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Endocrinology, doi:10.1210/en.2003-0024
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Endocrinology Vol. 144, No. 7 2845-2855
Copyright © 2003 by The Endocrine Society

Regulation of the Intronic Promoter of Rat Estrogen Receptor {alpha} Gene, Responsible for Truncated Estrogen Receptor Product-1 Expression

Diane Schausi, Christophe Tiffoche and Marie-Lise Thieulant

Université de Rennes I, Interactions Cellulaires et Moléculaires, Equipe Information et Programmation Cellulaires, Centre National de la Recherche Scientifique, Unité Mixte de Recherche 6026, Campus de Beaulieu, 35042 Rennes Cedex, France

Address all correspondence and requests for reprints to: Dr. M.-L. Thieulant, Equipe Information et Programmation Cellulaires, Unité Mixte de Recherche 6026, Bat 13, Campus de Beaulieu, 35042 Rennes Cedex, France. E-mail: Marie-Lise.Thieulant{at}univ-rennes1.fr.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We have characterized the intronic promoter of the rat estrogen receptor (ER) {alpha} gene, responsible for the lactotrope-specific truncated ER product (TERP)-1 isoform expression. Transcriptional regulation was investigated by transient transfections using 5'-deletion constructs. TERP promoter constructs were highly active in MMQ cells, a pure lactotrope cell line, whereas a low basal activity was detected in {alpha}T3–1 gonadotrope cells or in COS-7 monkey kidney cells. Serial deletion analysis revealed that 1) a minimal -693-bp region encompassing the TATA box is sufficient to allow lactotrope-specific expression; 2) the promoter contains strong positive cis-acting elements both in the distal and proximal regions, and 3) the region spanning the -1698/-1194 region includes repressor elements. Transient transfection studies, EMSAs, and gel shifts demonstrated that estrogen activates the TERP promoter via an estrogen-responsive element (ERE1) located within the proximal region. Mutation of ERE1 site completely abolishes the estradiol-dependent transcription, indicating that ERE1 site is sufficient to confer estrogen responsiveness to TERP promoter. In addition, ER{alpha} action was synergized by transfection of the pituitary-specific factor Pit-1. EMSAs showed that a single Pit-1 DNA binding element in the vicinity of the TATA box is sufficient to confer response by the TERP promoter. In conclusion, we demonstrated, for the first time, that TERP promoter regulation involves ERE and Pit-1 cis-elements and corresponding trans-acting factors, which could play a role in the physiological changes that occur in TERP-1 transcription in lactotrope cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ESTROGEN IS WELL known to exert pleiotropic effects on multiple tissues, including brain and pituitary gland. Most estrogen actions are mediated by two estrogen receptors (ER), ER{alpha} and ERß, encoded by two distinct genes (1, 2). The ERs belong to a large superfamily of nuclear receptors that function as ligand-inducible transcription factors (3). When activated through ligand binding, the ERs usually bind as homodimers to estrogen response elements (ERE), which are generally located in the 5'-flanking region of estrogen-responsive genes. The consensus ERE (GGTCAnnnTGACC) is an inverted palindromic sequence separated by three nucleotides (4). Transactivation by nuclear receptors is a complex process involving ligand-induced conformational changes, dissociation of corepressors, and recruitment of coactivators (5).

Multiple mRNA transcripts (6, 7, 8, 9) are generated through various mechanisms that include the use of alternate promoters (for review, see Ref. 10) and alternative splicing (11). These isoforms contribute to the tissue specificity of estrogen action. In the pituitary gland, an ER variant named TERP-1 (truncated ER product-1; Ref. 12) has been shown to be specifically expressed in the rat lactotrope cells (13). Expression of TERP-1 is dynamically regulated. We and others observed an up-regulation of TERP-1 mRNA level on the morning of proestrus and a decline by the afternoon of proestrus (13, 14). Both TERP-1 mRNA and protein levels dramatically increased throughout the second half of gestation and were abruptly inhibited by lactation (15). In both situations, serum estradiol (E2) increased concurrently to TERP-1 expression, suggesting that E2 could be implicated in transient expression patterns. Indeed, estrogen up-regulates TERP-1 expression, both in vivo and in lactotrope pituitary cell lines (13, 14, 16, 17). Although TERP-1 lacks the A/B, DNA binding, hinge region, and a portion of ligand binding domain, the truncated protein can form heterodimers with ER{alpha}, ERß, and androgen receptor (18, 19, 20). TERP-1 alone has no effect on transcription, but it suppresses ER-dependent transcription (18, 20, 21). In addition, Resnick et al. (19) indicated that TERP-1 might titrate coactivator of ER activity. Because ER{alpha} and ERß expression is not altered by E2 in the pituitary (15, 17), TERP-1 fluctuations may be a critical parameter regulating lactotrope cell responsiveness to E2 through protein-protein interactions. TERP-1 likely acts as a negative regulator of ER{alpha} activity in vivo and could exert a protective role against the high level of E2 observed at the proestrous stage or in late pregnancy. TERP-1 could also contribute to regulating the E2-induced activation of proliferation of lactotrope cells. Recently, we demonstrated that an intronic promoter in rat ER{alpha} gene controls TERP-1 expression (Ref. 20 ; GenBank accession no. AF169237). TERP-1 is spliced from a noncoding leader exon localized within intron 4 of the ER{alpha} gene. Although the transient patterns of TERP-1 expression suggest the existence of a specific transcriptional control through its promoter, the activating signals required to drive the intronic promoter remain unknown. To gain insight into the transcriptional regulation of TERP-1 gene expression, it is essential to undergo the characterization of its promoter. Neither the cis-elements nor the trans-acting factors of the ER{alpha} intronic promoter (hereafter named TERP promoter) have been identified yet. Computer analysis indicated that a number of putative binding sites are present within the upstream sequence of the TERP-1 exon (20).

