Sp1 Binding Sites and An Estrogen Response Element Half-Site Are Involved in Regulation of the Human Progesterone Receptor A Promoter
Larry N. Petz and
Ann M. Nardulli
Department of Molecular and Integrative Physiology University
of Illinois Urbana, Illinois 61801
 |
ABSTRACT
|
---|
Progesterone receptor gene expression is
induced by estrogen in MCF-7 human breast cancer cells. Although it is
generally thought that estrogen responsiveness is mediated through
estrogen response elements (EREs), the progesterone receptor gene lacks
an identifiable ERE. The progesterone receptor A promoter does,
however, contain a half-ERE/Sp1 binding site comprised of an ERE
half-site upstream of two Sp1 binding sites. We have used in
vivo deoxyribonuclease I (DNase I) footprinting to demonstrate
that the half-ERE/Sp1 binding site is more protected when MCF-7 cells
are treated with estrogen than when cells are not exposed to hormone,
suggesting that this region is involved in estrogen-regulated gene
expression. The ability of the half-ERE/Sp1 binding site to confer
estrogen responsiveness to a simple heterologous promoter was confirmed
in transient cotransfection assays. In vitro DNase I
footprinting and gel mobility shift assays demonstrated that Sp1
present in MCF-7 nuclear extracts and purified Sp1 protein bound to the
two Sp1 sites and that the estrogen receptor enhanced Sp1 binding. In
addition to its effects on Sp1 binding, the estrogen receptor also
bound directly to the ERE half-site. Taken together, these findings
suggest that the estrogen receptor and Sp1 play a role in activation of
the human progesterone receptor A promoter.
 |
INTRODUCTION
|
---|
Estrogen is a hormone of critical importance in the development
and maintenance of reproductive tissues and also plays an important
role in cardiovascular and bone physiology. Estrogens effects are
mediated through its interaction with the intracellular estrogen
receptor (ER). Numerous studies have demonstrated that the two ERs,
and ß, mediate their effects by binding to specific DNA sequences,
estrogen response elements (EREs), thereby initiating changes in
transcription of target genes (1 2 ).
It has become apparent that, in addition to binding directly to an ERE,
the ER may also modulate transcription indirectly by interacting with
other DNA-bound proteins. For example, ER interaction with AP1-bound
fos and jun proteins confers estrogen responsiveness to the ovalbumin
(3 ), c-fos (4 ), collagenase (5 ), and insulin-like growth
factor I (6 ) genes. In addition, a growing body of evidence suggests
that the ER may influence binding of Sp1 to its recognition site and
thereby confer estrogen responsiveness to the creatine kinase B (7 ),
c-myc (8 ), retinoic acid receptor
(9 ), heat shock
protein 27 (10 11 ), cathepsin D genes (12 ), and uteroglobin (13 )
genes.
The progesterone receptor (PR) gene is under estrogen control in normal
reproductive tissues (14 15 ) and in MCF-7 human breast cancer cells
(16 17 ). MCF-7 PR mRNA and protein increase and reach maximal levels
after 3 days of 17ß-estradiol (E2) treatment
(16 17 18 ). Like ER, two distinct PR forms are differentially expressed
in a tissue-specific manner (19 20 21 22 23 ). PR-B is a 120-kDa protein
containing a 164 amino acid amino-terminal region that is not present
in the 94-kDa PR-A. Two discrete promoters, A and B, which are thought
to be responsible for the production of PR-A and PR-B, respectively,
have also been defined (24 ). The activities of these two promoters are
increased by estrogen treatment of transiently transfected Hela cells.
Interestingly, no consensus EREs have been identified in either
promoter A (+464 to +1105) or promoter B (-711 to +31). Promoter A
does, however, contain an ERE half-site located upstream of two Sp1
sites (24 ). The presence of these adjacent binding sites suggests that
the ER might be able to influence PR expression directly by binding to
the ERE half-site, indirectly by interacting with proteins bound to the
putative Sp1 sites, or a combination of these two methods. To determine
whether the ERE half-site and the two Sp1 sites present in the human
PR-A promoter might impart estrogen responsiveness to the PR gene, a
series of in vivo and in vitro experiments were
carried out.
 |
RESULTS
|
---|
In Vivo Footprinting of the PR Gene
A number of studies have suggested that an Sp1 site alone or in
combination with an imperfect ERE or ERE half-site may be involved in
conferring estrogen responsiveness to target genes (7 8 9 10 11 12 13 ). To
determine whether the ERE half-site and two potential Sp1 sites
residing in the endogenous human PR gene (+571 to +595, Ref. 24 ) might
be involved in estrogen-regulated transactivation, in vivo
deoxyribonuclease I (DNase I) footprinting was carried out using MCF-7
cells. The region of the PR-A promoter containing the consensus ERE
half-site and two potential Sp1 sites is shown in Fig. 1
and will hereafter be referred to as
the half-ERE/Sp1 binding site.

View larger version (14K):
[in this window]
[in a new window]
|
Figure 1. Sequence of the Half-ERE/Sp1 Binding Site
The sequence of the half-ERE/Sp1 binding site in the PR-A promoter
originally reported by Kastner et al. (24 ) is shown. The
locations of the ERE half-site and the proximal and distal Sp1 sites
(Sp1P and Sp1D, respectively) are indicated.
|
|
To carry out in vivo footprinting assays, MCF-7 cells were
treated with ethanol vehicle or with E2 for 2 or
72 h and then exposed to DNase I. The cells were rapidly lysed,
DNA was isolated, and ligation-mediated PCR (LMPCR) procedures were
carried out (25 ). Naked genomic DNA, which had been treated in
vitro with DNase I, served as a reference in identifying sequences
that were susceptible to cleavage in the absence of proteins (Fig. 2
, Vt). When cells
were treated with E2 for 2 h, the protection
of the proximal Sp1 site (Sp1P), the distal Sp1
binding site (Sp1D), and the ERE half-site was
greater than seen in cells that had not been exposed to hormone. After
72 h of E2 treatment, a time when PR mRNA
and protein reach maximal levels (16 17 18 26 27 ), the protection of
the half-ERE/Sp1 binding site was sustained. E2
treatment also elicited protection of regions flanking the half-ERE/Sp1
binding site. Thus, we were able to detect distinct differences in
protection of the half-ERE/Sp1binding site on the coding strand of the
endogenous PR gene after E2 treatment. Despite
numerous attempts, we were unable to obtain a footprint of the
noncoding PR DNA strand in this region. The failure of these LMPCR
reactions may have been due to formation of an extensive stem loop
structure (
G = -11.5 kcal/mol) extending from +674 to +733
(24 ) that limited primer annealing or interfered with the ability of
polymerase to proceed through this region. Nonetheless, our in
vivo footprinting of the coding strand demonstrated that the
half-ERE/Sp1 binding site residing in the endogenous PR gene was
differentially protected in ethanol- and
E2-treated MCF-7 cells and suggested that the ERE
half-site as well as the proximal and distal Sp1 sites might be
involved in regulation of the endogenous PR gene in MCF-7 cells.

View larger version (46K):
[in this window]
[in a new window]
|
Figure 2. In Vivo DNase I Footprinting of the
Endogenous PR Gene in MCF-7 Cells
MCF-7 cells were maintained in serum-free medium for 5 days, treated
with ethanol control (0 h E2) or 1 nM
E2 for 2 or 72 h, and then exposed to DNase I. Genomic
DNA was isolated and used in in vivo footprinting. Naked
genomic DNA, which had been treated in vitro with either
DMS (G) or DNase I (Vt), was included as a reference. The
locations of the proximal Sp1 site (Sp1P), distal Sp1 site
(Sp1D), and ERE half-site are indicated.
|
|
Estrogen Enhances Transcription of a Reporter Plasmid Containing
the Half-ERE/Sp1 Binding Site
To determine whether the half-ERE/Sp1 binding site could confer
estrogen responsiveness to a heterologous promoter, transient
cotransfection experiments were carried out with a human ER expression
vector and a chloramphenicol acetyltransferase (CAT) reporter plasmid
containing either a TATA box alone (TATA CAT) or in combination with
the half-ERE/Sp1 binding site (ERE/Sp1-TATA CAT). Exposure of
transiently cotransfected CHO cells to E2
resulted in a 1.7-fold increase in CAT activity when the reporter
plasmid contained the half-ERE/Sp1 binding site (Fig. 3
, ERE/Sp1-TATA CAT). This difference was
statistically significant. In contrast, no statistical difference in
activity was observed with E2 treatment when the
parental TATA CAT reporter plasmid was used. These findings suggest
that the half-ERE/Sp1 binding site is involved in estrogen-mediated
activation of the PR-A promoter.

