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Gene Expression by Estrogen in Estrogen Receptor-Containing Breast Cancer Cells via Upstream Half-Palindromic Estrogen Response Element Motifs
Departments of Molecular and Integrative Physiology, Cell and Structural Biology, University of Illinois and College of Medicine, Urbana, Illinois 61801
Address all correspondence and requests for reprints to: Dr. Benita S. Katzenellenbogen, Department of Molecular and Integrative Physiology, 524 Burrill Hall, 407 South Goodwin Avenue, University of Illinois, Urbana, Illinois 61801-3704. E-mail: katzenel{at}uiuc.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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(PT), a protein associated with cell
proliferation and chromatin remodeling, and found to selectively
enhance ER transcriptional activity by interacting with a repressor of
ER activity, is shown to be a primary response gene to estrogen.
Prothymosin mRNA was rapidly increased by estrogen, followed by a
6-fold increase in prothymosin protein content in ER-containing
breast cancer cells. Analysis of the prothymosin promoter and
5'-flanking region, and electrophoretic gel mobility shift studies
showed the strong inducibility by the estradiol-ER complex to be
mediated by two consensus half-palindromic estrogen response elements
at -750 and -1051, which directly bind the ER. Estrogenic stimulation
of prothymosin required a DNA binding form of ER with a functional
activation function-2 domain. The prothymosin 5'-regulatory region
also contains multiple Sp1 sites. Although addition of Sp1 did not
further enhance estradiol-ER stimulated prothymosin transcriptional
activity in breast cancer cells, transfection and response element
mutagenesis studies using Drosophila cells, which are deficient in Sp1,
revealed that Sp1 and the estradiol occupied-ER can each activate the
prothymosin gene independently of the other and act in an additive
manner. These observations, documenting robust prothymosin
up-regulation by the estradiol-ER complex via widely spaced
half-palindromic estrogen response element motifs, are reminiscent of
those shown previously for the ovalbumin gene and suggest that the use
of multiple half response elements may be a more common mode for
regulation of gene expression by the ER than previously appreciated. In
addition, these observations suggest interrelationships between cell
proliferation and gene transcriptional activities and indicate a
positive mechanism by which PT, which increases ER transcriptional
effectiveness, is itself up-regulated by the estrogen-ER
complex. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and/or ß. In ER-positive breast cancer cells, estrogens promote
proliferation and invasiveness (2, 3, 4). Different studies
have shown that the two ERs mediate many of their effects by binding to
specific DNA sequences, the estrogen response elements (EREs),
initiating the transcription of target genes (5).
The magnitude of ER transcriptional activity is now well known to be
modulated by coregulators. In previous studies aimed at identifying
coregulators of the ER, we characterized a repressor of ER activity
(REA) that directly interacted with the liganded ER and competed with
coactivator for binding to ER (6, 7). Through two-hybrid
screening and additional studies, we subsequently identified
prothymosin (PT) as a binding partner of REA (8).
The interaction of PT with REA sequesters REA away from ER, enabling
coactivator association with ER and resulting in enhanced ER
transcriptional effectiveness (8).
PT, a 12.5-kDa protein, has been shown to be associated with cell
proliferation and has been proposed as a breast tumor prognostic marker
(9, 10, 11). Because estrogen stimulates the proliferation of
ER-containing breast cancer cells and, as noted above, PT
selectively enhances ER transcriptional activity via its interaction
with REA (8), we asked whether estrogen might up-regulate
PT in ER-positive breast cancer cells. In the study reported herein,
we found that PT gene expression is rapidly increased by estrogen.
