Effects of Multiple Estrogen Responsive Elements, Their Spacing, and Location on Estrogen Response of Reporter Genes
G. Sathya,
Wenzhuo Li,
Carolyn M. Klinge,
Jennifer H. Anolik,
Russell Hilf and
Robert A. Bambara
Department of Biochemistry and Biophysics and the Cancer Center
(G.S., W.L., J.H.A., R.H., R.A.B.) University of Rochester
School of Medicine and Dentistry Rochester, New York 14642
Department of Biochemistry (C.M.K.) University of Louisville
School of Medicine Louisville, Kentucky 40292
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ABSTRACT
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Most highly estrogen-responsive genes possess
multiple estrogen-responsive elements (EREs) that act synergistically
to activate expression. Synergism between EREs appears to depend on
structural features of the EREs and the promoter. To examine the
activation process, we cloned single or multiple tandem copies of the
consensus ERE into reporter plasmids. These plasmids contained either a
chloramphenicol acetyl transferase reporter gene driven by a minimal
promoter or a luciferase reporter gene driven by the Simian virus 40
(SV40) promoter. Using MCF-7 human breast cancer cells, we demonstrate
that synergism among EREs depends on the number of EREs, their spacing,
and the distance of the EREs from the promoter. The induction capacity
of EREs falls off slowly with distance from the promoter. Remarkably,
multiple EREs can induce effectively and synergize even when they are
located more than 2000 nucleotides from the promoter. For EREs located
immediately upstream of the promoter, both the distance separating the
EREs and the distance to the promoter have to be optimal for synergy.
Altering either distance changes the response from synergistic to
additive. For distant EREs, presumed to interact by a looping mechanism
at the promoter, the length of DNA between the EREs and the promoter is
not critical. Synergy among closely spaced EREs that are far from the
promoter only requires an optimal distance separating the ERE centers
of symmetry. Interestingly, very widely separated EREs can also
synergize, presumably also because of their ability to interact by
looping.
The estrogen response from single or multiple tandem copies of ERE
half-palindromes near the SV40 promoter was also tested. The negligible
induction capacity of a single half-site was not significantly
increased in multiple sites. The biological role of half-EREs is not
apparent in the system employed here.
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INTRODUCTION
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Estrogens belong to a family of classic steroid hormones that
include progesterone, androgens, glucocorticoids, and
mineralocorticoids. The major site of synthesis and secretion of
estrogens is the ovaries. Estrogens control a variety of physiological
processes by regulating the expression of specific genes at their
target sites. The hormone acts via the estrogen receptor protein, ER,
located predominantly in the nucleus. The hormone-activated receptor
binds to estrogen responsive elements (EREs) present in the regulatory
region of the target gene. By interacting with the basal transcription
machinery, the activated receptor modulates gene expression (1, 2, 3). The
minimal ERE that confers estrogen inducibilty in transfection assays
has been reported to be the 13-bp 5'-GGTCAnnnTGACC-3' (4, 5, 6). We
derived a consensus ERE from three highly estrogen-responsive genes. It
is a 38-bp sequence, comprising a 17-bp inverted repeat 5'-CAGGTCA nnn
TGACCTG-3' followed by an AT-rich sequence (7).
Most of the highly estrogen-responsive genes, including the
vitellogenins and the progesterone receptor, have multiple, variably
spaced EREs within their regulatory regions (8, 9). The presence of
multiple EREs results in synergistic activation of these genes.
Synergism in the vitellogenin B1 gene was reported to result from the
cooperative binding of the receptor to the two ERE sites (10). However,
Ponglikitmongkol et al. (11) reported that synergism may not
be primarily due to cooperative binding. The stereoalignment of ER-
bound EREs and their spacing influence synergistic response to
estradiol (11). Interestingly, another highly estrogen-responsive gene,
ovalbumin, is synergistically activated by a series of four
half-palindromic sites, all of which appear to be necessary for
induction (12, 13). The ovalbumin EREs appear to be cell type-specific,
indicating the requirement of certain additional factors.
In this report, we investigated how multiple EREs mediate reporter gene
expression in response to estradiol (E2), and whether
placing them in tandem or separating them from one another affects the
level of induction. We examined whether estrogen response from multiple
EREs was affected by the strength of the promoter and the relative
distance of the EREs from the promoter. We compared the levels of
induction by E2 in reporter genes having single or multiple
copies of EREs located upstream or downstream of the transcribed region
and positioned in tandem or widely separated.