View larger version (23K):
[in this window]
[in a new window]
|
Figure 3. Estrogen-Enhanced Activity of a Plasmid Containing
the Half-ERE/Sp1 Binding Site
CHO cells were transfected with TATA CAT or ERE/Sp1-TATA CAT reporter
plasmid, hER expression plasmid, ß- galactosidase expression
plasmid, and pTZ nonspecific DNA using the calcium phosphate
coprecipitation method as described in Materials and
Methods. Cells were treated with ethanol vehicle or 10
nM E2. Data represent the average of 9
independent experiments. Values are presented as the mean ±
SEM. *, P < 0.02 for E2
vs. ethanol control by Students t test
when ERE/Sp1-TATA CAT was used.
|
|
Proteins Present in MCF-7 Nuclear Extracts Bind to the Half-ERE/Sp1
Binding Site in Vitro
Our in vivo footprinting and transient transfection
experiments provided evidence for the involvement of the half-ERE/Sp1
binding site in mediating estrogens effects on the PR-A promoter.
However, these studies did not allow us to identify proteins that
interact with this DNA sequence. To begin to identify proteins that
bind to this site, gel mobility shift assays were carried out with
MCF-7 nuclear extracts. When 32P-labeled oligos,
each containing the half-ERE/Sp1 binding site, were combined with
nuclear extracts prepared from E2-treated MCF-7
cells, one major protein-DNA complex was formed (Fig. 4
, lane 1). Since we anticipated that ER
and Sp1 might bind to this region, antibodies to these proteins were
included in separate binding reactions. The major protein-DNA complex
was supershifted by the Sp1-specific antibody 1C6, which binds to Sp1,
but does not cross-react with Sp24 (lane 2), suggesting that Sp1 was
present in substantial amounts in our MCF-7 nuclear extracts and that
it bound efficiently to the half-ERE/Sp1 binding site. In contrast, the
major protein-DNA complex was not affected by the ER-specific antibody
H222 (lane 3).

View larger version (23K):
[in this window]
[in a new window]
|
Figure 4. Binding of MCF-7 Sp1 Protein to the Half-ERE/Sp1
Binding Site
32P-labeled oligos containing the half-ERE/Sp1 binding site
were incubated with nuclear extracts from E2-treated MCF-7
cells. The ER-specific antibody H222 (ER Ab) or the Sp1-specific
antibody IC6 (Sp1 Ab) was added to the binding reaction as indicated.
The 32P-labeled oligos were fractionated on a nondenaturing
gel and visualized by autoradiography.
|
|
Although our gel mobility shift experiments suggested that Sp1
was involved in regulation of the PR gene expression, they did not
provide evidence that the ER was involved in formation of a protein-DNA
complex. However, gel mobility shift experiments require the formation
of stable protein-DNA complexes, which must be maintained during
extended periods of electrophoresis. To determine whether a more
transient or lower affinity interaction might occur between MCF-7
nuclear proteins and the ERE half-site and/or either one or both of the
Sp1 binding sites, in vitro DNase I footprinting was carried
out. DNA fragments (181 bp), each containing the half-ERE/Sp1 binding
site and additional PR flanking sequence, were
32P-labeled on one end, incubated with increasing
amounts of MCF-7 nuclear extract, and exposed to limited DNase I
cleavage (Fig. 5
, lanes 35 and 810).
When DNA fragments that had been 32P-labeled on
the coding strand were used, the proximal and distal Sp1 sites were
partially protected by proteins present in the MCF-7 nuclear extracts
(lanes 35). Quantitative analysis of the coding strand revealed
slightly greater protection of the proximal Sp1 site than the distal
Sp1 site. Although the ERE half-site was not protected, nucleotides
within and immediately flanking the ERE half-site were hypersensitive
to DNase I cleavage upon addition of increasing concentrations of
nuclear proteins (lanes 35). When the noncoding DNA strand was
labeled and used in in vitro footprinting experiments with
MCF-7 nuclear extracts, the proximal Sp1 site was more extensively
protected than the distal Sp1 site (lanes 810). As seen with the
coding strand, hypersensitive sites were observed within and adjacent
to the ERE half-site on the noncoding strand. Control lanes containing
DNA fragments that had been exposed to dimethylsulfate (DMS) (lanes 1
and 6) or DNase I (lanes 2 and 7) in the absence of protein were
included for reference. The enhanced protection of the Sp1 sites
observed in our in vitro footprints in the presence of MCF-7
nuclear extracts was similar to the increased protection of the Sp1
sites in the endogenous gene upon E2 treatment of
MCF-7 cells. The ERE half-site was not protected in our in
vitro footprints as seen in the in vivo footprints, but
rather displayed hypersensitivity to DNase I cleavage on both strands.
Since DNase I hypersensitivity can result from binding of a protein to
the major groove of the DNA helix, making the minor groove more
accessible to DNase I cleavage (28 ), the hypersensitivity observed
within and adjacent to the ERE could result from binding of a protein
to the major groove in the region of the ERE.

View larger version (84K):
[in this window]
[in a new window]
|
Figure 5. In Vitro Footprinting of the
Half-ERE/Sp1 Binding Site with MCF-7 Nuclear Extracts
DNA fragments (181 bp) containing the half-ERE/Sp1 binding site were
end-labeled on either the coding or noncoding strand and incubated with
increasing concentrations of nuclear extract from
E2-treated MCF-7 cells (lanes 35 and 810). The binding
reactions were subjected to limited DNase I digestion, and the cleaved
DNA fragments were fractionated on a denaturing gel. DNA fragments,
which had been treated in vitro with either DMS (lanes 1
and 6) or DNase I (lanes 2 and 7), were included as references. The
locations of the proximal Sp1 site (Sp1P), distal Sp1 site
(Sp1D), and ERE half-site are indicated.
|
|
Purified Sp1 Binds to the Half-ERE/Sp1 Binding Site
Our antibody supershift experiments indicated that native Sp1
present in MCF-7 nuclear extracts was binding to the half-ERE/Sp1
binding site. However, the MCF-7 extracts used in these assays
contained a complex combination of nuclear proteins. To determine
whether Sp1 protein alone was capable of binding to the half-ERE/Sp1
binding site or whether other proteins present in MCF-7 nuclear
extracts were required for Sp1 binding, gel mobility shift experiments
were carried out with purified Sp1 protein.
32P-labeled oligos, each containing the
half-ERE/Sp1 binding site, were incubated with increasing
concentrations of purified Sp1 protein and fractionated on a
nondenaturing acrylamide gel (Fig. 6
, lanes 25). At the lowest Sp1 concentration used (1 ng), a single
gel-shifted band was observed (
1, lane 2). As increasing
concentrations of Sp1 were added to the binding reaction, there was a
dose-dependent increase in a second, higher mol wt complex (
2, lanes
35). These experiments demonstrate that purified Sp1 was capable of
forming a stable complex with the half-ERE/Sp1 binding site. Additional
gel shift assays demonstrated that the more rapidly migrating Sp1-DNA
complex had the same mobility as the complex formed with MCF-7 nuclear
extracts (data not shown).