To understand the mechanism of this hormonal regulation, we analyzed
the 5-kb promoter region of the PT gene (12) and have
identified half-palindromic estrogen response elements responsible for
the robust up-regulation by the estradiol-ER complex. It is intriguing
that PT, a protein that enhances estrogen-ER transcriptional
effectiveness, is itself up-regulated by estrogen.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Plasmids
The pCMV5 expression vectors for the human ER,
human ERß (530 residues), pCMV-PRB (progesterone receptor B), pRSV-GR
(glucocorticoid receptor), pCMV-REA (7), pCMV-ERDBDmut
(missing amino acids 185–251) and pCMV-ERS554fs (lacking activation
function-2 activity) have been described (13, 14). The
expression vector, pBK-CMV-SRC-1 (15), was kindly provided
by Drs. Ming Tsai and Bert O’Malley (Baylor College of Medicine,
Houston, TX). The plasmids pPst-PT-CAT and pApa-PT-CAT
(12) were kindly provided by Dr. Shelby Berger (NIH,
Bethesda, MD). The plasmids pPac Sp1 and pPac0 were kindly provided by
Dr. Robert Tjian (Howard Hughes Medical Institute, University of
California, Berkeley, CA). pPac-ER was cloned into pPac0 using
BamHI sites and pSp1-TK-CAT was cloned using an
oligonucleotide containing an Sp1 binding site into
HindIII/BamHI pTZ-TK-CAT. The plasmid pCMVß
(CLONTECH Laboratories, Inc., Palo Alto, CA) was
used as a ß-galactosidase internal control for transfection
efficiency, and all CAT activity measurements were corrected for
ß-galactosidase activity (16, 17).
The mutations of the proximal and the distal EREs of the PT promoter
region were generated using the Quick Change site-directed mutagenesis
kit (Stratagene, La Jolla, CA) and the following
oligos: forward (fw) 5'-CTG AGG CTG TCG CGA CGG CAG TGC TCG CTC GAG ACA
GAC CCT AAC TAG A-3', reverse (rv) 5'-TCT AGT TAG GGT CTG TCT CGA GCG
AGC ACT GCC GTC GCG ACA GCC TCA G-3', fw 5'-GGT GCC CGG GTC TCG GCC GCG
TGC TCT CGT TGC TCG TCG TGG GGC TGC C-3', rv 5'-GGC AGC CCC ACG ACG AGC
AAC GAG CAC GCG GCC GAG ACC CGG GCA CC-3'.
Cell culture and transfection
MCF-7 human breast cancer cells, Chinese hamster ovary (CHO)
cells, and MDA-MB-231 human breast cancer cells were maintained in cell
culture and transfected by lipofectin method (18) as
previously described (19). CHO cells were transfected in
24-well plates with 0.7 µg pPst-PT-CAT or 0.7 µg pApa-PT-CAT,
0.2 µg pCMVß (ß-galactosidase internal control plasmid), with
receptor expression plasmid, either 5 ng pCMV5-ER or 10 ng
pCMV5-ERß, and carrier DNA. MDA-MB-231 cells in 24-well plates were
transfected with 0.7 µg Pst-PT-CAT or 0.7 µg Apa-PT-CAT, 5 ng
CMV5-ER expression vector, 0.4 µg pCMVß, and carrier DNA. At
8 h after transfection, cells were treated with hormone or control
vehicle. Cells were harvested 24 h after hormone treatment and
cell extracts were prepared. ß-galactosidase activity, which was
measured to normalize for transfection efficiency, and CAT activity
were assayed as described (17).
Drosophila Schneider SL2 insect cells were purchased from
ATCC (Manassas, VA) and cultured as described
(20) with some modifications. Streptomycin (100 µg/ml)
and penicillin G (100 IU/ml) were added to the medium. Cells were
transfected in 24-well plates using Fugene (Roche
Diagnostics, Indianapolis, IN) according to the user manual provided,
with 0.7 µg pPst-PT-CAT or 0.7 µg pApa-PT-CAT or 0.7 µg
pSp1-TK-CAT, 100 ng pPac-ER, indicated amounts of pPac-Sp1 and 1
µg pCMVß (ß-galactosidase internal control plasmid). At 24 h
after transfection, cells were washed and treated with hormone for
48 h. Cells were then harvested and cell extracts assayed for
ß-galactosidase and CAT activity.
