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Promoter and Functional Regulation by Ovine Interferon-
1
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
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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(IFN), the ruminant pregnancy recognition signal,
inhibits transcription of the estrogen receptor 
(ER) gene in the
endometrial lumenal epithelium of the sheep uterus, thereby abrogating
production of luteolytic PGF2 pulses. The effects of
IFN are mediated in part by IFN-stimulated response elements (ISREs)
and IFN regulatory factor elements (IRFEs). The promoter/enhancer
region of the ovine ER 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 inhibited transcriptional activity of the 2.7-kb ovine ER
promoter in transfection assays using ovine lumenal epithelium cells.
Analyses of sequential 5'-deletion mutants of the ovine ER promoter
indicated that the effects of IFN 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 promoter containing an IRFE or an ISRE as well as some, but not
all, regions lacking these elements. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(ER) gene expression between days 11 and 13 (4, 5).
The increase in ER 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 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-
(IFN), 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 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 and OTR gene expression in the
LE and sGE (8, 10, 11, 12, 13). Intrauterine injections of
recombinant ovine IFN into cyclic ewes were sufficient to prevent
estrogen-induced increases in epithelial ER and OTR expression
(10, 12, 13). Available evidence in the ovine model
supports the hypothesis that IFN inhibits transcription of the ER
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 from the endometrium (13).
The cellular and molecular mechanisms mediating the inhibitory effects
of IFN 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
-activated factor that binds to 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/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 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 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 and OTR was detected in the contralateral uterine
horn receiving control proteins (14). Our working
hypothesis is that IFN inhibits expression of the ovine ER 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 on ovine ER (oER) gene transcription
by 1) cloning, sequencing, and analyzing the 5'-flanking
promoter/enhancer region of the oER gene; 2) identifying functional
IRF-Es and ISREs by electrophoretic mobility shift analyses (EMSAs);
and 3) determining the effects of oIFN, oIRF-1, and oIRF-2 on oER
promoter activity using transient transfection analyses of an
immortalized ovine endometrial LE cell line.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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gene 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 
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 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-luciferase (LUC) constructs
A HindIII site that ablated the ATG of the oER
gene was introduced by cloning a NotI-HindIII
adaptor oligonucleotide at the NotI site closest to the ATG
in the oER gene. This adapter maintained the oER 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-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-TK-LUC and -1492/-958-oER-TK-LUC
constructs were created by digesting the oER promoter with
EcoRI and subcloning the fragments into pBluescript. The
oER 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-TK-LUC construct was prepared by
digesting oER 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 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
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 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 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 (roIFN) was prepared and
assayed for biological (antiviral) activity as described previously
(36). The specific activity of roIFN was 1.8 x
108 AVU/mg protein.
Nuclear extract preparation and EMSAs
Ovine LE cells were treated with roIFN
(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 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 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
(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 and time of IFN treatment on
activity of the full-length oER 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 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 on oIRF-1 and
oIRF-2 overexpression on activity of oER 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.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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gene 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 gene being present in both
human and sheep genomes. An exon 1-specific fragment of the human ER
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 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 gene displayed high overall
sequence similarity (>85%) to the 5'-flanking region of the ER
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 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 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 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 1
, the 2.7-kb oER 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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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. 1). Specific
binding of IRF-1 was detected on radiolabeled oligonucleotides
containing a consensus IRFE, the oER IRFEs at positions -2110 and
-1877, and the oER ISRE at -1284 (Fig. 1A), whereas IRF-2 bound
only the -1877 and -1284 oER elements and the control consensus
oligonucleotide (Fig. 1B). 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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, A and B), -1877 IRFE (Fig. 2, C and
D), or -1284 ISRE (Fig. 2, 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. 2, 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. 2, 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-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 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 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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on IRF-2 protein expression in immortalized ovine
LE cells 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. 3). 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 treatment.
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on transcriptional activity of the oER
promoter on transcriptional activity,
immortalized, IFN-responsive ovine endometrial LE cells were
transiently transfected with a construct containing the entire 2.7-kb
oER promoter upstream of a luciferase (LUC) reporter gene and
treated for 48 h with increasing doses of roIFN
(102–106 AVU/ml) selected
to mimic the very high amounts of IFN (
2 x
107 AVU/conceptus) present in the ovine uterus
between days 13 and 17 of pregnancy. As illustrated in Fig. 4A, IFN inhibited basal activity of
promoter in a dose-dependent fashion (P < 0.01,
quadratic effect of dose). IFN did not affect basal activity of the
pGL3 luciferase vector (data not shown).