View larger version (39K):
[in this window]
[in a new window]
|
Figure 6. Gel Mobility Shift Assay of half-ERE/Sp1 Binding
Site-Containing Oligos and Purified Sp1 Protein
32P-labeled oligos containing the half-ERE/Sp1 binding site
were incubated alone (lane 1) or with increasing concentrations of
purified Sp1 protein (lanes 25) and fractionated on a nondenaturing
gel. The locations of the more rapidly ( 1) and more slowly ( 2)
migrating Sp1/DNA complexes are indicated.
|
|
It seemed likely that the formation of the higher order complex
in our gel shift experiments represented the simultaneous binding of
two Sp1 proteins to the two Sp1 sites and the more rapidly migrating
complex represented Sp1 binding to one of the two Sp1 sites. To
determine whether Sp1 was binding to one or both of the Sp1 sites and
whether it displayed any preference in binding to the proximal or the
distal Sp1 site, in vitro footprinting experiments were
carried out. DNA fragments (181 bp), each containing the half-ERE/Sp1
binding site, were 32P-labeled on the coding
strand and incubated with increasing concentrations of purified Sp1
protein. When 12.5 ng of purified Sp1 were included in the binding
reaction, the proximal and distal Sp1 sites were protected (Fig. 7
, lanes 3). Addition of 25 and 37.5 ng
of purified Sp1 protein further enhanced protection of the two Sp1
sites (lanes 4 and 5). When DNA fragments labeled on the noncoding
strand were incubated with increasing amounts of purified Sp1, the
proximal Sp1 site was more protected than the distal Sp1 site (lanes
810). This preference for the proximal Sp1 site was also evident in
the in vitro footprints of the noncoding strand in the
presence of MCF-7 nuclear extracts (Fig. 5
). Control lanes containing
DNA fragments that had been exposed to DMS (Fig. 7
, lanes 1 and 6) or
DNase I (lanes 2 and 7) in the absence of proteins were included for
reference. These data, combined with our gel mobility shift assays,
support the idea that Sp1 binds first to the proximal Sp1 site and then
to the distal Sp1 site.

View larger version (84K):
[in this window]
[in a new window]
|
Figure 7. In Vitro Footprinting of the
Half-ERE/Sp1 Binding Site with Purified Sp1
DNA fragments (181 bp) containing the half-ERE/Sp1 binding site and
flanking regions were end-labeled on either the coding or noncoding
strand and incubated with increasing concentrations of purified Sp1
protein (lanes 35 and 810). The binding reactions were subjected to
limited DNase I digestion, and the cleaved DNA fragments were
fractionated on a denaturing gel. DNA fragments that had been treated
in vitro with either DMS (lanes 1 and 6) or DNase I
(lanes 2 and 7) were included as references. The locations of the
proximal Sp1 site (Sp1P), distal Sp1 site
(Sp1D), and ERE half-site are indicated.
|
|
ER Enhances Sp1 Binding to the Half-ERE/Sp1 Binding Site
Our in vitro binding assays suggested that Sp1 was
involved in regulating the PR gene, but left some question about the
involvement of ER in this process. From previous studies examining
ER-mediated transcription activation, it seemed possible that ER could
increase transcription either directly by binding to the ERE half-site
or indirectly by enhancing Sp1 binding (7 8 9 10 11 12 29 30 ). To determine
whether ER affected protein-DNA complex formation, gel mobility shift
assays were carried out. When a 32P-labeled oligo
containing the half-ERE/Sp1 binding site was incubated with 3 ng of
purified Sp1, a single gel-shifted band was formed (Fig. 8A
, lane 1, Sp1
). When the amount of
purified Sp1 protein was decreased to 0.25 ng, a faint gel-shifted band
was barely visible (lanes 2 and 8). Addition of 150, 350, or 700 fmol
of purified, unoccupied (lanes 37), or
E2-occupied (lanes 913) receptor to 0.25 ng
purified Sp1 protein elicited a dose-dependent increase in Sp1 binding.
This ER-enhanced Sp1 binding was not due to an increase in protein
concentration since all reactions contained the same amount of total
protein. Incremental addition of ER also resulted in a dose-dependent
increase in a more rapidly migrating protein-DNA complex. The
ER-specific antibody H151 supershifted this more rapidly migrating
complex (ER
) but did not affect the Sp1-DNA complex (lanes 6 and
12). The Sp1-specific antibody IC6 supershifted the Sp1-DNA complex but
did not affect the more rapidly migrating ER-DNA complex (lanes 7 and
13). These findings demonstrate that ER enhances Sp1 binding and that
both ER and Sp1 can bind directly to the half-ERE/Sp1 binding site.

View larger version (73K):
[in this window]
[in a new window]
|
Figure 8. ER-Enhanced Binding of Sp1 to the Half-ERE/Sp1
Binding Site
32P-labeled oligos containing the half-ERE/Sp1 binding site
were incubated with purified Sp1, ER, or Sp1 and ER and fractionated on
nondenaturing gels. A, Binding reactions contained either 3 ng of Sp1
(lane 1) or 0.25 ng of Sp1 alone (lanes 2 and 8) or in combination with
150 (lanes 3 and 9), 350 (lanes 4 and 10), or 700 (lanes 57 and
1113) fmol of unoccupied (-E2) or
E2-occupied (+E2) ER. B, Binding reactions
contained 350 (lane 1) or 700 (lanes 24) fmol of ER, 3 ng of Sp1
(lane 5), or 1 ng of Sp1 (lanes 69) and 350 (lane 6) or 700 (lanes
79) fmol of ER. The ER-specific antibody H151 (ER Ab) or the
Sp1-specific antibody IC6 (Sp1 Ab) was added as indicated. The
locations of the ER/DNA ( ER), Sp1/DNA ( Sp1), and ER/Sp1/DNA
( ER/Sp1) complexes are indicated.
|
|
In addition to the major ER-DNA and Sp1-DNA complexes, three
minor, higher order protein-DNA complexes, which we thought might
reflect the formation of a trimeric ER-Sp1-DNA complex, were
consistently observed in our gel shifts (Fig. 8A
, lanes 5 and 11). To
determine whether this was the case, 32P-labeled
oligos containing the half-ERE/Sp1 binding site were incubated with ER
in the absence and in the presence of Sp1. When ER, but not Sp1, was
included in the binding reaction (Fig. 8B
, lanes 14), the major
ER-DNA complex was formed (
ER) as well as two minor, higher order
complexes, which most likely contained ER and Sf-9 proteins that
copurified with the receptor. When 1 ng Sp1 and 350 or 700 fmol ER were
included in the binding reaction (lanes 69), the major ER (
ER) and
Sp1 (
Sp1) complexes and three minor, higher order complexes were
formed. The one unique, higher order protein-DNA complex (lanes 6 and
7,
ER/Sp1) formed in the presence of both ER and Sp1 was supershifted
by ER- and Sp1-specific antibodies, demonstrating that both ER and Sp1
were present. A lane containing Sp1 alone was included as a reference
(lane 5). These combined experiments suggest that ER and Sp1 can form a
trimeric complex with DNA at the half-ERE/Sp1 binding site in the PR-A
promoter.
To determine how ER affected Sp1 protection of the half-ERE/Sp1 binding
site, in vitro DNase I footprinting experiments were carried
out with purified ER and Sp1 proteins. When 15 ng of purified Sp1 were
incubated with the 32P-labeled coding strand, the
proximal and distal Sp1 sites were protected (Fig. 9
, lane 3). Addition of 15 ng Sp1 and
0.331.3 pmol of purified ER incrementally enhanced the protection of
both the proximal and distal Sp1 sites (lanes 46). As suggested from
the gel mobility shift assays, the consensus ERE half-site was
protected in the presence of higher ER concentrations (lane 6). When
DNA fragments labeled with 32P on the noncoding
strand were incubated with 15 ng of purified Sp1 and increasing
concentrations of purified ER, enhanced protection of both the proximal
and distal Sp1 sites and the half-ERE was observed (lanes 912). As
seen in the in vitro footprints with MCF-7 nuclear extracts
and with purified Sp1, the proximal Sp1 site on the noncoding strand
was more extensively protected than the distal Sp1 site. The ERE
half-site was partially protected on the noncoding strand. Control
lanes containing DNA fragments that had been exposed to DMS (lanes 1
and 7) or DNase I (lanes 2 and 8) in the absence of proteins were
included for reference.