Additionally, we examined whether ERE half-site sequences could
have a biological role when present as single or multiple copies, by
measuring the levels of induction by E2 in reporters having
single or multiple copies of half-ERE sequences. Based on our
observations, we propose generalizations that describe the behavior of
multiple EREs in regulating gene expression.
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RESULTS
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Trimeric EREs Synergize Upstream of a Minimal Promoter
To understand how single or multiple EREs affected reporter gene
activation in response to E2, we used chloramphenicol
acetyltransferase (CAT) reporter constructs that contained a single
copy, or multiple tandem copies, of EREc38 upstream of a minimal
promoter containing the Xenopus vitellogenin B1 TATA box.
The EREs were located 52 nucleotides upstream of the TATA box. The
center-to-center distance between the EREs was 38 nucleotides. As shown
in Fig. 1
, the ratio of normalized CAT
activity with/without 1 nM of estradiol for one and two
copies of EREc38 was 1.2 and 1.88, respectively. For three copies of
the ERE we observed a very large increase in the ratio representing a
16-fold induction. Addition of a fourth ERE more than doubled the ratio
again. The specificity of the response to E2 was
demonstrated by the lack of reporter activation in response to
4-hydroxytamoxifen (4-OHT) even at concentrations up to
10-6 M (data not shown). The
E2-induced response was reduced to basal levels with the
addition of 100-fold excess of 4-OHT (Fig. 2
). 4-OHT is a metabolite of tamoxifen, a
type I antiestrogen used to treat patients with breast cancer (14, 15).
Our results demonstrate that 4-OHT inhibits E2-induced
reporter gene activation by single and multiple EREs in MCF-7
cells.

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Figure 1. Trimeric EREc38 Synergize Upstream of a Minimal
Promoter
pATC- 1, 2, 3, 4EREc38 constructs contain one, two, three, or four
tandem copies of the consensus EREc38 upstream of the vitellogenin B1
TATA box in the CAT reporter vector. 1.5 µg of each vector was
cotransfected with 0.3 µg of pCMVß-galactosidase internal control
plasmid into MCF-7 cells. Three hours after transfection, 1
nM E2 or an equal volume of ethanol (as
control) was added in duplicate. CAT and ß-galactosidase assays on
the cell lysates were performed after 24 h. Ratios of the mean
normalized CAT activities, with and without estradiol, from six
individual experiments are plotted; error bars are SEM.
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Figure 2. 4-OHT Does Not Induce Expression from the CAT
Reporters
CAT reporter constructs containing one, two, three, or four tandem
copies of EREc38 upstream of the minimal promoter were cotransfected
with pCMVß-gal internal control plasmid into MCF-7 cells. Three hours
after transfection, 1 nM E2 (gray
bars), 1 nM E2 plus 100 nM
4-OHT (white bars), or an equal volume of ethanol as
control (black bars) were added in duplicate. CAT and
ß-galactosidase assays were performed after 24 h. The normalized
CAT activity (mean with range) for each construct obtained from a
single experiment is illustrated.
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Multiple EREs Upstream of a Promoter Can Respond Linearly to
Estrogen
Single or multiple tandem copies of EREc38 were cloned 83
nucleotides upstream of the SV40 promoter (described in Materials
and Methods and Fig. 3A
). The
center-to-center spacing between the EREs in these constructs was 38
nucleotides. The ratio of normalized luciferase activity with and
without estrogen was 2.0 for 1EREc38, 3.7 for 2EREc38, 7.6 for 3EREc38,
and 5.1 for 4EREc38, as shown in Fig. 3B
. The numbers indicate a
slightly synergistic response for each increase in the number of EREs
in the construct up to three. That response was slightly reduced with
addition of a fourth ERE, showing that four leads to diminishing
returns. The overall appearance of the increase is approximately linear
with the number of EREs. This observation clearly differs from that
obtained with the CAT reporters, where we found a distinct synergistic
increase for three, and even four, copies of EREs. The altered pattern
of induction in the two reporters could result from differences in
either the promoter structures, or the distance of the EREs from the
promoter. The SV40 promoter contains several other cis
acting elements including six GC boxes, which bind Sp1 upstream of the
TATA box, causing higher basal activity than with the minimal promoter.
However, as considered below, we attribute differences in synergy to
the distance relationships among EREs and the TATA box.

