A Single Pitx1 Binding Site Is Essential for Activity of the LHß Promoter in Transgenic Mice

Christine C. Quirk, Kristen L. Lozada, Ruth A. Keri and John H. Nilson

Department of Pharmacology Case Western Reserve University School of Medicine Cleveland, Ohio 44106


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Reproduction depends on regulated expression of the LHß gene. Tandem copies of regulatory elements that bind early growth response protein 1 (Egr-1) and steroidogenic factor 1 (SF-1) are located in the proximal region of the LHß promoter and make essential contributions to its activity as well as mediate responsiveness to GnRH. Located between these tandem elements is a single site capable of binding the homeodomain protein Pitx1. From studies that employ overexpression paradigms performed in heterologous cell lines, it appears that Egr-1, SF-1, and Pitx1 interact cooperatively through a mechanism that does not require the binding of Pitx1 to its site. Since the physiological ramifications of these overexpression studies remain unclear, we reassessed the requirement for a Pitx1 element in the promoter of the LHß gene using homologous cell lines and transgenic mice, both of which obviate the need for overexpression of transcription factors. Our analysis indicated a striking requirement for the Pitx1 regulatory element. When assayed by transient transfection using a gonadotrope-derived cell line (LßT2), an LHß promoter construct harboring a mutant Pitx1 element displayed attenuated transcriptional activity but retained responsiveness to GnRH. In contrast, analysis of wild-type and mutant expression vectors in transgenic mice indicated that LHß promoter activity is completely dependent on the presence of a functional Pitx1 binding site. Indeed, the dependence on an intact Pitx1 binding site in transgenic mice is so strict that responsiveness to GnRH is also lost, suggesting that the mutant promoter is inactive. Collectively, our data reinforce the concept that activity of the LHß promoter is determined, in part, through highly cooperative interactions between SF-1, Egr-1, and Pitx1. While Egr-1 can be regarded as a key downstream effector of GnRH, and Pitx1 as a critical partner that activates SF-1, our data firmly establish that the Pitx1 element plays a vital role in permitting these functions to occur in vivo.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
LH, with its ability to stimulate gonadal steroidogenesis and gametogenesis, is essential for normal reproduction (1, 2). Synthesis and secretion of this glycoprotein hormone are limited to gonadotropes of the anterior pituitary gland (1) and are controlled by the binding of the hypothalamic neurohormone GnRH to specific, high-affinity GnRH receptors (1, 2). A naturally occurring strain of mice (hpg/hpg) harboring a spontaneous deletion of the GnRH gene illustrates the tight interrelationship between GnRH and LH biosynthesis (3). These hypogonadal mice produce no functional GnRH and therefore neither synthesize, nor secrete, LH or FSH. Daily administration of GnRH (4) or delivery of a functional GnRH transgene (5) restores both hormones, and hence, fertility. Thus, synthesis and secretion of LH in vivo require sustained pulses of GnRH.

Significant effort has been devoted toward understanding the regulatory mechanisms controlling the biosynthesis of LH, a heterodimeric protein composed of a common {alpha}-subunit and unique ß-subunit that confers biological specificity (1, 2). Many of the elements and factors that are essential for expression of the common {alpha}-subunit gene have been studied extensively in vitro (6, 7, 8, 9, 10, 11, 12) and in transgenic mice (6, 13, 14, 15). From this work, it is clear that a complex array of regulatory elements form a combinatorial code that directs expression of the {alpha}-subunit gene to gonadotropes and conveys responsiveness to GnRH. In contrast, while characterization of the LHß promoter has lagged behind that of the {alpha}-subunit promoter, recent evidence suggests that distinct arrays of regulatory elements confer gonadotrope specificity and GnRH responsiveness to each gene.

All of the regulatory elements that have been identified in the promoter of the LHß subunit gene lie within 500 bp of the start site of transcription. This broad region can be further subdivided into two distinct domains, proximal and distal (Fig. 1Go). Functional elements located in the distal domain appear to differ across species. For example, CCAAT boxes that bind NF-Y have been identified in the bovine LHß promoter (16), whereas Sp1 binding elements occupy the distal domain of the rat LHß promoter (17, 18, 19). These upstream elements of the distal domain enhance promoter activity and, in the case of Sp1, contribute to GnRH responsiveness (17, 18, 19, 20). The Sp1 elements do not, however, appear to be essential for promoter activity or GnRH responsiveness, since appreciable activity remains when they are deleted (17, 19).



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Figure 1. Several Elements Are Necessary for Expression of the LHß Gene

A schematic of the bovine LHß proximal promoter identifying DNA sequences of important cis-acting regulatory elements, many of which correspond to identical elements in the LHß promoters from other species. Residing within 130 bp of the transcriptional start site in the proximal domain of the LHß gene is a pair of GSEs that bind SF-1. Closely associated with the GSEs are the bipartite Egr-1 elements, whose cognate binding factor functionally cooperates with SF-1 to enhance transcription of LHß. Located between the pairs of GSEs and Egr-1-regulatory elements is a homeobox binding motif (ATTA) that binds the bicoid-related Pitx1 factor. Pitx1 functions synergistically with SF-1 and Egr-1 to activate LHß expression. Upstream of this elemental array in the distal domain of the LHß promoter is a pair of nearly perfect CCAAT box motifs found in opposing orientation approximately 60 bp apart from one another that bind NF-Y.