View larger version (95K):
[in this window]
[in a new window]
|
Figure 9. In Vitro DNase I Footprinting of the
Half-ERE/Sp1 Binding Site with Purified Sp1 and ER
DNA fragments containing the half-ERE/Sp1 binding site and
flanking regions were end-labeled on either the coding or noncoding
strand and incubated with 15 ng of purified Sp1 protein (lanes 36 and
912) and 0.33, 0.66, or 1.3 pmol of purified ER (lanes 46 and
1012). The binding reactions were subjected to limited DNase I
digestion, and the cleaved DNA fragments were fractionated on a
denaturing gel. DNA fragments that had been treated in
vitro with either DMS (lanes 1 and 7) or DNase I (lanes 2 and
8) were included as references. The locations of the proximal Sp1 site
(Sp1P), distal Sp1 site (Sp1D), and ERE
half-site are indicated.
|
|
Sp1 Does Not Enhance ER Binding to the ERE Half-Site
Our binding assays indicated that ER greatly enhanced binding of
Sp1 to the half-ERE/Sp1 binding site. To determine whether Sp1
influenced ER binding, gel mobility shift assays were carried out. When
32P-labeled oligos containing the half-ERE/Sp1
binding site were incubated with 150 fmol of purified ER, a single
major gel shifted band was formed (Fig. 10
, lane 1,
ER). When the amount of
purified ER protein was decreased to 50 fmol, a fainter gel shifted
band was produced (lanes 26,
ER). Addition of 0.25, 0.5, 1.5, or 3
ng of purified Sp1 protein (lanes 36,
Sp1) elicited a
dose-dependent increase in Sp1 binding. In contrast, incremental
addition of Sp1 slightly decreased ER binding to the half-ERE/Sp1
binding site. These combined findings demonstrate that although ER
greatly enhanced Sp1 binding, Sp1 did not enhance ER binding to the
half-ERE/Sp1 binding site.

View larger version (45K):
[in this window]
[in a new window]
|
Figure 10. Effect of Sp1 on ER Binding to the Half-ERE/Sp1
Binding Site
32P-labeled oligos containing the half-ERE/Sp1 binding site
were incubated with 150 fmol (lane 1) or 50 fmol of purified ER (lanes
26) and 0.25, 0.5, 1.5, or 3 ng of purified Sp1 (lanes 36) and
fractionated on nondenaturing gels. The locations of the ER-DNA ( ER)
and Sp1-DNA ( Sp1) complexes are indicated.
|
|
Purified Sp1 and ER Bind Differentially to Wild- Type and Mutant
Half-ERE/Sp1 Binding Sites
The in vitro footprinting experiments reproducibly
suggested a preference of Sp1 for the proximal Sp1 site. To determine
how each of the Sp1 sites and the ERE half-site contributed to
protein-DNA complex formation, each of the individual elements was
mutated and tested in gel mobility shift assays. Complementary oligos
containing the wild-type half-ERE/Sp1 binding site (wt) or mutations in
both Sp1 sites (mP/D), the distal Sp1 site (mD), the proximal Sp1 site
(mP), or the ERE half-site (mE) were synthesized, annealed, and labeled
with 32P. The labeled oligos were combined with
purified Sp1 (Fig. 11
, lanes 15) or
purified Sp1 and ER (lanes 610) and fractionated on nondenaturing
gels. Sp1 or Sp1 and ER bound effectively to the wt half-ERE/Sp1
binding site (lanes 1 and 6). As anticipated, Sp1 did not bind to the
oligo containing mutations in both Sp1 sites, in the absence (lane 2)
or in the presence of ER (lane 7). Sp1 alone or in combination with ER
bound to oligos containing a mutation in one of the two Sp1 sites, but
more protein-DNA complex was formed when the oligo contained an intact
proximal Sp1 site (mD; compare lanes 3 and 8 with lanes 4 and 9).
PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA)
analysis of four gel shift experiments demonstrated that there was a
30.8% (±7 SEM) increase in complex formation
with the proximal Sp1 site compared with the distal Sp1 site. These
findings corroborate the preferential binding of Sp1 to the proximal
Sp1 site observed in the in vitro footprinting studies. The
ability of ER to bind to oligos containing an intact ERE half-site
(lanes 69), but not to an oligo containing a mutated ERE half-site
(lane 10), further supports the idea that ER can bind to the ERE
half-site. When the ERE half-site was mutated, increased Sp1-DNA
complex formation was observed (lanes 5 and 10). The reason for this
apparent increase in Sp1 binding is unclear, but it was a reproducible
finding.

View larger version (62K):
[in this window]
[in a new window]
|
Figure 11. Interaction of Purified Sp1 and ER with wt and
Mutant Half-ERE/Sp1 Binding Sites
32P-labeled oligos containing the wild-type half-ERE/Sp1
binding site (wt), or mutations in both Sp1 binding sites (mP/D), the
distal Sp1 binding site (mD), the proximal Sp1 binding site (mP), or
the ERE half-site (mE) were incubated with 3 ng of purified Sp1 (lanes
15) or 3 ng of purified Sp1 and 0.33 pmol of purified ER (lanes
610). The 32P-labeled oligos were fractionated on a
nondenaturing gel and visualized by autoradiography. The locations of
the ER-DNA ( ER) and Sp1-DNA ( Sp1) complexes are indicated.
|
|
 |
DISCUSSION
|
---|
Sequence comparison of the PR gene from different species has been
used to identify cis elements that are involved in estrogen-regulated
transactivation. The rabbit PR gene contains an imperfect ERE, which
overlaps with the translation start site and is capable of conferring
estrogen responsiveness to a heterologous promoter in transient
transfection assays (31 ). Although a similar sequence is present in the
chicken PR gene (32 ), no homologous sequence has been identified in the
human PR gene (24 ). A number of studies have suggested that ER and Sp1
may be involved in conferring estrogen responsiveness to the creatine
kinase B (7 ), c-myc (8 ), retinoic acid receptor
(9 ),
heat shock protein 27 (10 11 ), cathepsin D (12 ), and uteroglobin (13 )
genes. The identification of an ERE half-site adjacent to two Sp1 sites
in the human PR gene (24 ) led us to investigate whether this region
might be involved in conferring estrogen-responsiveness to the human PR
gene. We initiated our studies by examining the endogenous PR gene in
MCF-7 cells. Unlike transient transfection assays, which examine the
ability of ER to activate transcription of synthetic promoters in
supercoiled plasmids, our in vivo DNase I footprinting
experiments allowed us to examine the endogenous PR gene as it exists
in native chromatin and assess whether the half-ERE/Sp1 binding site
might play a physiological role in gene expression.
E2 treatment of MCF-7 cells did elicit more
extensive protection of the half-ERE/Sp1 binding site than was observed
in the absence of hormone. The enhanced protection of the half-ERE/Sp1
binding site seen after 72 h of hormone treatment, a time when PR
mRNA and protein reach maximal levels (16 17 18 26 27 ), suggests that
sustained protein-DNA interactions are required for maximal production
of PR mRNA and protein. Furthermore, the ability of the half-ERE/Sp1
binding site to enhance transcription of a CAT reporter plasmid in the
presence of E2 suggests that this region is
involved in estrogen responsiveness of the PR-A promoter.