Isolation of RNA
Total RNA was isolated from MCF-7 and MDA-MB 231-ER stable cells
using the RNA Stat-60 extraction kit (Tel-Test, Inc.,
Friendswood, TX) following the manufacturer’s instructions.
Northern blot analysis
Gel purified PT and 36B4 cDNAs were random primer-labeled
using the Redi-Prime II DNA labeling kit from Amersham Pharmacia Biotech Hybridization of RNA from MCF-7 and 231-ER stably
transfected breast cancer cells was performed in Expresshyb
hybridization solution (CLONTECH Laboratories, Inc.) at 65
C for 18 h. Signal intensity was quantified by phosphorimager
analysis and normalized using 36B4 RNA as the internal control.
Western blot analysis
MCF-7 cells were harvested after hormone treatment from
100-mm dishes and resuspended in 100 µl of lysis buffer (150
mM NaCl, 1% NP-40, 50 mM Tris-HCl pH 8,
containing protease inhibitors: 0.2 mM PMSF, 5.0 µg/ml
aprotinin, 2.0 µg/ml leupeptin, 1.0 µg/ml pepstatin A). Whole cell
extracts were obtained by subjecting cells to three rounds of freezing
on dry ice and thawing at 37 C followed by centrifugation at
15,000 x g to remove cell debris. Approximately 200
µg of total cell extract was loaded on a 15% SDS-polyacrylamide gel.
Electrophoresis and Western blotting were done according to standard
methods (13). Nylon membranes were blocked with 0.5%
glutaraldehyde and were probed with human PT primary antibody
(ImmunDiagnostik, Bensheim, Germany) at 2.0 µg/ml and were then
incubated with goat antirabbit IgG at 1 µg/ml, and detected with the
ECL Plus Western blotting detection reagents (Amersham Pharmacia Biotech, Piscataway, NJ).
Electrophoretic gel mobility shift assay
The oligonucleotides for the electrophoretic mobility shift
assay and for the competition assay were as follows: proximal ERE,
5'-GAT CTC GGC CTC GTG ACC TCG TTG CTC GTC GA-3' and
complement 5'-GAT CTC GAC GAG CAA CGA GGT CAC GAG GCC GA-3', distal
ERE, 5'-GAT CTG ACG GCA GTG ACC GCT CGG GAC AGA CA-3' and
complement 5'-GAT CTG TCT GTC CCG AGC GGT CAC TGC CGT CA-3', half
nonconsensus ERE 5'-GAT CTA CCT ATT CTG GTC CTT TTT CCC ACA
CA-3' and complement 5'-GAT CTG TGT GGG AAA AAG GAC CAG AAT AGG TA-3'
and 55 bp of a consensus ERE (21). The single-stranded
oligomers, which contain either half-consensus ERE or half nonconsensus
ERE, were annealed to their complement. The resultant double-stranded
oligomers were gel purified and 32P-labeled as
described (22) and combined with 300 fmol of purified
E2-occupied ER made in baculovirus (22, 23) kindly
provided by Dr. Ann Nardulli (University of Illinois, Urbana, IL), in
binding reaction buffer (20 mM KCl, 50 ng
poly(dI-dC) in a final volume of 20 µl and incubated for 15 min at
room temperature. For the competition assay, a radiolabeled consensus
palindromic ERE was incubated with several concentrations of the
radioinert oligonucleotides described above. Free and complexed DNAs
were separated on nondenaturing acrylamide gel as described
(21), and signal intensity was visualized and quantified
by phosphorimager analysis.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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mRNA and protein levels are up-regulated by estrogen was a major protein
binding partner for the repressor of REA and that REA bound not only to
ER but also to PT, we investigated whether estrogen might regulate
the levels of PT in breast cancer cells. As seen in Fig. 1A
, estradiol exposure markedly increased
PT mRNA levels in MCF-7 or MDA-MB-231 ER- containing (231/ER+)
breast cancer cells, and it did so in a time-dependent manner. The
level of PT mRNA increased as early as 1–2 h after treatment with
hormone, peaked at 4 h, and declined slowly thereafter (Fig. 1A).
