Human Estrogen Receptor (ER) Gene Promoter-P1: Estradiol-Independent Activity and Estradiol Inducibility in ER+ and ER- Cells
Isabelle Treilleux,
Nadine Peloux,
Myles Brown and
Alain Sergeant
Laboratoire de Virologie Moléculaire (I.T., N.P., A.S.)
Unité INSERM U412 Ecole Normale Supérieure de Lyon
69364 Lyon Cédex 07, France
Département
dAnatomie et de Cytologie Pathologiques (I.T., N.P.) Centre
Léon Bérard 69373 Lyon Cédex 08, France
Division of Neoplastic Disease Mechanisms (M.B.) Dana Farber
Cancer Institute Boston, MA 02115
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ABSTRACT
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Estrogen receptor (ER) is expressed at a low level
in normal tissues such as breast and uterus but at a high level in
breast and endometrial carcinomas. A proximal element (ERF-1) located
between positions +133 and +204 relative to the promoter P1 major
initiation site has been recently identified in
ER+ cells and contributes to the differential
promoter activity between ER+ and
ER- cells. In this study, MCF7 and HeLa cells
were transfected with chloramphenicol acetyltransferase constructs
containing ER gene promoter P1 sequences. We show here that the
sequences lying between nucleotides +13 to +212 are also essential for
transcription at the ER gene promoter P1 in
ER- cells, which do not express ERF-1.
Interestingly, on gel shift experiments, a complex specific to
ER- cells forms in the region spanning
nucleotides +123 to +210. We also show that promoter P1 is responsive
to estradiol in cells expressing endogenous (MCF7) or exogenous ER. We
further demonstrate, using mutational analysis and gel retardation
assays, that the three half-estrogen response elements located between
nucleotides -420 and -892 are responsible for the estradiol
inducibility of promoter P1. Because estradiol has a mitogenic effect
on both breast and endometrial epithelial cells, our data would give an
insight into the role of estrogens in the occurence of breast and
endometrial carcinomas.
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INTRODUCTION
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Estrogen receptor (ER) is a transcription factor that
belongs to the steroid/thyroid hormone/retinoic acid receptor
superfamily, which, upon activation by ligand, increases transcription
of target genes via direct interaction with specific DNA elements (see
Ref. 1 for review). The estrogen response element (ERE) has been
described as a perfect palindromic sequence: 5'-GGTCAnnnTGACC-3' (2, 3). ER binds this sequence as a head to head homodimer in which each
half-ERE would contact one molecule of ER (4). In fact, most of
estradiol-regulated promoters or activators do not contain perfect
palindromic EREs, but include one or more GGTCA motifs, which are
sometimes widely spaced (5, 6, 7, 8, 9, 10, 11, 12, 13). Despite the relatively weak affinity
of ER for these half-sites (binding of ER as a monomer), they are
sufficient for estradiol induction, and the efficiency of induction is
comparable to that obtained with perfect palindromic sequences
(10).
The effects of estrogens in target tissues are mediated by ER.
This nuclear receptor is expressed at a low level in a wide variety of
normal tissues including brain (14, 15, 16), liver (16, 17), bone (18), and
pelvic peritoneum (19). More classic estrogen target tissues are
represented by the uterus (16, 20, 21), breast (22, 23, 24), ovary (25),
and sex skin (26). However, a high level of ER expression is a
characteristic of breast carcinomas (27) and, to a much lesser extent,
of endometrial carcinomas (21). Moreover, osteosarcomas (18),
fibromatoses (28), soft part sarcomas (liposarcomas, leiomyosarcomas,
neurosarcomas), and extramammary carcinoids (29) have also been
reported to express ER.
Regulation of ER expression includes transcriptional (30, 31, 32, 33, 34, 35) and
posttranscriptional (33, 36, 37) mechanisms. Transcription of this gene
is initiated at two separate promoters: the promoter P1 (38), which is
upstream of the major transcript (mRNA1) cap site (+1) and promoter P0
(18, 35, 39, 40, 41) located usptream of nucleotide -3067 relative to the
promoter P1 + 1, which drives the expression of two mRNAs differing by
their 5'-end (mRNA 2 and 3). De Coninck et al. (42)
demonstrated that an important transcriptional regulatory element lies
between nucleotides +133 to +204. This region contains two binding
sites for a DNA-binding protein that is only expressed in
ER+ cell lines from breast (T47D, BT20, MCF7) and
endometrial (RL952 and ECC1) carcinomas. This protein, named ERF-1,
is not expressed in ER-negative cell lines such as MDA-MB-231 and HeLa
cells. These authors suggest that ERF-1 might account for the
differential expression of ER in these cell lines and would be
responsible for ER overexpression in some breast cancers. Tang et
al. (43), however, described more recently a transcriptional
enhancer ER-EH0 located between positions -3778 and -3744, which acts
in the context of the full-length promoter as a dominant
cis-acting element in the differential ER expression.
