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Department of Molecular and Integrative Physiology University of Illinois Urbana, Illinois 61801
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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and will hereafter be referred to as
the half-ERE/Sp1 binding site.
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, 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.
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, 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.
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, 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 Sp2–4 (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).
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, lanes 3–5 and 8–10).
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 3–5). 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 3–5). 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 8–10). 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.
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, lanes 2–5). 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
3–5). 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).
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, 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
8–10). 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.
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, 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 3–7), or
E2-occupied (lanes 9–13) 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.
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, 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 1–4), 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 6–9), 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
0.33–1.3 pmol of purified ER incrementally enhanced the protection of
both the proximal and distal Sp1 sites (lanes 4–6). 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 9–12). 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.
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, lane 1, ER). When the amount of
purified ER protein was decreased to 50 fmol, a fainter gel shifted
band was produced (lanes 2–6,ER). Addition of 0.25, 0.5, 1.5, or 3
ng of purified Sp1 protein (lanes 3–6, 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.
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, lanes 1–5) or
purified Sp1 and ER (lanes 6–10) 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 6–9), 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.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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(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 Sp1’s
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 7–57 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.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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 30–60 µg of MCF-7 nuclear extract, 12.5–37.5 ng of
purified Sp1 protein (Promega Corp., Madison, WI), or 15
ng of purified Sp1 and 0.13–1.3 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 (1–2
U) (Promega Corp., Madison, WI) was added to each sample
and incubated at room temperature for 0.75–8 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, 0.25–3 ng of purified Sp1
protein, or 0.25 ng of purified Sp1 and 50–700 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.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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This research was supported by United States Army Medical Research and Materiel Command Grant DAMD17–96-1–6267 and NIH Grants R29 HD-31299 and DK-53884 (to A.M.N.). Postdoctoral support for L. Petz was provided by USMRMC Grant DAMD17–97-1–7201 and NIH Reproductive Training Grant PHS 2T32 HD-0728–19.
Received for publication September 7, 1999. Revision received April 6, 2000. Accepted for publication April 7, 2000.
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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and ß. Endocrinology 138:863–870This article has been cited by other articles:
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  S. Safe and K. Kim Non-classical genomic estrogen receptor (ER)/specificity protein and ER/activating protein-1 signaling pathways J. Mol. Endocrinol., November 1, 2008; 41(5): 263 - 275. [Abstract] [Full Text] [PDF]
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 A. K. Rao, Y. S. Ziegler, I. X. McLeod, J. R. Yates, and A. M. Nardulli Effects of Cu/Zn Superoxide Dismutase on Estrogen Responsiveness and Oxidative Stress in Human Breast Cancer Cells Mol. Endocrinol., May 1, 2008; 22(5): 1113 - 1124. [Abstract] [Full Text] [PDF]