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Figure 3. Multiple EREs Upstream of a Complex Promoter
Respond Additively to Estrogen
A, pGL31EREc38, 2EREc38, 3EREc38, and 4EREc38 contain one, two,
three, or four copies, respectively, of EREc38 at 83 nucleotides
upstream of the SV40 promoter. The center-to-center distance between
the EREs is 38 nucleotides. K, KpnI; S,
SacI; black box, SV40 promoter;
gray box, luciferase-coding region; EREc38 is
represented by a white rectangle, and the bar in
the center of the rectangle denotes the center nucleotide. B,
Luciferase reporter constructs containing one, two, three, or four
tandem copies of EREc38, 83 nucleotides upstream of the SV40 promoter,
were cotransfected with pCMVß-gal internal control plasmid into MCF-7
cells. Three hours after transfection, 1 nM E2,
or an equal volume of ethanol as control, was added in duplicate.
Luciferase and ß-galactosidase assays were performed after 24 h.
The ratio of normalized luciferase activity, with and without
estradiol, from three separate experiments is shown with
SEM.
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ERE Positioning and Spacing Influence Induction Levels
In the luciferase reporter constructs, we made two alterations in
an attempt to determine whether the additive estradiol response of
upstream dimeric EREs could be enhanced to a synergistic response. One
copy or two tandem copies of EREc38 were cloned into the
NheI site, 28 nucleotides upstream of the promoter, creating
the constructs pUP1 and pUP2, respectively (Fig. 4A
). The ERE centers for pUP1 and each of
pUP2 were 37, 60, and 83 nucleotides from the promoter, respectively.
The two EREs in pUP2 had a center-to-center spacing of 23 nucleotides
rather than 38 nucleotides as in pGL32EREc38. The ratios of
luciferase activity with and without estradiol were 1.7 for pUP1 and
4.5 for pUP2, with 1.0 being the basal uninduced level (Fig. 4B
). The
ratio of the induction level of pUP2 over pUP1 was considerably greater
than the ratio of the induction level for pGL32EREc38 over
pGL31EREc38 (Fig. 3B
). These observations led us to conclude that
moving two EREs with respect to each other and the promoter can alter
synergy. We also point out that the EREs in pGL32EREc38, separated by
38 nucleotides, are on opposite faces of the DNA helix, whereas in
pUP2, the two EREs are on the same face of the helix. The latter
orientation might have promoted synergistic interaction between the
receptor molecules bound at these sites.

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Figure 4. Appropriately Spaced Dimeric EREc38 Elements
Synergize Upstream of the SV40 Promoter
A, pUP1 contains a single copy of EREc38 at the NheI
site, 28 nucleotides upstream of the SV40 promoter. pUP2 contains two
tandem copies of EREc38 at the NheI site. The
center-to-center distance between the EREs is 23 nucleotides. N,
NheI; black box, SV40 promoter;
gray box, luciferase coding region; EREs are shown by
white rectangles with the bar inside representing the
center nucleotide. B, pUP1 and pUP2 constructs were cotransfected with
pCMVß-gal internal control plasmid into MCF-7 cells. Three hours
after transfection, 1 nM E2, or an equal volume
of ethanol as control, was added in duplicate to the cells. Luciferase
and ß-galactosidase assays were performed after 24 h. The ratio
(±SEM) of normalized luciferase activity with/without
estradiol from three separate experiments is shown.
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EREs Are Functional when Present Downstream of the Target Gene
We tested the ability of single or multiple EREs to activate an
estrogen-inducible gene by cloning EREs at the SalI site
downstream of the luciferase gene. At this position, the EREs are more
than 2000 nucleotides away from the promoter (Fig. 5A
). Figure 5B
shows the ratio of
normalized luciferase activity with and without 1 nM
estradiol for pLL1 and pLL2, which contain one and two copies of EREs,
respectively, downstream of the luciferase gene. The ratios for pLL1
and pLL2 are 1.3 and 3.0, respectively, where 1 represents the basal
level in the absence of estrogen. Therefore, the fold induction by
estrogen is 0.3 and 2.0, respectively. This indicates a synergistic
response for two copies of EREs. The response from pLL2 is lower than
the response from two EREs located upstream of the transcribed region.
Evidently, EREs may function well at a downstream site and may
synergize when the distance separating them is optimal, but proximity
to the promoter improves their induction capacity.

