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
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ABSTRACT
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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
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.
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INTRODUCTION
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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
(hER
). 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.
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RESULTS
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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. 1
). 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. 1
) 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. 1
). Sequence analysis indicated a potential ERE in
the downstream region between +174 and +237; therefore, we focused our
attention on this region (+197/+237).

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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 , 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.
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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. 2
). Deletion of the
AP1 site did not abolish estrogen-dependent transactivation (Fig. 2
)
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. 2
). 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. 2
). The everted ERE
(region E) did not contribute to estrogen inducibility. The construct
encompassing the imperfect ERE and the DR13 (constructs AD) was the
functional, minimal ERU. A single copy of this element exhibited an
approximately 6-fold increase in expression in response to moxestrol
(Fig. 2
). Analysis of deletions and/or mutations showed that all four
of the ERE half-sites (AD) 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. 2
). 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. 2
, 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.

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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.
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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
expression plasmid (Fig. 3
). 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. 3
).

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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. 2 , 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.
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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. 13

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. 4
). A smaller construct
containing -308/+237 also shows strong estrogen inducibility (Fig. 4
).
These data show that a single downstream ERU is sufficient for estrogen
inducibility.

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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.
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The ERU Interacts with Two ER Complexes in EMSA
Transfection data (Fig. 2
) 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. 2
) 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
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. 5A
, 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
, 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. 5A
, lanes 47, lower arrow) and a second,
slower-migrating up-shifted band progressively increased in intensity
(Fig. 5A
, lanes 47, middle arrow). Thus, at higher ER
concentrations, the ERU is presumably occupied by a second ER dimer.
Although we used purified recombinant hER
, 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. 5A
, lanes 811). 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. 5A
, lane 12). The two ER-ERU complexes were both
supershifted by the FLAG antibody (Fig. 5A
, 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. 5B
, lanes 35). 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. 5B
, 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. 5B
, lanes 47, with Fig. 5A
, lanes 27 and
Fig. 5B
, 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.

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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 . 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 to the cERE probe
showing a major up-shifted band (bottom arrow). In lanes
47, increasing amounts of purified hER (0.5, 1.5, 2.5, and 10 ng
of ER, respectively) were incubated with the PI-9 ERU probe. In lanes
811, 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 -cERE and
FLAG-hER -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 37 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 homodimer-cERE complex.
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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
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. 6
). 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. 6
).
Interestingly, the ER did not protect the entire 13-nucleotide spacer
region between the two half-sites in DR13 (Fig. 6
, 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.

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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 . 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.
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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. 7C
), 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. 7B
) 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. 7B
).
Additionally, primers that anneal approximately 1.5 kb from the ERU did
not amplify any product (Fig. 7
, 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. 7C
).

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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.
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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. 8A
). 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. 8A
, +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. 8B
, +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.

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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. 1 . 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. 2 , A, B, C, and D) was evaluated. The data represent the mean ±
SEM for three separate experiments.
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DISCUSSION
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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. 7
).
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. 3
) 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. 1
). 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. 2
) 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 260263 of hER
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. 5B
). The
absence of a faster-migrating monomer band (Fig. 5B
, 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. 5
, A and B). A diffuse, slower-migrating third band is
also visible in EMSAs with the ERU probe (Fig. 5A
, lanes 6 and 7).
Because this band appears at concentrations of ER 510 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. 6
, 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
|
---|
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 manufacturers
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 manufacturers 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
was
overexpressed and purified from transiently transfected CHO-S cells
(Life Technologies, Inc.). FLAG-hER
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 2001,000 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
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 manufacturers 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
, human ER
; PI-9, proteinase
inhibitor 9; SV40, simian virus 40.
Received for publication January 3, 2001.
Accepted for publication July 13, 2001.
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