Estrogens have multiple effects in vivo. They play a
crucial role for the establishment and maintenance of pregnancy. They
are also involved in the development of female secondary sexual
characteristics and maintenance of bone mass and would favor the
occurrence of breast and endometrial carcinomas. There is some evidence
that estradiol induces the expression of the ER in vivo at
both protein and mRNA levels during the menstrual cycle in the uterus
(20, 44, 45, 46), pelvic peritoneum (19), normal breast tissue (23), and
sex skin (26). Furthermore, estradiol administration to male
Xenopus laevis (17, 47) or ovariectomized female rats
(14, 16) induces liver (16, 17) and brain (14) ER mRNA expression,
respectively. In vitro experiments on MCF7 breast cancer
cells also showed a 2-fold increase in ER mRNA expression upon
estradiol treatment (34). Moreover, Saceda et al. (33)
demonstrated that estradiol addition to the same cells led to an
increased ER mRNA transcription as detected with nuclear run-on
assays.
In this paper, we demonstrate that the human ER gene promoter P1 is
responsive to estradiol through three half-EREs that are dispersed in
the promoter. We also show that although the ERF-1 binding sites
located between nucleotides +133 to +204 seems to be prevalent for the
promoter P1 activity in ER+ cells, this region is also
crucial for the estrogen-independent activity of promoter P1 in
ER-, ERF-1 negative HeLa cells.
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RESULTS
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Human ER Promoter P1 Activity Requires Sequences from +13 to +212
Both in HeLa and MCF7 Cells
Different fragments of the human ER gene promoter region (Fig. 1A
) were subcloned into the chloramphenicol
acetyltransferase (CAT) expression plasmid pBLCAT3del whose AP1 site
has been deleted to remove the GGTCA sequence, which is a half-ERE
(48). These constructs were transfected either in MCF7 cells or in HeLa
cells. The promoter activity was assessed by measuring the amount of
CAT protein expressed using an enzyme-linked immunosorbent assay. In
MCF7 cells, the ER promoter in construct ER -900 +13 exhibited a weak
activity (Fig. 1B
, lane 1) but had no detectable activity in HeLa cells
(Fig. 1C
, lane 1). However, for both cell lines, the amount of CAT
protein increased when the P1 promoter was extended to position +120
(Fig. 1
, B and C; compare lanes 1 and 3). The increase in the amount of
CAT protein was even higher when the ER promoter sequences were
extended to position +212 (Fig. 1
, B and C; compare lanes 3 and 5). As
has been shown by De Coninck et al. (42), it should be noted
that the activity of the region of the ER promoter containing ERF-1
sites seems to be greater in ER+ cells (Fig. 1B
) than in
ER- cells (Fig. 1C
). Moreover, although the addition of
sequences 3' to +13 to the CAT constructs increased CAT expression in
both MCF7 (Fig. 1B
) and HeLa cells (Fig. 1C
), CAT expression was
apparently much higher in HeLa cells (Fig. 1C
) than in MCF7 cells (Fig. 1B
). ER promoter activity due to sequences lying between +13 and +212
also increased in other ER+ cells (BT20, T47D) and
ER- cells (MDA-MB-231) (data not shown).

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Figure 1. Transcription Initiated at Promoter P1 Is Dependent
on TATA Box Downstream Elements and Is Inducible by Estradiol
A, Schematic representation of ER gene promoter P1 constructs.
The dark thick line corresponds to the DNA sequence
derived from the ER gene, and the arrow indicates the
location of the major transcription start site. The dark
box represents the binding sites for the transcription factor
ERF-1. All numbers correspond to distance from the major transcription
start site +1. Location of HindIII,
EcoRI, and BamHI sites is shown. B, MCF7
cells were transfected with the promoter P1 constructs depicted in
panel A. Estradiol (lanes 2, 4, and 6) or vehicle (cyclodextrins, CD)
(lanes 1, 3, and 5) was added to the medium to a final concentration of
10 nM. C, HeLa cells were cotransfected with the ER
promoter P1 constructs depicted in panel A and either pSG5 (lanes 1, 3,
5) or pSG5 expressing ER (HEO) (lanes 2, 4, and 6). Estradiol was added
to the medium in both cases at a final concentration of 10
nM. All data were corrected for transfection efficiency.
The results shown are the average of four to eight separate
transfection experiments performed in duplicate. They are expressed as
the amount of CAT protein per 100-mm diameter dish assayed with a CAT
enzyme-linked immunosorbent assay. The error bars
represent the SD.
