Identification of a Novel Transferable cis Element in the Promoter of an Estrogen-Responsive Gene that Modulates Sensitivity to Hormone and Antihormone
Monica M. Montano,
W. Lee Kraus1 and
Benita S. Katzenellenbogen
Department of Molecular and Integrative Physiology (M.M.M.,
W.L.K., B.S.K.) Department of Cell and Structural Biology
(B.S.K.) University of Illinois, Urbana, Illinois 61801
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ABSTRACT
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The estrogen receptor (ER) is a
ligand-regulated transcription factor that acts at the promoters of
estrogen-regulated genes to modulate their expression. In the present
study, we examined three estrogen-regulated promoters, namely the rat
progesterone receptor gene distal
(PRD) and proximal
(PRP) promoters and the human pS2 gene
promoter, and observed marked differences in their sensitivity to
stimulation by estrogen and repression of estrogen-stimulated
transcription by antiestrogen (AE)-occupied ER. ER-containing MCF-7
human breast cancer cells were transfected with reporter gene
constructs containing estrogen response elements upstream of the three
gene promoters. In this system, PRP and
PRD showed similar dose-response curves for
stimulation by estradiol whereas pS2 was activated by even lower
concentrations of estradiol. By contrast, PRD
was much less sensitive to repression of estrogen-stimulated activity
by all AEs studied, relative to the PRP and the
pS2 promoters. Using deletion and mutational analysis, we have
identified a transferable cis element at -131 to -94 bp
in PRD that is involved in modulating the
sensitivity of this promoter to both estrogens and AEs. The element
reduced the magnitude of estrogen-stimulated activity, enhanced the
ability of AEs to repress estrogen-stimulated activity, and elicited
similiar effects when transferred to the promoter of another
estrogen-responsive gene. Thus, removal of this region from
PRD further accentuated the insensitivity of
this promoter to AE while enhancing its sensitivity (both
EC50 and fold induction) to estrogen. Gel
mobility shift assays showed that proteins from nuclear extracts of
MCF-7 cells interact with this element and that the binding of these
proteins is inversely correlated with the transcriptional effectiveness
of the ER. The findings demonstrate that a specific cis
element from the promoter of an estrogen-responsive gene can alter the
transcriptional activity of hormone and antihormone-occupied receptor
bound at its response element near the promoter. Such ligand response
modulatory elements, and changes in the levels and activity of factors
that bind to such elements, may underlie the different sensitivities of
steroid hormone-regulated genes to both hormones and antihormones.
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INTRODUCTION
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Steroid hormones, such as estrogen, modulate gene expression via
intracellular receptors that belong to a large superfamily of
hormone-regulated transcription factors. In the case of the estrogen
receptor (ER), the binding of estrogen initiates a process of receptor
activation that includes the high-affinity binding of ER to specific
DNA sequences, termed estrogen response elements (EREs). The
interaction of ER with EREs results in the modulation of specific gene
expression, through which the physiological actions of estrogens are
manifested (for reviews, see Refs. 15). The regulatory actions of
estrogens on gene expression, which are generally stimulatory, can be
inhibited by potent synthetic ER antagonists (6) termed antiestrogens
(AEs).
The promoters of many known estrogen-regulated genes are complex, with
binding sites for other transcription factors in addition to ER.
Positive and negative interactions between ER and these transcription
factors, which may be promoter- or cell-specific, provide an important
step at which ER function may be regulated (reviewed in Refs. 1, 4, and
5). A number of studies from this laboratory and others have
demonstrated the significance of promoter and cell context in
modulating responses to both estrogens and AEs (7, 8, 9).
In the present study, we observed marked differences in the
sensitivities of three estrogen-regulated promoters to repression by
AEs, suggesting the involvement of promoter-specific factors capable of
modulating the activity of the ER. Using several approaches, we have
identified a transferable cis element in the rat
progesterone receptor (PR) gene distal promoter that is involved in
modulating promoter sensitivity to both estrogens and AEs. Gel mobility
shift assays have been used to show that proteins from nuclear extracts
of MCF-7 human breast cancer cells interact with this ligand response
modulatory element (LRME) and that the binding of these proteins is
inversely correlated with the transcriptional effectiveness of ER. Our
results demonstrate that cis-acting elements in the promotor
region of estrogen-responsive genes can alter the transcriptional
activity of estrogen- and AE-occupied ER bound at its response element
near a promoter. Such ligand response modulatory elements may be
broadly applicable in the actions of many nuclear receptors in which
gene-specific modulation of hormonal induction is known, but the
underlying basis is poorly understood.
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RESULTS
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Examination of the Differential Sensitivity of Several
Estrogen-Stimulated Promoters to Repression by AEs
As shown in Fig. 1
, we analyzed three
estrogen-regulated promoters [the PR gene distal and proximal
promoters (PRD and PRP) and the promoter of the
human pS2 gene; pS2] for their relative sensitivity to the stimulatory
actions of estrogen and the repressive actions of AEs. We previously
cloned the 5'-flanking region of the rat PR gene and demonstrated the
presence of two promoters, a distal promoter (-131 to +65;
PRD) and a proximal promoter (+461 to +675;
PRP) (10), and we have shown that these promoters are
functionally distinct with respect to activation by ER-dependent
pathways (11). The PR distal and proximal promoters control production
of the B and A isoforms of the PR (ca. 120- and 90-kDa,
respectively), and the pS2 promoter regulates production of a growth
factor-like secreted protein whose function is not completely known
(12).

