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 d’Anatomie 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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 (RL95–2 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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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. 1AGo) 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. 1BGo, lane 1) but had no detectable activity in HeLa cells (Fig. 1CGo, lane 1). However, for both cell lines, the amount of CAT protein increased when the P1 promoter was extended to position +120 (Fig. 1Go, 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. 1Go, 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. 1BGo) than in ER- cells (Fig. 1CGo). Moreover, although the addition of sequences 3' to +13 to the CAT constructs increased CAT expression in both MCF7 (Fig. 1BGo) and HeLa cells (Fig. 1CGo), CAT expression was apparently much higher in HeLa cells (Fig. 1CGo) than in MCF7 cells (Fig. 1BGo). 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.

 
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. 2AGo), together with nuclear extracts from HeLa and MCF7 cells. On a probe spanning nucleotides +7 to +120 (Fig. 2BGo), one specific complex formed in nuclear extracts from both MCF7 and HeLa cells (Fig. 2BGo, 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. 2CGo, lanes 2 and 5). However, a complex called B3 formed in MCF7 but not in HeLa nuclear extracts (Fig. 2CGo, 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. 2CGo, lane 10) but remained unchanged when a mutated ERF-1 site was added to the reaction (Fig. 2CGo, 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. 2CGo 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).

 
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. 1BGo, 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. 1CGo, 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. 1CGo 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. 3AGo). The mutants were transfected in HeLa cells and, as shown in Fig. 3BGo, 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. 4AGo). Estrogen-dependent transcriptional activity of these mutants was compared upon transfection in HeLa cells (Fig. 4BGo). 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.

 
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. 5AGo) and baculovirus-expressed ER. As shown in Fig. 5BGo, using ERE1 (lanes 1–9), ERE2 (lanes 10–13), or ERE3 (lanes 14–17) 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 ).

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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. 1BGo, lanes 3 and 5) than in ER- cells (Fig. 1CGo, 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
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 {lambda}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 [{alpha}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
 
Address requests for reprints to: Isabelle Treilleux, Département d’Anatomie 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.


    REFERENCES
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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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