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Different Protein-DNA Complexes Are Formed in the +7 to +210 Region
in Both HeLa and MCF7 Cells
We then searched for proteins that may bind to the region
encompassing nucleotides +13 to +212. For that purpose, we used two
double-stranded DNA fragments covering this region as probes in gel
shift experiments (Fig. 2A
), together with nuclear
extracts from HeLa and MCF7 cells. On a probe spanning nucleotides +7
to +120 (Fig. 2B
), one specific complex formed in nuclear extracts from
both MCF7 and HeLa cells (Fig. 2B
, lanes 2 and 5). On a probe spanning
nucleotides +123 to +210, a fast migrating complex B1 was observed
using extracts from both MCF7 and HeLa cells (Fig. 2C
, lanes 2 and 5).
However, a complex called B3 formed in MCF7 but not in HeLa nuclear
extracts (Fig. 2C
, lanes 5 and 2). This complex corresponded to the
ERF-1 DNA complex described by De Coninck et al. (42) since
it was competed away when the wild type ERF-1 distal site was added to
the reaction (Fig. 2C
, lane 10) but remained unchanged when a mutated
ERF-1 site was added to the reaction (Fig. 2C
, lane 11). Of interest, a
specific complex called B2 whose relative migration was intermediate
between B1 and B3 was only detected in HeLa nuclear extracts (Fig. 2C
lane 2). In conclusion, the region of the human ER gene promoter P1
spanning nucleotides +13 to +212 seems to play a key role in ER- HeLa
cells that do not express ERF-1 factor. Furthermore, a protein that
binds this region between nucleotides +123 to +210 might be important
for the transcription of the ER gene in these cells.

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Figure 2. Cell-Specific Factors Bind Downstream of the TATA
Box in Promoter P1
A, Schematic representation of the double-stranded DNA probes generated
from promoter P1 +7 to +210 DNA sequences and of the double-stranded
DNA oligonucleotides corresponding to the wild type and a mutated ERF-1
distal site. The dark box represents the binding sites
for the transcription factor ERF-1. All numbers correspond to the
distance from the major transcription start site +1. Gel shift assays
were performed with nuclear extracts from HeLa and MCF7 cells using
these DNA probes. The corresponding unlabeled full-length probe or
double-stranded oligonucleotides corresponding either to the wild type
ERF-1 distal site or a mutated ERF-1 site were used for specific
competition; a 100-bp unrelated DNA piece purified from pUC19 was used
for nonspecific competition. Ten molar excess of unlabeled probe or
nonspecific DNA or 250 molar excess of double-stranded oligonucleotide
was added to the reaction as indicated above panels B
and C. B, A radiolabeled DNA fragment encompassing nucleotides +7 to
+120 was used as a probe. An arrow indicates the common
specific complex observed in nuclear extracts from both HeLa and MCF7
cells. C, A radiolabeled DNA fragment spanning nucleotides +123 to +210
was used as a probe. Three specific complexes are formed: B1, which is
common to nuclear extracts from both cells; and B2 and B3, which are
restricted to HeLa and MCF7 cells, respectively. B3 is competed away by
wild type ERF-1 distal site but not by a mutated ERF-1 site
(right panel).
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Promoter P1 Is Inducible by Estradiol in Both MCF7 and HeLa Cells
Cotransfected with an ER-Expressing Vector
We next evaluated whether the ER gene promoter would be responsive
to estrogens in MCF7 cells expressing a high level of ER mRNA and in
HeLa cells that do not express detectable ER mRNA by Northern blot.
Therefore, HeLa cells were cotransfected with pSG5 or pSG5 ER
expression vector HEO and were treated with estradiol to a final
concentration of 10 nM. Since MCF7 cells already express
ER, they were treated either with estradiol or vehicle (cyclodextrins).
In MCF7 cells, the ER promoter activity in constructions ER -900 +13,
ER -900 +120 and ER -900 + 212 increased upon addition of estradiol
(Fig. 1B
, lanes 2, 4 and 6). In HeLa cells, although the ER gene
promoter activity was also increased by estradiol, the amplitude of
activation varied among the ER promoter constructs used: 600-fold for
ER -900 +13, 10-fold for ER -900 +120, and 20-fold for ER -900 +212
(Fig. 1C
, lanes 2, 4, and 6). However, there appears to be a complex
response to estradiol with the untranslated leader, which is seen in
HeLa cells but not in MCF7 cells. Indeed, when lanes 2, 4, and 6 in
Fig. 1C
are compared, the estradiol-induced transcription decreased
tremendously when sequences +13 to +120 were added to the P1 promoter.
Therefore, promoter P1 sequences located between positions +13 and +120
might contain a regulatory element acting negatively on estradiol
inducibility in HeLa cells.