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Figure 1. Differential Sensitivity of Three Promoters to the
Inhibitory Actions of AEs
MCF-7 cells were transfected with CAT reporter constructs containing
two consensus EREs upstream of the pS2 promoter [-90 to +10;
(ERE)2-pS2-CAT], the proximal promoter of the rat PR gene
[-131 to +65; (ERE)2-PRP-CAT], or the distal
promoter of the rat PR gene [+461 to + 675;
(ERE)2-PRD-CAT], and a ß-galactosidase
expression plasmid, used as an internal control to correct for
transfection efficiency, as described in Materials and
Methods. The cells were then treated for 24 h with the
estrogen E2 (10-9 M), or the AE
ICI 164,384 (ICI, 5 x 10-7 M) or LY
117018 (LY, 10-6 M) alone or in the
combinations as indicated. Cell extracts were prepared and analyzed for
CAT activity as described in Materials and Methods. The
activity for each construct was expressed as a percent of the activity
observed with E2 treatment alone, which is set at 100%.
Each bar represents the mean of three or more separate
determinations + SEM. The numbers above the
E2 bar show the fold induction observed with E2
alone for each of the three promoter constructs.
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For the studies in Fig. 1
, MCF-7 human breast cancer cells, which
contain high levels of endogenous ER, were transfected with
chloramphenicol acetyltransferase (CAT) reporter constructs containing
two consensus EREs upstream of the pS2, PRP or
PRD promoters. Extracts from the cells were analyzed for
CAT activity after treatment with the lowest maximally stimulatory
concentration of estradiol (E2; 10-9
M) in the absence or presence of a 500- or 1000-fold excess
of the AEs, ICI 164,384 (ICI) or LY 117018 (LY), respectively. As shown
in Fig. 1
, E2 stimulated large (i.e. 50- to
130-fold) increases in the activity of the three promoter-reporter gene
constructs. The AEs (ICI and LY) alone evoked essentially no activity,
and they were able to repress greater than 90% of the
E2-stimulated CAT activity from either the pS2 or
PRP promoter-containing reporters. Of note, the AEs were
substantially less effective at repressing E2-stimulated
activity from the PRD-containing reporter (Fig. 1
; and
further investigated in Fig. 3
below).

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Figure 3. Dose-Dependent Stimulatory Effects of
E2 (Panel A) and Dose-Dependent Inhibitory Effects of ICI
on E2 (10-9 M)-Stimulated CAT
Activity (Panel B) Using the Three Estrogen-Responsive
Promoter-Reporter Constructs
Constructs are defined in Figs. 1 and 2 . The truncated PRD
is denoted PRD, B/N and lacks the -131 to -94
XmnI/BsmI fragment of the PRD
promoter. Each value represents the mean of three or more separate
determinations ± SEM.
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Characterization of a Region of the PRD
Promoter that Modulates Sensitivity to Estrogen and AE: Deletion and
Mutational Analyses
We further analyzed PRD to identify region(s) involved
in modulating the sensitivity of the promoter to the suppressive
effects of AE on E2-stimulated activity. Reporter
constructs containing two consensus EREs upstream of the full-length
PRD, or deletion mutants of PRD, were analyzed
for stimulation by E2 and repression by ICI. The results
are shown in Fig. 2A
. Deletion of the -131 to -94
XmnI/BsmI fragment from PRD (to
generate a truncated promoter denoted
PRD, B/N, since this truncated promoter
contains the region -94 to +65, which spans from the BsmI
to the NheI restriction sites) resulted in approximately
2-fold higher induction by E2 than was observed with the
full-length promoter (Fig. 2A
; compare line 2 with line 1).
Surprisingly, we also observed a 2-fold decrease in the sensitivity of
the deleted promoter to the inhibitory actions of ICI relative to the
full-length promoter (Fig. 2A
). This indicates, as shown in Fig. 3
also (see below), that the -131 to -94 region plays
a role in modulating the sensitivity of PRD to both
estrogens and AEs and that in its absence (as in PRD,B/N),
PRD gene responsiveness to estrogen and AE is even more
discordant than that of PRP and pS2.

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Figure 2. Identification of a Region of PRD
Involved in ER-Ligand Sensitivity
Panel A, Estrogen stimulation and AE (ICI) repression were examined
using (ERE)2-PRD-CAT or CAT reporter constructs
containing two EREs upstream of PRD deletion mutants (lines
15), or CAT reporter constructs containing the five natural
ERE-containing estrogen-responsive regions of the PR gene linked
together and placed upstream of PRD (line 6), which were
transfected into MCF-7 cells as described in the legend of Fig. 1 .
Differential responsiveness of the reporter constructs to
E2 (10-9 M) and to repression of
E2-stimulated activity by ICI (5 x 10-7
M) was monitored. The magnitude of transactivation of the
full-length PRD in response to E2 was set at
100%. Percent repression with ICI indicates the percent inhibition of
E2-stimulated activity observed for each reporter construct
upon cotreatment with 10-9 M E2
and 5 x 10-7 M ICI. Each value
represents the mean of three or more separate determinations ±
SEM. Panel B, Mutagenesis of the region from -131 to -80
encompassing the XmnI/BsmI fragment of
PRD results in differential responsiveness to
E2 (10-9 M) and repression of
E2-stimulated activity by ICI (5 x
10-7M). Shown is the nucleotide sequence of the -131 to
-80 region of the PRD promoter. The mutated nucleotides
are indicated by the boxed regions in mutants 1 to 6.