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Figure 5. EREs Placed Downstream of the Gene Are Functional
A, One copy or two tandem copies of EREc38 were cloned into the
pGL3-Promoter vector at the downstream SalI site to
generate pLL1 and pLL2 constructs. The center-to-center distance
between the two EREs of pLL2 was 23 nucleotides. S,
SalI; black box, SV40 promoter;
gray box, luciferase-coding region; EREs are shown by
white rectangles with the bar inside representing
the center nucleotide. B, pLL1 and pLL2 constructs were cotransfected
with pCMVß-gal internal control plasmid into MCF-7 cells. Three hours
after transfection, 1 nM E2, or an equal volume
of ethanol as control, was added in duplicate to the cells. Luciferase
and ß-galactosidase assays were performed after 24 h. The ratio
(±SEM) of normalized luciferase activity with and without
estradiol from four to five separate experiments is plotted in the
graph.
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Multiple EREs Separated by Large Distances from Each Other Can
Contribute to Induction of a Single Promoter
The results presented above show that tandem copies of EREs with a
center-to-center distance of 23 nucleotides were synergistic when
placed downstream of the luciferase gene. EREs located upstream and
closer to the promoter were even more effective, but not by a large
amount. Since EREs can act at a large distance from the promoter, we
wanted to measure the effect of separating EREs from each other by more
than 2000 nucleotides. To test this, we cloned in EREs at distant sites
upstream and downstream of the luciferase gene as shown in Fig. 6A
. We compared the estrogen induction in
pUP1 (one upstream), pUP1-down1 (one upstream and one downstream),
pUP1-down2 (one up and two down), and pLL2 (two down). The ratios of
the normalized luciferase activity with and without estradiol for these
constructs were 1.7, 2.8, 5.8, and 3.0, respectively (Fig. 6B
). The
induction from two EREs was almost the same in the two constructs that
contain the EREs in tandem (pLL2) and nontandem (pUP1-down1) locations.
This shows that EREs separated by a large distance can both contribute
to induction of the same promoter. The pUP1-down2 construct has three
EREs, one located upstream and two tandem EREs located downstream of
the gene. The response from this construct (4.8-fold) was more than
additive of the induction produced by the single upstream ERE
(0.7-fold) plus the dual downstream EREs (2.0-fold). This shows that
EREs are capable of synergizing even when separated by very large
distances.

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Figure 6. EREs Synergize at Very Large Distances of
Separation
A, pUP1-down1 contains one ERE at the NheI site and one
ERE at the SalI site downstream of the luciferase gene.
pUP1-down2 contains a single ERE upstream and two tandem EREs
downstream. The center-to-center distance of the downstream EREs is 23
nucleotides. N, NheI; S,
SalI; black box, SV40 promoter;
gray box, luciferase-coding region; all EREs are shown
as white rectangles. The bar inside the
rectangle represents the center nucleotide. B, The pUP1
luciferase construct contained a single copy of the consensus EREc38 at
28 nucleotides upstream of the SV40 promoter in the PGL3-Promoter. A
single or two copies of EREc38 were inserted into the downstream
SalI site of pUP1 to generate pUP1-down1 and pUP1-down2
constructs. pLL2 had two tandem copies of EREc38 at the
SalI site but no EREs upstream. These constructs were
cotransfected with pCMVß-gal internal control plasmid into MCF-7
cells. Three hours after transfection, 1 nM E2,
or an equal volume of ethanol as control, was added in duplicate to the
cells. Luciferase and ß-galactosidase assays were performed after
24 h. The ratio (±SEM) of normalized luciferase
activity with/without estradiol from three to five separate experiments
is illustrated.
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Multiple Copies of Half-EREs Produce a Negligible Estrogen
Response
Genes regulated by estrogens often contain multiple EREs, having
sequences that vary from the core consensus palindrome by one or two
nucleotide changes (16, 17, 18). These nonconsensus EREs were reported to
have lower affinity for liganded ER in vitro (19).
Half-palindromic sequences are also frequently found near full-length
EREs in the regulatory regions of responsive genes. Measurements made
in vitro have also shown that individual half-sites have
very low affinity for the receptor; however, certain genes are
regulated by half-sites in vivo (20, 21, 22, 23). We next tested
whether half-EREs are functional in our transfection assay and whether
multiple tandem copies of half-EREs have any significant effect on
estrogen-induced gene expression. Single or multiple tandem copies of
half-ERE sequences were cloned upstream of the SV40 promoter in the
luciferase reporter plasmid. The distance between the half-sites was 38
nucleotides. Figure 7
shows the response
from single or multiple tandem copies of half-ERE sequences upstream of
the SV40 promoter of the luciferase gene. The ratios of normalized
luciferase activities with and without estrogen for one, two, and three
tandem copies of half-EREc38 are 1.4, 1.42, and 1.6, respectively.