A Role for Sp1 in Regulating Expression of the PR Gene
Sp1 was originally described as a trans-acting factor
that bound to a GC box (5'-GGGCGG-3') and activated transcription of
the SV40 promoter (33 34 ). Subsequent comparison of numerous Sp1
binding sites led to the identification of a higher affinity, consensus
Sp1 site, 5'-GGGGCGGGGC-3' (35 ), and the discovery that sequences that
varied from this consensus sequence displayed decreased affinities for
Sp1. While both of the Sp1 sites in the human PR half-ERE/Sp1 binding
site contain the GC box motif, only the proximal Sp1 site contains the
10-bp consensus Sp1 sequence (Fig. 1
). The increased affinity of Sp1
for the 10-bp proximal Sp1 site, when compared with the distal Sp1
site, was repeatedly observed in our in vitro footprinting
assays and was most obvious on the noncoding strand (Figs. 5
, 7
, and 9
). Gel mobility shift assays carried out with oligos containing
mutations in the proximal or distal Sp1 site confirmed Sp1s
preference for the proximal Sp1 site (Fig. 11
). Interestingly, the
centers of the two GC boxes present in the half-ERE/Sp1 binding site
are separated by 10 bp or one turn of the DNA helix
(Sp1D +580 to +585, Sp1P
+590 to +595). The periodicity of these elements could either favor
interaction between adjacent DNA-bound proteins resulting in
cooperative binding or sterically hinder binding of two Sp1
proteins. Our gel mobility shift and in vitro DNase I
footprinting assays provided evidence for additive, not cooperative,
binding of Sp1 to these sites and indicate that Sp1 binds first to the
proximal Sp1 site and then to the distal Sp1 site.
A Role for ER in Regulating Expression of the PR Gene
One way that estrogen might affect PR gene expression is through
direct binding of the receptor to the ERE half-site. The ERE was
protected in our in vitro footprinting experiments with ER
and Sp1, but not with Sp1 alone, demonstrating that the ER did bind to
the ERE half-site. Likewise, gel mobility shift experiments carried out
with purified ER alone or in combination with Sp1 indicated that the ER
bound surprisingly well to the ERE half-site and formed a stable
protein-DNA complex that was capable of withstanding the extensive
periods of electrophoresis required for gel mobility shift experiments.
Furthermore, the ERE half-site was protected in our in vivo
footprinting experiments after treatment of MCF-7 cells with
E2, suggesting that this element is involved in
regulation of the endogenous gene. Although we were unable to detect
protection of the ERE half-site in our in vitro binding
assays using MCF-7 nuclear extracts, the level of ER in these extracts
(0.42 fmol/µg protein) was significantly lower than the level present
in an intact cell nucleus. Assuming a nuclear radius of 6 µm and
150,000 ER sites per cell (36 ), the ER concentration in an MCF-7
nucleus would be 273 nM. These ER concentrations
are significantly higher than the 757 nM
concentrations used in our in vitro binding assays and would
most likely favor ER binding to the ERE half-site. The 10 bp separating
the ERE half-site and the distal Sp1 binding site would place the ER on
the same side of the DNA helix as the DNA-bound Sp1 proteins and could
help to foster protein-protein interactions.
Estrogen could also modulate PR gene expression through ER-enhanced Sp1
binding. ER effectively enhanced Sp1 binding to the two Sp1 sites in
the PR-A promoter and formed a trimeric complex with Sp1 and DNA in our
in vitro binding assays. Direct ER-Sp1 interaction has also
been documented in immunoprecipitation and
glutathione-S-transferase (GST) pulldown experiments (11 29 ).
We have considered only ER
in our studies since MCF-7 cells express
high levels of ER
, but do not express ERß (36 37 ). Although we
have not ruled out the possibility that another nuclear protein might
bind to the ERE half-site, the high levels of nuclear ER, the
differential protection of the ERE half-site in the presence and
absence of E2, and the demonstrated ability of ER
to bind to the ERE half-site in vitro suggest that it is
most likely the ER that interacts with this site in vivo and
helps to regulate transcription of the PR-A promoter.
Regulation of the PR-A Promoter in MCF-7 Cells
Estrogen treatment of transfected cells resulted in a modest, but
reproducible 1.7-fold increase in transcription of a plasmid containing
the ERE/Sp1 binding site. Since estrogen treatment of MCF-7 cells
results in a 2- to 10-fold increase in PR mRNA levels (16 17 18 ), it
seems likely that the ERE/Sp1 binding site may play a significant role
in mediating the estrogen responsiveness of this gene. However, the
ERE/Sp1 site represents a small part of the complex PR promoter, which
contains multiple regulatory elements. Unlike promoters that contain a
palindromic ERE, transcription of the estrogen-regulated PR gene may
require ER action at multiple cis elements. Preliminary
experiments from our laboratory suggest that additional Sp1 and AP1
sites in the PR promoter may also be involved in estrogen-regulated
gene expression (L. Petz and A. Nardulli, unpublished data). Thus, the
cooperative action of the multiple sites within the PR promoter
may be required for effective estrogen-regulated
transcription.
Our studies support the idea that ER and Sp1 are involved in
estrogen-regulated expression of the human PR-A promoter. The
protection of nucleotides flanking the half-ERE/Sp1 binding site in our
in vivo footprinting experiment suggests that other proteins
are associated with the promoter and are involved in transcription
activation. Interestingly, the E2-occupied ER,
but not the unoccupied ER, interacts with a number of coactivator
proteins, which participate in transcription activation and chromatin
remodeling (38 39 40 41 42 43 44 45 46 ). The recruitment of these proteins to the
DNA-bound, liganded receptor could account for protection of sequences
flanking the half-ERE/Sp1 binding site and serve as the initiating
event in the formation of an active transcription complex.
While models of DNA are typically drawn in a linear array, the
packaging of DNA and protein into the nucleus of a cell requires
tremendous compaction. This compaction could facilitate interaction
between trans acting factors bound to more distant
cis elements. Thus, the association of upstream activators,
such as ER and Sp1, with factors bound to downstream elements could be
fostered. In fact, both ER and Sp1 are known to directly associate with
TFIID components. ER interacts with TATA binding protein (TBP),
transcription factor IIB (TFIIB), and TBP-associated factor
(TAF)II30 (47 48 49 ), and Sp1 interacts with TBP,
TAFII130, and TAFII55
(50 51 52 53 ). The interaction of ER and Sp1 with TBP and its associated
proteins could foster formation of a protein-protein network that helps
to establish an active transcription complex. Furthermore, the
E2-dependent recruitment of coactivators such as
CBP/p300, which can function as a histone acetyltransferase (39 ), could
help remodel chromatin in the PR-A promoter and enhance formation of an
interconnected protein-protein and protein-DNA network involved in
activation of the human PR gene.
 |
MATERIALS AND METHODS
|
---|
Cell Culture
MCF-7 human breast cancer cells (54 ) were maintained in Eagles
MEM containing 5% heat-inactivated calf serum. Cells were seeded in
10-cm plates and transferred to phenol red-free, serum-free improved
MEM (55 ) 5 days before the experiments were conducted. Chinese Hamster
Ovary (CHO) cells were maintained in DMEM/F12 supplemented with 5%
charcoal dextran-stripped calf serum (27 ).
Oligonucleotides and Plasmid Constructions
The names and sequences of wild-type (wt) or mutant
half-ERE/Sp1 binding sites are listed. Nucleotides that differ from
the endogenous, wt half-ERE/Sp1 binding site are
underlined.
ERE/Sp1 wt: 5'-GATCTAGGAGCTGACCAGCGCCGCCCTCCCCCGCCCCCGACCA-3'
and 5'-GATCTGGTCGGGGGCGGGGGAGGGCGGCGCTGGTCAGCTCCTA-3',
ERE/Sp1 mP/D:
5'-GATCTAGGAGCTGACCAGCGTTGTACTCCCTTGTACCCGACCA-3'
and
5'-GATCTGGTCGGGTACAAGGGAGTACAACGCTGGTCAGCTCCTA-3',
ERE/Sp1 mD:
5'-GATCTAGGAGCTGACCAGCGTTGTACTCCCCCGCCCCCGACCA-3'
and
5'-GATCTGGTCGGGGGCGGGGGAGTACAACGCTGGTCAGCTCCTA-3',
ERE/Sp1 mP:
5'-GATCTAGGAGCTGACCAGCGCCGCCCTCCCTTGTACCCGACCA-3'
and
5'-GATCTGGTCGGGTACAAGGGAGGGCGGCGCTGGTCAGCTCCTA-3',
ERE/Sp1 mE:
5'-GATCTAGGAGCTGATTAGCGCCGCCCTCCCCCGCCCCCGACCA-3'
and 5'-GATCTGGTCGGGGGCGGGGGAGGGCGGCGCTAATCAGCTCCTA-3'.