 
Several closely associated cis-acting elements comprise a promoter-proximal domain that extends 150 bp upstream from the start site of transcription (19, 21, 22, 23, 24, 25, 26, 27, 28). These elements appear to be highly conserved across several species (27), suggesting their functional importance. First characterized were the tandem gonadotrope-specific elements (GSEs) that bind steroidogenic factor-1 (SF-1) (21, 24, 26). These sites are necessary for expression of the LHß gene, as mutation of the distal SF-1 binding element (dSF-1) or both the proximal (pSF-1) and distal GSEs within the context of the full-length LHß promoter severely attenuates promoter activity and eliminates its responsiveness to GnRH when assayed either in transgenic mice (21) or the gonadotrope-derived cell line, {alpha}T3-1 (23, 24). Like the element that binds SF-1, the transcription factor itself may also be vital for LHß gene expression (29) as underscored by recent evidence obtained from mice where the SF-1 gene was specifically deleted from gonadotropes (30). Although these animals are infertile and fail to develop secondary sexual characteristics, gonadal steroidogenesis can be restored upon treatment with exogenous gonadotropins (30).

Closely associated with the GSEs is a pair of regulatory elements that bind early growth response protein 1 (Egr-1), also known as NGFI-A, zif/268, and Krox-24. These cis-acting elements have been studied extensively in the bovine (27), racine (26), and equine (25) LHß promoters and are necessary for full transcriptional activity as well as GnRH responsiveness (22, 25, 27). Through Egr-1, LHß mRNA levels are increased after hormonal activation of protein kinase C (PKC), a mediator of GnRH action (22, 27). Egr-1 is up-regulated and phosphorylated by the GnRH signaling cascade (31), suggesting that it is a downstream effector of the neurohormone. Further, SF-1 and Egr-1 functionally cooperate to increase LHß promoter activity, and mutation of any of the corresponding response elements attenuates this synergism (22, 27, 31). Targeted inactivation of the Egr-1 gene in mice resulted in a specific failure to synthesize LHß, although the number of gonadotropes and concentration of FSH were similar to those of wild-type animals (32, 33). Together, these data suggest that Egr-1, although not involved in differentiation of gonadotropes, is essential for expression of the LHß gene (33) and probably a direct target of the GnRH signaling pathway.

Nested between the pairs of Egr-1 and SF-1 binding elements in the proximal domain of the LHß promoter is a single binding site for Pitx1 (34). Pitx1, also known as Ptx1 (pituitary homeobox 1), p-OTX (pituitary OTX-related factor), Bft (backfoot), and Brx2 (Rieg gene), is a member of the bicoid-related subclass of homeobox genes (35). The Pitx family of transcription factors includes three highly conserved vertebrate paralogs that have been cloned in multiple species: Pitx1, Pitx2, and Pitx3 (35). These transcription factors play crucial roles in several aspects of development such as limb patterning (Pitx1) (36, 37, 38), left-right axis determination (Pitx2) (39, 40, 41), and proper eye formation (Pitx2 and Pitx3) (42, 43). In humans, genetic mutations of Pitx1 are thought to be involved in development of Treacher Collins Franceschetti Syndrome (44). Mutations of Pitx2 cause Rieger’s syndrome (45), whereas Pitx3 mutations result in anterior segment mesenchymal dysgenesis and cataracts (43, 45). Although both Pitx1 and Pitx2 are early markers of pituitary development (35, 46), Pitx1 also activates transcription of a large number of pituitary target genes, including many that are active in gonadotropes, such as those that encode {alpha}, FSHß, LHß, and GnRH receptor (36, 47). Thus, normal gonadotrope function clearly requires Pitx1.

Since Pitx1 activates transcription of the LHß gene, it is tempting to assume that this occurs through a DNA-dependent interaction. In this regard, three potential Pitx1 binding sites have been identified in the bovine LHß promoter (34). Of these, the best studied is the most promoter-proximal site that resides between the tandem Egr-1 and SF-1 sites. Overexpression of Pitx1 in heterologous cell lines activates the LHß promoter through a cooperative mechanism that includes direct interactions with SF-1 and Egr-1 (27, 28). Interestingly, however, this cooperative interaction appears to occur independently of Pitx1 binding to DNA (28). Instead, Drouin and colleagues (28) suggest that Pitx1 functions as an activator of SF-1. In addition to interacting with SF-1, Pitx1 may also augment GnRH responsiveness since it can interact with Egr-1, one of the downstream effectors of this pathway.

Given the strong conservation of the Pitx1 binding site across several species, we speculated that overexpression studies in heterologous cell lines might mask its importance in contributing to activity of the LHß promoter in more physiological settings such as homologous cell lines or transgenic mice where transcription factors are expressed at normal levels. We also wanted to determine whether the Pitx1 binding site contributes to GnRH responsiveness directly (i.e. in the absence of additional factors) or indirectly by supporting the action of Egr-1. Herein, we report results that address both questions using transfection and transgenic approaches that obviate the need for overexpression paradigms.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Mutating the Pitx1 Element Attenuates Expression but Does Not Diminish GnRH-Responsive Activity of the LHß Promoter in LßT2 Cells
Pitx1 functionally cooperates with Egr-1 and SF-1 to activate the LHß promoter when overexpressed in CV-1 (monkey kidney fibroblast) cells (27, 28). In this paradigm, Pitx1 exerts its synergistic effects upon this promoter even in the absence of the Pitx1 DNA binding site (27, 28). To determine whether the apparent DNA independence was unique to the overexpression paradigm, we assayed activity of wild-type and Pitx1 mutant forms of the LHß promoter in LßT2 cells, a gonadotrope-derived cell line that expresses the endogenous LHß and Pitx1 genes (27, 48).

