Proximal cis-Acting Elements, Including Steroidogenic Factor 1, Mediate the Efficiency of a Distal Enhancer in the Promoter of the Rat Gonadotropin-Releasing Hormone Receptor Gene

Hanna Pincas, Karine Amoyel, Raymond Counis and Jean-Noël Laverrière

Endocrinologie Cellulaire et Moléculaire de la Reproduction Université Pierre et Marie Curie Centre National de la Recherche Scientifique ESA 7080, Paris, France


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The gonadotrope-specific and regulated expression of the GnRH receptor (GnRH-R) gene is dependent on multiple transcription factors that interact with the noncanonical GnRH-R activating sequence (GRAS), the activator protein-1 (AP-1) element, and the steroidogenic factor-1 (SF-1) binding site. However, these three elements are not sufficient to mediate the complete cell-specific expression of the rat GnRH-R gene. In the present study, we demonstrate, by transient transfection in gonadotrope-derived {alpha}T3–1 and LßT2 cell lines, the existence of a distal enhancer [GnRH-R- specific enhancer (GnSE)] that is highly active in the context of the GnRH-R gene promoter. We show that the GnSE activity (–1,135/–753) is mediated through a functional interaction with a proximal region (–275/–226) that includes the SF-1 response element. Regions of similar length containing either the AP-1 or GRAS elements are less active or inactive. Transfection assays using an artificial promoter containing two SF-1 elements fused to a minimal PRL promoter indicate that SF-1 is crucial in this interaction. In addition, by altering the promoter with deletion and block- replacement mutations, we have identified the active elements of GnSE within two distinct sequences at positions –983/–962 and –871/–862. Sequence analysis and electrophoretic mobility shift experiments suggest that GnSE response elements interact, in these two regions, with GATA- and LIM-related factors, respectively. Altogether, these data establish the importance of the GnSE in the GnRH-R gene expression and reveal a novel role for SF-1 as a mediator of enhancer activity, a mechanism that might regulate other SF-1 target genes.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The hypothalamic decapeptide, GnRH, interacts with high-affinity, cell-surface G protein-coupled receptors present in the anterior pituitary, resulting in the increased expression of at least three genes that encode the common {alpha}-subunit and the specific ß-subunit of LH and FSH. In addition to this action at the gene level, GnRH also stimulates the release of LH and FSH, which, in turn, through the systemic circulation, orchestrate gonadal function including steroid hormone synthesis and release, as well as gametogenesis (reviewed in Refs. 1, 2, 3, 4).

Both the specificity and magnitude of the pituitary response to GnRH are highly dependent on GnRH receptor (GnRH-R) gene expression. Although the mature anterior pituitary is composed of at least five different endocrine cell types, only gonadotropes express the GnRH-R, which confers cell-specific regulation of gonadotropin secretion. Furthermore, the sensitivity of the pituitary gland to hypothalamic GnRH inputs is dependent on the number of active cell-surface GnRH-Rs, which is itself regulated, at least in part, at the transcriptional level. Thus, molecular mechanisms underlying gonadotrope-specific expression and transcription efficacy of the GnRH-R gene are critical for the normal functioning of the pituitary- gonadal axis.

In addition, the GnRH-R gene is expressed, although to a lower extent, in other tissues such as the hippocampus and the hypothalamus (3) as well as in multiple rat ovarian compartments, especially the granulosa cells of atretic follicles (5, 6, 7). GnRH-R mRNAs have also been detected in Leydig cells, and mature and fetal testes and ovaries as well as in human breast and placental trophoblasts during pregnancy (8; Ref. 9 and references therein; 10–13). More recently, the presence of mRNA encoding GnRH-R has also been described in human peripheral blood mononuclear cells (14). Such findings raise questions about the nature of cis-regulatory elements and cognate trans-acting factors that confer either gonadotrope or extrapituitary expression to the GnRH-R gene.

To investigate this issue, the 5'-flanking sequences of the ovine, human, mouse, and rat GnRH-R gene have been isolated and partially characterized (15, 16, 17, 18, 19, 20, 21). The human, mouse, and rat promoters display strong sequence homology in a region extending over 1,200 bp upstream of the ATG codon. However, the rat and mouse promoters diverge extensively from the human promoter in the position of the transcription start sites. While transcription start sites have been identified within a region that extends over 110 bp upstream of the ATG codon of the mouse and rat promoters, the start sites of the human promoter are clustered at 0.7 and 1.4 kb upstream of the ATG codon.

The gonadotrope-derived {alpha}T3–1 cell line expresses the GnRH-R and the {alpha}-subunit of gonadotropin hormones and has been extensively used for testing the cell-specific expression of the GnRH-R gene by transient transfection assays. Analysis of the mouse promoter has led to the identification of cis-acting elements localized essentially within 500 bp upstream of the ATG codon. These elements include a new element termed the GnRH-R-activating sequence or GRAS, a consensus activator protein-1 element (AP-1), and the gonadotrope-specific element or GSE (22) that binds the nuclear orphan receptor, steroidogenic factor-1 (SF-1) (23, 24). This tripartite basal enhancer appears to be sufficient in vitro to ensure maximum gonadotrope-specific activity of the mouse promoter. The GRAS element has also been shown to mediate autocrine/paracrine stimulation of cell-specific expression of the GnRH-R gene by activin (25). In parallel, the AP-1 response element has been recently demonstrated to play a central role in conferring GnRH responsiveness in the murine GnRH-R gene (26, 27). The gonadotrope-specific activity of the human promoter is also dependent on an SF-1 response element located in the 5'-untranslated region downstream of the transcription start sites (28).

Elements analogous to the mouse GRAS, AP-1, and SF-1 are present at corresponding positions in the rat GnRH-R promoter gene. Nevertheless, despite the presence of these cis-acting elements, the promoter of the rat GnRH-R gene seems to be regulated in a different manner. For maximal gonadotrope-specific activity the presence of additional distal elements localized within the –1,150 to –750 bp region (21) are necessary. The distal elements appear to be active only in the context of the GnRH-R promoter. In fact, we have previously reported that these distal elements do not display any activity if they are directly fused to the heterologous minimal thymidine kinase (TK) promoter. However, when the full-length 1.2-kb promoter was fused to the TK promoter, cell-specific activity was recovered. We thus hypothesized that the activity of the upstream elements, thereafter referred to as the GnRH-R-specific enhancer (GnSE), necessitates the presence of promoter-specific elements located in the proximal part of the rat promoter.

In the present study, by using deletion and mutational analysis combined with functional transfection studies in the murine gonadotrope-derived {alpha}T3–1 and LßT2 cell lines (29, 30, 31), we show that the activity of the GnSE (–1,135/–750) requires sequences localized within the proximal region (–412 to –26). A critical element lies at position –245/–237 and contains the consensus sequence for the SF-1 element. The functional interaction between the GnSE and proximal elements is confirmed by the demonstration that while the GnSE and a proximal 50-bp region including the SF-1 element are capable of independently conferring a more or less modest activity to a heterologous minimal PRL promoter, both elements induced full synergistic stimulation. The regulatory elements within the GnSE have been restricted to two short sequences of 10 and 20 bp extending from –871 to –862 and from –983 to –962, respectively.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The Proximal Domain (–412 to –26) of the Rat Promoter Was Essential for Mediating the Stimulatory Effect of GnSE
To determine whether downstream elements in the rat GnRH-R gene were required for the activity of the distal positive regulatory region (–1,135/–753), we constructed a promoter fragment containing GnSE immediately upstream of the proximal region (–412/–26) that included the GRAS, AP-1, and SF-1 related elements (Fig. 1AGo). This promoter, thus deleted from an intermediate domain extending from –753 to –412, was inserted into the pCAT basic vector before the chloramphenicol acetyltransferase (CAT) gene. In addition, we designed two similar CAT constructs that contained either the 5' region (–1,135/–900) or the 3' region (–896/–753) of the GnSE. The constructs were then transiently transfected into {alpha}T3-1 cells in parallel with the promoterless vector pCAT-basic, which served as a control for basal levels of CAT activity. Promoter activities were compared with those of reference constructs containing either the full-length promoter (–1,135/–26) or a proximal region extending from –433 to –26 (Fig. 1BGo). Consistent with our previous studies (21), the construct containing the full-length 1.1-kb 5'-flanking region displayed a significantly higher CAT activity than the construct containing the proximal region only (15.8 ± 2.0- vs. 4.3 ± 1.1-fold increase in CAT expression over the promoterless vector). However, in contrast with data obtained in the study using the TK promoter (21), fusion of GnSE with the GnRH-R proximal region (distal/proximal fusion promoter) resulted in CAT activity equivalent to that of the full-length promoter (19.1 ± 3.0-fold). Furthermore, under these circumstances, the 5'-distal region of the GnSE as well as the 3'-end strongly stimulated activity of the proximal region (16.2 ± 2.5 and 11.7 ± 1.8-fold, respectively). Thus, these data suggest that the proximal region, which extends from –412 to –26, was capable of mediating the positive regulation exerted by the GnSE, whereas the intermediate domain (–753 to –412) was unnecessary. In addition, GnSE apparently contained active elements in both its 5' (–1,135/–900) and 3' (–896/–753) regions.