Because the effect of estradiol was the highest in HeLa cells
transfected with the ER -900 +13 construct, ER gene P1 promoter
5'-deletion mutants were generated using this construct (Fig. 3A
). The mutants were transfected in HeLa cells and, as
shown in Fig. 3B
, the estrogen-dependent activity of the ER gene
promoter decreased when the deletions increased. If one considers as
100% the estrogen-dependent activity of the ER gene promoter in the
construct ER -900 +13, then the estrogen-dependent activity of the ER
gene promoter in ER -887, ER -519, and ER -396 constructs would
represent 60%, 10%, and 1%, respectively. A meticulous search for
ER-binding sites in the sequence encompassing nucleotides -900 to +13
did not reveal any consensus canonical palindromic ERE site (2).
However, three half-ERE sites (GGTCA motifs) were found that did not
correspond to direct tandem repeats. We named them ERE1 (nucleotides
-424 to -420), ERE2 (nucleotides -864 to -860), and ERE3
(nucleotides -888 to -892). We then generated ER -900 +13 mutants in
which one or two half-EREs were mutated in various combinations as well
as a mutant with all three EREs mutated (Fig. 4A
).
Estrogen-dependent transcriptional activity of these mutants was
compared upon transfection in HeLa cells (Fig. 4B
). The ER gene
promoter constructs with one ERE mutated (lanes 2, 3, and 4) were less
active than the wild type promoter construct (lane 1) but were more
active than promoter constructs with two EREs mutated (lanes 5, 6, and
7) or three EREs mutated (lane 8). It is noteworthy that the deletions
of EREs had a more severe effect on estrogen induction than ERE
mutations. However, this was likely to be due to the fact that specific
mutations did not completely abolish ER binding whereas deletions of
the binding sites obviously did. It could also be that sequences in
addition to the EREs may be important for estrogen stimulation. In
conclusion, the three EREs contribute to the estrogen-dependent
activity of the ER gene promoter, both in ERF-1-expressing (MCF7) and
ERF-1-nonexpressing (HeLa) cells.

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Figure 3. The ER Gene Promoter P1 Contains Three Half-EREs
A, Schematic representation of the ER gene promoter P1 5'-deletion
constructs. The dark line corresponds to the DNA
sequence derived from the ER gene, and the arrow
corresponds to the major transcription start site. All numbers
correspond to distance from the major transcription start site +1.
Location of HindIII, EcoRI, and
BamHI sites is shown. The three half-EREs, ERE1, ERE2,
and ERE3, are indicated with a star. B, HeLa cells were
cotransfected with the ER gene promoter P1 constructs depicted in panel
A and either with pSG5 (lanes 1, 3, 5, and 7) or pSG5 expressing the ER
(HEO) (lanes 2, 4, 6, and 8). Estradiol was added to the medium in both
cases at a final concentration of 10 nM. All data were
corrected for transfection efficiency. The results shown are the
average of four to eight separate transfection experiments performed in
duplicate. They are expressed as the amount of CAT protein per 100-mm
diameter dish assayed with a CAT enzyme linked immunosorbent assay. The
error bars represent the SD.
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Figure 4. Three Half-EREs Contribute Additively to
Estrogen-Activated Transcription at Promoter P1
A, Schematic representation of the ERE mutants in ER gene promoter P1.
The wild type half-ERE sites are indicated in open
boxes; the mutated half-EREs are indicated by dark
boxes. The corresponding ERE-DNA sequences are represented
above (wild type) or on the right of the
figure for the mutants. The half-ERE sites are indicated
below: ERE1 (-424 to -420), ERE2 (-864 to -860), and
ERE3 (-888 to -892). B, HeLa cells were cotransfected with the ER
gene promoter P1 constructs depicted in panel A and ER-expressing
vector HEO. Estradiol was added to the medium at a final concentration
of 10 nM. All data were corrected for transfection
efficiency. The results shown are the average of four to eight separate
transfection experiments performed in duplicate. They are expressed as
the amount of CAT protein per 100-mm diameter dish assayed with a CAT
enzyme-linked immunosorbent assay. The error bars
represent the SD.
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Baculovirus-Expressed ER Is Able To Bind the Three Half-ERE Sites
Found in Promoter P1 but Not Their Mutated Counterparts
To demonstrate that ER could bind ERE1, ERE2, and ERE3 but could
not bind mutERE1, mutERE2, and mutERE3 in vitro, a gel shift
analysis was performed using double-stranded oligonucleotides
corresponding to ERE1, ERE2, or ERE3 sites as probes (Fig. 5A
) and baculovirus-expressed ER. As shown in Fig. 5B
, using ERE1 (lanes 19), ERE2 (lanes 1013), or ERE3 (lanes 1417) as
probes, a specific complex could form in extracts from SF9 cells
infected with a human ER recombinant baculovirus (lanes 2, 5, 11, and
15) but not in extracts from SF9 cells infected with the wild type
baculovirus (lanes 1, 10, 14). The ER complexes were observed as a
major complex that we named 1 for ERE1 probe, 2 for ERE2 probe, and 3
for ERE3 probe. Competitions were performed using the synthetic
double-stranded DNA fragments carrying the wild type or the mutant
half-EREs. ER complexes 1 were displaced when cold ERE1 (lane 3), cold
ERE2 (lane 6), or cold ERE3 (lane 7) was used as competitor.