Reporter activity was measured in LßT2 cells after transient transfection of a mutant form of the LHß promoter in which the core homeobox binding motif (ATTA) was converted to an RsaI restriction enzyme site (GTAC). The LßT2 cells were chosen for these analyses because they are one of the few cell lines that closely resemble a differentiated gonadotrope (48). As illustrated in Fig. 2AGo, the wild-type LHß promoter, which contains a functional Pitx1 binding site, displays activity 5-fold over the promoterless control vector in LßT2 cells incubated without GnRH. The Pitx1 cis-acting element contributes substantially to this activity because luciferase activity in cells transfected with the mutant Pitx1 LHß promoter was not different from that observed with the promoterless control (P < 0.05). Given the endogenous expression of SF-1 and Pitx1, these data suggest that the Pitx1 site may be necessary for maintaining their appropriate interaction (27).



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Figure 2. Mutating the Pitx1 Element Attenuates Basal Expression but Does Not Diminish GnRH-Responsive Activity of the LHß Promoter in LßT2 Cells

A, Cells were transiently transfected with promoter-driven luciferase constructs consisting of either the wild-type (-779/+10) LHß promoter, an LHß promoter in which the consensus homeobox motif (ATTA) was replaced with an RsaI restriction enzyme site (GTAC), or a promoterless luciferase plasmid. Cells transfected with the wild-type LHß promoter construct exhibited 5-fold higher luciferase activity than cells transfected with the mutant form of the promoter, which displayed luciferase activity at levels no different from the promoterless control. While the Pitx1 mutant promoter lacked basal expression in LßT2 cells, it maintained GnRH-responsive expression. Values are means ± SEM of firefly luciferase activity normalized with renilla luciferase activity (under control of the CMV promoter) and expressed as arbitrary light units. Bars bearing different letters indicate luciferase values that are significantly different (P < 0.05). B, Mutating the core homeobox binding motif in the Pitx1 element inhibits protein binding. EMSAs were performed with LßT2 nuclear extracts and wild-type Pitx1 probe. Unlabeled Pitx1 (wild-type) or µPitx1 competitors were added at 10- (lanes 2 and 6), 50- (lanes 3 and 7), 100- (lanes 4 and 8), and 250-fold (lanes 5 and 9) molar excess. For heterologous competition, unlabeled distal NF-Y was added at 250-fold (lane 10) molar excess. Numbered arrows designate nuclear proteins that bind the Pitx1 probe. Bands numbered 1, 2, and 4 represent specific binding events while band number 3 is a protein that binds nonspecifically. Unbound probe is labeled at the bottom of the gel (free).

 
Even in the absence of measurable activity in cells incubated without GnRH, the mutated LHß promoter construct, like the wild-type vector, appeared to retain its GnRH responsiveness (P < 0.05). In drawing this conclusion, it is important to emphasize that even though the fold induction by GnRH for the mutant promoter (approximately 40x) was greater than the wild-type promoter (15x), the absolute levels of luciferase activity for the mutant construct in the absence of GnRH were at the limits of detection. Thus, we are unable to accurately estimate fold-induction of the mutant promoter conferred by GnRH. Nevertheless, these results do underscore the ability of GnRH to reveal activity of an otherwise compromised LHß promoter.

To confirm that mutating the core motif (ATTA) within the Pitx1 element would inhibit the binding of Pitx1 or related proteins to DNA, electrophoretic mobility shift analyses (EMSAs) were performed using a double-stranded wild-type Pitx1 element as a radiolabeled probe and nuclear extracts from LßT2 cells (Fig. 2BGo). Three bands representing proteins that bind specifically to the Pitx1 element were observed (bands 1, 2, and 4). Formation of these complexes was blocked by addition of increasing molar concentrations (10x to 250x) of unlabeled homologous competitor (wild-type Pitx1; lanes 2–5). While the addition of increasing molar concentrations (10x to 250x) of the unlabeled mutated Pitx1 competitor (lanes 6–9) also inhibited binding to the labeled probe, the competition was not as efficient as the wild-type Pitx1 site, indicating that mutation of the core homeobox-binding motif substantially reduces its binding affinity. Indeed, EMSA using radiolabeled probe representing the mutant form of the Pitx1-regulatory element identified no protein from LßT2 cell nuclear extracts that bound specifically and with high affinity to this site (data not shown). To confirm the specificity of these binding events, a nonradiolabeled double-stranded DNA representing the distal NF-Y site that has been characterized in the LHß promoter (16) was included (lane 10); this heterologous competitor (250x) did not appear to affect any of the bound proteins. An additional band that is nonspecific was also observed (band 3); the intensity of this band did not change dramatically between any of the samples. Based on these observations, we conclude that mutation of the Pitx1 core sequence (ATTA) eliminates proteins that normally bind with high affinity to this site.