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Figure 1. The Proximal Region of the Rat GnRH-R Promoter Mediates the Activity of the Distal Domain

A, Structure of the full-length GnRH-R promoter and distal/proximal fusion constructs. The GRAS-, AP-1-, and SF-1-related elements are indicated by black boxes at their corresponding locations within the rat promoter sequence. B, The intermediate domain of the GnRH-R promoter was not necessary for full cell-specific activity. The {alpha}T3–1 cells were transfected with a full-length construct (–1,135/–26), a construct containing the proximal promoter region only (–433/–26), or three different distal/proximal fusion constructs containing the proximal region (–412/–26) directly fused either to the full-length GnSE (–1,135/–753), the 5'-region (–1,135/–900), or the 3'-region (–896/–753) of the GnSE. CAT activity was determined in cell extracts and corrected for transfection efficiency by normalizing to a cotransfected CMV-ß-galactosidase expression vector (CMVß). Normalized CAT activity was expressed as fold-stimulation over promoterless vector. Results are the mean of three independent transfection experiments performed in duplicate, with error bars representing the SD. Data represented by bars labeled with the same letter are not statistically different (P > 0.05). C, The importance of the SF-1-related element in mediating the positive effect of the distal domain. {alpha}T3–1 and LßT2 cells were transfected with full-length GnRH-R or the proximal promoter inserted upstream of the firefly luciferase reporter gene in pGL3 basic vector. Full-length and proximal promoters contain mutations that disrupted either GRAS (GRASmut), AP-1 (APmut), or SF-1 (SFmut) elements. Firefly luciferase activity was corrected for transfection efficiency by normalization to TK-Renilla luciferase expression vector. Normalized luciferase activity was expressed as fold stimulation over promoterless vector. The experiment was performed as in panel B.

 
Predominant Negative Effect of Block-Replacement Mutation of the SF-1 Site on GnSE Activity
Several studies in our laboratory, as well as in other laboratories, have consistently used the {alpha}T3–1 cell line as a gonadotrope-derived cell model. However, although the {alpha}T3–1 cells express the gonadotropin {alpha}-subunit and the GnRH-R gene, unlike normal gonadotropes no expression of the gonadotropin ß-subunit has been observed. We, therefore, extended our investigations to the gonadotrope-derived cell line, LßT2, which has been shown to express the gonadotropin {alpha}-subunit and the GnRH-R gene, as well as the LHß-subunit gene (30, 31) and, under activin treatment, the FSHß-subunit gene (32). The use of this cell line required an optimization in the sensitivity of the transfection assay. For this, the reference constructs were subcloned into the pGL3 basic vector containing the luciferase reporter gene.

To analyze the relative contribution of the cis-active elements within the proximal region that could potentially mediate the positive action of GnSE, we altered the full-length and proximal promoter by introducing three block-replacement mutations within the related GRAS, AP-1, or SF-1 elements. These mutant promoters were compared with the wild-type promoter and reference constructs (Fig. 1CGo). As a consequence of the presence of the GnSE, the entire promoter elicited significantly higher activity than the proximal region alone, in both {alpha}T3–1 and LßT2 cells. Mutation of the related GRAS element (GRASmut) caused a weak or nonsignificant decrease in the transcription efficacy of the proximal and the full-length promoter in both cell lines. Alignment of the rat (–412/–395) and mouse (–395/–378) GRAS sequences revealed a single base pair modification at position –399 (A to G) that might be responsible for the weak efficacy of the rat element. Indeed, a similar modification (AA to CC) decreased by 60% the activity of the mouse GRAS element (24). In contrast, block-replacement mutation of the related AP-1 site (APmut) markedly affected luciferase activity, decreasing proximal and full-length promoter activity by 85% and 68%, respectively, in {alpha}T3–1 cells. Similar decreases were observed in LßT2 cells (63% and 40%, respectively). Nevertheless, AP-1 mutation had no effect on GnSE activity since the efficiency of the full-length promoter remained significantly higher than that of the proximal promoter. More importantly, with regard to the mediation of GnSE activity, mutation of the SF-1 site (SFmut) displayed stronger efficiency in the full-length than in the proximal promoter context. Disruption of the SF-1 element abolished the differences in activity observed between the full-length and the proximal promoter. This was particularly evident in LßT-2 cells, suggesting that the SF-1 mutation affected both SF-1- and GnSE-dependent cis-acting efficiencies. The SF-1- and the AP-1-related elements were thus crucial for gonadotrope-specific activity of the rat promoter. In addition, SF-1, rather than AP-1, could mediate the effect of the GnSE.

A 50-bp Sequence, Which Included the SF-1 Element, Was Capable of Mediating the GnSE Effect
The cell models were used to determine first, if a restricted part of the proximal region (–275/–226) encompassing the SF-1-related element could effectively mediate the stimulatory effect of the GnSE and second, if this activation was gonadotrope specific. To this aim, we designed three artificial promoters, based on the heterologous minimal PRL promoter (Fig. 2AGo). The first construct contained the GnSE fused to a single copy of the –275/–226 region containing the SF-1 element (SF-1 50 bp module) with both elements placed upstream of the PRL promoter. Similar constructs containing a 50-bp module that included either the GRAS (–412/–362) or AP-1 (–370/–321) element were also generated. Constructs containing the PRL promoter alone, the 50-bp modules placed upstream of the PRL promoter, the GnSE fused immediately upstream of the PRL promoter, or the promoterless vector were tested in parallel by transient transfection assays in {alpha}T3–1, LßT2, and Chinese hamster ovary (CHO) cells.



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Figure 2. The –275/–226 Region Mediates Synergistic Activation of Transcription by the GnSE

A, Structure of the artificial promoter constructs. The GnSE (–1,135/–753) was placed upstream of one copy of either the –275/–226 region (SF-1 module), the –370/–321 (AP-1 module) or the –412/–362 (GRAS module) regions themselves fused to the minimal PRL promoter (–35 to +36). B, To test the ability of the GnSE to enhance the activity of the minimal PRL promoter in the presence or in the absence of the 50-bp modules, the corresponding reporter constructs were transfected into {alpha}T3–1, LßT2, and CHO cells. To assess the importance of the SF-1 site in mediating the effect of the GnSE, {alpha}T3–1, LßT2, and CHO cells were also transfected with constructs containing the GnSE fused to a 50-bp module with a single-point mutation (5'-TT*GCCTTCA 3'–SF*) or a block-replacement mutation (5'-TGGTACCCA 3'-SFmut) in the SF-1 element. All other details are as described in the legend of Fig. 1CGo.