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M. C. Velarde, Z. Zeng, J. R. McQuown, F. A. Simmen, and R. C. M. Simmen Kruppel-Like Factor 9 Is a Negative Regulator of Ligand-Dependent Estrogen Receptor {alpha} Signaling in Ishikawa Endometrial Adenocarcinoma Cells Mol. Endocrinol., December 1, 2007; 21(12): 2988 - 3001. [Abstract] [Full Text] [PDF]
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 J. Cheng, D. V. Yu, J.-H. Zhou, and D. J. Shapiro Tamoxifen Induction of CCAAT Enhancer-binding Protein {alpha} Is Required for Tamoxifen-induced Apoptosis J. Biol. Chem., October 19, 2007; 282(42): 30535 - 30543. [Abstract] [Full Text] [PDF]
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S. El Marzouk, J. R Schultz-Norton, V. S Likhite, I. X McLeod, J. R Yates, and A. M Nardulli Rho GDP dissociation inhibitor {alpha} interacts with estrogen receptor {alpha} and influences estrogen responsiveness J. Mol. Endocrinol., October 1, 2007; 39(4): 249 - 259. [Abstract] [Full Text] [PDF]
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 E. Higashi, T. Fukami, M. Itoh, S. Kyo, M. Inoue, T. Yokoi, and M. Nakajima Human CYP2A6 Is Induced by Estrogen via Estrogen Receptor Drug Metab. Dispos., October 1, 2007; 35(10): 1935 - 1941. [Abstract] [Full Text] [PDF]
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 M. H. Hassan, S. A. Salama, H. M. M. Arafa, F. M. A. Hamada, and A. Al-Hendy Adenovirus-Mediated Delivery of a Dominant-Negative Estrogen Receptor Gene in Uterine Leiomyoma Cells Abrogates Estrogen- and Progesterone-Regulated Gene Expression J. Clin. Endocrinol. Metab., October 1, 2007; 92(10): 3949 - 3957. [Abstract] [Full Text] [PDF]
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 J. R. Schultz-Norton, V. A. Gabisi, Y. S. Ziegler, I. X. McLeod, J. R. Yates, and A. M. Nardulli Interaction of estrogen receptor {alpha} with proliferating cell nuclear antigen Nucleic Acids Res., August 1, 2007; 35(15): 5028 - 5038. [Abstract] [Full Text] [PDF]
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J. R. Schultz-Norton, K. A. Walt, Y. S. Ziegler, I. X. McLeod, J. R. Yates, L. T. Raetzman, and A. M. Nardulli The Deoxyribonucleic Acid Repair Protein Flap Endonuclease-1 Modulates Estrogen-Responsive Gene Expression Mol. Endocrinol., July 1, 2007; 21(7): 1569 - 1580. [Abstract] [Full Text] [PDF]
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 L. Mauro, S. Catalano, G. Bossi, M. Pellegrino, I. Barone, S. Morales, C. Giordano, V. Bartella, I. Casaburi, and S. Ando Evidences that Leptin Up-regulates E-Cadherin Expression in Breast Cancer: Effects on Tumor Growth and Progression Cancer Res., April 1, 2007; 67(7): 3412 - 3421. [Abstract] [Full Text] [PDF]
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A. Romano, B. Delvoux, D.-C. Fischer, and P. Groothuis The PROGINS polymorphism of the human progesterone receptor diminishes the response to progesterone J. Mol. Endocrinol., February 1, 2007; 38(2): 331 - 350. [Abstract] [Full Text] [PDF]
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G. E. Weitsman, L. Li, G. P. Skliris, J. R. Davie, K. Ung, Y. Niu, L. Curtis-Snell, L. Tomes, P. H. Watson, and L. C. Murphy Estrogen Receptor-{alpha} Phosphorylated at Ser118 Is Present at the Promoters of Estrogen-Regulated Genes and Is Not Altered Due to HER-2 Overexpression. Cancer Res., October 15, 2006; 66(20): 10162 - 10170. [Abstract] [Full Text] [PDF]
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J. R. Schultz-Norton, W. H. McDonald, J. R. Yates, and A. M. Nardulli Protein Disulfide Isomerase Serves as a Molecular Chaperone to Maintain Estrogen Receptor {alpha} Structure and Function Mol. Endocrinol., September 1, 2006; 20(9): 1982 - 1995. [Abstract] [Full Text] [PDF]
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Y. Zhu, L. L. Sullivan, S. S. Nair, C. C. Williams, A. K. Pandey, L. Marrero, R. K. Vadlamudi, and F. E. Jones Coregulation of Estrogen Receptor by ERBB4/HER4 Establishes a Growth-Promoting Autocrine Signal in Breast Tumor Cells Cancer Res., August 15, 2006; 66(16): 7991 - 7998. [Abstract] [Full Text] [PDF]
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 G. B Silberstein, K. Van Horn, E. Hrabeta-Robinson, and J. Compton Estrogen-triggered delays in mammary gland gene expression during the estrous cycle: evidence for a novel timing system. J. Endocrinol., August 1, 2006; 190(2): 225 - 239. [Abstract] [Full Text] [PDF]
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 P. Haeger, M. E. Andres, M. I. Forray, C. Daza, S. Araneda, and K. Gysling Estrogen receptors alpha and beta differentially regulate the transcriptional activity of the Urocortin gene. J. Neurosci., May 3, 2006; 26(18): 4908 - 4916. [Abstract] [Full Text] [PDF]