It is unclear which shifted band represents Pitx1, as our attempts at detection with Pitx1 antibodies were unsuccessful for reasons that remain unexplained. However, because bacterially produced Pitx1 has been shown previously to bind the proximal Pitx1 site of the bovine LHß gene (28), we presume that at least one of the three bands represents Pitx1 and/or one of its isoforms. Indeed, several members of this family have been shown to have in vitro DNA binding specificities similar to that of Pitx1 (49) and can activate transcription of pituitary promoters, including that of the LHß gene (49). In fact, it is also possible that additional homeodomain proteins found in LßT2 cells can bind the Pitx1 site and affect transcription. While additional experiments will be necessary to define the identity of each protein complex detected by EMSA, the tight correlation between the absence of detectable binding with the mutant Pitx1 site and its loss of activity after transfection suggests that the binding of at least one of them is required for promoter activity.

The apparent lack of impact of the Pitx1 mutation on GnRH responsiveness of the LHß promoter contrasts with other LHß promoter mutations that have been assessed in either heterologous or gonadotrope- derived cell lines (19, 25, 27). Individual mutations in SF-1, Egr-1, or Sp1 binding sites often attenuate GnRH responsiveness while pairwise mutations have an even greater impact (17, 19, 25). In contrast, our data suggest that GnRH responsiveness of the LHß promoter in LßT2 cells does not require a functional Pitx1 binding site and that Pitx1 may not be a direct target of the signaling pathway. This is further supported by the unchanging expression of the endogenous Pitx1 gene in {alpha}T3-1 cells treated with forskolin, cyclic ADP-ribose, and GnRH (27). Although this does not rule out the possibility of post-transcriptional or translational effects of GnRH on Pitx1 mRNA or protein, it does distinguish Pitx1 and its cis-acting element from other downstream effectors that may be direct targets of the GnRH signaling pathway.

The Pitx1-Regulatory Element of the LHß Promoter Fails to Function as an Autonomous GnRH-Responsive Element in LßT2 Cells
In cell culture, the Pitx1 site may not be necessary for GnRH responsiveness of the LHß gene. However, it has previously been shown that Pitx1 functionally interacts with Egr-1 and SF-1, transcription factors that bind the proximal promoter of the LHß gene and mediate its response to GnRH. This bridging occurs even in the absence of the consensus Pitx1 binding site in CV-1 cells (27). Thus, although Pitx1 may be necessary for induction of the hormonal response of GnRH on the LHß gene, its cognate binding site appears to be dispensable. To further clarify the role of the Pitx1 site as well as other regulatory elements located in the proximal domain in mediating GnRH-induced expression of the LHß gene, we determined which elements could function autonomously as GnRH-responsive elements when attached to a minimal heterologous promoter. Tandem copies of individual elements that have been characterized in the proximal domain of the LHß promoter were subcloned upstream of the PRL minimal promoter (50) within a luciferase reporter vector. The gonadotrope-derived LßT2 cell line was transiently transfected with each construct and GnRH responsiveness was measured (Fig. 3Go).



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Figure 3. The Pitx1-Regulatory Element of the LHß Promoter Fails to Function as an Autonomous GnRH-Responsive Element in LßT2 Cells

LßT2 cells were transiently transfected with constructs containing tandem copies of each element located in the proximal domain of the bovine LHß promoter (pEgr-1, pSF-1, Pitx1, dEgr-1, and dSF-1) cloned upstream of the PRL minimal promoter within a luciferase reporter vector. Luciferase activity was measured in untreated and GnRH-treated cells, and values are expressed as fold-increase in luciferase activity upon treatment with GnRH. An asterisk (*) represents luciferase values that are significantly greater than observed with the minimal PRL promoter, which has been set to 1 (P < 0.01).

 
PKC-mediated induction of Egr-1 gene expression as well as phosphorylation of Egr-1 constitutes at least part of the intracellular signaling cascade induced by GnRH in gonadotropes (22, 27). Thus, it was not surprising that the proximal Egr-1 element (pEgr-1), that has previously been shown to be critical for GnRH induction of LHß promoter activity, as well as the distal Egr-1 site (dEgr-1), function as autonomous GnRH response elements (P < 0.01) (Fig. 3Go). It is important to note, however, that while the response of each Egr-1 element to GnRH was significant, neither one produced the robust response to GnRH that was observed for the full-length LHß promoter (Fig. 2AGo). Given the clear synergy among the transcription factors that bind elements in the proximal domain of the LHß promoter, it is not surprising that isolation of a single component from its native context would be ineffective in conferring the degree of GnRH responsiveness observed with a full-length promoter.

Neither the proximal nor the distal SF-1 binding elements in the LHß promoter appear to function as autonomous GnRH response elements (Fig. 3Go). In contrast, mutation of the distal SF-1 site renders the cognate promoter nonresponsive to GnRH in transgenic mice (21), and mutation of one or both SF-1 sites substantially decreases the GnRH-mediated response in {alpha}T3-1 and LßT2 cells (25, 31). This suggests that while SF-1 may not be a direct target of the GnRH signaling pathway, it must play an indirect role in mediating the effects of the hormone through its cooperative interaction with Egr-1 and Pitx1.

Tandem copies of the Pitx1 element linked to the PRL minimal promoter were also nonresponsive to GnRH treatment in transiently transfected LßT2 cells. Thus, like the elements that bind SF-1, the homeobox motif that binds Pitx1 may not function as an autonomous GnRH-responsive element. Based on the transfection studies described above, and coupled with the observation that Pitx1, whose expression levels do not appear to correlate with GnRH induction of gonadotropes (27), it is tempting to speculate that the Pitx1 site is not essential for GnRH-stimulated expression of the LHß gene. To test this notion further, we examined the activity of our LHß promoter constructs in transgenic mice where selected contributions of the hypothalamic-pituitary-gonadal axis could be assessed in a more physiological setting.