In the present study, we elucidated for the first time the genetic basis of transcriptional regulation of TERP-1 in the rat pituitary. We have analyzed the basal activity of the rat TERP promoter in pituitary or nonpituitary cell lines. We showed that the TERP-1 promoter was transcriptionally the most active in the lactotrope cell lines (MMQ). We defined the most critical regions of the promoter by deletion and mutational analyses. Using transient transfection of serial 5'-flanking regions as well as EMSAs, we analyzed elements that could confer the pituitary-specific expression and the estrogen regulation of TERP-1. We demonstrated that both ER{alpha} and the pituitary-specific transcription factor Pit-1 (22) act at the proximal promoter region and are essential for full promoter activity.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture
Cells were grown at 37 C in 5% CO2. Chinese hamster ovary (CHO-K1) cells, COS-7, and MCF-7 cells were maintained in DMEM supplemented with 10% fetal calf serum (FCS). Pure lactotrope MMQ cells (23) were cultured in RPMI 1640 medium with 7.5% horse serum and 2.5% FCS. The gonadotrope-derived clonal cell line {alpha}-T3–1 (24), kindly provided by Dr. P. Mellon, was grown in DMEM in high glucose with L-glutamine, sodium pyruvate (Life Technologies, Inc., Cergy-Pontoise, France), and 10% FBS. Culture media were purchased from Life Technologies, Inc.

Reporter plasmids and expression vectors
The TERP promoter 5'-deletion constructs were prepared by PCR amplification of the designated TERP promoter longest sequence, RDL-4 (20), with MluI/XhoI sites induced by the primers (Proligo, Paris, France; Table 1Go). Amplification conditions were 94 C for 45 sec, annealing for 45 sec at 63 C, 72 C for 1 min 30 sec for 30 cycles, and a final cycle of a 7-min extension step. The PCR products were then purified, electrophoresed on a 1.2% agarose gel, visualized by ethidium bromide, and subcloned into MluI/XhoI polylinker restriction sites in pGL2 (Promega Corp., Charbonnières, France).


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  		Table 1. Primers used for generation of rat TERP promoter 5'-deletion constructs

 
Transient transfection assays
All transfections were done in triplicate. For cell specific activity, 1–5 x 105 cells were plated on six-well plates 24 h before transfection in medium supplemented with complete serum. Cells were transfected with 0.7 µg of TERP-reporter vector and 0.3 µg galactosidase expression plasmid (pCMV-gal, Promega Corp.) using FuGENE 6 according to the manufacturer’s instructions (Roche Diagnostics, Meylan, France). For rER{alpha} (25), Pit-1 (26), Pitx-1 (27), and Pitx-2a (27) transient transfection assays in COS-7 cells, the amount of plasmids transfected was 0.7 µg of TERP-reporter vector, 0.15 µg of ß-galactosidase expression plasmid, 50 ng of rat ER expression vector, 100 ng of Pit-1, Pitx-1, and/or Pitx-2a expression vector. The amount of transfected plasmids was maintained constant (1 µg) with Bluescript plasmid (Stratagene, Amsterdam Zuidoost, The Netherlands). For all studies, fresh medium (DMEM-F12 medium with 2.5% dextran-coated charcoal-treated FCS for ER studies, in presence or not of 100 nM E2, or 1 µM ICI 182,780) was added 15 h after transfection. Cells were harvested 24 h after change of media. Protein concentrations in cell lysates were determined using the colorimetric protein assay system (Bio-Rad Laboratories, Inc., Ivry/Seine, France). ß-Galactosidase assays were performed using the method essentially as described (20). Luciferase activity was determined with reagents according to the manufacturer’s instructions (Promega Corp.) using a reporter microplate luminometer system (Promega Corp.).

ERE mutation
The ERE1 site (-205/-190) was muted on both -272/+34 pGL2 and -693/+34 TERP/Luc plasmids, using QuickChange Site-Directed Mutagenesis Kit (Stratagene) following the manufacturer’s guidelines. Oligonucleotides used for ERE1 site mutation were: mut ERE1 sense (S), 5'-GGATCTCTCTGTGTTCCAGTACAAACTAAATTTTATAGAGAGTTCC-3'; mut ERE1 antisense (AS), 5'GGAACTCTCTATAAAATTTAGTTTGTACTGGAACACAGAGAGATCC-3' (underlined base represents mutated base).

EMSA
Electrophoretic assays were performed in a 20-µl binding reaction buffer [10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.5 mM dithiothreitol, 5% glycerol, and 50 mM NaCl). Each binding reaction contains 12 µg MMQ whole cell extract, 1 µg polydeoxyinosinic-deoxycytidylic acid, and 20,000 cpm of ER{alpha} consensus, Pit-1, or potential sites probes (Table 2Go) labeled with [{alpha}-32P]dCTP (>3000 Ci/mmol; ICN Biomedicals, Orsay, France) using Klenow fragment of DNA polymerase I (Roche Diagnostics, Meylan, France). For supershift Pit-1 studies and for competition studies, 6 µl of Pit-1 rabbit polyclonal antibody (Activ Motif, Rixensart, Belgium) or an excess unlabeled DNA was added 30 min before the addition of probe at room temperature. Protein-DNA complexes were resolved on a 5.5% native polyacrylamide gel using 0.5x Tris-borate-EDTA buffer. For supershift assays, 1x Tris-glycine-EDTA buffer was used. Gels were then dried and subjected to autoradiography.