 
In contrast to the CHO cells in which all constructs were deprived of any specific action compared with the promoterless vector, a substantial variation was observed depending on the construction in {alpha}T3–1 and LßT2 cells (Fig. 2Go). In both cell lines, the relative luciferase activity of cells transfected with the minimal PRL promoter was not significantly different (P > 0.05) from that observed with the promoterless vector. In addition, the GnSE alone induced a moderate increase in luciferase activity (2- to 3-fold stimulation over the promoterless vector). In the absence of the GnSE, the three modules containing either the GRAS-, AP-1-, and SF-1-related elements induced either no increase (GRAS module in LßT2 cells) or modest increases in luciferase activity, and maximal stimulation was observed with the AP-1 module in {alpha}T3–1 cells (4.1 ± 1.1-fold stimulation). The promoter activity of the GnSE associated with the GRAS module was equivalent to that of the GRAS module alone in the two gonadotrope cell lines. Combination of the AP-1 module with the GnSE resulted in stimulation of luciferase activity that was approximately equivalent to the sum of the stimulation induced by the individual elements in {alpha}T3–1 and LßT2 cells (6.4 ± 1.1- and 4.3 ± 1.4-fold, respectively). Finally, the association of GnSE with the –275/–226 region containing the SF-1-related element induced a dramatic increase in luciferase activity in both {alpha}T3–1 and LßT2 cells indicative of a synergistic stimulation of the minimal PRL promoter in these cells (13.0 ± 1.6-fold and 33.1 ± 7.9-fold, respectively). A single-point mutation in the SF-1 element in this promoter (G to A at position –244, SF*) resulted in a distinct reduction in promoter activity of 48% and 66% in {alpha}T3–1 and LßT2 cells, respectively. Complete disruption of the SF-1 element (SFmut) further decreased the GnSE/SF-1 module efficiency by 65% in {alpha}T3–1 cells and 86% in LßT2 cells. The GnSE activity, therefore, required the presence of gonadotrope-specific elements within the –275/–226 region, in particular the SF-1 element.

Gel-Shift Experiments with Oligonucleotide Probes Overlapping the –275/–226 Region Revealed a Single Major Complex That Involved the Potential SF-1 Element
To identify the possible factors that could interact with the –275/–226 region, gel retardation assays were performed with nuclear extracts isolated from {alpha}T3–1 cells (Fig. 3Go). We used two overlapping oligonucleotide probes that extended from either –277 to –240 or –264 to –231. With the labeled –277/–240 probe (Probe-1, Fig. 3CGo), no specific complex was detected under the conditions used (data not shown). In contrast, using the –264/–231 probe (Wild, Fig. 3CGo), a major shifted complex was observed, the specificity of which was confirmed by homologous competition with an excess of unlabeled probe (10-, 100-, or 1,000-fold molar excess). To localize more precisely the cis-element involved in the complex formation, three oligonucleotides spanning the –264/–231 region and containing 8-bp block-replacement mutations at positions –260/ –253 (mut A), –251/–244 (mut B), and –244/–237 (mut C) were designed. The A, B, and C mutant oligonucleotides (see Fig. 3CGo) were then used together with the labeled wild-type oligonucleotide in competition experiments using {alpha}T3–1 nuclear extracts. The A and B mutants were able to abrogate complex formation in a dose-dependent manner and with a similar apparent affinity as compared with the unlabeled wild-type oligonucleotide. In contrast, in the presence of an excess of unlabeled mutant C, complex formation was unmodified, indicating that the major shifted complex, obtained with the wild-type oligonucleotide, involved the sequence extending from –244 to –237, which corresponded to the potential SF-1 binding site. These results were confirmed using labeled mutant oligonucleotides (not illustrated). Heterologous competition using an unlabeled oligonucleotide identical to the sequence containing the SF-1 element of the rat aromatase gene (SFArom, Fig. 3CGo) was performed. Although this oligonucleotide exhibited substantial sequence differences with the GnRH-R proximal region, complete abrogated complex formation was obtained at a 100-fold molar excess.



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Figure 3. EMSA Reveals a Single Major Complex within the 50-bp Proximal Region That Corresponds to the SF-1-Related Element

A, Wild-type oligonucleotide that corresponded to the sequence extending from –264/–231 (WildA/S, see Table 1Go, and panel C) was 32P-labeled and incubated with {alpha}T3–1 nuclear extract (9 µg) in the absence (–) or in the presence of 10-, 100- or 1,000-fold molar excess of unlabeled homologous competitor (Wild), mutant A (MutA), mutant B (MutB), or mutant C (MutC) oligonucleotides. After the binding reaction, the DNA and protein complexes were resolved in 5% native polyacrylamide gels. The major SF-1 complex is indicated (arrow). B, The experiment was performed as in panel A except that the heterologous competitor was an oligonucleotide corresponding to a sequence in the aromatase promoter that contained the binding site for SF-1 (SFArom). C, Sequence of wild-type and oligonucleotide probes. Only the sense strand is illustrated.

 
Altogether these data suggest that the –275/–226 region bound with high affinity a nuclear factor at position –244/–237, which could correspond to the orphan nuclear receptor SF-1 present in {alpha}T3–1 cells.

The SF-1 Site Was a Determinant for Mediating the Effect of the GnSE
To investigate the importance of the SF-1 site directly, the previously described 50-bp modules were replaced by two copies of the SF-1 site, combining wild-type or mutated elements (Fig. 4AGo). In addition, overexpression of SF-1 was performed by cotransfecting {alpha}T3–1, LßT2, and CHO cells with an SF-1 expression vector. To evaluate potential squelching effects, control cells were cotransfected in parallel with identical amounts of cytomegalovirus-ß (CMVß). Under these control conditions and as expected, none of the constructs displayed greater activity than the minimal PRL promoter in CHO cells (Fig. 4BGo, right panel, open bars). In transfected {alpha}T3–1 cells, two wild-type SF-1 copies induced a modest increase in the activity of the minimal PRL promoter but failed to mediate the effect of the GnSE (Fig. 4BGo, left panel, open bars). In contrast, in control LßT2 cells, the duplicated wild-type SF-1 sites were able to mediate the effect of the GnSE, and the level of promoter activity attained 5.2 ± 0.5-fold that of the minimal PRL promoter (Fig. 4BGo, middle panel, open bars). Disruption of one or two SF-1 elements drastically reduced the promoter activity and resulted in a construct that displayed the same activity as the minimal PRL promoter, suggesting that the SF-1 mutations concomitantly abolished SF-1- and GnSE-dependent promoter activity in LßT2 cells.



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Figure 4. Overexpression of SF-1 Enhances in LßT2 and Restores in {alpha}T3–1 Cells the Capacity of the GnSE to Stimulate the SF-1/PRL Chimeric Promoter

A, Structure of the artificial promoter construct containing two copies of the SF-1 element instead of the 50-bp module. To create the GnSE/SF-1 fusion construct, the GnSE (–1,135/–753) was placed upstream of SF-1/PRL chimeric promoter composed of a 26-bp sequence containing two copies of wild-type SF-1 element (SFx2) fused to the minimal PRL promoter. B {alpha}T3–1, LßT2, and CHO cells were transfected with wild-type GnSE/SF-1 fusion construct (GnSE/SFx2) or mutated constructs either combining wild-type (5'-TGGCCTTCA-3') and mutated SF-1 element (5'-TGGTACCCA-3') (GnSE/SFmut 1/2) or containing two copies of the mutated SF-1 element (GnSE/SFmut 2/2) and cotransfected with SF-1 expression vector (50 ng/well) (CMV/SF-1, closed bars) or with equivalent amounts of control ß-galactosidase expression vector (CMVß, open bars). All other details are as described in the legend of Fig. 1CGo.