The CCAAT motif, Sp1-binding site, and putative binding site for
CTF/NFI are also indicated. CAT reporter constructs containing two EREs
upstream of the mutated PRD were examined for
E2 responsiveness and for suppression of
E2-mediated transactivation by ICI as described in the
legend of Fig. 1 . The magnitude of transactivation of wild type
PRD in response to E2 was set at 100%. Percent
repression with ICI indicates the percent inhibition of
E2-stimulated activity observed for each reporter construct
upon cotreatment with 10-9 M E2
and 5 x 10-7 M ICI. Each value
represents the mean of three or more separate determinations ±
SEM.
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Further deletion of the PRD promoter to -67 resulted in a
substantial loss of estrogen-inducible activity (line 3). Deletion from
the 3'-end of PRD (the +25 to +65 region) similarly reduced
promoter activity (line 4 vs. line 1), and further deletion
to +1 almost fully destroyed PRD activity (line 5), as
expected. We also tested PRD in the context of the natural
estrogen-responsive sequences of the PR gene (line 6). With the
ERE-like sequences contained in the five estrogen-responsive fragments
of the rat PR gene linked together and placed upstream of
PRD [5E-PRD-CAT (11)], we observed good
stimulation by E2 [50% of the magnitude observed with
(ERE)2-PRD-CAT, line 1] and the same poor
sensitivity to repression by ICI (Fig. 2A
, line 6, only 60%
repression) as seen with 2ERE-PRD-CAT (line 1). This
indicates that the unusual resistance of PRD to AE
inhibition is a function of the 5'-flanking region to -131 and not the
nature of the estrogen-response element regions.
Mutagenesis of the -131 to -84 region of PRD (Fig. 2B
)
identified a nucleotide sequence that appears to be involved in
conferring differential sensitivity to stimulation by estrogen and
repression by AEs. Introduction of mutations at -115 to -110 of the
PR gene distal promoter (Mut3) increased the magnitude of the response
to E2 (2-fold) and decreased the ability of ICI to suppress
E2-mediated transactivation with respect to the wild type
promoter construct, reproducing what was observed upon deletion of the
-131 to -94 region of PRD (Fig. 2A
, line 2). Mutations in
the nucleotide sequence corresponding to the putative CTF/NF-1 site
(Mut 4 and Mut 5) decreased E2-mediated transactivation
from PRD to 4050% of the wild type promoter, suggesting
an involvement of CTF/NF-1 nuclear factors in estrogen regulation of
PRD, possibly similar to that observed previously for the
vitellogenin B1 gene (13). Mutations at other sites within the -131 to
-84 region (Mut 1, 2, and 6) had relatively little effect on the
response to E2 or ICI.
Analysis of the Relationship between Estrogen Stimulation of the
Three Promoters and the Sensitivity of the Three Promoters to AE
Repression
We performed E2 and AE (ICI) dose-response experiments
using the estrogen-responsive reporter constructs containing the three
different promoters (PRD, PRP, and pS2). We
also assessed whether the decreased sensitivity of the -131 to -94
deleted PRD (i.e. the PRD,B/N
promoter) to AE was attributable to the greater sensitivity of
PRD, B/N to stimulation by E2
relative to PRD.
The PRP and PRD gene promoter constructs showed
similar dose-response curves for stimulation by E2 (Fig. 3A
), with half-maximal stimulation at approximately 3 x
10-11 M E2. The pS2
promoter-containing construct [(ERE)2-pS2-CAT] showed a
2- to 3-fold greater maximal CAT activity, and half-maximal activity
required 5- to 10-fold less E2 (
5 x
10-12 M E2, Fig. 3A
). The pS2 and
PRP promoter constructs were equally and highly sensitive
to suppression of E2-stimulated CAT activity by the AE ICI,
whereas the PRD promoter was much less sensitive to ICI
suppression over the entire concentration range tested (Fig. 3B
),
consistent with the data shown in Fig. 1
, in which only a single
concentration of ICI was used to inhibit E2 activation.
Thus, promoters that have similar sensitivities to the stimulatory
actions of estrogens can have very different sensitivities to the
inhibitory actions of AEs.
Shown in Fig. 3
, panels A and B, is our observation that deletion of
the -131 to -94 portion of the PRD promoter [to give
(ERE)2-PRD,B/N-CAT] resulted in a 3-fold
increase in the magnitude of CAT activity in response to E2
(Fig. 3A
) and also resulted in a reduced sensitivity to suppression by
ICI relative to that shown by the intact PRD promoter
construct [(ERE)2-PRD-CAT] (Fig. 3B
). In
addition, the truncated promoter construct
[(ERE)2-PRD,B/N-CAT] required 10-fold less
E2 (i.e.
3 x 10-12
M E2) for half-maximal activity compared with
the PRD-containing reporter
[(ERE)2-PRD-CAT] (Fig. 3A
).
It is of note that although the PRD,B/N and the pS2
promoter constructs required similar E2 concentrations for
half-maximal activity, they had very different dose-response curves for
suppression by ICI (Fig. 3B
). These results illustrate the lack of
correlation between the estrogen and AE sensitivities of a particular
promoter and suggest that the decreased sensitivity of
PRD,B/N to repression by ICI relative to
PRD was not related to its increased sensitivity to the
stimulatory actions of E2.