These results show that as the number of half- EREs is increased from
one to three, there is little increase in the response. Also, the
levels of induced expression by half-sites are significantly lower than
expression levels from multiple consensus EREs.

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Figure 7. Half-EREs Minimally Affect Estrogen Response
One, two, or three tandem copies of 1/2 EREc38 were cloned 83
nucleotides upstream of the SV40 promoter in the pGL3-Promoter vector.
There is a 38-nucleotide separation between the half-sites.
Cotransfections with pCMVß-gal internal control plasmid into MCF-7
cells were performed. Three hours after transfection, 1 nM
E2, or an equal volume of ethanol as control, was added in
duplicate to the cells. Luciferase and ß-galactosidase assays were
performed after 24 h. The ratio (±SEM) of normalized
luciferase activity with and without estradiol from three separate
experiments is presented.
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DISCUSSION
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The main objective of our study was to analyze the influence of
selected factors on the induction of gene expression by multiple EREs.
The parameters considered were: the number of EREs, the distance
separating them, their stereoalignment; the distance between the EREs
and the promoter; the presence of additional cis-acting
elements; and the effect of adjacent ERE half-sites. We used two
reporter systems containing different promoters to ascertain and
compare the effects of single or multiple tandem copies of consensus
EREs on estrogen-induced gene expression. The data show that the
percent induction varies based on the strength of the promoter and the
presence of additional cis-acting elements. The
E2-induced reporter gene activity was lower in the presence
of the SV40 promoter because of its higher basal activity.
When two EREs were placed at 83 nucleotides from the SV40 promoter and
their center-to-center distance was 38 nucleotides, they responded
approximately additively to the hormone. When they were moved closer,
to a distance of 28 nucleotides from the promoter and their spacing
reduced to 23 nucleotides, they activated expression synergistically.
When the EREs and promoter are close, linear separation on the DNA has
a major influence on the process of synergy. In this situation, it
appears that physical structure and bending constraints of the
protein-DNA complexes control the ability of components to interact
appropriately for synergy.
We then tested whether EREs were functional when located downstream of
the gene, at a distance of about 2400 nucleotides from the promoter. As
expected, the response levels from the downstream EREs were not as high
as seen with the upstream EREs. However, the response from downstream
tandem copies of EREs separated by 23 nucleotides remained synergistic.
This suggests that when EREs are located close to each other, and
spaced at an optimal distance, they can synergize at a promoter located
far away.
In the situation in which multiple EREs were located upstream and
downstream of the reporter gene, there was remarkably effective
interaction among them for induction of the promoter. There was
moderate synergy in the construct having one ERE upstream and one
downstream. The fold of induction in this construct (1.8) was more than
the sum of induction from a single upstream ERE in pUP1 (0.7-fold) and
a single downstream ERE in pLL1 (0.3). The presence of one additional
ERE downstream in pUP1-down2 construct demonstrated a strong synergy
with the upstream ERE.
Based on these observations, can we define rules that describe the
efficiency of induction of gene expression by multiple EREs? Certainly,
two or more EREs can synergize for induction. Synergistic interactions
among EREs can occur at distances close to the promoter or at more than
2000 nucleotides from the promoter. EREs located close to the promoter
are constrained by two parameters: the distance between the ERE centers
and the distance between the EREs and the TATA box. Both of these
parameters must be optimal for synergy. EREs located far away from the
promoter presumably interact with the promoter by a looping mechanism
(24). In this way, the ER-ERE complexes could physically interact with
the promoter, irrespective of their distances from the promoter along
the DNA. Therefore, for EREs located far away from the promoter, the
distance separating the EREs is the only requirement for synergy.
Presumably, two EREs located far from each other, could also interact
for synergy by looping.
When EREs are located both close to and far away from the promoter of a
single gene, the situation is more complex but still completely
consistent with the rules described above. The EREs can interact with
each other for synergy, because looping can bring them together. The
ERE that is far away interacts with the promoter by looping. However,
the ERE that is close to the promoter is constrained by its position on
the DNA. The capacity of this ERE for synergy with the downstream
promoter depends on whether or not it is at the optimal distance from
the promoter. This reasoning would explain why we observed strong
synergy in the pUP1-down2 construct, which had a single ERE at 28
nucleotides upstream of the promoter and two tandem EREs separated by a
center-to-center distance of 23 nucleotides located downstream of the
luciferase gene. The downstream EREs were at an optimal distance to
interact with each other and could loop to the promoter. The pUP1 ERE
was at an optimal distance from the promoter to enable it to interact
with both it and the looped EREs.