 
Cotransfection of SF-1 expression vector significantly enhanced the activity of the construct containing two SF-1 copies fused to the minimal PRL promoter in the three cell lines (Fig. 4Go). The SF-1 element of the GnRH-R promoter was thus functional and sufficient for mediating SF-1-dependent transactivation in gonadotrope-derived as well as in CHO cells. When SF-1 was overexpressed, GnSE significantly enhanced the activity of the promoter containing the wild-type SF-1 elements in {alpha}T3–1 and LßT2 cells (3.5 ± 0.4 vs. 2.3 ± 0.2 and 8.4 ± 0.5 vs. 4.9 ± 0.6-fold over the minimal PRL promoter vector, respectively) but not in CHO cells (2.4 ± 0.2 vs. 3.3 ± 0.3). Disruption of the two SF-1 elements abrogated both GnSE- and SF-1-dependent transactivations. Overexpression of SF-1 thus increased the efficiency of the GnSE in LßT2 cells and was sufficient to restore the capacity of the SF-1 element to mediate GnSE activity in {alpha}T3–1 cells. However, the isolated SF-1 elements displayed lower efficiency than the proximal 50-bp region in mediating GnSE activity. The sequence surrounding the SF-1 element might be necessary for promoting the efficient recruitment of SF-1 to its cognate element, thereby facilitating binding and transactivation. In contrast, the GnSE remained inactive in CHO cells overexpressing SF-1 despite the potency of the SF-1 elements to transactivate the minimal PRL promoter implying that other factors, absent in these cells, could be required for interaction with the GnSE.

The Potency of the GnSE Resided within Two Separate Regions of Approximately 30 bp
In an attempt to localize the active elements of the GnSE, serial 5'- and 3'-deletion mutants of the full-length GnSE were placed upstream of an artificial promoter containing duplicated copies of the SF-1 50-bp module fused to the PRL promoter. This artificial promoter was able to mediate GnSE activity 3 to 7 times as much as the previous promoter containing a single SF-1 module (see Fig. 2Go) and thus was used to ensure accuracy in the analysis. As illustrated in Fig. 5Go, deletions that extended from –1135 to –1063 were inefficient in both {alpha}T3–1 and LßT2 cells (P > 0.01). In contrast, 5'-deletions in a 114-bp region situated between –1063 and –950 severely affected the efficiency of the GnSE in {alpha}T3–1 cells. Further deletion of a 71-bp region extending from –896 to –826 resulted in a promoter activity equivalent to that of the duplicated 50-bp module (2.4 ± 0.3 vs. 2.8 ± 1.5-fold over the promoterless vector, P > 0.05). The importance of the 114- and 71-bp sequences was further attested by independent results obtained with 5'-/3'-deleted constructs. In fact, 3'-deletion of the –900/–753 region, which included the 71-bp sequence identified above, yielded a substantial reduction (~60%) in the efficiency of the GnSE as compared with the full-length construct (8.8 ± 0.8 vs. 20.0 ± 1.1-fold over the promoterless vector, P < 0.001). Additional 5'-deletion of the 114-bp region completely abrogated GnSE activity, resulting in promoter activity that was equivalent to that of the duplicated 50-bp module.



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Figure 5. The Activity of the GnSE Resides in Two Nonadjacent Regions

A, Schematic representation of the artificial promoter construct containing two copies of the –275/–226 region (SF-1 module). B, To localize the response elements that underlie the activity of the GnSE, {alpha}T3–1 and LßT2 cells were transfected with 5'- and 3'-truncated GnSE in the context of the artificial promoter construct containing two copies of the SF-1 module. The numbers beside each construct or associated with an arrow indicate endpoints of GnSE 5'- and/or 3'-deletion mutants, respectively. All other details are as described in the legend of Fig. 1CGo.

 
Transfection of LßT2 cells with the same constructs gave a similar pattern of luciferase activity. Some divergence in the relative importance of the 114- and 71-bp sequences was observed. A decrease in promoter efficiency induced by a deletion encompassing the –1063/–950 region was attenuated in LßT2 as compared with {alpha}T3–1 cell line. The 114-bp region accounted for approximately 45% (range 35%–60%) of the overall efficiency of the GnSE in LßT2 cells as compared with 70% (range 66%–75%) in {alpha}T3–1 cells. On the other hand, the decrease in promoter efficiency induced by a deletion of the –896/–826 region was more obvious in LßT2 than in {alpha}T3–1 cells. The 71-bp region accounted for 55% (range 43%–63%) as compared with 30% (range 25%–34%) in the respective cell lines.

Additional 5'- and 3'-deletions were created in the context of the artificial promoter and {alpha}T3–1 cells were transfected with the resulting constructs. The active elements of the 114-bp sequence (5'GnSE) were further delimited to a region extending from –983 to –950 whereas those of the 71-bp sequence (3'GnSE) were found to be located between –896 and –859 (data not illustrated).

Block-Replacement Mutagenesis within the Wild-Type Promoter Confirmed the Bipartite Organization of the GnSE
These active regions were then scanned by adjacent 10-bp block-replacement mutations in the wild-type promoter, and the mutant constructs were tested by transient transfection in both gonadotrope cell lines (Fig. 6AGo). Wild-type full-length and proximal promoter constructs were transfected in parallel. In {alpha}T3–1 cells, mutations extending from –983 to –974 (GA) and from –971 to –962 (GB) altered significantly promoter activity, inducing a 45–50% decrease in the stimulation induced by GnSE (Fig. 6BGo). This suggested that the DNA sequence overlapped by these two mutations contained positive regulatory elements that were most likely responsible for the activity of the 5'-region of GnSE. Among the three block-replacement mutations that altered the 3'-region of GnSE only the GF mutation (–871 to –862) was proficient and reduced GnSE efficiency by 37%. Similar results were obtained after transfection of LßT2 cells: the GA, GB and GF mutations decreased the GnSE-induced stimulation by 47%, 63%, and 62%, respectively. In addition, the GD mutation (–895/–886), which was ineffective in {alpha}T3–1 cells, induced a 47% decrease in LßT2 transfected cells.



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Figure 6. Delineation of the Active Elements of GnSE by Block Replacement Mutations

A, Schematic representation of the 10-bp adjacent mutations that cover the upstream (GA, GB, GC) and downstream (GD, GE, GF) active domains of GnSE in the context of the full-length promoter. B, The promoter activity of the mutant constructs was evaluated by transfection in both {alpha}T3–1 and LßT2 cells and compared with that of full-length and proximal wild-type promoter. Stars beside the bars indicate statistically significant differences with the activity of the full-length wild-type promoter construct (P < 0.05). All details are as in Fig. 1CGo. C, Analysis of sequences overlaid by mutation GA, GB, GD, and GF reveals potential response elements for NFY, GATA, Ptx1, USF, and LIM-homeodomain transcription factors. The core sequence of the binding sites is mentioned with the sense or antisense orientation indicated by arrows.

 
Analysis of the sequence overlaid by mutations GA and GB revealed the presence of binding sites for nuclear factor Y (NFY) in the antisense orientation and a consensus element for the pituitary homeobox 1 (Ptx1) transcription factor in the sense strand (Fig. 6CGo). In addition, two GATA elements were present in the sense and antisense strand, one of which corresponded to the canonical motif WGATAR. The sequence altered by mutation GF harbored a palindrome element containing consensus half-sites that are recognized by the LIM-homeodomain factors Lhx2 (also designated LH-2), Isl I, and Lmx I (33). Finally, the sequence overlaid by mutation GD encompassed an upstream stimulatory factor (USF) element that matched perfectly with the consensus binding site (CACGTG). However, since GD mutation appeared to affect promoter activity in LßT2 cells only while GnSE was active in both gonadotrope cell lines, we restricted further analyses to GnSE elements altered by mutation GA, GB, and GF.