Mutating the Pitx1 Element within the LHß Promoter Abrogates Pituitary Chloramphenicol Acetyltransferase (CAT) Activity in Transgenic Mice
To assess the functional importance of the Pitx1 homeobox core motif in the context of the full-length LHß promoter within a physiological context, the same mutant and wild-type promoter constructs used in vitro were linked to a CAT reporter and used to develop several lines of transgenic mice. We have shown previously that the full-length wild-type LHß promoter targets expression of reporter genes specifically to gonadotropes in transgenic mice and renders them responsive to GnRH and gonadal steroids (16, 21, 51). In contrast to the robust activity observed with the wild-type promoter (wt-2), pituitary CAT activity in the Pitx1 mutant lines of transgenic mice was indistinguishable from activity in the pituitaries of nontransgenic mice (Fig. 4Go). This pattern of expression, or lack thereof, was seen in both males and females (P < 0.01).



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Figure 4. Mutating the Pitx1 Element within the LHß Promoter Abrogates Pituitary CAT Activity in Transgenic Mice

Pituitary CAT activity was measured in four lines of transgenic mice harboring a Pitx1 mutant form of the LHß promoter linked to CAT (µPitx1–1 through µPitx1–4) as well as one line of mice harboring the wild-type LHß promoter linked to CAT (wt-2). Values are means ± SEM of pituitary CAT activity (percent conversion/µg protein/h) in sexually mature males (white bars) and females (black bars). The following are numbers of male and female animals per group, respectively, that were used: 4/4 (µPitx1–1), 4/1 (µPitx1–2), 4/4 (µPitx1–3), 4/4 (µPitx1–4), and 7/11 (wt-2). Background pituitary CAT activity in nontransgenic littermates (4 males and 4 females) was measured and is represented by the gray box. In all transgenic animals in which liver CAT activity was examined, it was at a level no different from pituitary levels in nontransgenic mice. Bars bearing different letters indicate values that are significantly different (P < 0.01).

 
A sex-specific pattern of pituitary expression was observed in the wild-type line of mice in which CAT activity was 10-fold higher in the pituitaries of females than males, and, as a result, we used female animals for further characterization of the Pitx1 element as their high activity allows for detection of a wider range of responses. This dichotomy has been observed previously (21) and its cause is currently unknown. We speculate that elements important to activation of the LHß promoter in males, or perhaps elements necessary for repression of LHß promoter activity in females, must lie outside the approximately 780 bp of 5'-flanking sequence that have been used to date.

Increasing Endogenous GnRH by Ovariectomy Does Not Rescue Pituitary CAT Activity in Transgenic Mice Harboring the Mutated Pitx1 Element within the LHß Promoter
Expression of the LHß gene responds robustly to GnRH and the homeobox binding motif appears to be unnecessary for this response in cultured cells. Thus, we reasoned that increasing endogenous GnRH within physiological limits might increase pituitary transgene activity to detectable levels in animals harboring the mutated form of the LHß construct. Because GnRH is secreted in regular pulses, administration of exogenous pulsatile GnRH is difficult. Therefore, we elected to increase GnRH levels through ovariectomy of randomly cycling transgenic females (Fig. 5Go). Removal of the ovaries eliminates steroid negative feedback, allowing a sustained increase in synthesis and secretion of GnRH in a pulsatile manner from the hypothalamus (2). Intact animals were not included in these experiments because their asynchronous reproductive cycles result in wide variations in serum LH concentrations that would require large numbers of animals to assure statistical differences at each stage of the estrous cycle. Instead, we treated a subset of gonadectomized females with a specific GnRH antagonist, antide, to provide an indirect measure of GnRH responsiveness. As shown in Fig. 5AGo, serum LH concentrations were considerably higher in all ovariectomized transgenic mice when compared with their antide-treated ovariectomized counterparts. This provides evidence for the effectiveness of antide in blocking events normally mediated by GnRH.



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Figure 5. Increasing Endogenous GnRH by Ovariectomy Does Not Rescue Pituitary CAT Activity in Transgenic Mice Harboring the Mutated Pitx1 Element within the LHß Promoter

A, Serum LH was measured in one line of mice harboring the wild-type LHß promoter linked to CAT (wt-3) and two lines of transgenic mice harboring a Pitx1 mutant form of the LHß promoter linked to CAT (µPitx1–1 and µPitx1–3) 10 days after ovariectomy (white bars) or ovariectomy plus antide treatment (black bars). Values are means ± SEM of serum LH concentrations (ng/ml) as calculated by RIA. B, CAT activity was also measured in these animals. Values are means ± SEM of pituitary CAT activity (percent conversion/µg protein/h). Liver CAT activity was measured in all animals and was found to be no different from levels of pituitary CAT activity in the Pitx1 mutant transgenic animals; this value is represented by the gray box. Bars bearing different letters indicate values that are significantly different (P < 0.01). The following are numbers of ovariectomized or ovariectomized plus antide-treated animals used per group, respectively: 7/9 (wt-3), 6/6 (µPitx1–1), and 4/3 (µPitx1–3).