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  		Table 2. Oligonucleotides used for ER and Pit-1 EMSA

 
RT-PCR
For RT-PCR conditions, MMQ cells were preincubated in RPMI medium with 1 µM ICI 182,780 during 24 h. Then, cells were treated with 10 nM E2, 1 µM ICI 182,780, or control medium for 24 h. MMQ-treated cells were collected, and total RNA was extracted using Trizol solution (Invitrogen, Gröningen, The Netherlands) according to the manufacturer’s instructions. Three micrograms of total RNA were reverse transcripted (200 U Moloney murine leukemia virus reverse transcriptase, Invitrogen) as previously described (15). Briefly, amplification conditions were 94 C for 30 sec, annealing for 45 sec, 72 C for 1 min 30 for 27 cycles, and a final cycle of a 5-min extension step. The oligos used to amplify respectively the TERP-1, ER{alpha}, and P0 mRNA as previously described (15) were TERP-1 S, 5'-CCATTTCTTGAGCTTGTTG-3'; TERP-1 AS, 5'-CGTTTCAGGGATTCGCAG-3'; ER{alpha} S, 5'-CAGCAGCGAGAAGGGAAACA-3'; ER{alpha} AS, 5'-GGGCGGGGCTATTCTTCTTA-3'; P0 S, 5'-AAYGTGGGCTCCAAGCAGATG-3'; and P0 AS, 5'-GAGATGTTCAGCATGTTCAGCAG-3'. The PCR products were electrophoresed on a 1.2% agarose gel, visualized by ethidium bromide.

Data analyses
Results are expressed as the mean ± SEM of 3–10 experiments each performed in triplicate. ANOVA analyses were performed using StatView software (SAS Inc., Cary, NC). Significant differences between treatment groups were determined by the Fisher’s test or t test. Statistical significances were inferred at P value less than 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Cell-specific activity of the TERP 5'-flanking region
We recently isolated the 5'-flanking region of the TERP exon (20). We showed that this sequence contains a major transcriptional start site, a canonical TATA box, located in close proximity to the major transcription initiation site, and several putative sites for transcription factors (Ref. 20 ; Fig. 1Go). In the present study, the cell-specific influence on the promoter activity of the TERP 5'-flanking regions was examined. The promoter region -2001/+34 and its serial 5'-deletions (-1698/+34, -1194/+34, -693/+34, and -272/+34) were placed upstream to the Luciferase (Luc) reporter gene in the pGL2-basic vector (Fig. 2Go). These constructs were transiently transfected into MMQ pure lactotrope (23) or {alpha}T3–1 gonadotrope cell lines (24). Basal transcriptional activity was also examined in nonpituitary cell lines: MCF-7 human mammary tumor cells (ER-positive), COS-7, and CHO-K1 cells (ER-negative). The promoterless vector pGL2-basic was used as a control for basal Luc expression. As illustrated in Fig. 2AGo, the full-length promoter construct (-2001/+34) displayed an activity 60- to 70-fold higher than the activity of the empty vector in MMQ cells. On the opposite, reduction of promoter region from -2001/+34 to -1698/+34 bp dramatically decreased basal activity in MMQ cells by 83%. Further truncation of the -1698/-1194-bp sequence restored the full promoter activity, suggesting the presence of negative regulatory element(s) in this region. Because deletion from -2001 to -1791 bp retained the high activity of the full-length construct (data not shown), the basal transcriptional activity of TERP promoter is regulated by strong positive elements in the distal promoter region (-1791 to -1698 bp). Basal activity obtained with the construct -693/+34 was similar to that of the full-length construct. Deletion between -693 and -272 bp caused a serious reduction in promoter activity. The activity of the -272/+34 construct was indeed reduced to about 30% of the full activity, indicating that the maximum activity in the lactotrope cell line was conferred by the region spanning from -693 to +34 bp. To test further the role of the silencer region -1698 to -1194 bp in mediating TERP promoter transcriptional inactivation, an internal deletion mutant was generated, deleting -1177 bp/-273 bp from -1698/+34/Luc. Transfection of the construct into MMQ cells led to a basal activation similar to that obtained with -272/+34/Luc (Fig. 2AGo). Therefore, data could suggest that silencer element(s) in the region -1698 to -1194 bp may require other elements located downstream (-1177 to -273 bp). Of importance, transfection of the full-length promoter construct into {alpha}T3–1 gonadotrope cell line or MCF-7 cells only induced a low activity compared with that of MMQ cells (Fig. 2BGo). This activity varied from 2- to 8-fold depending upon the constructs (data not shown). A very low basal activity of the full-length (Fig. 2BGo) or any TERP promoter deletion constructs (data not shown) was observed in COS-7 or CHO cells (1- to 3-fold). As a result, these experiments identified an upstream promoter of the TERP exon with lactotrope-specific activity. The cell-specific promoter activities are strictly dependent on the presence of the -693/+34 sequence. Data also suggest the existence of potent silencer element(s) in the region -1698 to -1194 bp and strong positive elements in both the distal and proximal promoter regions.