ERE/Sp1 wt oligos with BglII compatible ends were subcloned
into the BglII-cut, dephosphorylated CAT reporter plasmid,
TATA CAT (56 ), to create ERE/Sp1-TATA CAT. The ligated vector was
transformed into the DH5
strain of Escherichia coli,
sequenced, and purified on two cesium chloride gradients.
In Vitro and in Vivo Treatment of Genomic
DNA
MCF-7 cells were exposed to ethanol vehicle or 1 nM
E2 for 0, 2, or 72 h before DNase I
treatment. Cells were permeabilized with 0.4% NP-40 and treated with
750 U DNase I/ml (Roche Molecular Biochemicals,
Indianapolis, IN) for 3 min at 25 C. Isolation of genomic DNA was
carried out as described by Mueller and Wold (25 ). The genomic DNA was
purified, incubated with RNase A, resuspended in TE (10 mM
Tris, pH 7.5, 1 mM EDTA) and stored at -20 C.
Naked genomic DNA was treated in vitro with DMS as described
(25 ). In vitro DNase I-treated DNA was prepared by adjusting
100 µg of protein-free, RNase A-treated DNA to 175 µl with TE. DNA
was incubated with 2.5 x 10-5 U DNase I
for 5 min at 37 C. The reaction was stopped by the addition of 10
mM EDTA and processed as described for in
vivo-treated genomic DNA.
In Vivo Footprinting
Ligation-mediated PCR (LMPCR) footprinting was carried out
essentially as described by Mueller and Wold (25 57 ). Two micrograms
of genomic DNA were subjected to LMPCR procedures using nested primers,
which annealed to sequences upstream of the half-ERE/Sp1 binding site
(+571 to +595) in the human PR gene. The primer sequences were: primer
1, 5'-TCCCCGAGTTAGGAGACGAGAT-3'; primer 2, 5'-CGCTCCCCACTTGCCGCTC-3';
and primer 3, 5'-GCTCCCCACTTGCCGCTCGCTG-3'. The annealing temperatures
for the primers were 55 C, 62 C, and 69 C, respectively. The linker
primers LMPCR 1 and LMPCR 2 described by Mueller and Wold (57 ) were
also used, except that LMPCR 1 was modified by removing the two
5'-nucleotides to eliminate potential secondary structure.
In Vitro DNase I Footprinting
Primers, which annealed 88 bp upstream (primer 3) or 79 bp
downstream (primer 4, 5'-TCGGGAATATAGGGGCAGAGGGAGGAGAA-3') of the
half-ERE/Sp1 binding site, were subjected to 30 rounds of PCR
amplification with 30 ng of the PR-(+464/+1105) CAT (24 ). Labeling of
the coding and noncoding strands was carried out with
32P-labeled primer 3 or primer 4, respectively.
The 181-bp singly end-labeled amplified fragments were fractionated on
an acrylamide gel and isolated. End-labeled DNA fragments (100,000 cpm)
containing the half-ERE/Sp1 binding site were incubated for 15 min at
room temperature in a buffer containing 10% glycerol, 50
mM KCl, 15 mM Tris, pH 7.9, 0.2 mM
EDTA, 1 mM MgCl2, 50 ng of poly dIdC,
and 0.4 mM dithiothreitol (DTT) in a final volume of 50
µl with either 3060 µg of MCF-7 nuclear extract, 12.537.5 ng of
purified Sp1 protein (Promega Corp., Madison, WI), or 15
ng of purified Sp1 and 0.131.3 pmol of purified Flag-tagged ER, which
had been expressed and purified as described by Kraus and Kadonaga
(58 ). E2 (10 nM) was included in
binding reactions containing the purified ER. BSA, ovalbumin, and KCl
were added as needed to maintain constant protein and salt
concentrations. When MCF-7 nuclear extracts were used, poly dI/dC was
increased to 1 µg per reaction. RQ1 ribonuclease-free DNase I (12
U) (Promega Corp., Madison, WI) was added to each sample
and incubated at room temperature for 0.758 min. The DNase I
digestion was terminated by addition of stop solution (200
mM NaCl, 1% SDS, 30 mM EDTA, and 100 ng/µl
tRNA). The DNA was phenol/chloroform extracted, precipitated, and
resuspended in formamide loading buffer (59 ). Samples were fractionated
on an 8% denaturing acrylamide gel. Radioactive bands were visualized
by autoradiography and quantitated with a PhosphorImager and ImageQuant
software (Molecular Dynamics, Inc.).
Gel Mobility Shift Assays
Gel mobility shift assays were carried out essentially as
described previously (60 61 ). 32P-labeled
(10,000 cpm) half-ERE/Sp1-containing wt or mutant oligos were incubated
for 15 min at room temperature in a buffer containing 10% glycerol, 50
mM KCl, 15 mM Tris, pH 7.9, 0.2 mM
EDTA, 1 mM MgCl2, 50 ng of poly
dI/dC, and 0.4 mM DTT in a final volume of 20 µl with
either 20 µg of MCF-7 nuclear extract, 0.253 ng of purified Sp1
protein, or 0.25 ng of purified Sp1 and 50700 fmol of purified ER.
E2 (10 nM) was included
in all binding reactions containing ER unless otherwise indicated. BSA,
ovalbumin, and KCl were added as needed to maintain constant protein
and salt concentrations. When MCF-7 nuclear extracts were used, the
nonspecific DNA for each reaction included 1 µg of salmon sperm DNA,
and poly dI/dC was increased to 2 µg. For antibody supershift
experiments, the Sp1-specific monoclonal antibody, 1C6 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) or the ER-specific
monoclonal antibody H222 or H151 (kindly provided by Drs. Geoffrey
Greene, The University of Chicago, Chicago, IL and Dean Edwards,
University of Colorado Health Science Center, Denver, CO, respectively)
was added to the protein-DNA mixture and incubated for 10 min at room
temperature. Low ionic strength gels and buffers were prepared as
described previously (59 ). Radioactive bands were visualized by
autoradiography and quantitated with a PhosphorImager and ImageQuant
software (Molecular Dynamics, Inc.).
Transient Transfections
CHO cell transfections were performed using the calcium
phosphate method (62 ). Crystals were formed in the presence of 3 µg
of the indicated CAT reporter, 200 ng of the ß-galactosidase vector
pCH110 (Pharmacia Biotech, Piscataway, NJ), 5 ng of the
human ER
expression vector pCMVhER (63 ), and 4.8 µg of pTZ18U and
incubated with CHO cells for 16 h followed by a 2 min 20%
glycerol shock. Cells were maintained in media containing ethanol
vehicle or 10 nM E2 for 24 h.
Protein concentration was determined using the Bio-Rad protein assay
(Bio-Rad Laboratories, Inc. Hercules, CA) with BSA as a
standard. Mixed-phase CAT assays were performed using 35 µg protein
as previously described (64 ). The ß-galactosidase activity was
determined at room temperature as previously described (65 ) and used to
normalize the amount of CAT activity in each sample.
 |
ACKNOWLEDGMENTS
|
---|
We are very grateful to Jennifer Wood for providing MCF-7
nuclear extracts and purified ER; W. Lee Kraus and James T. Kadonaga
for providing the viral stock used in ER production; Geoffrey Greene
and Dean Edwards for the ER antibodies; and Pierre Chambon for the
PR-(+464/+1105) CAT. We also thank Jongsook Kim for providing technical
expertise and Robin Dodson for helpful comments during the preparation
of this manuscript.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Ann M. Nardulli, Department of Molecular and Integrative Physiology, University of Illinois at Urbana-Champaign, 524 Burrill Hall, 407 South Goodwin Avenue, Urbana, Illinois 61801.
This research was supported by United States Army Medical Research and
Materiel Command Grant DAMD1796-16267 and NIH Grants R29 HD-31299
and DK-53884 (to A.M.N.). Postdoctoral support for L. Petz was provided
by USMRMC Grant DAMD1797-17201 and NIH Reproductive Training Grant
PHS 2T32 HD-072819.
Received for publication September 7, 1999.
Revision received April 6, 2000.