Gel-Shift Experiments with Oligonucleotide Probes Overlapping the GA/GB Region Revealed the Potential Implication of GATA-Related Factors in GnSE Activity
To examine whether the GA/GB region could bind specific transcription factors, we designed a double-stranded oligonucleotide (AB probe) which extended from –985 to –957 and gel retardation assays were performed with nuclear extracts isolated from both {alpha}T3–1 and LßT2 cell lines. Using a labeled AB probe, two distinct shifted complexes (complexes 1 and 2) were observed in {alpha}T3–1 and LßT2 nuclear extracts, and the specificity was confirmed by homologous competition with an excess unlabeled probe (5-, 10-, 50-, and 100-fold molar excess) (Fig. 7AGo). Depending on the gel resolution, the faster-migrating complex (complex 2) was actually a composite of two distinct complexes (Fig. 7AGo, right panel, and Fig. 7Go, panels B and C). We then designed two mutant double-stranded oligonucleotides that differed from the wild-type AB probe by 3 bp located at position –977 to –975 (mut1) and at position –970 to –968 (mut 2). These mutations disrupted either the NFY binding site or the GATA antisense and Ptx1 binding site, respectively. The mut1 and mut2 oligonucleotides were then used together with the labeled AB probe in competition experiments. The mut1 oligonucleotide with the disrupted NFY binding site was able to abrogate complex formation in a dose-dependent manner and with a similar apparent affinity as compared with the unlabeled wild-type oligonucleotide, suggesting that NFY factor was not involved in complex formation (not illustrated). In contrast, the mut2 oligonucleotide displayed binding capacity that differed from wild-type AB probe. Only the faster-migrating complex (complex 2) was detected (Fig. 7BGo), indicating that the –970/ –968 mutation within the GATA antisense/Ptx-1 binding site abrogated complex 1 formation. These data were confirmed by using labeled AB probe and unlabeled mut2 oligonucleotide in competition experiments (not illustrated).



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Figure 7. EMSA Identifies Several Complexes within the Upstream Element of GnSE That Could Be Related to Members of the GATA Family of Transcription Factors

A, The upstream sequence of GnSE forms specific complexes with gonadotrope nuclear extract. Nuclear extracts from LßT2 or {alpha}T3–1 cells were incubated with a 32P-labeled oligonucleotide that overlaps the sequence covered by GA and GB mutations (AB probe) in the presence or in the absence of increasing concentrations (5- to 100-fold molar excess) of unlabeled AB probe. After the binding reaction, the DNA and protein complexes were resolved in 5% native polyacrylamide gels. The shifted complexes are indicated (arrow). B, A 3-bp pair mutation within the antisense GATA and sense Ptx 1 element abrogates complex 1 formation. Nuclear extracts of LßT2 cells were incubated with 32P-labeled mut 2 oligonucleotide in the absence or in the presence of heterologous unlabeled GATA or Ptx1 oligonucleotides and processed as in panel A. C, Evidence that GATA-related factors are involved in complex formation. Nuclear extracts of LßT2 cells were incubated with 32P-labeled AB oligonucleotide in the absence or presence of heterologous unlabeled GATA or Ptx1 oligonucleotides (5- to 100-fold molar excess) and processed as in panel A.

 
To further discriminate between GATA and Ptx1 factors, we performed heterologous competitions with unlabeled oligonucleotides harboring consensus binding sites for each factor. An oligonucleotide corresponding to sequence extending from –317 to –287 of the rat POMC promoter was designed to be used as a Ptx1 probe. The GATA probe was derived from the mouse {alpha}-globin promoter. In parallel, we also tested the capacity of a NFY oligonucleotide, derived from the rat albumin promoter to abrogate complex formation. As expected and consistent with the results presented above, complex formation was unmodified in the presence of an excess of unlabeled NFY oligonucleotide (not shown). Interestingly, the POMC Ptx-1 oligonucleotide was unable to abrogate the shifted complexes obtained with either the labeled AB or the mut2 probe (Fig. 7Go, B and C). In contrast, the binding of the AB and mut2 probe was clearly competed by a 100-fold molar excess of the GATA oligonucleotide (Fig. 7Go, B and C), suggesting that GATA-related factors interacted with the 5'-region of GnSE. Furthermore, since a mutation at position –970 to –968 abrogated the shifted complex 1, the antisense GATA element (–965/–969) was likely involved in its formation. Consequently, since neither mutation at position –977 to –975 nor at position –970 to –968 affected the faster-migrating complex (complex 2), it could involve the canonical GATA element in the sense orientation. Similar results were obtained with {alpha}T3–1 nuclear extracts. Altogether these findings suggest that the AB probe contained two binding sites for GATA-related factors that might account for the activity of the 5'-region of GnSE.

The Binding Activity of the 3'-Region of GnSE Was Abolished by Mutation of the Palindrome LIM-Homeodomain Element
To determine whether the sequence overlaid by mutation GF could also bind specific factors, a labeled double-stranded oligonucleotide (F) corresponding to sequence –880 to –852 of GnSE was used as a probe with nuclear extracts prepared from {alpha}T3–1 and LßT2 cells. A single shifted complex was detected, which was competed with a 100-fold molar excess of the unlabeled oligonucleotide F but not by a double-stranded oligonucleotide bearing the GF mutation (Fig. 8Go). In addition, this DNA binding activity was not competed by heterologous GATA or Ptx1 probes. Enhancer activity of the 3'-region of GnSE thus correlated with a binding activity that was different from that in the 5'-region of GnSE.



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Figure 8. EMSA Identifies a Single Specific Complex within the Downstream Element of GnSE That Is Abrogated by GF Mutation

Nuclear extracts from {alpha}T3–1 cells were incubated with a 32P-labeled oligonucleotide that overlapped with the sequence covered by GF mutations (F probe) in the presence or in the absence of increasing concentrations (5- to 100-fold molar excess) of wild-type unlabeled F probe or F probe altered by GF mutation (mutF). The unlabeled heterologous GATA and Ptx1 oligonucleotides (see Fig. 7Go) were also tested in competition experiments (50- and 100-fold molar excess). After the binding reaction, the DNA and protein complexes were resolved in 5% native polyacrylamide gels. An arrow indicates the single specific complex.

 
The cognate transcription factor might be related to LIM-homeodomain factors since disruption of the sense and antisense consensus half-sites by the GF mutation abolished both binding (Fig. 8Go) and transactivation (see Fig. 6Go). Among the members of the LIM- homeodomain family of transcription factors, Lhx2 was shown to be expressed in {alpha}T3–1 cells and to stimulate constitutive transcription and hormonally regulated expression of the mouse gonadotropin {alpha}-subunit gene (33). Lhx2 could bind to a pituitary-specific enhancer designated the pituitary glycoprotein basal element (PGBE) which is present in the {alpha}-subunit gene promoter. Alignment of the –871/–859 sequence of the GnSE with the –350/–323 sequence of the PGBE indicated that the perfect palindrome CTAATTAG of GnSE was strongly homologous to both the upstream and downstream region of the binding site of Lhx2 within the PGBE (Fig. 9AGo). We, therefore, evaluated the ability of a PGBE oligonucleotide (Fig. 9BGo) to abrogate the specific complex obtained with the labeled F probe. A 50- and 100-fold molar excess of unlabeled PGBE clearly decreased the abundance of the specific complex, suggesting that the palindrome sequence of the GnSE interacted with protein(s) that were related to PGBE binding factor such as Lhx2. Mutations in PGBE that were previously shown to abolish binding of Lhx2 (33) also abrogated the ability of the heterologous oligonucleotide to compete with F probe binding (Fig. 9BGo).