Conversely, cold ERE1 competed away ER complexes formed on ERE2 (lane
12) or ERE3 (lane 16) probes. However, MutERE1 was unable to compete
with ER binding on ERE1 (lane 4), ERE2 (lane 13), or ERE3 (lane 17)
probes. Finally, MutERE2 (lane 8) and MutERE3 (lane 9) did not displace
ER complexes formed on ERE1 probe. Those data together indicate that ER
is able to bind in vitro the three half-ERE sites found in
promoter P1. Furthermore, the deleterious effects of the ERE mutations
on the estradiol response of promoter P1 construct ER -900 +13 are
likely to be due to the inability of ER to bind the mutated ERE
sites.

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Figure 5. The ER Binds in Vitro to Promoter P1
Half-EREs but Not to Their Mutated Counterparts
A, Sequences of the three half-EREs in promoter P1: for each half-ERE,
the wild type sequences are listed on the left and the
corresponding mutants on the right. Wild type ERE sites
and their mutants are in bold. These double-stranded DNA
fragments were used the for the gel shifts shown in panel B. B,
Double-stranded DNA probes ERE1, ERE2, or ERE3 were incubated with
lysates of SF9 insect cells infected with recombinant ER baculovirus
(SF9ER) or with SF9 insect cells infected with wild type baculovirus
(SF9wt) as indicated at the top of each panel. As
indicated on the top of each panel, 50 molar excess of
cold ERE1 (lanes 3, 12, and 16), cold ERE2 (lane 6), or cold ERE3 (lane
7) or 50 molar excess of the mutated half-EREs, MutERE1 (lanes 4, 13
and 17), MutERE2 (lane 8), or MutERE3 (lane 9) were added to the
reactions. The numbers adjacent to the arrows refer to
ER complexes using ERE1 probe (1 ), ERE2 probe (2 ), or ERE3 probe (3 ).
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DISCUSSION
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ER is expressed in a wide variety of normal tissues and tumors but
with dramatic differences in the level of expression. According to the
level of ER expression, these tissues can basically be classified as
low (<20 fmol/mg of cytosol protein) or high (>100 fmol/mg of cytosol
protein) ER-expressing tissues. The high ER expression group includes
mainly breast carcinomas (50% of breast cancers) and some endometrial
carcinomas, whereas the low ER expression group is represented by
normal tissues and some tumors for which the benefit of antihormonal
treatment, such as tamoxifen, is debatable (e.g.
fibromatoses). The molecular mechanisms underlying the differential
expression of the ER gene in ER+ and ER- cells
are not fully elucidated. However, it is known that the ER gene CpG
islands are hypermethylated in ER- cells (49) and that ER
gene expression can be reactivated upon demethylation (50).
Differential promoter utilization has been studied by Weigel et
al. (35), who suggested that in cells overexpressing ER (MCF7 and
T47D) both promoters P1 (mRNA 1) and P0 (mRNA 2 and 3) were functional,
whereas in normal breast cells only promoter P1 would be functional.
Grandien et al. (18) found similar results in breast cancer
cell lines but not in normal breast. It should be pointed out that
those discrepancies might arise from the different sources of human
mammary epithelial cells. In fact, promoter usage may be affected by
hormonal status (menstrual cycle, pre- or postmenopausal status) as is
suggested by our data. In any case, ER mRNA 1 is the major transcript
even in breast cancer cells such as MCF7 and T47D expressing a high
level of ER. It has also been published that two widely separated
cis-regulatory elements are involved in differential ER
expression. However, the contribution of the upstream element ER-EH0
(43) located between positions -3778 and -1744 seems to be
predominant compared with the two ERF-1-binding sites (42) located
between positions +133 and +204.