 
When examining activity of the Pitx1 site in intact (Fig. 4Go) and ovariectomized (Fig. 5Go) mice, two lines of wild-type LHß transgenic mice representing two different integration sites were used for comparison. These lines of mice have been used previously and have been shown to have similar levels of transgene expression, indicating that minimal integration effects occur with this promoter (16). Since expression is indistinguishable between these two lines, both were used in these experiments due to the numbers of animals from each line that were available. These lines are referred to as wt-2 and wt-3 as previously described (16).

Antide also lowered activity of the wild-type transgene in ovariectomized females when compared with their vehicle-treated counterparts (Fig. 5BGo; P < 0.01). This provides an index of the GnRH responsiveness conferred by the transgenic wild-type LHß promoter. In contrast, the Pitx1 mutant transgene remained inactive in both lines of ovariectomized females (Fig. 5BGo). This suggests that the mutated LHß promoter could not be rescued by high physiological concentrations of GnRH in transgenic mice. In fact, levels of expression of the mutant transgene remained undetectable as demonstrated by comparison to activity of the wild-type transgene in nonexpressing tissues. Thus, in the absence of the 4-bp core homeobox motif, activity of the LHß promoter is minimal and cannot be rescued by physiologically elevated concentrations of GnRH.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Results presented herein indicate that LHß promoter activity in transgenic mice is completely dependent upon a functional Pitx1-regulatory element that resides in the promoter-proximal region. Although the Pitx1 element is essential for expression of the LHß gene, it is clearly not sufficient. As summarized in Fig. 6Go, there is now a large body of evidence from several laboratories (19, 26, 27, 31) indicating that full activity of the LHß promoter, including its responsiveness to GnRH, requires contributions from a constellation of regulatory elements that cluster into proximal and distal domains of the 5'-flanking region in addition to elements that may as yet be unidentified. There is also substantial evidence indicating that most of these regulatory elements serve as critical docking sites that allow their cognate DNA-binding proteins to interact directly and cooperatively with one another. Despite previous reports indicating that Pitx1 acts in a DNA-independent manner (27, 28), our data indicate that the proximal Pitx1 element can be added to the list of critical sites necessary for defining activity of the LHß promoter.



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Figure 6. Model of the "Composite GnRH Response Element" in the Proximal Promoter of the LHß Gene

An array of five closely associated cis-acting elements located in the proximal domain of the LHß promoter (pEgr-1, pSF-1, Pitx1, dEgr-1, and dSF-1) comprise the composite GnRH-response element. Transcription factors (puzzle pieces) that bind the corresponding elements in this complex enhancer functionally cooperate to allow basal as well as GnRH-mediated expression of the LHß gene, presumably through interactions with one or more adapter molecules. The NF-Y binding sites in the distal promoter may represent species-specific regulatory elements whose role in the GnRH response have not yet been fully elucidated. The NF-Y factors may also influence GnRH-mediated expression of the LHß subunit gene through interactions with adapters common to factors associated with the composite GnRH-response element, providing the bridge that serves to efficiently integrate the signal from the transcription factors to the basal transcription complex.

 
The best characterized functional interactions between the DNA-binding proteins that regulate the LHß gene occur between Egr-1, Pitx1, and SF-1 (19, 26, 27, 28, 31). In the context of this report, these interactions are notable because previous work indicates that contributions from Pitx1 can occur independently of its binding to DNA (27). The ramifications of transcriptional activity in the absence of DNA binding imply that Pitx1 may reside in gonadotropes in a constitutively active conformation where it could serve as an efficient ligand for activating SF-1 (28) and as a critical partner of Egr-1 (27), a key downstream effector that transduces the signal from GnRH to the LHß promoter. Given the strong spatial conservation of the proximal Pitx1 element in LHß promoters from several species, however, we wondered whether overexpression of its cognate DNA-binding protein in heterologous cell lines could mask the importance of the element.

We have reassessed the importance of this Pitx1 site using two experimental models in which expression of the endogenous LHß gene occurs in concert with the three DNA-binding proteins of interest: Egr-1, Pitx1, and SF-1. Collectively, our data indicate that the proximal Pitx1 site is essential for LHß promoter activity. Mechanistically, this suggests that interaction with DNA causes an allosteric change that endows endogenous Pitx1 with the ability to carry out a transcriptional program specifically adapted for the LHß promoter. This also indicates that while the other putative Pitx1 binding sites in the LHß gene may be functional, they are clearly not sufficient to support Pitx1-mediated activation of the promoter. Even physiologically high concentrations of endogenous GnRH reached by removal of gonadal steroid negative feedback could not activate the Pitx1 mutant form of the promoter in transgenic mice.

Because expression of the endogenous LHß gene is exquisitely dependent upon GnRH, the role of the Pitx1 binding site in mediating responsiveness to this neurohormone requires further consideration. While our experimental approaches support the importance of the Pitx1 site in defining a fully functional LHß promoter, we also uncovered an unexpected dichotomy between the necessity for the Pitx1 element in conferring a GnRH response of the LHß promoter in cell lines vs. transgenic mice. There are many possible explanations for this contrariety including 1) differences in pulse patterns of GnRH and its concentration (52, 53); 2) differences in thresholds of detection associated with transfection and transgenics; 3) a generalized variation in the expression of transcription factors between the in vitro and in vivo settings; and 4) simple differences in mass action. Based on these possibilities, we suggest that the transfection assays reveal the potential for GnRH to rescue at least some activity of the mutant LHß promoter, possibly through the action of Egr-1, but that this rescued activity remains below the limit of detection in transgenic mice.