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Figure 9. Factors That Bind the Downstream Element of GnSE Are Related to PGBE Binding Proteins

A, The palindrome sequence CTAATTAG of the GnSE matches with the downstream and upstream sequences of the PGBE of the mouse gonadotropin {alpha}-subunit (–350/–323) that binds the Lhx2 transcription factor. B, Nuclear extracts from LßT2 cells were incubated with a 32P-labeled oligonucleotide that overlapped with the sequence covered by GF mutations (F probe) in the presence or absence of increasing concentrations (5- to 100-fold molar excess) of wild-type unlabeled F probe, PGBE probe, or PGBE probe altered by mutations (PGBEmut). All others details are as described in the legend of Fig. 8BGo.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The cell-specific expression of marker genes of the gonadotrope lineage is controlled by multiple regulators. The combinatorial actions of transcription factors such as SF-1, GATA-2, bicoid-related homeobox transcription factor Ptx1, early growth response 1 (Egr-1 also designed as Krox24, NGF1-A, or zif/268) or the LIM homeobox gene Lhx2 and Lhx3 are decisive in the early or late stages of gonadotrope differentiation (34, 35, 36, 37, 38). During this process, the orphan nuclear receptor SF-1 plays a central role, being involved in the cell-specific expression of the genes encoding the gonadotropin {alpha}-subunit, the LHß-subunit, and the GnRH-R (see review in Ref. 39). It has been shown, using transient transfection assays in {alpha}T3–1 cells that, in addition to SF-1, constitutive expression of the GnRH-R gene requires AP-1 and, an as yet unknown, GRAS-binding protein. These factors interact with a tripartite basal enhancer located in the proximal region of the promoter (Refs 23, 24 ; see Introduction). In the present study, we have shown that the cell-specific expression of the rat GnRH-R gene is also dependent on the activity of two novel positive regulatory regions in GnSE localized further upstream of the tripartite enhancer.

The GnSE exhibits an attractive feature that is likely related to mechanisms underlying the expression of the GnRH-R gene in cells of the gonadotrope lineage. Convergent data in fact indicate that the SF-1 response element is crucial for mediating the effect of GnSE. First, mutations that disrupt the putative SF-1 response element diminished, most effectively, the activity of the full-length promoter, as compared with mutations in GRAS or AP-1 elements. This was particularly evident in LßT2 cells. Second, the GnSE could cooperate in a synergistic manner with a limited part of the proximal promoter region that includes the putative SF-1 binding site but not with modules of similar length that contain either GRAS or AP-1 element. In this proximal region, a single specific protein/DNA complex detected in gel-shift experiments was shown to implicate the SF-1 response element and a factor that was closely related to SF-1. Finally, in SF-1 overexpressing {alpha}T3–1 and LßT2 cells, two SF-1 elements were able to mediate the effect of GnSE, and mutations in SF-1 elements completely abrogated this capacity.

Altogether, these data demonstrate the existence of a functional interaction between the GnSE and the SF-1 response element, suggesting possible protein/protein interactions with transcription factors that bind to GnSE. Block-replacement mutations within the 5'- and 3'-region of GnSE combined with gel retardation experiments suggest the implication of GATA and LIM homeodomain-related factors. Proteins such as Lhx2 belonging to the LIM family as well as GATA factors are present in gonadotrope cells and have been shown to participate in the constitutive and/or GnRH-regulated expression of the gonadotropin {alpha}-subunit in {alpha}T3–1 cells (33, 40). However, a physical interaction between Lhx2 and SF-1 has not yet been demonstrated, although an increasing list of transcription factors have been shown to have a functional cooperation with SF-1 including stimulating protein 1 (Sp1) (41), CCAAT/enhancer-binding protein ß (C/EBPß) (42), Wilm’s tumor 1 (WT-1) (43), SRY-box containing gene 9 (SOX9) (44), and c- JUN (45). Cooperation with SF-1 was also observed with GATA-4, resulting in a synergistic stimulation of the antimullerian hormone/mullerian inhibiting substance (AMH/MIS) promoter (46). Furthermore, several factors belonging to the GATA family of transcription factors display similar capacity. In addition, GATA-2 appears to be decisive in the early stages of gonadotrope cell differentiation. In transgenic mice that express GATA-2 under the control of the Pit-1 promoter, the gonadotrope population was dramatically expanded, reaching 90% instead of the usual 10% of the total pituitary cell population, with a concomitant failure in the differentiation of lactotropes, somatotropes, and, to a minor extent, thyrotropes (34). This suggests that GATA-2 controls the expression of several marker genes of the gonadotrope lineage such as the GnRH-R gene. Our data are, therefore, consistent with a model in which members of the GATA family mediate GnSE activity in cooperation with SF-1 as suggested in the present study by gel retardation experiments.

It was previously reported that promoter activity of the GnRH-R gene is enhanced in transient transfection assays by cotransfection of a Ptx1 expression vector and, correspondingly, the expression of the mouse GnRH-R gene is decreased in Ptx1 knock-down {alpha}T3–1 cells (47). However, despite the presence of a consensus binding site on the sense strand at position –971/–966, Ptx1 does not appear to be implicated in GnSE activity. Nevertheless, it may regulate GnRH-R gene by interacting with other potential Ptx1 binding sites or by modulating SF-1 activity through direct protein-protein interaction. Indeed, synergistic activation of transcription by Ptx1 and SF-1 of the LHß-subunit promoter is not prevented by disruption of the Ptx1 binding site, indicating that Ptx1 can also cooperate with SF-1 through DNA-independent interaction (48).

In the case of the LHß-subunit and AMH/MIS promoters, the binding sites for SF-1 and interacting transcription factors are close to each other. Binding sites for SF-1, Ptx1, and Egr-1 are indeed clustered within approximately 80 bp in the proximal region of the LHß-subunit promoter (49). Likewise, in the proximal AMH/MIS promoter, the SF-1 and GATA response elements are 10 bp apart (46). The situation is very different in the GnRH-R promoter since the GnSE upstream element is separated from the SF-1 site by approximately 700 bp in the wild-type promoter. Thus, the GnSE belongs to a class of promoter-specific enhancers, capable of acting from a distance as classical enhancers, but requiring a specific promoter context.

In addition to its ability to interact with several transcription factors, SF-1 is able to bind at least two coactivators, the cAMP regulatory element-binding protein (CREB) binding protein (CBP/p300) (50) and the steroid receptor coactivator 1 (SRC-1) (51, 52) and to recruit the general transcription factor TFIIB (44, 53). SRC-1 is known to interact with several nuclear hormone receptors and augment ligand-dependent transactivation. SRC-1 is also capable of interacting with CBP/p300 via a separate domain (reviewed in Ref. 54). In addition, our data demonstrated that, in the context of the minimal PRL promoter, SF-1 in the absence of any other additional element enhances transcription (Fig. 4Go). SF-1 is thus likely capable of recruiting the general transcription factors and the RNA polymerase II through direct interaction with CBP/p300 or TFIIB or indirectly through SRC-1. In contrast, GnSE behaves differently since it requires the presence of proximal elements such as those located in the 50-bp proximal region, namely SF-1, to activate the minimal PRL promoter in a synergistic manner. This is reminiscent of the differential properties of the coactivator CBP/p300 as compared with its associated factor pCAF (p300/CBP associated factor) in their capacity to activate transcription (55). To be efficient, CBP/p300 must be recruited to the vicinity of the core promoter region whereas pCAF activates transcription from a pro-moter-distant position provided an upstream activator element is present in the vicinity of the core promoter region. Similarly, it might be hypothesized that GATA- and/or Lhx2-related factors that bind the GnSE recruit a co-activator such as, or with similar properties as, pCAF. This putative coactivator would subsequently interact with CBP recruited to the vicinity of the core promoter region through SF-1/SRC-1 interactions. Recently, evidence has been obtained that the melanocyte-specific gene-related gene 1 (MRG1) may bind Lhx2 in vitro and form a complex with Lhx2 in {alpha}T3–1 cells (56). Furthermore, MRG1 is also able to bind CBP/p300. Therefore, the functional link that occurs between the GnSE and the GnRH-R proximal promoter region, which includes the SF-1 response element, might be established through coactivator interaction.

