as described in Materials and Methods. The asterisk denotes a significant effect of IRF-2 (P < 0.05).

 
Overexpression of oIRF-1 did not affect the expression any of the constructs. In contrast, IRF-2 overexpression decreased the activity (P < 0.05) of several oER{alpha}-TK-LUC constructs containing -1877 IRFE or -1284 ISRE. IRF-2 also decreased (P < 0.05) the activity of the -732 to -266 construct that lacks an IRF-binding element. The activities of the other oER{alpha}-TK-LUC, TK-LUC, and pEF1-Myc/His-lacZ constructs were not affected by IRF-2 overexpression.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
IFN{tau} suppresses transcription of the ER{alpha} and OTR genes in the endometrial epithelium in vivo (13, 24). Available evidence supports the working hypothesis that IFN{tau} directly suppresses transcription of the ER{alpha} gene, thereby precluding the up-regulation of OTR gene transcription (10, 11, 12, 13). This working hypothesis is supported by findings of the present study that IFN{tau} inhibits the transcriptional activity of the 2.7-kb oER{alpha} promoter transfected into an ovine endometrial LE cell line. Moreover, the results of the present study suggest that the inhibitory effects of IFN{tau} are mediated by several regions of the oER{alpha} promoter/enhancer DNA. In vivo studies found that IRF-1 protein was up-regulated before up-regulation of IRF-2 protein in pregnant ewes and in cyclic ewes receiving intrauterine injections of roIFN{tau} (24). In other model systems, both IRF-1 and IRF-2 were demonstrated to bind ISREs and IRFEs present in the promoter of IFN-regulated genes (29, 30, 38, 39). In the present study IRF-1 and IRF-2 binding could be detected on -1877 IRFE and -1284 ISRE in the oER{alpha} gene promoter. This finding is consistent with reports that IRFs can bind to either IRF-Es or ISREs; however, ISGF3 can only bind to ISREs and not IRFEs (38, 39). Interestingly, the -2110 IRFE bound IRF-1, but not IRF-2. The IRF family of transcription factors is comprised of at least 10 members that share sequence homology in the N-terminal DNA-binding region. The inferred amino acid sequence of oIRF-2 is almost identical to that of the human and mouse proteins. Therefore, the weak half-site structure of -2110 IRFE compared with that of -1877 IRFE or consensus IRFEs may account for the differential binding of IRF-1 and IRF-2.

Although IRF-2 expression in the endometrial epithelium is up-regulated during the pregnancy recognition period in sheep, Western blot analyses and EMSAs found no increase in IRF-2 protein expression in immortalized ovine endometrial LE cells in the present study. In spontaneously immortalized bovine endometrial cells, Perry et al. (16) also observed that IRF-2 protein expression was not affected by IFN{tau}. The inability of IFN{tau} to increase IRF-2 expression in ovine LE cells is not clear, but could be due to the immortalization procedure. Therefore, we overexpressed IRF-2 in transient transfection assays to mimic the up-regulation of IRF-2 expression observed in the endometrial epithelium during early pregnancy in ewes. The results clearly demonstrated that IRF-2 overexpression inhibited the activity of regions of the oER{alpha} promoter containing a functional IRF-2-binding element. However, the activity of the -958/-736 oER{alpha} promoter construct was also inhibited by IRF-2. These results support the idea that the increase in endometrial epithelial IRF-2 expression during early pregnancy is responsible in part for suppression of increases in ER{alpha} gene transcription. Recently, Senger et al. (33) demonstrated that IRF-2 can compete with IRF-1 for DNA binding to an IRFE and that IRF-1 and IRF-2 formed a heterocomplex in which IRF-2 decreased IRF-1’s transcriptional activity. Further, experiments strongly suggested that IRF-2 represses transcription by inhibiting recruitment of coactivators into enhanceosomes by transcription factors, such as cAMP response element binding-binding protein or p65 (33). Interestingly, the region of the oER{alpha} promoter from -958 to -736 contains several transcription factor-binding elements, such as cAMP response element binding-binding protein and nuclear factor-{kappa}B, which are demonstrated targets for IRF-2 inhibition of coactivator binding (33). The effects of IRF-2 to inhibit transcription of the oER{alpha} gene may be more readily observed on promoter DNA in a chromatin-dependent transcription assay compared with the transient transfection assay used in the present study.

The factors and pathways mediating IFN{tau} inhibition of oER{alpha} promoter regions that lack detectable IRF binding remain to be determined. Computer-assisted transcription factor binding site analyses predict a multitude of different binding elements. Recent studies indicate that STAT1 dimers can mediate both activation and suppression of different genes by type I and II IFNs in a promoter context-dependent manner (40, 41, 42). For example, IFNß suppression of c-myc gene transcription is mediated by activated STAT1 dimer and a GAS element in the promoter/enhancer region (40). Our transcription factor-binding site analyses also predict GAS elements at positions -2644, -2556, -2143, -1860, -1418, and -394 in the oER{alpha} promoter/enhancer DNA with high identity to the consensus sequence (TTNCNNNAA). Although necessary, the GAS element is not sufficient to mediate IFN{gamma} or IFNß suppression of c-myc, because it lacks intrinsic repressor activity. Therefore, STAT1 probably recruits a corepressor or interacts with a repressor bound to another site in the promoter to inhibit expression (40, 41, 42). In the present study IFN{tau} inhibited the activity of a number of different reporter constructs containing regions of the oER{alpha} gene with putative GAS elements. Indeed, the IFN{tau} activates STAT1 in ovine endometrial LE cells and also suppresses the activity of a reporter construct containing three copies of the rat IRF-1 GAS element fused to the TK promoter (17). Future studies will be directed toward defining the functionality of putative GAS elements in the oER{alpha} promoter and determining their role, if any, in IFN{tau} inhibition of oER{alpha} promoter activity.

In summary, this study represents the first report of the structure of the promoter/enhancer region of the ovine ER{alpha} gene. Transfection assays established that IFN{tau} inhibited the activity of the oER{alpha} promoter transfected into an immortalized ovine endometrial LE cell line. Structure-function studies indicated that an IRFE at -1877 and an ISRE at -1284 were sufficient to confer IRF-2 inhibition of oER{alpha} activity. Our findings indicate that IFN{tau} inhibition of oER{alpha} gene transcription may involve additional mechanisms distinct from those involved in gene repression by IRF-2.


   Acknowledgments
 
The authors appreciate the gift of plasmids for murine IRF-1 and IRF-2 from Drs. Richard Ivell and Ross Bathgate (Institute for Hormone and Fertility Research, University of Hamburg, Hamburg, Germany).


   Footnotes
 
1 This work was supported by NIH Grant HD-32534 (to F.W.B. and T.E.S.) and in part by NIH Grant P30-ES-09106. Back

2 J.-A.G.W.F. and Y.C. contributed equally and should be considered co-first authors. Back

Received January 11, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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