Our model explains synergy in other constructs as well. The EREs in the
CAT reporter were located at about five helical turns (52 nucleotides)
from the TATA box, which was apparently an optimal distance. In the
trimer construct, the first ERE and the third ERE were separated by 76
nucleotides, with the observation of synergy defining this as an
optimal distance. The first ERE of the dimer construct was located at
the same distance (52 nucleotides) from the promoter. However, the
distance between the two EREs of this construct was a nonoptimal 38
nucleotides. The result was a linear, nonsynergistic response. The
luciferase reporter had single or multiple copies of EREc38 at 83
nucleotides from the start of the SV40 promoter region. The SV40 TATA
box was located at 191 nucleotides from the EREs. Although the distance
separating the EREs in the luciferase reporter was identical to that in
the CAT reporter, the distance between EREs and the TATA box was
suboptimal. This resulted in a response that was not highly synergistic
from three EREs upstream of the luciferase gene. The response from four
copies of EREs also appeared to be linear instead of synergistic.
Although the complex structure of the SV40 promoter increased the basal
levels of expression in the constructs, the requirement for synergism
between multiple EREs was consistent for the two kinds of
promoters.
The synergistic response from dimeric EREs separated by 23 nucleotides
placed 28 nucleotides upstream of the SV40 promoter was also consistent
with our proposed rules for multiple EREs, as the structure of these
EREs satisfied the two distance requirements for synergy. The
requirements were also met by the dimeric EREs with optimal
center-to-center distance located downstream of the luciferase
gene.
The induction of gene expression by estradiol in each of our plasmids
is normalized to the basal expression in the absence of added hormone,
a value that we have set to 1.0. In fact, the basal level of expression
from a promoter in this system is influenced by the presence and number
of EREs in each plasmid. Generally, we find that the introduction of
increasing numbers of EREs into a plasmid progressively increases the
amount of reporter gene expression, even in the absence of added
hormone. Furthermore, the increase in basal expression appears to show
some synergy with the increase in the numbers of EREs. This phenomenon
might result from a low level of receptor-mediated induction, occurring
in the absence of added estradiol. The observed variations in basal
expression do not affect the conclusions concerning synergism of
estradiol-mediated induction. In fact, the estradiol-dependent synergy
we measure would be enhanced in many cases if we had measured all
induction against a single basal level of expression, e.g.
that of a plasmid with no EREs.
Our observations, leading to the above generalizations, are consistent
with the DNA-bending phenomenon proposed for ER-ERE interactions.
Nardulli and co-workers (25, 26, 27, 28, 29) have shown that when ER binds to an
ERE, it induces a conformational change in the DNA, resulting in a bend
of about 65°. The helical separation between EREs and the TATA box
may influence the bending induced by both ER binding to the ERE and the
interaction of transcription factor IID with the TATA box (30, 31). In
a situation of close proximity, the helix orientation of the binding
sites for each protein and the degree of DNA bending are expected to
exert a strong influence on the induction process. Incorrect
positioning of proteins on the DNA helix may prevent interactions
necessary for synergy. These structural considerations are not critical
for EREs located far from the promoter region, since looping allows
interactions to occur with much less constraint. Our results are also
consistent with the observations of others that EREs can act at very
large distances from the promoter (32, 33).
Many estrogen-regulated genes possess both full palindromic EREs and
half-site EREs (12, 34, 35, 36, 37). We tested the effect of single and tandem
copies of half-ERE sequences located upstream of the luciferase gene on
enhancement of estrogen-induced gene expression. Half-sites were found
to have negligible induction capacity. Comparison of multiple tandem
copies of half-EREs did not show a progressively increasing effect
on estrogen response. Also, when we inserted the half-site adjacent to
dimeric consensus EREs downstream of the luciferase gene, we did not
detect a significant increase in estrogen response from the
half-ERE-containing construct (data not shown). It is unclear whether
the orientation and spacing between half-sites and full consensus EREs
can affect their induction capacity. If so, there may be induction from
a configuration that we have not explored. Although there have been
several reports on genes regulated by half-EREs, in this reporter
system half-EREs are unable to significantly enhance estrogen response
from full EREs.