Treatment of cells with cycloheximide under conditions that block
>95% of protein synthesis failed to reduce the estradiol stimulation
of PT mRNA (Fig. 1B), implying that the increase in PT mRNA by
estrogen is a primary response not dependent on prior protein
synthesis. The increase in mRNA levels for PT was associated with
substantial elevation of PT protein level, as observed in Western
blot analysis using a polyclonal antibody made to PT (Fig. 1C).
PT protein level was increased 3-fold by 12–24 h and continued to
increase, reaching over 6-fold that of the control cells by 72
h.
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promoter by estrogen gene
expression regulation by estrogen, we examined the PT promoter and
5'-flanking region. Both the 5-kb promoter region, denoted PstPT,
and the shorter region, denoted ApaPT (Fig. 2A) were analyzed. Although no
palindromic ERE could be identified in this region, this 5-kb region
(as shown in Fig. 2A) contains three half-estrogen response elements,
the most proximal of which is present within the smaller -990
ApaI fragment. The estrogen inducibility of these
promoter-5' region constructs was investigated (Fig. 2B) in
transfection experiments in ER-negative MDA-MB-231 breast cancer cells
(or in CHO cells, where very similar results were obtained). Both the
PstPT and ApaPT constructs showed significant estradiol
concentration-dependence for inducibility by the ER (Fig. 2B). Of note,
the ApaPT construct showed stimulation by estrogen of a magnitude
only approximately half that observed in the 5-kb PstPT construct,
an effect that we found to be correlated with the involvement of both
the proximal and distal half-ERE sites in determining the degree of
estrogen stimulation of PT, as investigated further below. The
stimulation by estradiol was reversed by the antiestrogens
trans-hydroxytamoxifen and ICI182,780 (Fig. 2C).
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, was able to stimulate PT gene promoter
activity, and the stimulation via either ER was repressed by REA and
was enhanced by the coactivator SRC-1 (Fig. 3A). Furthermore, stimulation of either
the 5-kb PstPT or the shorter 0.99-kb ApaPT construct was
selective for the estrogen occupied-ER; no stimulation was observed
with the liganded progesterone receptor or glucocorticoid receptor
(Fig. 3B).
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promoter promoter. We used two different mutants of the ER for these
studies, the ERDBDmut missing amino acids 185–251, which lacks most of
the DNA binding domain of the ER, and the ER S554fs, which contains a
frameshift mutation at the end of the ligand binding domain and lacks
activation function-2 activity. As shown in Fig. 4, we observed no estrogen inducibility
of the PT promoter using either the 5-kb PstPT region or the
990-bp ApaPT region with either receptor mutant, suggesting that
transcriptional activation of PT requires direct binding of the ER
to DNA and that a functional AF-2 activity is required.
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promoter and
assessment of their involvement in estrogen inducibility of PT 5'-flanking region sequence has no
palindromic ERE. However, this region contains 3 widely spaced
half-EREs at positions -750, -1051, and -1437, as shown in Fig. 5A. Two of the half-EREs are consensus
(-750 and -1051, 5'-TGACC-3') but the one at position -1437
(5'-GGTCC-3', whose complement is 5'-GGACC-3') is not. In gel mobility
shift assay experiments (shown in Fig. 5B), only the two consensus
half-EREs, the proximal at -750 and the distal at -1051, were found
to bind to the ER, though less strongly than did a consensus full
ERE.
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). The proximal half-ERE and the
distal half-ERE showed significant abilities to compete for receptor
binding to the radiolabeled consensus ERE, while the nonconsensus
half-ERE showed no competition, even at extremely high levels of
competitor oligonucleotide. However, as seen in Fig. 5, right panel,
the half-EREs, whether the distal or proximal half-ERE, showed a
competitive ability only 1–2% that observed with the consensus
palindromic ERE. Hence, these gel shift assays indicate that binding of
the ER occurs at both proximal and distal half-ERE sites, possibly with
a slight affinity preference for the proximal half-ERE.