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-LUC
construct, treated with roIFN (104 AVU/ml), or
left untreated as a control and harvested at 0, 6, 12, 24, or 48 h
posttreatment (Fig. 4B
). The basal activity of the 2.7-kb oER-LUC
construct increased 3.7-fold between 0 and 48 h in untreated
control LE cells (P < 0.01, quadratic effect of time).
IFN inhibition of oER promoter activity was evident by 6 h
and continued to 48 h after IFN 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 at any time point (data not shown).
Effects of IFN on sequential 5' deletion mutants of the oER
promoter
A series of 5' oER promoter deletions was generated using
convenient restriction enzyme sites (Fig. 5). Ovine LE cells were transiently
transfected with the 2.7-kb oER-LUC or each 5'-deletion construct
and treated for 24 h with roIFN (104
AVU/ml). Transcriptional activity of the oER promoter varied in
untreated controls as the promoter was sequentially truncated, but
IFN decreased expression from all except the shortest promoter
construct. IFN inhibited (P < 0.0001) the activity
of the full-length 2.7-kb oER-LUC construct. Deletion to -2163 or
-1620 increased promoter activity, but this activity was still
inhibited by roIFN (P = 0.003 and P
= 0.006, respectively). Further 5'-truncation in the -958 deletion
mutant decreased the activity of the remaining oER promoter,
although the level of activity was much greater than that for the pGL3
vector. Nevertheless, IFN 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. The -268 deletion had high basal activity that was not
affected (P = 0.08) by IFN. The activity of the pGL3
backbone vector was not affected (P = 0.39) by IFN
treatment.
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on heterologous fusions of the oER gene and TK
promoter promoter fragments containing an ISRE
and/or IRFEs were able to confer IFN regulation of a heterologous
promoter, selected regions of the oER promoter were cloned upstream
of the TK promoter in a luciferase reporter construct (TK-LUC; Fig. 6). Ovine LE cells were transiently
transfected with these oER-TK-LUC constructs or the TK-LUC backbone
vector. Transfected cells were left untreated (control) or were treated
with roIFN (104 AVU/ml) and harvested at
24 h posttreatment.
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inhibited the activity of all oER 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-TK-LUC and -958 to -736 oER promoter TK-LUC construct were
also inhibited by IFN. IFN treatment did not affect the activity
of the -732 to -266 oER-TK-LUC or the basal activity of the TK-LUC
backbone vector construct. The inhibitory actions of IFN on
promoter-reporter constructs are specific, because the activity of a
5xISRE-TK-LUC construct was increased 3.1-fold by IFN 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 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 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. 7).
Transfected cells were left untreated and harvested at 24 h
posttransfection.
|
-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-TK-LUC, TK-LUC, and pEF1-Myc/His-lacZ
constructs were not affected by IRF-2 overexpression. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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suppresses transcription of the ER and OTR genes in the
endometrial epithelium in vivo (13, 24).
Available evidence supports the working hypothesis that IFN directly
suppresses transcription of the ER 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 inhibits the transcriptional activity of the 2.7-kb oER
promoter transfected into an ovine endometrial LE cell line. Moreover,
the results of the present study suggest that the inhibitory effects of
IFN are mediated by several regions of the oER 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
(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 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. The inability of IFN 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 promoter
containing a functional IRF-2-binding element. However, the activity of
the -958/-736 oER 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 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 promoter from -958 to -736
contains several transcription factor-binding elements, such as cAMP
response element binding-binding protein and nuclear factor-
B,
which are demonstrated targets for IRF-2 inhibition of coactivator
binding (33). The effects of IRF-2 to inhibit
transcription of the oER 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 inhibition of oER 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 promoter/enhancer DNA with high
identity to the consensus sequence (TTNCNNNAA). Although necessary, the
GAS element is not sufficient to mediate IFN 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 inhibited the
activity of a number of different reporter constructs containing
regions of the oER gene with putative GAS elements. Indeed, the
IFN 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 promoter and
determining their role, if any, in IFN inhibition of oER promoter
activity.
In summary, this study represents the first report of the structure of
the promoter/enhancer region of the ovine ER gene. Transfection
assays established that IFN inhibited the activity of the oER
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
activity. Our findings indicate that IFN inhibition of oER gene
transcription may involve additional mechanisms distinct from those
involved in gene repression by IRF-2.
|
Acknowledgments |
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|
Footnotes |
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2 J.-A.G.W.F. and Y.C. contributed equally and should be considered
co-first authors.
Received January 11, 2001.
|
References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
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associated with antigen processing 1 gene expression. J Biol Chem 273:16177–16183This article has been cited by other articles:
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