While analyzing the activity of promoter P1 in ER- and
ER+ cell lines, we observed that the region spanning
nucleotides +13 to +212 was also fundamental for the transcription
initiation at that promoter in ERF-1-negative HeLa cells. However, when
one compares the activity of the promoter P1 between ER-
(HeLa and MDA-MB-231) and ER+ (MCF7, T47D) cells, promoter
P1 does not function at all in HeLa (ER -900 + 13) or MDA-MB-231 cells
(construct ER -3500 +1) (42) when the transfected constructs do not
contain the sequences spanning nucleotides +13 to +212; in contrast, a
weak activity is detected in ER+ cells such as MCF7, T47D
(42), or BT20 (data not shown). Furthermore, as has been described by
De Coninck et al. (42), the increased activity of promoter
P1 construct ER -900 +120 compared with construct ER -900 +212 was
greater in ER+ cells (Fig. 1B
, lanes 3 and 5) than in
ER- cells (Fig. 1C
, lanes 3 and 5). The weak activity
observed with ER -900 +13 in ER+ cells is not due to the
induction of the promoter by traces of estradiol in the serum and/or
estrogenic activity of red phenol (51) because similar results were
obtained with BT20 cells that do not express a functional ER (52).
Thus, the extension of the ER gene promoter sequence downstream of
nucleotide +13 allows detection of P1 promoter activity in
ER- cells and allows ERF-1-induced promoter activity in
ER+ cells.
Four protein-DNA complexes can form within the ER gene promoter region
spanning nucleotides +7 to +210: two are common to both ER+
(MCF7) and ER- (HeLa) cells, one is specific to MCF7 and
is due to ERF-1, and one is specific to HeLa cells and called complex
B2. Those results, together with the data from the transfection
experiments, suggest that transcription factors might bind to this
region and thus would contribute to the activity of promoter P1 in MCF7
and HeLa cells. Because it has been demonstrated that ERF-1 element
plays a role in the increased activity of promoter P1 in
ER+ cells as compared with ER- cells (42), it
could be speculated from our data that the protein-DNA complexes that
are common to MCF7 and HeLa cells would represent cellular factors that
contribute to the constitutive transcription initiated at promoter P1,
whereas the complexes restricted to each cell line would contribute to
activated transcription. It remains to be seen, however, whether
complex B2 can form in nuclear extracts from other ER-
cells. Moreover, although we do not know the exact DNA sequence to
which the factors(s) involved in complex B2 bind, it seems to be
located between nucleotides +168 to +210 (data not shown). Finally, the
search in the data bank for known transcription factors that could bind
this region remained negative.
Promoter P1 is also inducible by estradiol in ER+ cells,
and this inducibility is confirmed in ER- cells
cotransfected with an ER-expressing vector. Estradiol induction
required a functional ER either endogenous (MCF7 and T47D cells) or
exogenous (HeLa or MDA-MB-231). Like other workers (7, 53, 54), we used
ER expression vector HEO although ER expressed from this vector
contains a point mutation in codon 400 (substitution of a glycine to a
valine) that makes its binding on ERE strictly dependent on estradiol
at least in vitro (55). In addition, to obtain estradiol
induction in BT20 cells, HEO was mandatory (data not shown) since these
cells express a truncated ER (52). The induction of promoter P1 by
estradiol does not require the sequences lying between +13 to +212 DNA.
Moreover, the sequences between nucleotides +13 and +120 might contain
a negative regulatory element acting on estradiol inducibility, which
seems to be predominant in ER- cells than in
ER+ cells. Furthermore, we identified within promoter P1,
three cis-acting regulatory elements that are able to confer
estrogen inducibility. They are represented by three half-EREs that we
named ERE1 (-424 to -420), ERE2 (-864 to -860), and ERE3 (-888 to
-892). As compared with the most proximal site (ERE1), the two sites
ERE2 and ERE3 are separated by 23 bp. Because ERE2 and ERE3 are in
inverted orientation, they could form a perfect palindromic ERE.
However, in estrogen-responsive promoters exhibiting perfect
palindromes, the GGTCA motifs are usually separated by 3 bp (1)
suggesting that ERE2 and ERE3 in the ER gene promoter are too widely
separated for the ER to bind as a head-to-head homodimer. The ER would
rather bind as a monomer (10) contacting other regulatory proteins (56, 57) including both DNA and non-DNA-binding proteins. Indeed, the
mutation of the ERE half-sites in promoter P1 clearly demonstrates that
the relative estrogen-induced activity attributable to each half-ERE is
quite similar. These results suggest that the three half-EREs in ER
gene promoter P1 do not act synergistically but rather additively. For
these reasons, the GGTCA motifs in promoter P1 should be considered as
half-EREs to which ER would bind as a monomer. Our data from the gel
retardation assays show that ER can bind each of these half-sites and
support the idea of ER binding as a monomer as has been previously
demonstrated for half-EREs by Murdoch et al. (10).