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M L Panno, L Mauro, S Marsico, D Bellizzi, P Rizza, C Morelli, M Salerno, F Giordano, and S Ando' Evidence that the mouse insulin receptor substrate-1 belongs to the gene family on which the promoter is activated by estrogen receptor {alpha} through its interaction with Sp1 J. Mol. Endocrinol., February 1, 2006; 36(1): 91 - 105. [Abstract] [Full Text] [PDF]
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 W. M. Bryant, M. A. Gibson, and M. A. Shupnik Stimulation of the Novel Estrogen Receptor-{alpha} Intronic TERP-1 Promoter by Estrogens, Androgen, Pituitary Adenylate Cyclase-Activating Peptide, and Forskolin, and Autoregulation by TERP-1 Protein Endocrinology, January 1, 2006; 147(1): 543 - 551. [Abstract] [Full Text] [PDF]
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L. Bjornstrom and M. Sjoberg Mechanisms of Estrogen Receptor Signaling: Convergence of Genomic and Nongenomic Actions on Target Genes Mol. Endocrinol., April 1, 2005; 19(4): 833 - 842. [Abstract] [Full Text] [PDF]
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 A. Fritah, C. Saucier, J. Mester, G. Redeuilh, and M. Sabbah p21WAF1/CIP1 Selectively Controls the Transcriptional Activity of Estrogen Receptor {alpha} Mol. Cell. Biol., March 15, 2005; 25(6): 2419 - 2430. [Abstract] [Full Text] [PDF]
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Y-L Zhao, W-D Han, Q Li, Y-M Mu, X-C Lu, L Yu, H-J Song, X Li, J-M Lu, and C-Y Pan Mechanism of transcriptional regulation of LRP16 gene expression by 17-{beta} estradiol in MCF-7 human breast cancer cells J. Mol. Endocrinol., February 1, 2005; 34(1): 77 - 89. [Abstract] [Full Text] [PDF]
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J. R. Schultz, L. N. Petz, and A. M. Nardulli Cell- and Ligand-specific Regulation of Promoters Containing Activator Protein-1 and Sp1 Sites by Estrogen Receptors {alpha} and {beta} J. Biol. Chem., January 7, 2005; 280(1): 347 - 354. [Abstract] [Full Text] [PDF]
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N. Fujita, M. Kajita, P. Taysavang, and P. A. Wade Hormonal Regulation of Metastasis-Associated Protein 3 Transcription in Breast Cancer Cells Mol. Endocrinol., December 1, 2004; 18(12): 2937 - 2949. [Abstract] [Full Text] [PDF]
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R. O'Lone, M. C. Frith, E. K. Karlsson, and U. Hansen Genomic Targets of Nuclear Estrogen Receptors Mol. Endocrinol., August 1, 2004; 18(8): 1859 - 1875. [Abstract] [Full Text] [PDF]
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Y. Tsuchiya, M. Nakajima, S. Kyo, T. Kanaya, M. Inoue, and T. Yokoi Human CYP1B1 Is Regulated by Estradiol via Estrogen Receptor Cancer Res., May 1, 2004; 64(9): 3119 - 3125. [Abstract] [Full Text] [PDF]
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 J. Palacios, E. T. Marusic, N. C. Lopez, M. Gonzalez, and L. Michea Estradiol-induced expression of Na+-K+-ATPase catalytic isoforms in rat arteries: gender differences in activity mediated by nitric oxide donors Am J Physiol Heart Circ Physiol, May 1, 2004; 286(5): H1793 - H1800. [Abstract] [Full Text] [PDF]
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J. Cao, M. Wood, Y. Liu, T. Hoffman, J. Hyde, O.-K. Park-Sarge, and M. Vore Estradiol Represses Prolactin-Induced Expression of Na+/Taurocholate Cotransporting Polypeptide in Liver Cells through Estrogen Receptor-{alpha} and Signal Transducers and Activators of Transcription 5a Endocrinology, April 1, 2004; 145(4): 1739 - 1749. [Abstract] [Full Text] [PDF]
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L. N. Petz, Y. S. Ziegler, J. R. Schultz, and A. M. Nardulli Fos and Jun Inhibit Estrogen-Induced Transcription of the Human Progesterone Receptor Gene through an Activator Protein-1 Site Mol. Endocrinol., March 1, 2004; 18(3): 521 - 532. [Abstract] [Full Text] [PDF]
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A. J. Krieg, S. A. Krieg, B. S. Ahn, and D. J. Shapiro Interplay between Estrogen Response Element Sequence and Ligands Controls in Vivo Binding of Estrogen Receptor to Regulated Genes J. Biol. Chem., February 6, 2004; 279(6): 5025 - 5034. [Abstract] [Full Text] [PDF]
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V. Sriraman and J. S. Richards Cathepsin L Gene Expression and Promoter Activation in Rodent Granulosa Cells Endocrinology, February 1, 2004; 145(2): 582 - 591. [Abstract] [Full Text] [PDF]
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 J-C Lambert, N Coyle, and C Lendon The allelic modulation of apolipoprotein E expression by oestrogen: potential relevance for Alzheimer's disease J. Med. Genet., February 1, 2004; 41(2): 104 - 112. [Abstract] [Full Text] [PDF]
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S. Khan, M. Abdelrahim, I. Samudio, and S. Safe Estrogen Receptor/Sp1 Complexes Are Required for Induction of cad Gene Expression by 17{beta}-Estradiol in Breast Cancer Cells Endocrinology, June 1, 2003; 144(6): 2325 - 2335. [Abstract] [Full Text] [PDF]