One parameter not addressed in experiments presented here is the role
of cooperative binding of the receptor to multiple nearby EREs. It is
not known whether cooperative binding is the basis for the synergism of
vitellogenin EREs (10, 11). We previously reported that trimeric and
tetrameric head-to-tail tandem constructs of EREc38 appeared to bind
estradiol-liganded ER (E2-ER) cooperatively (19). The
measured cooperativity depended on stereoalignment of EREs, the
presence of a natural AT-rich flanking sequence, and the nature of the
ligand (38, 39, 40, 41, 42, 43). In transfection studies with CAT reporters, we
detected synergistic response from a trimeric ERE containing plasmid,
but not from a CAT reporter containing dimeric EREs that displayed
cooperativity of binding (data not shown). From these observations, we
cannot conclude whether cooperative binding is essential for
synergistic gene expression from EREs.
The physiological sequelae in response to estrogens is modulated by the
intracellular concentrations of ligand, ER, and ligand-specific
coactivator proteins. Our data show that the degree of synergy between
EREs is modulated by the physical distance between EREs and by the
separation between EREs and the TATA box. It is also clear that
multiple EREs in estrogen-responsive genes can collaborate to control
the level of expression. They can carry out this collaboration at great
linear distances from the promoter and each other. Hence, the cell can
control the degree to which these elements synergize for induction of
expression by altering their relative positions.
 |
MATERIALS AND METHODS
|
---|
Cell Culture
MCF-7 cells were maintained in Iscoves modified Dulbeccos
media (IMDM) (Life Technologies Inc., Gaithersburg, MD) supplemented
with 10% FBS (Atlanta Biologicals, Norcross, GA) and 1% penicillin
and streptomycin (Life Technologies Inc.). Cells used for transfection
were grown in IMDM without phenol red supplemented with 10%
charcoal-dextran treated FBS and antibiotics for 24 h before
transfection.
Sequences of EREs
EREc38
5'-CCAGGTCAGAGTGACCTGAGCTAAAATAACACATTCAG-3'
1/2EREc38
5'-CCA-GGTCAGAGCATTTCGAGCTAAAATAACACATTCAG-3'
Plasmids
Single or multiple tandem copies of EREc38 and 1/2EREc38 were
isolated by KpnI and SacI double digestion from
pGEM7Zf(+) plasmid constructs (described in Ref.19) and cloned
directly into the upstream multiple cloning site of pGL3-Promoter
vector (Promega, Madison, WI) to place the closest ERE 83 nucleotides
upstream of the SV40 promoter driving the luciferase reporter gene. The
center-to-center spacing of the consensus EREs and the distance between
adjacent half-EREs in these constructs is 38 nucleotides. The
constructs containing EREc38 were called pGL31EREc38, 2EREc38,
3EREc38, and 4EREc38 (Fig. 3A
). The constructs containing 1/2EREc38
were called pGL31/2EREc38, 2(1/2)EREc38, and 3(1/2)EREc38.
EREc38 double-stranded oligomer with a NheI cohesive
end was synthesized and ligated end-to-end and cloned upstream of the
luciferase-coding region at the NheI site. The
NheI site was located at 28 nucleotides from the SV40
promoter, and the ERE center-to-center spacing was 23 nucleotides. The
constructs containing one or two EREs at the NheI site, with
no EREs downstream of the luciferase gene, were called pUP1 and pUP2
(Fig. 4A
). Double-stranded EREc38 with a SalI cohesive end
was synthesized (Genosys, The Woodlands, TX), ligated end-to-end using
T4 DNA ligase (Boehringer Mannheim, Indianapolis, IN), and cloned into
the SalI site located downstream of the luciferase-coding
region of pGL3-Promoter vector. The resulting constructs containing one
and two copies of the ERE, at 2400 nucleotides downstream of the
promoter, were called pLL1 and pLL2, respectively. The
center-to-center spacing between the dimeric EREs of pLL2 is 23
nucleotides (Fig. 5A
).The construct containing one ERE upstream and one
ERE downstream was called pUP1-down1. The construct containing one ERE
upstream and two EREs downstream was called pUP1-down2 (Fig. 6A
).
CAT reporter constructs were made by removing the two EREs from the
pATC2 vector (provided by Dr. David Shapiro, University of Illinois,
Urbana, IL), and inserting the XbaI/HindIII
fragments containing single or multiple tandem copies of EREc38 from
pGEM7Zf(+) plasmid. These constructs were called pATC-1EREc38, 2EREc38,
3EREc38, and 4EREc38. The closest ERE in each construct was located at
52 nucleotides from the minimal promoter, which contained the
Xenopus vitellogenin B1 TATA box. The ERE center-to-center
distance in these constructs was 38 nucleotides. The orientation and
nucleotide sequence of the ERE(s) in each reporter construct were
verified by dideoxynucleotide sequencing (Sequenase version 2.0, USB).