In conclusion, we have shown here that, in transient expression assay,
the ER gene promoter P1 has a constitutive activity in ER-
cells that could be merely attributed to the binding of regulatory
proteins to elements located in the +7 +210 region. In ER+
cells, the high activity of the P1 promoter is probably due to the
binding of ERF-1 to the +133 to +204 region and can even increase upon
estradiol addition through ER binding to ERE1 to ERE3. From a clinical
point of view, the induction of ER expression by estradiol would
provide some answers about the role of estradiol in the occurrence of
breast and/or endometrial carcinomas (27). Two major arguments support
this idea: 1) the proliferative effects of estrogens on breast
epithelial cells (23, 58, 59, 60) and 2) the existence of a relative
hyperestrogenic impregnation in breast cancer patients (27).
Interestingly, some authors (22) have found that normal breast tissue
surrounding breast carcinomas expresses a higher level of ER than
normal breast surrounding benign lesions. The induction of ER by
estradiol would explain both the increased ER expression in the normal
breast tissue and the occurrence of a carcinoma: a relative
hyperestrogenic impregnation would induce ER expression in the normal
breast, which might further potentiate the proliferative effects of
estrogens. The occurrence of mitotic events would thereby provide
opportunities for genetic mishaps and initiation of malignancy.
Conversely, our data would explain why estrogen removal using
castration, for example, can improve a subset of breast cancer patients
especially with metastatic disease. Estradiol withdrawal, indeed, would
both suppress the induction of ER expression and thus lower the
proliferative effects of the remaining traces of estradiol (from the
adrenal glands for example).
 |
MATERIALS AND METHODS
|
---|
Cell Lines
HeLa and MCF7 cell lines were obtained from the American Type
Culture Collection (Rockville MD). Cells were maintained in DMEM (GIBCO
BRL, Gaithersburg, MD) supplemented with 10% FBS (GIBCO BRL),
penicillin (100 U/ml), and streptomycin (100 µg/ml) at 37 C in a 5%
CO2 incubator. Water-soluble estradiol or vehicle
(cyclodextrins, Sigma Chemical Co., St. Louis, MO) was added to the
medium to a final concentration of 10 nM.
Plasmid Constructions
A human ER genomic clone (EcoRI fragment of
GHER2
centered by the first exon) spanning the major transcriptional start
site to nucleotide -750 was kindly provided by P. Chambon (61). In
addition, from a human placenta genomic library obtained from CLONTECH
(Palo Alto, CA), we have cloned and sequenced approximatively 4 kb of
genomic sequence 5' to the major ER mRNA initiation site and the
downstream sequences (43).
pSG5 and human ER expression vector in pSG5 (HEO) were both provided by
P. Chambon (38). The promoterless CAT expression plasmid pBLCAT3 (62)
was digested with Eco0109 and NdeI to remove the AP1 site
(48) located upstream of the CAT sequence and then religated to obtain
pBLCAT3del. A set of primers was constructed, all containing a
HindIII site. All 3'-oligonucleotides contained a
XhoI site. These oligonucleotide primers were used in PCR
with genomic cloned DNA as the template. Primers used to generate
5'-deletion constructs were ER -519 (5'-GGAAGCTTCCCAGCTGCTAAATATAGC)
and ER -396 (5'-GGAAGCTTCTAGCCAACGAGGAGG). Primers used to generate
specific 3' ends were ER +13 (5'-GGCTCGAGTCCGCCAGCTCCTG), ER +120
(5'-GGCTCGAGGTCCCGCCGACACG), and ER +212
(5'-GGCTCGAGGCAGACCGTGTCCCCGGAGG). PCR products were then subcloned
into the HindIII-XhoI sites of pBLCAT3del. The ER
-900 +13 construct was obtained by digesting both a genomic clone and
ER -519 +13 plasmid with HindIII and BamHI and
ligating the insert from the genomic clone together with the vector
from the CAT construct. To get the ER -887 +13 plasmid, ER -900 +13
construct was digested with Bsu36I and HindIII and
religated.
Transfections
Plasmid DNA was prepared by alkaline lysis and doubly purified
by equilibrium centrifugation in cesium chloride-ethidium bromide
gradients (63). All transfections were done by the calcium phosphate
precipitation method (64). For each transfection, DNA included the
cloned plasmid (5 µg for HeLa cells, 14.5 µg for MCF7 cells), 0.5
µg of HEO or pSG5 (for HeLa cells), 0.5 µg of CMV driven
ß-galactosidase for use as an internal control, and pUC vector (New
England Biolabs, Beverly, MA) if required to bring the final DNA
content to 15 µg.
Eight hours before transfection, cells were seeded into 100-mm diameter
dishes to 1 million cells per dish. Estradiol or cyclodextrins were
added twice to the medium, once with the DNA precipitate and once
18 h after transfection. Transfected cells were collected 48
h after transfection. CAT protein was quantified in the extracts using
an enzyme-linked immunosorbent assay (Boehringer Mannheim,
Indianapolis, IN) and corrected for transfection efficiency as
determined by the ß-galactosidase assay.