Our initial expectation in deletion and mutagenesis studies in the
PRD promoter was that we would identify a region conferring
the resistance that this promoter shows to AE antagonism. We failed to
find such a region as far as we were able to study through deletions
and mutations. Although this aspect merits further study, we have found
that further deletions (Fig. 2
, lines 35) reduced estrogen
responsiveness of the promoter, complicating such an approach. On the
other hand, in the PRD gene, we have made the unusual
observation that a small region in PRD has a strong
modulatory effect on estrogen and AE responsiveness, and its presence
confers higher estrogen sensitivity and higher AE repression, even
though overall the PRD is less estrogen and AE responsive
than other genes such as pS2 and PRD. Therefore, we
investigated this modulatory element further.
Evaluation of the -131 to -94 Region of the PRD
Promoter as a Transferable cis Element
To determine whether the -131 to -94
XmnI/BsmI fragment of PRD had the
properties of a cis element, one or two copies of the
fragment were cloned 50 bp upstream of the EREs in both the
(ERE)2-PRD-CAT and
(ERE)2-PRD,B/N-CAT reporter
constructs (Fig. 4
). In the context of the full- length
PRD, which contains the -131 to -94 region in its natural
location, the presence of additional XmnI/BsmI
(i.e. -131 to -94) fragments resulted in no substantial
change in magnitude of E2-stimulated activity or ICI
repression (Fig. 4
, lines 13). Deletion of the -131 to -94 fragment
from PRD caused a doubling of the level of induction by
E2 and a reduction in the magnitude of repression by ICI
(Fig. 4
, line 4), as noted earlier in Figs. 2
and 3
. The addition of
one XmnI/BsmI fragment 50 bp upstream of the two
EREs in (ERE)2-PRD,B/N-CAT reduced
E2-inducibility by 50% with no change in AE repression
(Fig. 4
, line 5 vs. line 4). Two
XmnI/BsmI fragments had the same effect on the
level of E2 induction and gave a greater (67%) repression
by the AE ICI (Fig. 4
, line 7), as seen with the intact PRD
(Fig. 4
, line 1). The effect was not seen with the mutated (Mut 3) form
of the XmnI/BsmI fragment (Fig. 4
, line 6).
Therefore, the effect of the -131 to -94 fragment was to reduce
E2 stimulation and increase ICI repression.

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Figure 4. The -131 to -94 Region of PRD Acts as
a Transferable cis Element
CAT reporter constructs containing two EREs upstream of either
PRD (-131 to +65 bp) or the truncated PRD
(-94 to +65 bp) denoted PRD,B/N [lines 17] or pS2
(-90 to +10 bp) [lines 810] with one or two copies of the -131 to
-94 XmnI/BsmI fragment of
PRD cloned 50 bp upstream of the EREs, were examined for
E2 responsiveness and for repression of
E2-stimulated CAT activity by ICI in MCF-7 cells as
described in the legend of Fig. 1 . For line 6 and line 10, one copy of
the -131 to -94 XmnI/BsmI fragment
containing the mutated nucleotides in mut3 (see Fig. 2B ) was cloned
upstream of ERE-containing PRD,B/N or pS2, respectively.
The magnitude of transactivation of wild type PRD or wild
type pS2 (without an XmnI/BsmI fragment
cloned upstream of the ERE) was set at 100%. The percent repression
with ICI indicates the percent inhibition of E2-stimulated
activity observed for each construct upon cotreatment with
10-9 M E2 and 5 x
10-7 M ICI. Each value represents the mean of
three or more separate determinations ± SEM.
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The activity of the -131 to -94 fragment was also observed in the
context of the promoter of another estrogen-responsive gene. When the
fragment was cloned upstream of the EREs in the
(ERE)2-pS2-CAT reporter construct,
E2-stimulated CAT activity was reduced to approximately
30% when compared with (ERE)2-pS2-CAT lacking the fragment
(Fig. 4
, lines 89). ICI suppression, which in (ERE)2-pS2
itself was greater than 90%, remained strong and unaffected, as
expected (since ICI repression was complete and could not be increased
further). This effect on E2 stimulation was specific for
the intact -131 to -94 XmnI/BsmI fragment and
was not observed when the XmnI/BsmI fragment
contained the Mut3 mutations (Fig. 4
, line 10). Thus, the -131 to -94
XmnI/BsmI fragment satisfies two criteria of a
regulatory cis element in that it is positionally
independent and is transferable, being active in the context of a
heterologous promoter, in this case, the promoter of another
estrogen-responsive gene.
Analysis of the Effects of the -131 to -94 Region of the
PRD Promoter on Another Mediator of ER
Action
The preceding studies examined the effects of the -131 to -94
region of PRD on the actions of a typical positive
regulator of ER function, namely E2, and a typical fully
negative regulator of ER function, namely the AE ICI. Since the ligand
trans-hydroxytamoxifen (TOT) is a partial agonist/antagonist
that can exhibit agonistic activity in certain promoter contexts
(5, 6, 7, 8, 9), we wanted to determine whether the -131 to -94 region of
PRD could also modulate the agonistic effect of TOT.
As shown in Fig. 5
, we examined the ability of the -131
to -94 region of PRD to reduce the agonistic actions of
TOT on different promoters, similar to the way it reduced the agonistic
actions of E2 as described above. Reporter constructs
lacking the -131 to -94 XmnI/BsmI fragment from
PRD [i.e.