Accepted for publication April 7, 2000.
 |
REFERENCES
|
---|
-
Beato M, Herrlich P, Schutz G 1995 Steroid hormone
receptors: many actors in search of a plot. Cell 83:851857[Medline]
-
Kuiper GGJM, Gustafsson J-A 1997 The novel estrogen
receptor-ß subtype: potential role in the cell- and
promoter-specific actions of estrogens and anti-estrogens. FEBS Lett 410:8790[CrossRef][Medline]
-
Gaub M-P, Bellard M, Scheuer I, Chambon P, Sassone-Corsi P 1990 Activation of the ovalbumin gene by the estrogen receptor involves
the Fos-Jun complex. Cell 63:12671676[Medline]
-
Weisz A, Rosales R 1990 Identification of an estrogen
response element upstream of the human c-fos gene that binds
the estrogen receptor and the AP-1 transcription factor. Nucleic Acids
Res 18:50975106[Abstract]
-
Webb P, Lopez GN, Greene GL, Baxter JD, Kushner PJ 1992 The
limits of the cellular capacity to mediate an estrogen response. Mol
Endocrinol 6:157167[Abstract]
-
Umayahara Y, Kawamori R, Watada H, Imano E, Iwama N,
Morishima T, Yamasaki Y, Kajimoto Y, Kamada T 1994 Estrogen regulation
of the insulin-like growth factor I gene transcription involves an AP-1
enhancer. J Biol Chem 269:1643316442[Abstract/Free Full Text]
-
Wu-Peng X, Pugliese T, Dickerman H, Pentecost B 1992 Delineation of sites mediating estrogen regulation of the rat creatine
kinase B gene. Mol Endocrinol 6:231240[Abstract]
-
Dubik D, Shiu R 1992 Mechanism of estrogen activation of
c-myc oncogene expression. Oncogene 7:15871594[Medline]
-
Rishi A, Hhao Z-M, Baumann R, Li X-S, Sheikh S, Kimura S,
Bashirelahi N, Fontana J 1995 Estradiol regulation of the human
retinoic acid receptor gene in human breast carcinoma cells is mediated
via an imperfect half-palindromic estrogen response element and Sp1
motifs. Cancer Res 55:49995006[Abstract]
-
Porter W, Wang F, Wang W, Duan R, Safe S 1996 Role of estrogen
receptor/Sp1 complexes in estrogen-induced heat shock protein 27 gene
expression. Mol Endocrinol 10:13711378[Abstract]
-
Porter W, Saville B, Hoivik D, Safe S 1997 Functional synergy
between the transcription factor Sp1 and the estrogen receptor. Mol
Endocrinol 11:15691580[Abstract/Free Full Text]
-
Krishnan V, Wang X, Safe S 1994 Estrogen receptor-Sp1
complexes mediate estrogen-induced cathepsin D gene expression in MCF-7
human breast cancer cells. J Biol Chem 269:1591215917[Abstract/Free Full Text]
-
Scholz A, Truss M, Beato M 1998 Hormone-induced recruitment of
Sp1 mediates estrogen activation of the rabbit uteroglobin gene in
endometrial epithelium. J Biol Chem 273:43604366[Abstract/Free Full Text]
-
Kontula K, Janne O, Vihko R 1975 Steroidal regulation of the
progesterone receptor in human myometrium. Acta Endocrinol Suppl
(Copenh) 199:215
-
Luu Thi M, Baulieu E, Milgrom E 1975 Comparison of the
characteristics and of the hormonal control of endometrial and
myometrial progesterone receptor. J Endocrinol 66:349356[Abstract]
-
Nardulli AM, Greene GL, OMalley BW, Katzenellenbogen BS 1988 Regulation of progesterone receptor message ribonucleic acid and
protein levels in MCF-7 cells by estradiol: analysis of estrogens
effect on progesterone receptor synthesis and degradation.
Endocrinology 122:935944[Abstract]
-
Wei LL, Krett NL, Francis MD, Gordon DF, Wood WM, OMalley
BW, Horwitz KB 1988 Multiple human progesterone receptor message
ribonucleic acids and their autoregulation by progestin agonists and
antagonists in breast cancer cells. Mol Endocrinol 2:6272[Abstract]
-
Read LD, Snider CE, Miller JS, Greene GL, Katzenellenbogen BS 1988 Ligand-modulated regulation of progesterone receptor messenger
ribonucleic acid and protein in human breast cancer cell lines. Mol
Endocrinol 2:263271[Abstract]
-
Schrader WT, OMalley BW 1972 Progesterone-binding components
of chick oviduct: characterization of purified subunits. J Biol
Chem 247:5159[Abstract/Free Full Text]
-
Horwitz KB, Alexander PS 1983 In situ photolinked
nuclear progesterone receptors of human breast cancer cells: subunit
molecular weights after transformation and translocation. Endocrinology 113:21952201[Abstract]
-
Vegeto E, Shahbaz MM, Wen DX, Godman ME, OMalley BW,
McDonnell DP 1993 Human progesterone receptor A form is a cell- and
promoter- specific repressor of human progesterone receptor B function.
Mol Endocrinol 7:12441255[Abstract]
-
Tung L, Mohamed MK, Hoeffler JP, Takimoto GS, Horwitz KB 1993 Antagonist-occupied human progesterone B-receptors activate
transcription without binding to progesterone response elements and are
dominantly inhibited by A-receptors. Mol Endocrinol 7:12561265[Abstract]
-
Mohamed MK, Tung L, Takimoto GS, Horwitz KB 1994 The leucine
zippers of c-fos and c-jun for progesterone receptor dimerization:
A-dominance in the A/B heterdimer. J Steroid Biochem Mol Biol 51:241250[CrossRef][Medline]
-
Kastner P, Kurst A, Turcotte B, Stropp U, Tora L, Gronemeyer
H, Chambon P 1990 Two distinct estrogen-regulated promoters generate
transcripts encoding two functionally different human progesterone
receptor forms A and B. EMBO J 9:16031614[Abstract]
-
Mueller PR, Wold B 1992 Ligation-mediated PCR for genomic
sequencing and footprinting. In: Ausubel F, Brent R, Kingston R,
Moore D, Seidman J, Smith J, Struhl K (eds) Current Protocols in
Molecular Biology. John Wiley & Sons, Inc, New York, pp
15.15.1115.15.26
-
Graham J, Yeates C, Balleine R, Harvey S, Milliken J, Bilous
M, Clarke C 1996 Progesterone receptor A and B protein expression in
human breast cancer. J Steroid Biochem Mol Biol 56:9398[CrossRef][Medline]
-
Eckert RL, Katzenellenbogen BS 1982 Effects of estrogens and
antiestrogens on estrogen receptor dynamics and the induction of
progesterone receptor in MCF-7 breast cancer cells. Cancer Res 42:139144[Medline]
-
Suck D 1994 DNA recognition by DNase I. J Mol Recognit 7:6570[Medline]
-
Wang F, Hoivik D, Pollenz R, Safe S 1998 Functional and
physical interactions between the estrogen receptor Sp1 and nuclear
arcyl hydrocarbon receptor complexes. Nucleic Acids Res 26:30443052[Abstract/Free Full Text]
-
Duan R, Porter W, Safe S 1998 Estrogen-induced c-fos
protooncogene expression in MCF-7 human breast cancer cells: role of
estrogen receptor Sp1 complex formation. Endocrinology 139:19811989[Abstract/Free Full Text]
-
Savouret J, Bailly A, Misrahi M, Rauch C, Redeuilh G,
Chauchereau A, Milgrom E 1991 Characterization of the hormone
responsive element involved in the regulation of the progesterone
receptor gene. EMBO J 10:18751883[Abstract]
-
Gronemeyer H, Turcotte C, Quirin-Stricker C, Bocquel M, Meyer
M, Krozowski Z, Jeltsch J, Lerouge T, Garnier J, Chambon P 1987 The
chicken progesterone receptor: sequence, expression and functional
analysis. EMBO J 6:39853994[Abstract]
-
Dynan WS, Tjian R 1983 The promoter-specific transcription
factor Sp1 binds to upstream sequences in the SV40 early promoter. Cell 35:7987[Medline]
-
Gidoni D, Dynan WS, Tjian R 1984 Multiple specific contacts
between a mammalian transcription factor and its cognate promoters.