Site-directed mutagenesis reveals that two of the half-EREs are
responsible for PT inducibility by the ER
We mutated two base pairs known to eliminate ERE activity
(24) in the proximal, the distal, or both half-EREs in the
PstPT promoter region, and we mutated the proximal half-ERE
contained in the short ApaPT portion of the PT promoter region
(Fig. 6A). In assessing the estrogen
inducibility of these constructs, we observed that the single, proximal
half-ERE was fully responsible for the estrogen inducibility of the
ApaPT construct. In the full-length PstPT construct, both the
proximal and distal EREs contributed to the greater estrogen
stimulation observed with this full-length construct. Mutation of the
proximal half-ERE eliminated almost all of the activity of the PstPT
construct, whereas mutation of the distal half-ERE significantly
reduced estrogen inducibility, but less markedly than that observed
with mutation of the proximal site. Mutation of both of these EREs
completely eliminated estrogen inducibility, indicating that both are
required for the inducibility observed. These data support the findings
of the gel mobility shift assays in indicating that the nonconsensus
-1437 upstream half-ERE does not contribute to PT inducibility by
the ER.
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by the ER promoter region contains several Sp1 sites, in
addition to ERE half-sites, and the ER has been shown to cooperate with
Sp1 in promoting transcription activation of some genes such as the
progesterone receptor (25, 26), we investigated the
involvement of Sp1 in PT transcriptional regulation. For these
transfection experiments, we used Drosophila Schneider insect cells,
which are deficient in Sp1, and compared the results with similar
experiments conducted in MDA-MB-231 human breast cancer cells.
As shown in Fig. 7, Sp1 expression
plasmid transfection into Drosophila cells resulted in significant
stimulation of Sp1-TK-CAT, a control Sp1-responsive reporter plasmid,
indicating that Sp1 levels were indeed being increased in these cells.
In addition, Sp1 transfection resulted in a concentration-dependent
increase in PstPT-CAT activity, and this Sp1 stimulation of
PstPT-CAT activity was observed in the absence of any ER, consistent
with a process in which Sp1 is activating transcription via interaction
with its own Sp1 binding sites. Experiments with mutated half-EREs in
the PT construct confirmed this, because this construct
(PstPT-CAT, both EREs mut) showed equally good inducibility in
response to Sp1, but completely lost inducibility in response to the
estradiol-ER complex. Likewise, estradiol-occupied ER was able to
markedly stimulate PT activity in the absence of any Sp1 protein;
9-fold stimulation was observed with
10-10 M
estradiol and 10-8
M estradiol gave approximately 12-fold stimulation.
Interestingly, addition of Sp1 along with ER resulted in increases in
activity that were additive or slightly greater than additive at the
lower E2 concentration (Fig. 7). Furthermore, stimulation by the ER in
Drosophila cells, as shown previously in mammalian cells (Fig. 6),
required intact proximal and distal half-ERE sites. In contrast, Sp1
stimulation did not require intact wild-type half-ERE sites, suggesting
that Sp1 stimulation occurs independently of the ERE sites via its own
Sp1 response elements (Fig. 7).
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), we observed no significant effect of
transfected Sp1 on PT promoter activity, probably due to the high
endogenous Sp1 expression in mammalian cells (20). As seen
in Fig. 8, Sp1-TK-CAT activity and PstPT-CAT activity basal levels
were only marginally increased by transfected Sp1 expression plasmid.
Likewise, Sp1 transfection had no effect on PT promoter activity
above that due to the E2-occupied ER alone in these cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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gene expression in breast cancer cells, and that this estrogen
inducibility requires functional ER and is mediated via two upstream
half-palindromic 5'-TGACC-3' motifs. Our mutagenesis studies, in which
the role of each TGACC box half-ERE was investigated by mutating each
individually, revealed that the proximal and distal half-ERE each
contributed to the full estrogen induction, with the proximal motif
being of somewhat greater importance. Mutation of both the proximal and
distal half-EREs completely abolished estrogen induction, confirming
that good activation of transcription by the ER can be mediated by two
widely separated half-palindromic motifs. This is of interest as
estrogen target genes have been considered to most often contain
nonconsensus or consensus palindromic EREs in their 5'-regulatory
regions consisting of two half-estrogen response elements separated by
3 bp. Increasingly, however, gene regulation by estrogen has been found
to involve multiple half-estrogen response elements, such as in the
ovalbumin gene (27) and now observed for PT also.