(ERE)2-PRD,B/N-CAT and
(ERE)2-pS2-CAT] showed dose-dependent increases in CAT
activity in response to treatment with TOT (Fig. 5
, panels A and B)
that were greater in magnitude than that of constructs containing the
-131 to -94 XmnI/BsmI fragment
[(ERE)2-PRD-CAT, Fig. 5A
]. In addition,
deletion of the -131 to -94 fragment from PRD resulted in
a promoter construct in which the EC50 was lowered about
10-fold, from 10-10 M for
(ERE)2-PRD-CAT to approximately
10-11 M for
(ERE)2-PRD,B/N-CAT. Also, addition of the
XmnI/BsmI fragment to PRD,B/N (to
give X/B-(ERE)2-PRD,B/N) reduced TOT
stimulation back to that of the intact PRD construct (Fig. 5A
). The same effect was seen with a different promoter (the pS2 gene
promoter, Fig. 5B
), in which addition of the
XmnI/BsmI fragment [i.e.
X/B-(ERE)2-pS2-CAT] resulted in a great reduction in the
response to TOT, whereas addition of a mutated
XmnI/BsmI fragment
[X/Bmut3(ERE)2-pS2-CAT] resulted in virtually
no change in the response to TOT (Fig. 5B
). Thus, the -131 to -94
region of PRD reduced the ability of ER to respond
positively to at least two different types of stimulatory signals,
namely E2 and TOT acting as an estrogen agonist.

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Figure 5. The Effects of the -131 to -94 Region of
PRD on the Estrogen Agonist Activity of TOT
The (ERE)2-PRD-CAT,
(ERE)2-PRD,B/N-CAT,
(ERE)2-PRP-CAT, or (ERE)2-pS2-CAT
reporters were examined for responsiveness to the stimulatory
(agonistic) actions of TOT (panels A and B) using MCF-7 cells as
described in Materials and Methods. The activity for
each construct is expressed as a percent of the maximal stimulation
observed with 10-9 M E2. Each
value represents the mean of three or more separate determinations
± SEM.
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Interaction of MCF-7 Cell Factors with the -131 to -94 Region of
PRD
The identification of a region of PRD (i.e.
the -131 to -94 region) that can alter the sensitivity of the
promoter to the stimulatory actions of estrogens and the inhibitory
actions of AEs suggested the presence of a specific
trans-acting factor(s) that could interact with this region.
One major band (indicated in Fig. 6B
) was detected in
gel mobility shift assays using extracts from MCF-7 cells and a
radiolabeled double-stranded oligomer containing the -131 to -94
sequence (shown in Fig. 6A
). The band was competed by a 50-fold or
25-fold excess amount of unlabeled oligomer (Fig. 6B
, lanes 7 and 11
vs. no competitor, lanes 6 and 10), but not by an excess of
unlabeled -131/-94 oligonucleotide with a 6-bp mutation from -115 to
-110 (mut3) (Fig. 6B
, lane 8), indicating that the protein-DNA
interaction producing the band was specific. Since a portion of the
-131 to -94 sequence shows some homology to an NF-1 binding site (see
Fig. 2B
), an unlabeled double-stranded oligonucleotide containing an
NF-1 binding site was also tested for its ability to compete for
binding to the -131 to -94 oligomer. However, the complex was not
competed by the NF-1 oligomer (not shown).

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Figure 6. Analysis of Protein Interactions with the -131 to
-94 Region of PRD
A, The sequence of the coding strand of the -131 to -94 region
of PRD and of the double- stranded oligomer used in the gel
mobility shift assay. B, Gel mobility shift assays were performed using
a double-stranded oligomer containing the -131 to -94 sequence of
PRD and extracts from MCF-7 cells as described in
Materials and Methods. With extracts treated with
E2 (10-8 M, lane 1), or ICI
(10-7 M, lane 2), or TOT (10-7
M, lane 3) for 15 min on ice; with extract + 200-fold
excess unlabeled GRE (lane 4); with extract + 200-fold excess unlabeled
mutated ERE (lane 5); extract alone (lane 6); with extract + 50-fold
excess unlabeled -131 to -94 double stranded oligo (lane 7); with
extract + 50-fold excess unlabeled -131 to -94 double-stranded oligo
with a 6-bp mutation from -115 to -110 (mut3, lane 8); with extract +
50-fold excess unlabeled ERE (lane 9); extract alone (lane 10); with
extract + 25-fold excess unlabeled -131 to -94 double-stranded oligo
(lane 11); with extract + 25-fold excess unlabeled ERE (lane 12); no
extract (lane 13). The positions of the shifted complex and the free
probe are indicated. The autoradiograph is representative of three
separate experiments.
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Interestingly, although the -131 to -94 sequence shares no homology
with an ERE, the band was competed by a 25-fold excess of unlabeled ERE
(Fig. 6B
, lane 12) and more fully by a 50-fold excess of unlabeled ERE
(lane 9), but was not competed by 200-fold excess mutated ERE (lane 5)
or consensus glucocorticoid response element (GRE) (lane 4). The band
was not supershifted in the presence of an anti-ER antibody (not
shown), suggesting that ER was not present in the complex. These
results suggest that a protein in the complex is also capable of
interacting with ERE or with ER when it is bound to its response
element in a manner that disrupts the binding of the labeled oligo.
Because ER is present in the MCF-7 cell nuclear extract, this protein
may be titrated away from the complex upon the addition of excess
competing ERE.
The effect of ligand treatments on the formation of the DNA-protein
complex was also examined (Fig. 6B
). There was no difference in complex
formation using nontreated (lanes 6 and 10) vs. ICI-treated
cell extracts (Fig. 6
, lane 2). Notably, using cell extracts treated
with E2, there was a marked decrease in the intensity of
the shifted complex (lane 1). Cell extracts treated with TOT (lane 3)
showed a gel shift pattern similar to, but slightly less strong than,
that observed with ICI treatment. Therefore, differential binding of
factors to the XmnI/BsmI fragment may occur in
the presence of estrogens vs. AEs, and disappearance of the
DNA-protein complex is correlated with the presence of
transcriptionally productive, E2-liganded ER.