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  		Figure 1. Schematic representation and localization of TERP promoter. Pictorial representation of the rat ER{alpha} gene showing location of introns and exons. The TERP promoter region is contained within intron 4. The leader exon TERP is indicated as a black box. Putative cis-elements and position of deletion constructs are indicated. An arrow indicates the transcription start site.

 


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  		Figure 2. Analysis of promoter activity of deletion constructs of the TERP 5'-flanking region in various cells. A, The schematic diagram on the left represents a series of TERP promoter-luciferase gene constructs with variable 5'-ends (from -2001 bp to -272 bp) and the same 3'-end (+34 bp) including the TATA box. The constructs were all cloned into a Luc reporter vector (pGL2-Basic). Each construct and the promoterless vector were transiently transfected into MMQ cells. B, The full-length construct (-2001/+34/Luc) was transiently transfected into lactotrope MMQ, gonadotrope {alpha}T3–1, MCF-7, CHO, and COS-7 cells. Data were normalized to ß-galactosidase activity and expressed relative to the normalized activity of the pGL2-Basic. They are given as the mean ± SEM of three to seven independent experiments performed in triplicate.

 
The TERP promoter contains one ERE that binds ER{alpha}
To determine the promoter elements required for the E2 response, we previously mapped the ER binding sites in the TERP promoter by computer analysis. A putative consensus palindromic ERE was found at position -205/-190 and designated as ERE1. It is similar to the consensus ERE described in the vitellogenin A2 gene of Xenopus laevis (4) for 11 of 12 base pairs. Four perfect half-EREs were also found at positions -1645, -1375, -633, and -315.

To investigate whether the ERE1 site can efficiently function as an ER-binding site, EMSAs were performed with in vitro translated ER{alpha}. As shown in Fig. 3AGo, ER{alpha} forms DNA-protein complex with a 33-bp probe (-184/-216) containing the ERE1 sequence (Fig. 3AGo, lane 3). An ideal consensus ERE (25-fold excess) competed for ER binding (Fig. 3AGo, lane 4), whereas a 100-fold excess of a unrelated sequence [the glucocorticoid response element (GRE)] had no effect on ER binding (Fig. 3AGo, lane 5). As illustrated in Fig. 3BGo (lane 2), the probe bound to proteins from MMQ cell extracts. These protein-DNA complexes were effectively competed at least partly by the probe (lane 3) or by the ERE consensus oligonucleotide (lane 4), but not by a 100-fold excess of a GRE (lane 5). These results indicate that the entire potential ERE1 sequence in the TERP promoter is sufficient to efficiently bind ER{alpha} at least in vitro.



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  		Figure 3. Gel mobility shift assays using ERE1-containing oligonucleotides. A, In vitro translated ER{alpha} protein was incubated with radiolabeled oligonucleotides encompassing the ERE1 sequence. Competition experiments were performed in the presence of a 25- or 100-fold excess of a consensus ERE (cERE) or GRE, respectively. B, The [32P]ERE1 oligonucleotides (ERE1*) were incubated either alone (lane 1) or with whole-cell extracts (WCE) from MMQ cells (lanes 2–5). Competition was performed using a 50-fold molar excess of unlabeled ERE1 (lane 3) or consensus ERE (lane 4) or a 100-fold excess of a consensus GRE (lane 5). The DNA-protein complexes were fractionated on a nondenaturing polyacrylamide gel electrophoresis and visualized by autoradiography. The mobility of the ER complex is indicated by arrowheads. NS, Nonspecific complexes. The experiment was repeated twice and led to similar results each time.

 
Transcriptional activation by estrogen
In rats, E2 increases TERP-1 abundance in pituitary gland (13, 14, 16). Induction of TERP-1 protein is also correlated with high levels of circulating E2 (15). We previously showed that MMQ cells express appreciable amounts of TERP-1 mRNA (20). Herein, the inducibility of TERP-1 mRNA by E2 was assessed in MMQ cells by studying the E2 effect on expression of the endogenous TERP gene. Treatment of MMQ cells with ICI 182,780, a pure antiestrogen, completely inhibited basal expression of TERP-1 mRNA. In contrast, addition of 100 nM E2 highly increased TERP-1 mRNA level (Fig. 4Go), suggesting that estrogen induces expression of the TERP gene at the transcriptional level.



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  		Figure 4. Transcriptional regulation of rat endogenous TERP expression by estrogen in MMQ cells. The cells were cultured in RPMI 1640 medium (C, control medium) or in presence of 100 nM E2 or 1 µM ICI 182,780 for 24 h. RT-PCR was performed as described in Materials and Methods, using P0 as internal control. A representative PCR is shown. PCR without RT products was included as a negative control.

 
To further study transcriptional activation by E2, the TERP promoter constructs (Fig 5AGo) were transiently cotransfected with the rat ER{alpha} expression vector either into COS-7 cell line, which is devoid of ER{alpha}, or in MMQ cells, which are ER{alpha} positive. COS-7 cells were cultured in dextran-coated charcoal-treated serum with or without E2 (100 nM) or ICI 182,780 (1 µM). As illustrated in Fig. 5BGo, all constructs enhanced reporter gene transcription in the presence of E2, whereas the pGL2-basic vector was not reactive to E2/ER treatment. The constructs -2001/+34, -1194/+34, and -693/+34 gave a similar 4- to 4.5-fold induction, ruling out the involvement of the distal half EREs (Fig. 5AGo). When transfections were performed with the -272/+34/Luc construct, which includes the ERE1 sequence, the ligand-dependent reporter gene activation was significantly lower than with the -693/+34 region (2.6 ± 0.05-fold; n = 3; P < 0.005) in presence of E2. These results demonstrate that E2 activates the promoter activity through the proximal ERE1 and potentially through the two half EREs within the -693/-272 region.