Although GnRH-R promoter fusion constructs as well as artificial promoter constructs displayed rather similar patterns of activity after transfection in gonadotrope-derived cell lines, differences were consistently observed between LßT2 and {alpha}T3–1 cells. The proximal –412/–26 region as well as the duplicated copies of the GnRH-R-specific SF-1 element are significantly more efficient in LßT2 than in {alpha}T3–1 cells (Figs. 1Go and 4Go). Furthermore, mutations in SF-1 element affected, to a larger degree, the activity of the artificial promoter in LßT2 than in {alpha}T3–1 cells (Fig. 2Go). Such differences may result from differential representation of transcription factors, especially SF-1, which, in turn, may be related to the way in which the two cell lines have been selected (29). The {alpha}T3–1 cells were derived from pituitary tumors induced in transgenic mice by directed oncogenesis with the simian virus 40 T antigen under the control of the human gonadotropin {alpha}-subunit promoter. They express the gonadotropin {alpha}-subunit and GnRH-R genes. The LßT2 cells were obtained by a similar approach except that the rat LHß-subunit promoter was used for directing the expression of the oncogene (30, 31). These cells express not only the gonadotropin {alpha}-subunit and GnRH-R genes but also the LHß- and, under activin treatment (32), the FSHß-subunit genes. Interestingly, the gonadotropin {alpha}-subunit and the GnRH-R genes are expressed in the early stages of pituitary ontogenesis whereas the ß-subunits of LH and FSH are expressed in the late stages (Refs. 57, 58, 59, 60, 61, 62 ; also reviewed in Refs. 34, 63). It could be thus suggested that the {alpha}T3–1 cells are representative of immature gonadotrope cells whereas LßT2 cells are almost fully differentiated gonadotrope cells. Our results indicate that the SF-1 element alone is capable of mediating the effect of the GnSE in LßT2 cells whereas it appears insufficient in {alpha}T3–1 cells. In this cell line, overexpression of SF-1 is necessary to detect the enhancer capacity of the GnSE. Based on these findings, it may be hypothesized that the SF-1-dependent regulation of the GnRH-R gene is correlated with the stage of gonadotrope differentiation, playing a more significant role in differentiated than in immature gonadotrope cells. This may be achieved through variations in the intracellular concentration of SF-1 as well as through posttranslational modifications such as phosphorylation (64) that might affect SF-1 transactivation efficacy. This is more consistent with data showing that the expression of the GnRH-R gene is not strictly dependent on SF-1. Indeed, the gonadotrope lineage is not ablated after targeted disruption of the gene encoding SF-1 in mice since treatment with GnRH restores gonadotrope function (65).

In {alpha}T3–1 cells, the 50-bp region is efficient for mediating the effect of the GnSE and we thus propose that it promotes the recruitment of SF-1 to its binding site. Our observation that overexpression of SF-1 restored the capacity of the SF-1 element to mediate the activity of GnSE is consistent with this hypothesis. An alternative but nonexclusive possibility could be that additional transcription factors interact with the 50- bp region and mediate in place of, and/or in cooperation with, SF-1 the effect of the GnSE. These putative factors, however, were not detected in gel-shift experiments, suggesting that they interact weakly with their cognate DNA response elements. Nevertheless, they would provide a means by which, ultimately, the GnSE-bound factors may bypass the deficiency in SF-1, either under pathological conditions such as those artificially created in SF-1-disrupted mice, or under physiological conditions, e.g. during the early stages of pituitary ontogenesis that precede the appearance of SF-1 in the pituitary.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Oligonucleotides and PCR Amplification
The oligonucleotides used for the construction of reporter plasmids, mutagenesis, and electrophoretic mobility shift assays (EMSAs) were obtained from Eurogentec (Seraing, Belgium) or Prolabo (Paris, France) and are listed in Table 1Go or illustrated in Figs. 3Go, 7Go, 8Go, and 9Go. The nucleotide sequence of the GnRH-R gene is numbered relative to the translational initiation codon [GenBank accession numbers Z99955 and Z99956 (21)]. The rat PRL gene is numbered relative to the transcription start site (66). All PCR amplifications were performed with either Deep Vent DNA polymerase (New England Biolabs, Inc., Montigny-le Bretonneux, France) or Expand High Fidelity PCR System (Roche Molecular Biochemicals, Meylan, France).


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Table 1. Oligonucleotides Used for Generation of GnRH-R Promoter Constructs

 
Reporter Plasmids Used in Transfection Studies
The construct containing the full-length 1.1-kb 5'-flanking region of the rat GnRH-R gene (–1,135/–26) inserted upstream of the CAT gene into the pCAT-Basic vector (Promega Corp., Charbonnières, France) has been described previously (21). The proximal region of the rat promoter (–436/–26) was generated by PCR amplification from the GnRH-R promoter template using sense primer –426Hind and antisense primer –26Sal. The proximal promoter construct was then created by inserting the PCR product into the pCAT-Basic vector using HindIII and SalI restriction sites introduced by the primers. Subsequently, a KpnI restriction site was introduced at positions –430/–425 by site-directed mutagenesis. To this end, PCR products were generated from GnRH-R promoter template in separate reactions using either sense primer –412KpnS and antisense primer –26Sal or antisense primer –412KpnA and sense primer –436Hind. Sense primer –412KpnS and antisense primer –412KpnA included sufficient 5'- and 3'-flanking sequence to assure specific annealing. The overlapping PCR fragments were then self annealed and DNA double-strands were reconstituted in a third PCR reaction using sense primer –436Hind and antisense primer –26Sal. The resulting PCR products were HindIII and SalI digested, gel purified, and subcloned into the HindIII-SalI sites of the pCAT-Basic vector. This construction was then used to elaborate the distal/proximal fusion construct. The GnSE (–1,135/–753) was synthesized from GnRH-R promoter template by PCR using sense primer –1,135Hind and antisense primer –753Kpn. The PCR products were digested with HindIII and KpnI, gel purified, and subcloned into the HindIII/KpnI site of the proximal promoter construct. A similar procedure was used to construct the expression vectors containing the 5'- and 3'-regions of the GnSE using either sense primer –1,135Hind and antisense primer –900Kpn or sense primer –896Hind and antisense primer –753Kpn, respectively.

To subclone the GnRH-R promoter fragments upstream of the luciferase reporter gene into pGL3-Basic vector (Promega Corp.), the multiple cloning site of the pGL3-Basic vector was modified to provide compatible restriction sites in the appropriate orientation. The selected sense and antisense oligonucleotides MCS-S and MCS-A were annealed, and the resulting double-stranded oligonucleotide with 5'- and 3'-protruding ends was inserted into the KpnI/HindIII sites of the pGL3-Basic vector. The multiple cloning site of the modified pGL3-Basic vector thus included, in the 5'- to 3'-direction, restriction sites for HindIII, KpnI, BstEII, and XhoI. Full-length and proximal promoter fragment were generated by PCR amplification from GnRH-R promoter template using sense primers –1,135Hind and –475BstE, respectively, and antisense –26Xho, gel purified, and subcloned into the HindIII/XhoI or BstEII/XhoI sites of the modified pGL3-Basic vector. The GRAS, AP-1, and SF-1 mutant constructs were prepared following the same protocol used for insertion of the KpnI site into the proximal promoter construct. The GRAS, AP-1, and SF-1 elements were modified so that a restriction site for KpnI was created using mutagenic sense primers GRAS-MutS, AP1-MutS, or SF1-MutS and antisense primer Luc-A in one PCR and using sense primer Luc-S and mutagenic antisense primers GRAS-MutA, AP1-MutA, or SF1-MutA in another PCR; both PCR reactions were performed with the pGL3 plasmid containing the full-length GnRH-R promoter as a template. Overlapping PCR products were then self annealed, and DNA double-strands were reconstituted in a third PCR using sense primer Luc-S and antisense primer Luc-A.