While the use of overexpression paradigms in heterologous cells appear to have masked the importance of the Pitx1 site, they have been extremely valuable in demonstrating potential interactions that occur between Pitx1, Egr-1, and SF-1, contributions that cannot be ascertained from the experimental approaches we used. For example, Tremblay et al. (28) showed direct cooperative interactions between Pitx1 and SF-1 and proposed that Pitx1 may serve as an efficient ligand for SF-1 by modulating its activity in gonadotropes. A constitutively-active form of SF-1 in which the ligand-binding domain has been deleted (SF-1{Delta}LBD) activated the LHß promoter more than the wild-type SF-1, and, unlike wild-type SF-1, SF-1{Delta}LBD was unable to synergize with Pitx1, although the transcription factors were still shown to interact. In fact, the activities of SF-1 with Pitx1 compared with SF-1{Delta}LBD alone were identical. Collectively, the extent of the interaction between the transcription factors offers broad support for the possibility that Pitx1 serves as a physiological ligand that activates SF-1. If Pitx1 is a functional ligand for SF-1, then our data suggest that this property may only occur when Pitx1 is bound to its element.

Like the Pitx1 element, the SF-1 element does not act as an autonomous GnRH response element when linked in tandem to a heterologous promoter. In fact, neither the proximal nor the distal SF-1 binding elements in the LHß promoter appear to confer autonomous GnRH responsiveness. In contrast, our previous studies in transgenic mice indicated that the distal SF-1 binding site, like the proximal Pitx1 site, may be essential for GnRH-regulated expression of LHß (21) because activity of a transgene harboring a mutated distal SF-1 site remained refractory to a postcastration rise in GnRH. Taken together, these apparently contradicting data may indicate that while the SF-1 elements do not act as autonomous GnRH response elements, they may be regarded as critical accessory elements indirectly involved in the response. Indeed, it has been shown that while SF-1 does not have a direct effect on GnRH responsiveness of the LHß promoter, the ability of Egr-1 to activate the LHß promoter in response to GnRH requires an direct interaction between Egr-1 and DNA-bound SF-1 (19, 26, 27, 31). In this regard, the SF-1 sites, like the Pitx1 element, may be critical for a normal GnRH response even though they do not serve as direct downstream targets of the signaling pathway.

In contrast to the Pitx1 and SF-1 elements, the Egr-1 binding sites act as autonomous GnRH response elements when placed in tandem on a heterologous promoter. Binding of GnRH to receptors at the cell surface of gonadotropes activates the PKC signaling pathway (54, 55), which leads to elevated Egr-1 mRNA levels (22, 31) as well as phosphorylation of Egr-1 protein (27). In the absence of GnRH, only low levels of monophosphorylated Egr-1 can be detected (27). Egr-1 is vital for expression of LHß, as mice lacking this transcription factor are devoid of LH, but have normal serum FSH concentrations and gonadotrope numbers (32, 33). SF-1 and Pitx1 are also essential for GnRH responsiveness, because enhancement of Egr-1 action by superphysiological levels of GnRH (seen postcastration) is insufficient for expression of LHß in the face of a mutant binding site for either SF-1 (21) or Pitx1. Several groups have shown the importance of synergy between Egr-1 and SF-1 for activation of the LHß gene by GnRH (19, 22, 25, 27, 31). In addition, Tremblay and Drouin (27) have firmly established the role of Pitx1 in enhancing this cooperative interaction. However, while the site at which Pitx1 binds does not appear to be necessary for this synergism when transcription factors are overexpressed in heterologous cell lines, the site is crucial for expression of the LHß promoter in transgenic mice, as observed by the absence of detectable transgene expression in mice presented herein, even in the presence of high endogenous GnRH, and presumably increased Egr-1.

In contrast to the regulatory elements in the proximal domain, upstream DNA cis-acting elements in the distal domain display species-specific diversity, suggesting that multiple configurations are capable of achieving the same end point. For example, the distal domain of the rat LHß gene harbors two adjacent Sp1 binding sites (17). The ubiquitous Sp1 transcription factor binds these sites and enhances GnRH-stimulated expression (17) through interactions with factors that bind elements in the proximal promoter, including SF-1 and Egr-1 (18, 19). Indeed, Kaiser et al. (19) have described these elements as forming a "tripartite GnRH response element." In considering the proposed role for Sp1, it is important to note that the Sp1 elements are less conserved than the proximal LHß promoter elements, suggesting that their importance might be limited to the rat LHß promoter. For example, the bovine LHß promoter lacks Sp1 sites at positions comparable to those found in the rat LHß promoter. Rather, a CCAAT box that binds NF-Y is located in the distal domain of the bovine LHß promoter that contributes to its activity. This site, however, does not appear to be required for GnRH responsiveness (16). Therefore, from studies performed to date, the role of the distal domain in mediating responsiveness to GnRH appears species specific and requires further investigation.