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  		Figure 5. Effects of estrogen on the promoter activity of the TERP 5'-flanking deletion regions in COS-7 cells. A, Schematic representation of the various deletion constructs of the TERP 5'-flanking region is shown. Putative ER binding sites are indicated (half ERE and ERE1). B, COS-7 cells were transiently transfected with the -2001/+34, -1194/+34, -693/+34, and -272/+34 TERP/Luc constructs or with the empty vector (pGL2-basic). Each construct was cotransfected with ER{alpha}. ß-Galactosidase was cotransfected as an internal control. After transfection, cells were cultured in phenol red-free DMEM/F12 supplemented with charcoal-stripped serum, with or without 100 nM E2, for 24 h. Data were normalized to ß-galactosidase activity. They are given as the mean ± SEM of three to seven independent experiments performed in triplicate. The relative luciferase activities presented as fold induction over that obtained in the absence of E2 are indicated.

 
Similar experiments were then performed using MMQ cell line (ER positive). TERP constructs were transiently transfected into MMQ cells in the presence of complete serum supplemented with E2 (100 nM) or ICI 182,780 (1 µM). Figure 6Go shows that the five constructs (-2001/+34, -1698/34, -1194/+34, -693/+34, and -272/+34) enhanced reporter gene transcription in the presence of E2. The activation of transcription by estrogen was maximal with the full-length construct, which gave a 4.4-fold induction. Similar E2-dependent activation was observed for -1698, -1194, and -693 constructs, indicating no major role of distal half EREs. However, activation of the full-length and -1194 constructs was significantly higher than that of construct -272 (P < 0.01), suggesting that proximal half EREs could be involved in transcriptional regulation by E2.



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  		Figure 6. Effects of estrogen on the promoter activity of the TERP 5'-flanking deletion regions in MMQ cells. See the schematic representation of the various deletion constructs of the TERP 5'-flanking region on Fig 5AGo. MMQ cells were transiently transfected with the -2001/+34, -1898/+34, -1194/+34, -693/+34, -272/+34 TERP/Luc constructs, or with empty vector (pGL2-basic) and ß-galactosidase as an internal control. After transfection, cells were cultured in RPMI 1640 containing complete serum in the presence of 1 µM ICI 182,780 or 100 nM E2 for 24 h. Data were normalized to ß-galactosidase activity. They are given as the mean ± SEM of three independent experiments performed in triplicate. The relative luciferase activities presented as fold induction are indicated.

 
Identification of cis-acting estrogen element by site-directed mutagenesis
To determine whether proximal half EREs could be involved in estrogen responsiveness, MMQ cells were transfected with the -272/+34 TERP/Luc construct, the -693/+34 TERP/Luc, or the same constructs mutated at the ERE1 site. Figure 7Go shows that mutation introduced at the ERE1 site within the -272/+34 TERP/Luc construct or -693/+34 TERP/Luc constructs dramatically reduced up to basal activity the Luc activities in MMQ cells. Interestingly, this finding suggests that ERE1 element is important for full promoter activity and sufficient to mediate E2-dependent activation of TERP promoter.



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  		Figure 7. Effect of ERE1 mutation on TERP promoter activity. Schematics of the -272/+34 TERP, -693/+34 TERP, and -mut ERE1 constructs are presented on the left. The mutated sites are indicated by X. MMQ cells were transiently transfected with the respective TERP promoter constructs followed by treatment with 1 µM ICI 182,780 or 100 nM E2. Data were normalized to ß-galactosidase activity. The data are given as the mean ± SEM of two independent experiments performed in triplicate.

 
Transcriptional activation by Pit-1 and Pitx-1/2
The pituitary-specific protein Pit-1 is restrictedly expressed in somatotrope, thyrotrope, and lactotrope cells (28). Pitx-1 and Pitx-2 genes are abundantly transcribed in pituitary cells or pituitary adenomas (27, 29, 30, 31, 32, 33). In lactotrope cell lines like GC and MMQ, Pitx-2 mRNA expression predominates (33). The TERP promoter contains nine putative Pit-1-binding sites (Fig. 8AGo) and five putative Pitx-1/Pitx-2a -binding sites (Fig. 1Go). Pitx proteins recognize the same consensus TAA(T/G)CC DNA element (34). Accordingly, the ability of Pit-1 or Pitx-1/Pitx-2a to stimulate transcriptional activity of promoter was assessed by transfection in COS-7 cells. Pit-1 expression vector increased by 4.5 ± 1.2-fold (n = 10) the reporter activity of -2001/+34 construct (Fig. 8BGo). Similar results were obtained with the -693/+34 construct (Fig. 8BGo). Thus, data suggest that the TERP promoter is a target gene of Pit-1 in its proximal region.