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V. Sriraman, S. C. Sharma, and J. S. Richards Transactivation of the Progesterone Receptor Gene in Granulosa Cells: Evidence that Sp1/Sp3 Binding Sites in the Proximal Promoter Play a Key Role in Luteinizing Hormone Inducibility Mol. Endocrinol., March 1, 2003; 17(3): 436 - 449. [Abstract] [Full Text] [PDF]
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L. N. Petz, Y. S. Ziegler, M. A. Loven, and A. M. Nardulli Estrogen Receptor {alpha} and Activating Protein-1 Mediate Estrogen Responsiveness of the Progesterone Receptor Gene in MCF-7 Breast Cancer Cells Endocrinology, December 1, 2002; 143(12): 4583 - 4591. [Abstract] [Full Text] [PDF]
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S. L. Grimm, T. N. Seagroves, E. B. Kabotyanski, R. C. Hovey, B. K. Vonderhaar, J. P. Lydon, K. Miyoshi, L. Hennighausen, C. J. Ormandy, A. V. Lee, et al. Disruption of Steroid and Prolactin Receptor Patterning in the Mammary Gland Correlates with a Block in Lobuloalveolar Development Mol. Endocrinol., December 1, 2002; 16(12): 2675 - 2691. [Abstract] [Full Text] [PDF]
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 I. De Vivo, G. S. Huggins, S. E. Hankinson, P. J. Lescault, M. Boezen, G. A. Colditz, and D. J. Hunter A functional polymorphism in the promoter of the progesterone receptor gene associated with endometrial cancer risk PNAS, September 17, 2002; 99(19): 12263 - 12268. [Abstract] [Full Text] [PDF]
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M. Abdelrahim, I. Samudio, R. Smith III, R. Burghardt, and S. Safe Small Inhibitory RNA Duplexes for Sp1 mRNA Block Basal and Estrogen-induced Gene Expression and Cell Cycle Progression in MCF-7 Breast Cancer Cells J. Biol. Chem., August 2, 2002; 277(32): 28815 - 28822. [Abstract] [Full Text] [PDF]
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T. R. Ediger, S.-E. Park, and B. S. Katzenellenbogen Estrogen Receptor Inducibility of the Human Na+/H+ Exchanger Regulatory Factor/Ezrin-Radixin-Moesin Binding Protein 50 (NHE-RF/EBP50) Gene Involving Multiple Half-Estrogen Response Elements Mol. Endocrinol., August 1, 2002; 16(8): 1828 - 1839. [Abstract] [Full Text] [PDF]
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T. Bouras, M. C. Southey, A. C. Chang, R. R. Reddel, D. Willhite, R. Glynne, M. A. Henderson, J. E. Armes, and D. J. Venter Stanniocalcin 2 Is an Estrogen-responsive Gene Coexpressed with the Estrogen Receptor in Human Breast Cancer Cancer Res., March 1, 2002; 62(5): 1289 - 1295. [Abstract] [Full Text] [PDF]
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D. Zhang, X.-L. Zhang, F. J. Michel, J. L. Blum, F. A. Simmen, and R. C. M. Simmen Direct Interaction of the Kruppel-like Family (KLF) Member, BTEB1, and PR Mediates Progesterone-Responsive Gene Expression in Endometrial Epithelial Cells Endocrinology, January 1, 2002; 143(1): 62 - 73. [Abstract] [Full Text] [PDF]
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P. G. V. Martini and B. S. Katzenellenbogen Regulation of Prothymosin {alpha} Gene Expression by Estrogen in Estrogen Receptor-Containing Breast Cancer Cells via Upstream Half-Palindromic Estrogen Response Element Motifs Endocrinology, August 1, 2001; 142(8): 3493 - 3501. [Abstract] [Full Text] [PDF]
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C. M. Klinge Estrogen receptor interaction with estrogen response elements Nucleic Acids Res., July 15, 2001; 29(14): 2905 - 2919. [Abstract] [Full Text] [PDF]
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I. Samudio, C. Vyhlidal, F. Wang, M. Stoner, I. Chen, M. Kladde, R. Barhoumi, R. Burghardt, and S. Safe Transcriptional Activation of Deoxyribonucleic Acid Polymerase {{alpha}} Gene Expression in MCF-7 Cells by 17{{beta}}-Estradiol Endocrinology, March 1, 2001; 142(3): 1000 - 1008. [Abstract] [Full Text] [PDF]
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G. Giannoukos, D. Szapary, C. L. Smith, J. E. W. Meeker, and S. S. Simons Jr. New Antiprogestins with Partial Agonist Activity: Potential Selective Progesterone Receptor Modulators (SPRMs) and Probes for Receptor- and Coregulator-Induced Changes in Progesterone Receptor Induction Properties Mol. Endocrinol., February 1, 2001; 15(2): 255 - 270. [Abstract] [Full Text]
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E. Castro-Rivera, I. Samudio, and S. Safe Estrogen Regulation of Cyclin D1 Gene Expression in ZR-75 Breast Cancer Cells Involves Multiple Enhancer Elements J. Biol. Chem., August 10, 2001; 276(33): 30853 - 30861. [Abstract] [Full Text] [PDF]
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