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DISCUSSION
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Differential Sensitivity of Estrogen-Stimulated Promoters to the
Inhibitory Actions of AEs
The experiments described herein have demonstrated the
differential sensitivity of a number of estrogen-regulated promoters,
namely PRD, PRP and the pS2 promoter, to the
actions of estrogens and AEs. In general, there was no correlation
between the sensitivity of a given promoter to stimulation by estrogens
when compared with its sensitivity to inhibition by AEs. For example,
although the PRD and the pS2 promoters required relatively
comparable levels of E2 for half-maximal stimulation, they
showed markedly different dose-response profiles to the inhibitory
actions of AEs. These findings suggest that the magnitude of estrogen
responsiveness of a particular promoter is intrinsic to the nature of
the promoter and that promoter responsiveness to the actions of
estrogen- and AE-occupied ERs are separable. Furthermore, they
implicate the involvement of inhibitory cis elements and
promoter-specific factors acting to modulate the response of each
promoter to different ER-ligand complexes. These findings are
consistent with earlier reports in which it has been noted that
reporter constructs containing EREs upstream of different promoters are
differentially activated by estrogen in transient transfection assays,
even when other experimental variables remain constant (10, 14), and
with increasing evidence for promoter-specific actions of estrogens and
AEs (5, 7, 8, 9, 14, 15).
Identification of an Inhibitory cis Element that
Modulates the Sensitivity of Promoters to Estrogen- and AE-Occupied
ER
Our search for ligand response modulatory elements began with our
observation that the PRD promoter showed reduced
sensitivity to suppression by the AE-ER complex relative to the two
other promoters, PRP and pS2, examined. Deletion and
mutational analyses led to the identification of a region in
PRD that, in fact, made the PRD and a different
estrogen-responsive (pS2) promoter more sensitive to inhibition by AE.
Although these studies have allowed us to identify this novel element,
which directs the AE sensitivity that PRD does have, it is
evident that the reduced AE sensitivity of PRD overall must
derive from activities from other portions of the promoter, which may
normally act in concert with this element. Further analysis of
PRD would be of interest but may be complicated by our
observation that more extensive deletions in the 5'-flanking region
reduced activity altogether.
The identified element in PRD had the following properties:
1) it reduced the magnitude and sensitivity of estrogen-stimulated
activity, 2) it enhanced the ability of AEs to repress
estrogen-stimulated activity, and 3) it elicited similiar effects when
transferred to the promoter of another estrogen-responsive gene. This
LRME appears to have no clear homology to previously identified
cis elements. Gel mobility shift assays showed that a
cellular factor or factors were capable of binding to the element.
Although we know very little about the nature of these factors, changes
in the level or activity of these trans-acting factors would
be predicted to play important roles in the gene-selective actions of
hormone- and antihormone-receptor complexes.
Relevant to our findings are reports from the Simons laboratory (16, 17) of a cis-acting glucocorticoid modulatory element that,
like the element we identify here in the PR gene, alters the
sensitivity of the tyrosine aminotransferase (TAT) gene to
glucocorticoid and to mixed agonist/antagonist antiglucocorticoids. The
element differs, however, from the one we have identified in that it is
located much further away from the promoter (3646 bp upstream of the
start of TAT gene transcription). In addition, our LRME reduces the
magnitude of E2 stimulation or TOT agonism and increases
the EC50 for E2 stimulation or TOT agonism,
while the glucocorticoid-modulatory element enhances the sensitivity of
the TAT gene to glucocorticoid (lower EC50, i.e.
left-shifted dose-response curve) and confers greater agonistic
activity with partial agonist/antagonist antiglucocorticoids. However,
the magnitudes of the shift in the dose-response curves (
10-fold)
and the maximum activity levels (
2- to 3-fold) effected by the
glucocorticoid-modulatory element and our LRME are very similar.
Of note, PRD is a TATA-less promoter. However, the reduced
AE sensitivity and the activity of the -131/-94 element is not
exclusive to TATA-less promoters. For example PRP, which is
also TATA-less, shows strong sensitivity to AEs. Furthermore, the
-131/-94 element can be transferred to the pS2 promoter, which is
TATA-containing, and elicits the same activity.
Implications for Gene-Specific Regulation by Estrogens and AEs
Our results suggest that the sensitivity of a given promoter to
the stimulatory actions of estrogens is not necessarily correlated with
its sensitivity to the inhibitory actions of AEs. Furthermore, we have
demonstrated that the presence of a modulatory cis element
in the promoter region of a gene can dramatically influence the
response of that promoter to agonist- and antagonist-occupied receptor.
Ligand response-modulatory elements, such as we have identified in the
PRD promoter, may participate in regulating the activity of
different estrogen-responsive genes by altering the pharmacology of
estrogen and AE ligands that regulate these genes. They may thus be
important in selectively modulating the properties of gene induction by
estrogen agonists and antagonists and may underlie the known
differences in dose-response curves for estrogen induction of different
genes (5).
A BLAST search for this 38-bp cis element sequence in other
genes revealed a related sequence (26-bp sequence, 84% identity)
in the vinculin gene. Interestingly, vinculin, which encodes an
actin-binding cytoskeletal protein, is also known to be under estrogen
regulation (18). Thus, this sequence may potentially influence estrogen
and AE sensitivity of several genes.
Our findings add to the growing list of modulators of ER activity.