Nature 312:409413[Medline]
-
Briggs MR, Kadonaga JT, Bell SP, Tjian R 1986 Purification and
biochemical characterization of the promoter-specific transcription
factor, Sp1. Science 234:4752[Medline]
-
Clarke R, Brünner N, Katzenellenbogen BS, Thompson EW,
Norman MJ, Koppi C, Paik S, Lippman ME, Dickson RB 1989 Progression of
human breast cancer cells from hormone-dependent to hormone-independent
growth both in vitro and in vivo. Proc Natl Acad
Sci USA 86:36493653[Abstract]
-
Kuiper GGJM, Carlsson B, Grandien K, Enmark E, Haggblad J,
Nilsson S, Gustafsson J-A 1997 Comparison of the ligand binding
specificity and transcript tissue distribution of estrogen
receptors
and ß. Endocrinology 138:863870[Abstract/Free Full Text]
-
Oñate SA, Tsai SY, Tsai M-J, OMalley BW 1995 Sequence
and characterization of a coactivator for the steroid hormone receptor
superfamily. Science 270:13541357[Abstract]
-
Ogryzko VV, Schiltz RL, Russanova V, Howard BH, Nakatani Y 1996 The transcriptional coactivators p300 and CBP are histone
acetyltransferases. Cell 87:953959[Medline]
-
Smith CL, Oñate SA, Tsai M-J, OMalley BW 1996 CREB
binding protein acts synergistically with steroid receptor
coactivator-1 to enhance steroid receptor-dependent transcription. Proc
Natl Acad Sci USA 93:88848888[Abstract/Free Full Text]
-
Thenot S, Henriquet C, Rochefort H, Cavailles V 1997 Differential interaction of nuclear receptors with the putative human
transcriptional coactivator hTIF1. J Biol Chem 272:1206212068[Abstract/Free Full Text]
-
Hong H, Kulwant K, Trivedi A, Johnson DL, Stallcup MR 1996 GRIP1, a novel mouse protein that serves as a transcriptional
coactivator in yeast for the hormone binding domains of steroid
receptors. Proc Natl Acad Sci USA 93:49484952[Abstract/Free Full Text]
-
Anzick SL, Kononen J, Walker RL, Azorsa DO, Tanner MM, Guan
X-Y, Sauter G, Kallioniemi O-P, Trent JM, Meltzer PS 1997 AIB1, a
steroid receptor coactivator amplified in breast and ovarian cancer.
Science 277:965968[Abstract/Free Full Text]
-
Norris JD, Fan D, Stallcup MR, McDonnell DP 1998 Enhancement
of estrogen receptor transcriptional activity by the coactivator GRIP-1
highlights the role of activation function 2 in determining estrogen
receptor pharmacology. J Biol Chem 273:66796688[Abstract/Free Full Text]
-
Torchia J, Rose D, Inostroza J, Kamei Y, Westin S, Glass C,
Rosenfeld M 1997 The transcriptional co-activator p/CIP binds CBP and
mediates nuclear-receptor function. Nature 387:677684[CrossRef][Medline]
-
Hamstein B, Eckner R, DiRenzo J, Halachmi S, Liu H, Searcy B,
Kurokawa R, Brown M 1996 p300 is a component of an estrogen receptor
coactivator complex. Proc Natl Acad Sci USA 21:1154011545[CrossRef]
-
Ing NH, Beekman JM, Tsai SY, Tsai M-J, OMalley BW 1992 Members of the steroid hormone receptor superfamily interact with TFIIB
(S300-II). J Biol Chem 267:1761717623[Abstract/Free Full Text]
-
Jacq X, Brou C, Lutz Y, Davidson I, Chambon P, Tora L 1994 Human TAFII30 is present in a distinct TFIID
complex and is required for transcriptional activation by the estrogen
receptor. Cell 79:107117[Medline]
-
Sabbah M, Kang K, Tora L, Redeuilh G 1998 Oestrogen receptor
facilitates the formation of preinitiation complex assembly:
involvement of the general transcription factor TFIIB. Biochem J 336:639646[Medline]
-
Emili A, Greenblatt J, Ingles J 1994 Species-specific
interaction of the glutamine-rich activation domains of Sp1 with the
TATA box-binding protein. Mol Cell Biol 14:15821593[Abstract]
-
Tanese N, Saluja D, Vassallo M, Chen J-L, Admon A 1996 Molecular cloning and analysis of two subunits of the human TFIID
complex: hTAFII130 and h
TAFII100. Proc Natl Acad Sci USA 93:1361113616[Abstract/Free Full Text]
-
Chiang C-M, Roeder RG 1995 Cloning of an intrinsic human TFIID
subunit that interacts with multiple transcription activators. Science 267:531536[Medline]
-
Gill G, Pascal E, Tseng ZH, Tjian R 1994 A glutamine-rich
hydrophobic patch in transcription factor Sp1 contacts the
dTAFII110 component of the Drosophila
TFIID complex and mediates transcriptional activation. Proc Natl Acad
Sci 91:192196[Abstract]
-
Soule H, Vasquez J, Long A, Albert S, Brennan M 1973 A human
cell line from a pleural effusion derived from breast carcinoma. J
Natl Cancer Inst 51:14091416[Medline]
-
Katzenellenbogen BS, Norman MJ 1990 Multihormonal regulation
of the progesterone receptor in MCF-7 human breast cancer cells:
interrelationships among insulin/ insulin-like growth factor-I,
serum, and estrogen. Endocrinology 126:891898[Abstract]
-
Chang T-C, Nardulli AM, Lew D, Shapiro DJ 1992 The role of
estrogen response elements in expression of the Xenopus
laevis vitellogenin B1 gene. Mol Endocrinol 6:346354[Abstract]
-
Mueller PR, Wold B 1989 In vivo footprinting of a
muscle specific enhancer by ligation mediated PCR. Science 246:780786[Medline]
-
Kraus WL, Kadonaga JT 1998 p300 and estrogen receptor
cooperatively activate transcription via differential enhancement of
initiation and reinitiation. Genes Dev 12:331342[Abstract/Free Full Text]
-
Chodosh LA 1989 Mobility shift DNA-binding assay using gel
electrophoresis. In: Current Protocols in Molecular
Biology. Greene Publishing Associates and Wiley Interscience, New York,
pp 12.12.1112.12.10
-
Nardulli AM, Lew D, Erijman L, Shapiro DJ 1991 Purified
estrogen receptor DNA binding domain expressed in Escherichia
coli activates transcription of an estrogen-responsive promoter in
cultured cells. J Biol Chem 266:2407024076[Abstract/Free Full Text]
-
Petz L, Nardulli A, Kim J, Horwitz K, Freedman L, Shapiro D 1997 DNA bending is induced by binding of the glucocorticoid receptor
DNA binding domain and progesterone receptor to their response element.
J Steroid Biochem Mol Biol 60:3141[CrossRef][Medline]
-
Nardulli AM, Grobner C, Cotter D 1995 Estrogen
receptor-induced DNA bending: orientation of the bend and replacement
of an estrogen response element with an intrinsic DNA bending sequence.
Mol Endocrinol 9:10641076[Abstract]
-
Reese JC, Katzenellenbogen BS 1991 Differential DNA-binding
abilities of estrogen receptor occupied with two classes of
antiestrogens: studies using human estrogen receptor overexpressed in
mammalian cells. Nucleic Acids Res 19:65956602[Abstract]
-
Nielsen DA, Chang T-C, Shapiro DJ 1989 A highly sensitive
mixed-phase assay for chloramphenicol acetyl transferase activity in
cultured cells. Ann Biochem 179:1923
-
Herbomel P, Bourachot B, Yaniv M 1984 Two distinct enhancers
with different cell specificities coexist in the regulatory region of
polyoma. Cell 39:653662[Medline]