Gel Retardation Assays
To prepare nuclear extracts, cultured HeLa and MCF7 cells were
washed twice with PBS, collected with a scraper and expanded at 4 C in
hypotonic buffer (10 mM HEPES, pH 7.9, 10 mM
KCl). The cytoplasmic membranes were ruptured mechanically using a
Dounce B homogeneizer. Nuclei were then pelleted and washed once with
the same buffer. Nuclear proteins were extracted by incubation in high
salt buffer [20 mM HEPES pH 7.9, 420 mM NaCl,
0.2 mM EDTA, 0.5 mM dithiothreitol (DTT), 25%
glycerol, 1.5 mM MgCl2, 0.5 mM
Pefabloc (Interchim, Montlugon, France)] for 30 min at 4 C. The nuclei
were removed by centrifugation at 10,000 rpm in a microcentrifuge.
Protein concentration of the supernatant was determined using a Bio-Rad
(Hercules, CA) protein assay and was adjusted to 5 mg/ml with the above
high-salt buffer. Extracts were stored at -80 C until use. The probes
encompassing the region spanning nucleotides +13 to +210 were obtained
by PCR using a genomic ER clone as a template and the following
primers. For the +7 to +120 ER probe, the 5' primer was
5'-GGAGATCTTGGCGGAGGGCGTTCG and the 3' primer was
5'-GGCTCGAGGTCCCGCCGACACG. For the +123 to +210 ER probe, the upstream
primer was 5'-GCCGCTCGAGCTGCGTCGCCTCTAACCTCGG, and the downstream
primer was 5'-GCCGCTCGAGTGCAGACCGTGTCCC. Both probes were radiolabeled
at their 5'-end with [
32P]dCTP (3000 Ci/mmol) and T4
DNA polymerase. They were then purified on a 12% nondenaturating
acrylamide gel in 1x Tris-borate-EDTA (TBE). Ten micrograms of nuclear
extracts were incubated with 1 ng of labeled probe (5 x
107 cpm/µg) in 1x binding buffer (20 mM
HEPES, pH 7.9, 40 mM KCl, 0.4 mM DTT, 0.1
mM EDTA, 10% glycerol, 1 mM MgCl2)
containing 1 µg poly(deoxyinosinic-deoxycytidylic)acid. Specific
competitors included the unlabeled probes but also two double-stranded
oligonucleotides corresponding either to ERF-1 distal site (dwt) or a
mutated ERF-1 distal site (d1) as described by De Coninck et
al. (42). Nonspecific competitor (100 bp DNA piece from pUC19
backbone) and unlabeled probes were added at 10 molar excess whereas
double stranded oligonucleotides competitors were added at 250 molar
excess. The 20-µl reaction mixture was incubated at room temperature
for 30 min. The DNA-protein complexes were separated from the
uncomplexed DNA on a 4% acrylamide gel in 0.25x TBE.
Lysates of SF9 insect cells infected with recombinant ER baculovirus
and wild type baculovirus were kindly provided by M. Brown (65). The
wild type and the mutated ERE1, ERE2, and ERE3 that have been used in
gel shift experiments were double-stranded DNA oligonucleotides. They
were prepared by mixing both strands in equal molar ratio in water,
boiling for 10 min, and cooled slowly to room temperature.
Double-stranded oligonucleotides were then purified on a 10%
nondenaturing acrylamide gel in 1x TBE. Twenty four micrograms of SF9
insect cell lysates were incubated with 0.5 ng of radiolabeled ERE1,
ERE2, or ERE3 probes (2 x 108 cpm/µg) in 1x
binding buffer (8 mM Tris-HCl, pH 7.5, 0.5 mM
HEPES, pH 7.9, 82.5 mM KCl, 0.85 mM DTT, 0.8
mM EDTA, 8.5% glycerol) containing 1 µg
poly(deoxyinosinic-deoxycytidylic)acid and 10 µg BSA. Competitors
were added at 50 molar excess. The 20-µl reaction mixture was
incubated for 20 min at 25 C. The DNA-protein complexes were separated
from the uncomplexed DNA on a 4.5% acrylamide gel in 0.2x TBE. Gels
were dried and exposed to x-ray films.
 |
FOOTNOTES
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---|
Address requests for reprints to: Isabelle Treilleux, Département dAnatomie et de Cytologie Pathologiques, Centre Léon Bérard, 28 rue Laënnec, 69373 Lyon Cédex 08, France.
This work was supported by funds from Institut National de la
Santé et de la Recherche Médicale (INSERM) and by
Association pour la Recherche contre le Cancer (ARC).
Received for publication February 4, 1997.
Revision received April 22, 1997.
Accepted for publication May 7, 1997.
 |
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