Modulators at almost every step in the process of transcriptional
activation by ER have been identified: the type of ligand, receptor
phosphorylation (19, 20, 21), the sequence of the estrogen response element
(Refs. 22 and 23 for reviews), coactivator proteins (such as TIF-1,
SRC-1, SPT-6, and others) (24, 25, 26, 27, 28, 29), some other nuclear hormone
receptors (30, 31, 32, 33), and chromatin structure (34, 35). The
identification of so many potential modulators of ER activity suggests
that transcriptional activation by ER is not a simple process and that
there are many checkpoints in the process suitable for regulation.
The modulatory cis element that we have identified,
which is capable of increasing the sensitivity of a promoter to the
inhibitory actions of AEs, is especially interesting in light of the
therapeutic uses of AEs in the treatment of breast cancer. A detailed
understanding of the mechanisms by which this element and the factors
that bind to it alter responsiveness to AEs may assist ultimately in
the development of more effective therapeutic agents. In addition,
since the ER is a member of a large superfamily of structurally and
functionally related ligand-activated transcription factors, it is
likely that similar cis elements, as identified previously
in the glucocorticoid-regulated TAT gene (16, 17), will be found to
modulate the sensitivity of genes regulated by other steroid receptors,
and thyroid and retinoic acid receptors, to their agonist and
antagonist ligands.
 |
MATERIALS AND METHODS
|
---|
Reagents and Radioisotopes
Cell culture media and antibiotics were purchased from GIBCO
(Grand Island, NY). Calf serum was from Hyclone Laboratories (Logan,
UT) and FCS from Sigma Chemical Company (St. Louis, MO).
35S- and 32P-radiolabeled nucleotides and
[dichloroacetyl-1,2-14C]-chloramphenicol (5060 Ci/mmol)
were from Dupont/NEN Research Products (Boston, MA). Custom
oligonucleotides were purchased from National Biosciences Inc.
(Plymouth, MN). DNA restriction and modifying enzymes were from New
England Biolabs (Beverly, MA), GIBCO/Bethesda Research Laboratory
(Gaithersburg, MD), and U.S. Biochemicals (Cleveland, OH). DNA
sequencing reagents were from U.S Biochemicals. E2 was from
Sigma. The AEs ICI 164,384 and TOT were kindly provided by Alan
Wakeling (Zeneca Pharmaceuticals, Macclesfield, U.K.). The AE LY
117,018 was kindly provided by Eli Lilly & Co. (Indianapolis, IN). All
general reagents were of molecular biology grade and were purchased
from Sigma Chemical Co., U.S. Biochemicals, or Fisher Scientific
(Houston, TX).
Plasmid Construction and Mutagenesis
All cloning was done using standard techniques (36, 37). When
necessary to make termini compatible, 3'- and 5'-overhangs generated by
restriction digestion were blunted with T4 DNA polymerase and the
Klenow fragment of Escherichia coli DNA polymerase,
respectively. The insertion of double-stranded oligonucleotides and the
deletion of DNA fragments were confirmed by dideoxy chain termination
DNA sequencing. Other manipulations were confirmed by restriction
digest analyses.
The construction of pTZ-TK-CAT, PRD-CAT,
PRP-CAT, and (ERE)2-PRP-CAT (10),
and 5E-PRD-CAT (11), as well as that of pS2-CAT (38), has
been described previously. PRD,B/N-CAT was constructed
by releasing and blunting the BsmI/NheI fragment
from the rat PR genomic clone EE(3.1)3Z (10) and cloning it into
SalI/BglII-digested pTZ-TK-CAT.
(ERE)2-PRD, B/N-CAT and
(ERE/HindIII)2-PRD-CAT were made by
annealing the single-stranded oligomers
5'-AATTAGTCAGGTCACAGTGACCTGATC-3' and
5'-AATTGATCAGGT-CACTGTGACCTGACT-3' and cloning two copies of the
resultant double-stranded oligomer into the HindIII
sites of PRD,B/N-CAT and PRD-CAT, respectively.
(ERE)2-pS2-CAT was made by annealing the single-stranded
oligomers
5'-GATCCAAAGTCAGGTCACAGTGACCTGATCAAAGA-3'
and 5'-GATCTCTTTGATCAGGTCACTGTGACCTGACTT-TG-3' and cloning two
copies of the resultant double-stranded oligomer into the
BamHI site of pS2-CAT.
(ERE/HindIII)2-pS2-CAT was made by replacing the
BamHI/NcoI fragment from
(ERE/HindIII)2-PRD-CAT with the
BamHI/NcoI fragment from pS2-CAT.
(X/B)N-(ERE)2-PRD-CAT and
(X/B)N-(ERE)2-PRD, B/N-CAT were
made by annealing the single-stranded oligomer
5'-TTTTCTTCTCGAAGTCTGATGTTCCAGGTGGAATGCC-3' with its complement
and cloning one or two copies of the resultant double-stranded oligomer
into EagI-digested and blunted
(ERE/HindIII)2-PRD-CAT and
(ERE/HindIII)2-PRD, B/N-CAT,
respectively. (X/B)2-(ERE)2-pS2-CAT was made by
replacing the BamHI/NcoI fragment from
(ERE)2-PRD-CAT with the
BamHI/NcoI fragment from pS2-CAT.
Six reporter constructs, each containing 6-bp mutations introduced
sequentially from the -131 to -84 region of the rat PR gene distal
promoter, were constructed by site-directed mutagenesis (39) with
modifications (40). The EcoRI fragment of
(ERE/HindIII)2-PRD-CAT was first
inserted into the EcoRI site of Bluescript II
SK+ (Stratagene, La Jolla, CA) to make
(ERE/HindIII)2-PRD-BSK+.