The need for estrogen receptors to contain an intact DNA binding domain
for stimulation of PT transcription, and the failure of
cycloheximide to affect the rapid stimulation of PT mRNA by
estrogen, suggest that ER regulation of PT involves the direct
binding of receptor to these half-EREs, as demonstrated in our gel
shift studies. Interestingly, our competitive gel shift assays indicate
that ER binds to the proximal and distal half-EREs with an affinity
only approximately 1–2% of that for binding to a consensus
palindromic ERE. Nonetheless, these sites are clearly capable of
mediating activation of PT transcription by the ER. Similar
consensus half-EREs upstream of the ovalbumin gene are also able to
confer the strong estrogen inducibility of the ovalbumin gene
(27).
Because nonconsensus palindromic full EREs as in the
estrogen-responsive progesterone receptor gene or pS2 gene, show
substantially reduced affinity for binding the ER (24), it
is not surprising that ER binding to the nonconsensus half-ERE in the
PT promoter was not observable in direct EMSAs or competitive gel
shift assays. Our mutagenesis studies, in which estrogen stimulation
was completely abolished by mutation of the proximal and distal
consensus half-EREs, also indicate that this nonconsensus half site is
not involved in ER regulation of the PT gene.
In addition to containing half-palindromic ERE motifs, the PT
promoter also contains multiple Sp1 sites. Interesting studies have
demonstrated that Sp1 and ERE half-site motifs play a role in the
regulation of several estrogen-inducible genes including cathepsin D,
RAR-, heat shock protein 27, IGF-binding protein-4
(28), c-myc, creatine kinase B, and
progesterone receptor A (25, 26); this regulation does not
involve direct DNA binding by the ER, but instead is associated with ER
binding to the Sp1 protein and enhancement of Sp1 binding at Sp1 sites.
We, therefore, investigated the possible role of ER-Sp1 interactions in
estrogen stimulation of PT.
Our data indicate that estrogen up-regulation of PT occurs by a
different mechanism that 1) requires the two half-EREs in the PT
promoter and is eliminated by mutation of these response elements; 2)
requires an ER with an intact DNA binding domain; and 3) is associated
with ER binding to the two consensus half-ERE sites in gel mobility
shift and competitive binding studies. Further, it is of interest that
ER/Sp1 synergy is known to be promoted by ER but only weakly or not
at all by ERß (29), whereas we observe that PT
stimulation by E2 is capable of being mediated by ERß as well as by
ER.
Our studies in Drosophila SL cells, which are deficient in Sp1,
indicate that Sp1 plays a role in regulating the PT promoter. The
estradiol-occupied ER in the absence of Sp1 can markedly stimulate
PT promoter activity, and the PT promoter in the absence of ER
and estrogen can be stimulated by high concentrations of Sp1,
presumably via the multiple Sp1 sites in the promoter. However, when
both ER and Sp1 were present, their effects on PT were additive. In
addition, mutation of the proximal and distal consensus half-EREs in
the 5'-regulatory region completely eliminated the response to the
E2-ER complex but did not affect the stimulation by Sp1. These findings
suggest that both transcription regulators, ER and Sp1, mediate their
effects on PT independently, with the relative regulation by these
two proteins being determined by the relative levels of ER, estrogen
ligand, and Sp1, the activity of each being mediated via its own ERE or
Sp1 binding sites. Interestingly, in breast cancer cells where
endogenous Sp1 levels are high, we could not demonstrate any effect of
additional transfected Sp1 on PT-reporter constructs, or on
Sp1-reporter constructs.