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  		Figure 8. Effects of Pit-1 on the promoter activity of the TERP 5'-flanking deletion regions in COS-7 cells. A, Putative ER (half ERE and ERE1) and Pit-1 binding sites are indicated. Position of the two deletion constructs of the TERP 5'-flanking region is shown (-2001/+34 or -693/+34 TERP/Luc). B, Each TERP/Luc construct or empty vector (data not shown) was cotransfected with the Pit-1 plasmid and/or ER{alpha} plasmid into COS-7 cells. ß-Galactosidase was cotransfected as an internal control. After transfection, cells were cultured in phenol red-free DMEM/F12 supplemented with charcoal-stripped serum, with or without 100 nM E2, for 24 h. The relative luciferase activities normalized to ß-galactosidase activity were calculated relative to that of each TERP Luc construct transfected alone, which was normalized to 1. The data are given as the mean ± SEM of 3–10 independent experiments performed in triplicate. ***, P < 0.0001

 
When the Pit-1 expression vector was cotransfected with ER{alpha} vector in COS-7 cells, the promoter activity of the -2001/+34/Luc construct was activated more dramatically than by Pit-1 alone. Figure 8BGo shows that the cotransfection of Pit-1 and ER{alpha} expression vectors in the presence of E2 significantly increased the activity of the -2001/+34/Luc construct by 11-fold (n = 3), whereas the activation of transcription by estrogen in the absence of Pit-1 was only 4-fold. The Pit-1 action was not affected by cotransfection of ER{alpha} in E2 absence (data not shown). Strictly similar results were obtained with the -693/+34 construct (Fig. 8BGo). The coexpression of Pit-1 and ER{alpha} induced a nearly 3-fold increase in the response compared with that seen with E2-bound ER{alpha} alone or Pit alone, suggesting a cooperative interaction between Pit-1 and ER{alpha} as observed for prolactin (PRL) gene (28, 35).

When the Pitx-1 expression vector was transfected along with -2001/+34/Luc construct or the -693/+34/Luc construct in COS-7 cells, both constructs were activated 3.6 ± 0.5-fold (n = 7), compared with the Luc construct alone (data not shown). Similar data were obtained with the Pitx-2a vector, suggesting that Pitx-1/2 may enhance the activity of the TERP promoter at the most proximal Pitx-binding sites. Neither Pit-1 nor ER{alpha} affected the increased promoter activity by Pitx1 or Pitx-2a (data not shown). Then, the possibility that Pitx1 could exert trans-acting activity through interaction with Pit-1 as reported for PRL and GH promoters (for review, see Ref. 36) or ER{alpha} may be ruled out.

Pit-1 responses can be conferred by a single proximal element
Sequence analysis of the DNA region between -693 and +34 revealed the presence of six DNA sites with significant homology to the consensus Pit-1 binding, located at positions -677/-665, -645/-633, -479/-456, -442/-430, -376/-363, -93/-82 (Fig. 8Go). The more proximal putative element [ATGAATTTTCAA] located at -93/-82 is referred to as P1. EMSAs were performed using oligonucleotide probe corresponding to P1 sequence and whole cell extracts derived from MMQ cells (Fig. 9Go). Two major protein-DNA complexes are observed when protein extracts are incubated with P1 (Fig. 9Go, lane 2). A supershift of the two protein-DNA complexes is observed with the addition of a Pit-1-specific antibody (Fig. 9Go, lane 3), indicating that they consist of Pit-1. Binding of these complexes to P1 site is also inhibited by a 50-fold excess of unlabeled P1 (Fig. 9Go, lane 4), suggesting that these complexes are specific. Moreover, the complexes are not eliminated by a 50-fold excess of nonspecific DNA (chimeric intron; Fig. 9Go, lane 6). Interestingly, one of the protein-DNA complexes is partially inhibited by an excess of ERE1 oligonucleotide sequence (Fig. 9Go, lane 5), confirming our results that protein-protein interactions between ER and Pit-1 occur to the Pit-1 element. Therefore, these results indicate specific binding of a protein(s) in the MMQ nuclear extracts to the P1 oligonucleotide. Antibody supershift assays identify Pit-1 as a component of the DNA-protein complex formed by MMQ nuclear extracts on P1 and confirm that P1 is the Pit-1 recognition site. On the other hand, binding of MMQ protein extracts to the other (-677, -645, -479, -442, -376) oligonucleotide sequences is nonspecific. It is not competed by excess unlabeled oligonucleotide and is not supershifted by a Pit-1-specific antibody (data not shown).



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  		Figure 9. Gel mobility shift assays using Pit-1-containing oligos and proteins from MMQ cells. The [32P]P1 oligonucleotide was incubated either alone (lane 1) or with whole-cell extracts (WCE) from MMQ cells (lanes 2–6). A Pit-1-specific antibody was used for supershift studies (lane 3). Competition was performed using 50-fold molar excess of unlabeled P1 or 100-fold molar excess of consensus ERE (lane 5), or 100-fold excess of nonspecific intronic DNA (lane 6), respectively. The 32 P-labeled oligos were fractionated on a nondenaturing polyacrylamide gel electrophoresis and visualized by autoradiography. The experiment was repeated twice with similar results. Upper arrow, Complex supershifted with the Pit-1-specific antibody; middle and lower arrow, specific protein-DNA complexes.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this paper, we characterized the intronic promoter of the rat ER{alpha} gene responsible for the transcription of TERP-1. To examine the functionality and cell-specific regulation of the TERP promoter, we introduced a series of TERP promoter constructs into both pituitary and nonpituitary cells. We found a very low activity of 5'-flanking region of the TERP exon in COS-7 or MCF-7 cells. Similarly, the mouse gonadotrope cell line, {alpha}T3–1, did not allow high activity of the promoter. In contrast, we found a strong activity of the TERP promoter in the rat lactotrope cell line MMQ, showing the cell specificity of this promoter. The highest transcriptional activity was observed with the full-length construct (2.0 kb) as well as with the 1.2- and 0.7-kb constructs, indicating that the 0.7-kb region adjacent to the TATA box contains responsive elements that ensure a maximal transcriptional activity in lactotropes. MMQ cells express ER{alpha} and Pit-1, whereas {alpha}T3–1 cells express ER{alpha} but not Pit-1, suggesting that this factor could be an important regulator. However, it cannot be excluded that {alpha}T3–1 cells may lack some rat-specific factor(s). The low transcriptional activity observed with the 1.7-kb construct suggests the existence of a potent silencing element located in the -1698/-1194 region because deletion of this region restored the promoter activity. In addition, this decrease in transcriptional activity points to the existence of strong positive elements between the -1791 and -1698 and the -1194 and -693 regions, respectively. Consequently, data from transfection studies indicate that the promoter is functional and that it has strong positive and negative cis-acting elements. However, the negative effect may in vivo be suppressed because the presence of a positive sequence downstream induces the enhancement. No site of interest was found when TRANSFAC or other available databases screened the -1791/-1698 sequence. The negative regulatory sites in the distal promoter region and the mechanism of the negative regulation are totally unknown at present and remain to be investigated in the future.