Mutagenic oligonucleotides were then annealed to single-stranded DNA
generated using the f1 origin of replication in Bluescript II
SK+. The mutagenic oligonucleotides used in six separate
mutagenesis reactions were:
5'-ATCAGACTTCGATTCTGCAGTCGACTCTAGAG-3'
5'-CCTGGAACATCACCATGGACGAAGAAAATCGA-3'
5'-GCATTCCACCTGAAGATATCGACTTCGAGAAG-3'
5'-TGGAGTTGGCATGGATCCAAGAACATCAGACT-3'
5'-TCCAAAACTGGACAAGATCTTCCACCTGGAAC-3'
5'-TGGCGAGATCCATTCATATGGTTGGCATTCCA-3'
To make each of the six
(ERE/HindIII)2-PRD,mut-CAT reporter
constructs, the EcoRI/EcoRI fragment of
(ERE/HindIII)2-PRD-CAT was then
replaced with the mutated EcoRI/EcoRI
frag-ment of
(ERE/HindIII)2-PRD,mut-BSK+.
To simplify the reporter construct nomenclature used, we will refer to
(ERE/HindIII)2-PRD-CAT,
(ERE/HindIII)2-PRD, B/N-CAT,
(ERE/HindIII)2-PRD, mut CAT, and
(ERE/HindIII)2-pS2-CAT as
(ERE)2-PRD-CAT, (ERE)2-PRD,
B/N-CAT, (ERE)2-PRD, mut CAT, and
(ERE)2-pS2-CAT, respectively.
The plasmid pCMVß, which constitutively expresses
ß-galactosidase, was obtained from Clonetech (Palo Alto, CA) and was
used as an internal control for transfection efficiency in all
experiments. The plasmid pTZ19, used as a carrier DNA, was provided by
Dr. Byron Kemper of the University of Illinois.
DNA Preparation
Plasmid DNA for transfections was prepared on CsCl gradients as
previously described (8, 37) or with a plasmid preparation kit (Qiagen,
Chatsworth, CA).
Cell Culture and Transfections
MCF-7 cells (K1 subline, see Ref.41) were maintained in MEM
plus phenol red supplemented with 5% calf serum. For transfection
experiments, the cells were switched to MEM plus phenol red
supplemented with 5% charcoal-dextran-treated calf serum for 2 days,
and then to MEM without phenol red plus 5% charcoal-dextran-treated
calf serum for 6 days before plating for transfection. All media
included penicillin (100 U/ml) and streptomycin (100 µg/ml). For
transfections, the cells were plated at a density of 3.5 x
106 per 100-mm diameter dish and were given fresh medium
about 30 h after plating. The cells were transfected by the
calcium phosphate coprecipitation method (42) 16 h later with 15
µg of CAT reporter plasmid plus 400 ng of pCMVß. The cells remained
in contact with the precipitates for 6 h and were then subjected
to a 3-min shock (25% glycerol in culture medium), which was followed
by a rinse with HBSS. Treatments were added in fresh medium after the
rinse.
ß-Galactosidase and CAT Assays
All cells were harvested 24 h after hormone treatment.
Extracts were prepared in 200 µl of 250 mM Tris-HCl (pH
7.5) using three freeze-thaw cycles. ß-Galactosidase activity, which
was measured to normalize for transfection efficiency, and CAT activity
were assayed as previously described (43).
Gel Shift Assays
Whole cell extracts from MCF-7 cells for use in the gel
shift assays were prepared by freeze-thaw lysis as described previously
for transfected COS-1 cells (44). The single-stranded oligomer
5'-TTTTCTTCTCGAAGTCTGATGTTCCAGGTGGAATGCC-3', which represents the -131
to -94 region of the rat PR gene, was annealed with its complement.
The resultant double-stranded oligomer was gel purified on a
nondenaturing 10% polyacrylamide gel run in 0.5x Tris-borate-EDTA.
The ability of extract protein(s) to bind to the -131 to -94 fragment
was analyzed using standard gel mobility shift assays. Briefly, 2 µl
(
5 µg) of MCF-7 whole cell extract was incubated with 1 ng of
end-labeled -131/-94 oligomer, under conditions described previously
(11). The specificity of binding was assessed by competition with
excess unlabeled double-stranded -131/-94 oligomer or with excess
unlabeled double-stranded -131/-94 oligonucleotide with a 6-bp
mutation from -115 to -110 (mut3; single-stranded oligomer
5'-TTTTCTTCTCGAAGTCgatatcttCAGGTGGAATGCC-3'annealed to its
complement) as well as with excess unlabeled double-stranded oligomers
containing the consensus ERE, mutated ERE, or consensus GRE sequence.
The nondenaturing gels used to analyze the protein-DNA complexes were
run as described previously (11, 44).
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dr. Benita S. Katzenellenbogen, Department of Molecular and Integrative Physiology, University of Illinois, 524 Burrill Hall, 407 South Goodwin Avenue, Urbana, Illinois 61801.
This work was supported by NIH Grant CA-18119 and US Army Grant
DAMD1794-J-4205. M.M.M. is the recipient of a postdoctoral fellowship
from the Susan G. Komen Foundation.
1 Present address: Department of Biology, University of
California, San Diego, La Jolla, California 92093. 
Received for publication October 14, 1996.
Revision received December 17, 1996.
Accepted for publication December 19, 1996.
 |
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