Large-scale purification of plasmids was performed using an Endo-free
Qiagen maxiprep kit (Chatsworth, CA).
Transfection
Cells (2.5 x 105) were plated in each well of
a 12-well plate in IMDM without phenol red supplemented with 10%
stripped serum and 1% antibiotics. After 24 h, at 50%
confluency, the cells were transfected using a liposome-mediated
transfection protocol (44). Either 0.3 µg of pCMVßGal (CLONTECH,
Palo Alto, CA) and 1.5 µg of CAT reporter construct, or 0.6 µg of
pCMVßGal and 0.6 µg of luciferase reporter DNA, were transfected
into each well using a DNA to liposome ratio of 1 µg:10 nmol. The DNA
and liposomes were mixed in Opti-MEM I without insulin, estrogen, and
other growth factors (Life Technologies). Three hours after
transfection, 1 nM 17ß-estradiol (Sigma, St. Louis, MO),
100 nM 4-OHT (Research Biochemicals International, Natick,
MA) or an equal volume of ethanol was added to the wells in duplicate.
The cells were maintained in medium containing 1% stripped FBS
post-transfection. The cells were lysed after 24 h in 200 µl of
1x reporter lysis buffer (Promega), and the extracts were assayed for
activity.
CAT Assays
Sixty microliters of extracts were assayed in a total volume of
125 µl containing 0.25 µCi of [3H] chloramphenicol
(DuPont NEN, Boston, MA) diluted in 0.25 M Tris-HCl, pH
8.0, and 5 µl n-butyryl Coenzyme A, 5 mg/ml (Sigma), at 37 C for 90
min. The reaction was stopped with 300 µl of mixed xylenes (J.T
Baker, Inc., Phillipsburg, NJ) and mixed on a vortexer for 30 sec. The
upper xylene phase was extracted twice with 0.25 M
Tris-HCl, pH 8.0, and 100 µl of each sample were counted in Ecoscint
A (National Diagnostics, Atlanta, GA).
ß-Galactosidase Assays
Fifty microliters of extracts were assayed in a total volume of
300 µl containing 1x reporter lysis buffer and 2x assay buffer
(protocol from Promega). In parallel, several serial dilutions of the
ß-galactosidase (Promega) were assayed for activity to obtain a
standard curve. The samples were incubated at 37 C until yellow color
developed. The reaction was stopped with 500 µl of 1 M
sodium carbonate. The activity was plotted as a function of the
absorbance at 420 nm to obtain enzyme activity in the extracts.
Luciferase Assays
Twenty microliters of extracts were used for luciferase assay
(Promega) with 100 µl of reconstituted substrate. The activity was
determined using a luminometer (Turner Designs, Sunnyvale, CA, model
20E) for 15 sec for each sample.
Luciferase and CAT activities of each transfection were normalized
using ß-galactosidase activity, and the ratio of normalized activity
with and without estradiol was calculated. A value of 1.0 indicates the
basal normalized activity without any added hormone, and values greater
than 1 represent the fold of induction for each reporter construct.
Each experiment was performed at least three times to obtain mean and
SEM values.
 |
ACKNOWLEDGMENTS
|
---|
The authors wish to thank Dr. David Shapiro, University of
Illinois, for the gift of the pATC2 vector; Dr. Barry Stripp,
University of Rochester, for the use of the luminometer to measure
luciferase activity; Jianghong Zhou and Julia Rozenblit for all
their assistance; and Dr. Mark Driscoll for his critical review of this
manuscript.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Robert A. Bambara, Department of Biochemistry and Biophysics and the Cancer Center, University of Rochester, School of Medicine and Dentistry, Rochester, New York 14642.
This work was supported by NIH Grant HD-24459, in part by NIH Grant
CA-16660 (to R.H.), and in part by NIEHS IP20 ES06832 and Veterans
Administration Center for the Study of Environmental Hazards to
Reproductive Health Grant 0006, Department of Veterans Affairs Medical
Center, Louisville, KY (to C.M.K.).
Received for publication June 12, 1997.
Revision received September 16, 1997.
Accepted for publication September 23, 1997.
 |
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