Screening of potential regulatory factors previously indicated that the promoter sequence contains response elements that may mediate the stimulation by ER{alpha}. Indeed, we found that treatment of MMQ cells with E2 resulted in TERP promoter induction and TERP-1 mRNA expression. In addition, E2 enhancement in COS-7 cells was dependent upon coexpression with ER{alpha} and was inhibited in the presence of the pure antiestrogen, indicating that ER{alpha} can activate the TERP promoter. The EMSAs identified the ERE (termed ERE1) at -205/-190 in the vicinity of the TATA box as functional. In addition, site-directed mutagenesis showed that ERE1 sequence is sufficient to confer full estrogen responsiveness, ruling out a high induction by putative half EREs detected in proximal region. However, the full-length promoter activity was significantly enhanced by E2 treatment in both MMQ or ER{alpha}-transfected COS-7 cells, suggesting the cooperation with other factors not highlighted yet. Estrogen induction of -272/+34 construct in MMQ cells was in the magnitude of that with a single consensus ERE element. When ERE1 was placed in tandem with itself, synergistic activation of transcription by E2 (9-fold induction, data not shown) was observed. The presence of a functional ERE sequence suggests that estrogens have a direct effect on the promoter activity and may play a role in the regulation of TERP-1 expression. To our knowledge, the ERE of the TERP promoter is the first ERE characterized as a functional ERE in a natural promoter of ER{alpha} in mammals. Therefore, our data demonstrate that physiological effects of estrogens can be mediated directly at the level of the TERP promoter via the ER{alpha}. The presence of a functional ERE is consistent with earlier studies showing an induction of TERP-1 expression by E2, in vivo (12, 13, 16). E2 injection to rats elicited a very rapid increase in TERP-1 levels (12, 13, 16), suggesting the existence of a direct transcriptional mechanism. This possibility is reinforced by our results showing the response of the gene to E2 in MMQ cells.

The TERP promoter has potential binding sites for pituitary transcription factors, such as Pit-1 and Pitx-1/2. The transcription factor Pit-1 is a pituitary-specific POU domain factor, which is necessary for development of somatotroph, lactotroph, and thyrotroph cells (28). In addition to its developmental role in the pituitary gland, Pit-1 also has an important function in the hormonal regulation of pituitary hormone gene expression (for review, see Ref. 36). Numerous studies have shown that Pit-1 binds to the promoter regions of the GH and PRL genes as well as to Pit-1 gene itself, controlling their pituitary-specific expression (36). Because several potential Pit-1 binding sites were identified in the promoter region of the rat TERP gene, we evaluated the ability of Pit-1 to stimulate the promoter activity in transfected COS-7 cells (devoid of any endogenous Pit-1). We observed that cotransfection of a Pit-1 expression vector increased the activity of the full-length promoter, which contains eight potential Pit-1 binding sites. However, the 0.7-kb construct that contains six potential Pit-1 binding sites was sufficient to ensure the maximal activity. Of importance, Pit-1 action is synergized by ER{alpha}, indicating that Pit-1 may exert a cooperative interaction with ER{alpha} in regulating TERP-1 expression. Similar cooperative interactions between Pit-1 and ER{alpha} have been shown to be important in the regulation of the PRL gene transcription (28, 35, 37). To determine the ability of six potential Pit-1 sites of the 0.7-kb region, EMSAs studies were performed using whole cell extracts from MMQ cells. Data demonstrated specific binding to the proximal Pit-1 site (P1) but not to the other putative sites. The protein(s) bound to the P1 site was recognized in supershift assays with a Pit-1-specific antibody, indicating that the complex contains Pit-1. It remains possible that proteins other than Pit-1 may also bind to Pit-1 element and play an additional role in activating the TERP promoter. Indeed, one of the Pit-1-DNA complexes is partially inhibited by an excess of ERE1 oligonucleotide sequence, confirming our results that protein-protein interactions occur between ER{alpha} and Pit-1. Data from Nowakowski and Maurer (38) demonstrating that ER{alpha} can bind to Pit-1 immobilized on glutathione agarose beads may explain this result. However, most of the interaction between Pit-1 and the ER{alpha} appears to be DNA dependent. Therefore, a single proximal Pit-1 binding site, located at -93/-82, is sufficient to confer responsiveness to TERP promoter, which may play a central role in regulating the TERP promoter. Both ER{alpha} and Pit-1 activated the promoter, and they acted synergistically.