PT is a chromatin remodeling protein known to be induced upon
growth stimulation (30, 31) and to promote cell
proliferation when introduced into cells (9). PT
modulates the interaction of histone H1 with chromatin (32, 33), and it has been proposed to show activity in nucleosome
assembly assays (34) and to modulate histone
acetyltransferase activity (35). Because it is a marker of
cell proliferation and cell cycle regulation (10), PT
is of prognostic value in breast cancer (36, 37). It is
perhaps not surprising then that estrogen, a major stimulator of
proliferation of ER-containing breast cancer cells, would also be a
significant regulator of the expression of PT. Our findings of rapid
estrogen up-regulation of PT mirror those of rapid estrogen
stimulation of PT observed in an ER-containing neuroblastoma cell
line by differential RNA display methods (38).
We recently reported that PT interacts with a repressor of
estrogen receptor activity, denoted REA (7, 39), and that
PT selectively enhances ER transcriptional activity but not that of
other nuclear receptors (8). PT does so by selectively
binding to REA and sequestering it away from ER, thereby allowing
increased association of coactivators with the ER with resultant
enhancement of ER transcriptional activity. Our current observation
that estrogen exposure increases PT levels in ER-containing
cells suggests a process by which the transcriptional effectiveness of
ER could be increased by a positive feed forward mechanism. Thus, under
estrogen dominated conditions, elevated PT levels would enhance the
recruitment of coactivators vs. corepressors to the ER,
resulting in enhanced transcriptional activity of the ER, including
increased transcription of the PT gene. This association of
increased ER transcriptional activity with proliferative activity of
cells is a fascinating one, that will require additional confirmation
in the future (40). Our findings reported herein reveal
the involvement of half-EREs that cooperate in estrogen regulation of
gene expression that is reminiscent of similar findings for the
ovalbumin gene (27). As more estrogen-regulated genes are
identified and their regulatory elements characterized, this paradigm
for gene regulation by estrogen may prove to be more common than
previously appreciated.
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Acknowledgments |
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Footnotes |
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Abbreviations: CHO, Chinese hamster ovary; ERE, estrogen
response element; fw, forward; GR, glucocorticoid receptor; PRB,
progesterone receptor B; PT, prothymosin ; REA, repressor
of ER activity; rv, reverse.
Received January 17, 2001.
Accepted for publication April 5, 2001.
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References |
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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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V. Bourdeau, J. Deschenes, R. Metivier, Y. Nagai, D. Nguyen, N. Bretschneider, F. Gannon, J. H. White, and S. Mader Genome-Wide Identification of High-Affinity Estrogen Response Elements in Human and Mouse Mol. Endocrinol., June 1, 2004; 18(6): 1411 - 1427. [Abstract] [Full Text] [PDF]
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 K. Lai, D. C. Harnish, and M. J. Evans Estrogen Receptor {alpha} Regulates Expression of the Orphan Receptor Small Heterodimer Partner J. Biol. Chem., September 19, 2003; 278(38): 36418 - 36429. [Abstract] [Full Text] [PDF]
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P. Ciana, S. Ghisletti, P. Mussi, I. Eberini, E. Vegeto, and A. Maggi Estrogen Receptor {alpha}, a Molecular Switch Converting Transforming Growth Factor-{alpha}-mediated Proliferation into Differentiation in Neuroblastoma Cells J. Biol. Chem., August 22, 2003; 278(34): 31737 - 31744. [Abstract] [Full Text] [PDF]
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S. Harendza, D. H. Lovett, U. Panzer, Z. Lukacs, P. Kuhnl, and R. A. K. Stahl Linked Common Polymorphisms in the Gelatinase A Promoter Are Associated with Diminished Transcriptional Response to Estrogen and Genetic Fitness J. Biol. Chem., May 30, 2003; 278(23): 20490 - 20499. [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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J. Sun, Y. R. Huang, W. R. Harrington, S. Sheng, J. A. Katzenellenbogen, and B. S. Katzenellenbogen Antagonists Selective for Estrogen Receptor {alpha} Endocrinology, March 1, 2002; 143(3): 941 - 947. [Abstract] [Full Text] [PDF]
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