The artificial promoter constructs were generated in successive steps. First, the minimal PRL promoter (–35/+36) was amplified by PCR from the p0.4 kb PRL-CAT vector (67) using sense primer BstE-S and antisense primer Xho-A, digested with XhoI and BstEII restriction enzymes, gel purified, and inserted into the XhoI-BstEII site of the modified pGL3-Basic vector. Second, single 50-bp modules containing the GRAS, AP-1, or SF-1 elements were amplified using sense primer –412Kpn-S, –370Kpn-S, or –275Kpn-S, respectively, and antisense primer –362BstE-A, –315BstE-A, or –229BstE-A, respectively, and processed as above to generate constructs containing a single copy of the –412/–362, –370/–321, or –275/–226 region, respectively. Two copies of the 50-bp module containing the SF-1 site were generated from the GnRH-R promoter template in a separate PCR using sense primer –275Bam-S or –275Kpn-S and antisense primer –229BstE-A or –226Bam-S, respectively. These were digested by restriction enzymes corresponding to the sites introduced by the primers and gel purified. Two 50-bp modules were created containing either 5'-KpnI and 3'-BamHI or 5'-BamHI and 3'-BstEII cohesive ends inserted together at the KpnI-BstEII site into the modified pGL3 vector containing the minimal PRL promoter. DNA fragments containing two copies of the wild-type or mutated SF-1 elements (–245/–237) were obtained by the hybridization of SFx2S and SFx2A complementary oligonucleotides, SFMut1/2S and SFMut1/2A or SFMut2/2S and SFMut2/2A. The resulting double-stranded DNA fragments with 5'-BstEII recessing and 3'-KpnI recessing ends were then subcloned in place of the 50-bp region. Finally, the full-length GnSE as well as the 5'- and/or 3'-deleted constructs were generated by amplification using selected sense primers –1,135Hind, –1,063Hind, –950Hind, –896Hind, or –826Hind and antisense primers –753Kpn or –900Kpn. The resulting amplified products containing the desired 5'- and/or 3'-deletions were then digested with HindIII and KpnI restriction enzymes and inserted into the HindIII-KpnI site of the modified pGL3-Basic vector containing the PRL promoter and the 50-bp modules. Block-replacement mutation (GA to GF) within the upstream and downstream region of GnSE were introduced after the same three-step PCR protocol as that used for generating previous mutants. The sense primers GA-S to GF-S were combined with antisense primer Luc-A, and the antisense primers GA-A to GF-A were combined with Luc-S. The identity of all reporter constructs was confirmed by sequencing using the dideoxynucleotide chain termination method.

Cell Culture and Transient Transfection
LßT2 and {alpha}T3–1 cells were maintained in monolayer cultures in high glucose DMEM supplemented with 10% FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin sulfate (Sigma, St. Louis, MO) at 37 C in humidified 5% CO2/95% air. CHO cells were cultured as described (68). For transient transfection experiments with CAT reporter constructs, cells were plated at 8 x 105 cells per 60-mm dish the day before transfection. On the day of transfection, equivalent molar amounts of reporter constructs were combined with 4 µl PLUS reagent and 6 µl lipofectAMINE reagent (Life Technologies, Inc., Gaithersburg, MD) in 500 µl OptiMEM medium (Life Technologies, Inc.) according to manufacturer’s instructions. In each experiment the total quantity of DNA per dish was standardized to 3 µg with pUC19 plasmid DNA. A vector expressing ß-galactosidase under the control of the CMV promoter (0.4 µg per dish) (CMVß , CLONTECH Laboratories, Inc. Palo Alto, CA) was cotransfected to serve as an internal standard for transfection efficiency. Transfection mixture was incubated for 30 min at room temperature, diluted to 2.5 ml with OptiMEM medium, and applied to the cells previously washed with the same serum-free medium. After a 6 h-incubation, the medium was replaced by DMEM supplemented with 2% FBS, 10 U/ml penicillin, and 10 µg/ml streptomycin sulfate, and 16 h later, the medium was aspirated and cells were processed as previously described for ß-galactosidase and CAT assays (21).

For transient transfection experiments with luciferase reporter constructs, cells were plated at 105 cells per well in 24-well plates. They were then processed according to the same protocol as for CAT reporter constructs, except that all quantities and volumes were scaled down 10-fold and Renilla luciferase expression vector (pRL-TK, 10 ng/dish) was used as an internal control to normalize gene expression measurements instead of CMVß. Firefly and Renilla luciferase activities were then measured using the Dual-Luciferase Reporter Assay System (Promega Corp.). Cells were washed once with PBS, pH 7.4, and lysed by the addition of 100 µl passive lysis buffer. Firefly luciferase activity was assayed using 1–4 µl cell extract combined with 5–20 µl luciferase assay reagent, and luminescence was measured with a 15-sec delay and a 15-sec measurement in a Wallach scintillation counter from which the coincidence circuit was turned off. An equal volume of Stop & Glo reagent was then added, and Renilla luciferase activity was determined under the same conditions.

EMSA
Cells were seeded at 3 x 106 ({alpha}T3–1) or 6 x 106 cells (LßT2) per 100-mm tissue culture dish in triplicate and cultured for 24 h in DMEM supplemented with 10% FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin sulfate. The culture medium was then replaced by OptiMEM medium and cells were cultured for an additional 6 h. The serum-free medium was then replaced by DMEM supplemented with 2% FBS, 10 U/ml penicillin, and 10 µg/ml streptomycin sulfate, and incubation was continued for a further 16 h. Thereafter, cells were harvested and nuclear extracts were prepared by the method of Andrews and Faller (69).

Double-stranded oligonucleotides were designed to contain 5'-protruding ends (see Table 1Go and Figs. 3Go, 7Go, and 8Go). They were thus end labeled (5 fmol) by filling-in the recessed 3'-termini with Klenow fragment of Escherichia coli DNA polymerase I and 50 µCi {alpha}[32P] dCTP (3000 Ci/mmol, Amersham Pharmacia Biotech, Les Ulis, France), and purified with a Sephadex G50 fine column.

Nuclear extracts (9 µg) and polydIdC (1 µg) were incubated in binding buffer [20 mM HEPES, pH 7.9, 60 mM KCl, 60 mM NaCl, 1 mM EDTA, 300 µg/ml BSA, and 12% (vol/vol) glycerol] for 15 min at 4C. Thereafter, 20,000 to 50,000 cpm of oligonucleotide probe ({approx}10 fmol) were added with or without an excess of unlabeled oligonucleotide, and incubation was continued at 20 C for 30 min. Protein DNA complexes were resolved on a 5% nondenaturing polyacrylamide gel in 0.5x Tris-borate-EDTA buffer. Gels were then dried and subjected to autoradiography.

Statistical Analysis
Data were analyzed by one-way ANOVA. If the F test was significant, then means were compared using Tukey-Kramer’s method of multiple comparisons.


    ACKNOWLEDGMENTS
 
We thank Dr. Pamela Mellon and the University of California, San Francisco, for kindly providing the LßT2 cell line and Dr. Keith Parker (University of Texas Southwestern Medical Center, Dallas, TX) and Dr. Bon-chu Chung (Institute of Molecular Biology, Taipei, Taiwan) for the SF-1 expression vector. We thank Dr Michel Raymondjean (ESA CNRS 7079, Paris, France) for helpful discussion and for kindly providing the GATA and NFY probes. Special thanks to Mrs. Danielle Duchene, Marie-Claude Chenut, and Mr. Philippe Nguyen for their contribution in the maintenance of cell lines, preparation of this manuscript, and illustrations, respectively. We gratefully acknowledge the contribution of Dr. Lisa Oliver (U-419 INSERM, Nantes, France) for the correction of English text and editorial assistance. We are indebted to Mr. Yves Brossas for his help in automated DNA sequencing and Drs. Jacques Treton and Yves Courtois (U-450 INSERM, Paris) for kindly giving us free access to the LI-COR DNA sequencer.


    FOOTNOTES
 
Address requests for reprints to: Dr. Jean-Noël Laverrière or Dr. Raymond Counis, Endocrinologie Cellulaire et Moléculaire de la Reproduction, Université Pierre and Marie Curie, CNRS ESA 7080, Case 244, 75252 Paris cedex 05, France. E-mail: Raymond.Counis{at}snv.jussieu.fr E-mail: Jean-Noel.

This work was supported by grants from the CNRS and the Université Pierre et Marie Curie. H. Pincas is a recipient of the Ministère de l’Education Nationale, de la Recherche et de la Technologie, and of the Fondation pour la Recherche Médicale.

Received for publication February 21, 2000. Revision received September 22, 2000. Accepted for publication October 23, 2000.


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 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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