The bicoid-related homeoprotein Pitx-1 (27, 29, 30) is expressed in all adult pituitary cell lineages and in pituitary adenomas (31, 33, 39, 40). Pitx-1 factor is known as the pan-pituitary activator of transcription and activates the transcription of most pituitary hormone genes (32). One other protein related to Pitx-l, named Pitx-2 (also known as Otlx2 and Rieg; Refs. 29, 30, 31 and 41) is expressed in pure lactotrope adenomas but not in somatotrope adenomas (42). Several Pitx2 isoforms have been described that can form homodimers or heterodimers (43). Our data indicate that Pitx-1 or -2a is capable alone to activate the TERP promoter. Synergism between Pitx-1 and Pit-1 was observed for PRL or GH promoters but not ßTSH promoter (32, 44). However, neither ER{alpha} nor Pit-1 enhanced the transcriptional activity of TERP promoter induced by Pitx-1 or -2a (data not shown). Because activation was not additive, a mutually exclusive binding of the Pit-1 and Pitx factors cannot be ruled out. The recruiting cofactors to the Pit-1 binding site (45, 46) or the DNA bending (47) caused by interaction of Pit-1 with DNA recognition sites could prevent Pitx binding to DNA. Pitx2 isoforms could differentially regulate the TERP promoter. A recent paper demonstrates that Pitx 2b activates the PRL promoter at higher levels than either Pitx 2a or Pitx 2c (43). Future studies will further assess the contribution of other Pitx 2 isoforms to regulate TERP promoter.

The present investigation provided, for the first time, the molecular basis of TERP regulation at the transcriptional level. We demonstrate the transcriptional regulation of TERP promoter by ER{alpha} and Pit-1 factors, which could play a role in the physiological changes that occur in TERP-1 transcription in lactotrope cells. The existence of a specific transcriptional control of the expression of the TERP-1 through the TERP promoter was previously suggested by the patterns of transient expression during estrous cycle (13, 14) and pregnancy (15). The increase in the expression of TERP-1 at proestrus and late gestation may be correlated to the high estrogen concentration at these periods. We and others (12, 13, 16) showed a role for estrogen in the in vivo induction of TERP-1 mRNA and protein expression in the female rat. Our data demonstrate that estrogen exert a direct effect on TERP promoter, possibly explaining the ability of estrogen to induce TERP-1 transient expression in lactotrope cells. A potentially complex cascade of events could occur; ER{alpha} with other factors may activate the internal promoter of ER{alpha} gene triggering TERP-1 expression, which may interact with ER to modulate its action, suggesting a regulating feedback loop. The present study revealed that both ER{alpha} and Pit-1 are required for full expression of the TERP gene, suggesting TERP involvement in the development and function of lactotrope cells. The pituitary-specific homeodomain proteins Pitx-1 or -2 may also regulate transcription of the TERP gene at least in part through specific sites in the promoter. Probably, the combined activity of identified factors with other undetermined cell-specific or ubiquitous transcription factors is needed to regulate TERP transcription in a cell-specific manner. Therefore, data suggest that TERP promoter may be regulated by multiple factors.

In summary, our initial characterization of an ER{alpha} gene intronic promoter demonstrates that this gene contains a promoter region with a strong specific activity in lactotrope cell lines. The present study allowed us to identify a region located at 0.7 kb upstream of the transcription start site. This sequence was shown to be crucial to confer a basal promoter activity in lactotrope cell lines. Gel shift and mutation analyses revealed functional cis-elements for the transcription factors, ER{alpha} and Pit-1. Transfection experiments showed regulation of the TERP promoter by estrogen in synergy with Pit-1. The factors involved in the repression of gene expression were not identified in this study. Therefore, further studies are necessary and will provide insights into the global mechanism that regulates TERP expression.


   Acknowledgments
 
We thank Dr. P. Mellon (University of California, La Jolla, CA) for kindly providing the {alpha}T3–1 pituitary cells and Drs. C. Pasqualini and P. Vernier (Institut A. Fessard, Gif-sur-Yvette, France) for generous provision of the MMQ cells generated by Dr. R. M. MacLeod (University of Virginia, Charlottesville, VA). Our thanks go to Dr. J. Drouin (University of Montréal, Montréal, Québec, Canada) for the generous gift of the rat Pitx-1 and Pitx-2a factor plasmids, Dr. H. P. Elsholtz (Research Institute, Toronto, Ontario, Canada) for the Pit-1, and Dr. M. Muramatsu (University of Tokyo, Tokyo, Japan) for the rat ER{alpha}. We are grateful to Dr. F. Demay and Dr. F. Chesnel (University of Rennes I, Rennes, France) for critical reading of this manuscript.


   Footnotes
 
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank Data Bank with accession no. AF169237.

Abbreviations: AS, Antisense; CHO, Chinese hamster ovary; E2, estradiol; ER, estrogen receptor; ERE, estrogen response element; FCS, fetal calf serum; GRE, glucocorticoid response element; PRL, prolactin; S, sense; TERP, truncated ER product.

Received January 6, 2003.

Accepted for publication April 4, 2003.


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 Discussion
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W. M. Bryant, M. A. Gibson, and M. A. Shupnik
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