In summary, we suggest that the strong conservation of regulatory elements in the proximal region of the LHß promoter across several species underscores their importance in regulating transcription, and, in particular, responsiveness to GnRH. In Fig. 6Go we diagram a model that builds on the work from several laboratories (22, 26, 27, 28, 31) as well as the work reported herein. We propose that the promoter-proximal elements define a core "composite GnRH response element" that includes the essential Pitx1 binding site as well as four other highly conserved regulatory elements (pEgr-1, pSF-1, dEgr-1, and dSF-1). Transcription factors that bind to this region interact directly with one another and most likely with transcription factors that bind to elements in the distal region such as Sp1 for the rat promoter (17, 19) and NF-Y for the bovine promoter (16). While Pitx1 has yet to be tested for functional synergism with proteins that bind to the distal elements, it can cooperatively interact with both Egr-1 and SF-1 (27) and may serve as a physiological ligand for the latter (28). More importantly, activity of the LHß promoter requires the homeobox binding motif that defines the core of the Pitx1 cis-acting element. Binding of Pitx1, and perhaps other related homeobox proteins, to this element increases its effectiveness in acting as a functional partner for SF-1 and Egr-1. Although Egr-1 can be regarded as a direct downstream effector of the GnRH signal pathway, DNA-bound Pitx1 and SF-1 are required accessory factors as elimination of their binding sites renders the promoter inactive and, in vivo, unresponsive to increased physiological levels of the neurohormone. Thus, all of the aforementioned elements serve as vital docking sites required for the formation of a higher-order transcriptional complex that directs spatial expression and hormone responsiveness of the LHß gene.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Plasmid Vectors
The vectors pGL2, pGL3, and PRLpGL3, as well as the wild-type LHß promoter construct [(-776/+10)bLHßLUC, herein referred to as wt-LHßLUC because of an error in sequence identification that was recently identified, the 5'-end of this promoter does not represent -776, but instead, -779] have been described elsewhere (21, 56). A construct similar to the wild-type LHß promoter vector that contained a mutated Pitx1 element (µPitx1-LHßLUC) in place of the wild-type sequence was generated by PCR utilizing the wt-LHßLUC construct. The sense primer used in the reaction annealed to -610/-581 (5'-AAA GAG CCT AAA TCA TGC TCT TTG CTG GGT-3') in the bovine LHß promoter while the antisense primer, which contained an RsaI site in place of the consensus homeobox binding motif (5'-TTT TTG GTA ACC TGG ACA CGT ACC TCC CCG GGG), with the mutant Pitx1 site underlined, annealed to the region -102/-78. Utilizing a BalI site at -480 and a BstEII site at -84, the mutation-containing insert was subcloned into wt-LHßLUC, replacing the wild-type sequence in the BalI/BstEII region.

Multimerized regulatory elements in the forward direction linked to PRLpGL3 were generated by ligating double-stranded oligos containing the various regulatory sequences flanked by HindIII sites into the same site in the multiple cloning region of PRLpGL3: 2xpEgr1PRLpGL3 (5'-AGC TTT GCC GCC CCC ACA GCA-3'; 5'- AGC TTG CTG TGG GGG CGG CAA-3'), 2xpSF1PRLpGL3 (5'-AGC TTC GGC GGC CTT GCC GCA-3'; 5'-AGC TTG CGG CAA GGC CGC CGA-3'), 2xPitx1PRLpGL3 (AGC TTG GGA GAT TAG TGA AGC TTG GGA GAT TAG TGA-3'; 5'-AGC TTC ACT AAT CTC CCA AGC TTC ACT AAT CTC CCA-3'), 2xdEgr1PRLpGL3 (5'-AGC TTT CTC GCC CCC GGG GAG A-3'; 5'-AGC TTC TCC CCG GGG GCG AGA A-3'), and 2xdSF1PRLpGL3 (5'-AGC TTC CCT GAC CTT GTC TA-3'; 5'-AGC TTA GAC AAG GTC AGG GA-3').

The Pitx1 mutant form of an LHß promoter-driven CAT reporter (µPitx1-LHßCAT) vector that was used to generate transgenic mice in these experiments was constructed by subcloning the bovine LHß promoter insert that contained the mutated Pitx1 site (as described above) into the HindIII site of the BSK-(-776/+10)bLHßCAT vector, as has been described previously (51), replacing the wild-type promoter with the mutated form. All clones were confirmed with dideoxynucleotide sequencing.

EMSAs
LßT2 cells were treated with GnRH (100 nM) for 24 h before extraction of nuclear proteins. Nuclear extracts were prepared as described previously (8). EMSAs were performed essentially as described (21) except with 23 µg of LßT2 nuclear protein. The following oligonucleotides were used: Pitx1(+), 5'-CCG GGG AGA TTA GTG TCC AGG TTA CCC CAC-3' (core homeobox motif underlined); Pitx1(-), 5'-GTG GGG TAA CCT GGA CAC TAA TCT CCC CGG 3' (core homeobox motif underlined); µPitx1(+), 5'-CCG GGG AGG TAC GTG TCC AGG TTA CCC CAC- 3' (mutated motif underlined); µPitx1(-), 5'-GTG GGG TAA CCT GGA CAC GTA CCT CCC CGG-3' (mutated motif underlined); dNF-Y(+), 5'-CTG CAG CCA ATC ACC ATC GGA AAA TGG AGC T-3'; dNF-Y(-), 5'-CCA TTT TCC GAT GGT GAT TGG CTG CAG AGC T-3' (16). Double-stranded oligodeoxynucleotides were end labeled with [{gamma}-32P] ATP (NEN Life Science Products, Boston, MA) using T4 polynucleotide kinase (Life Technologies, Inc., Gaithersburg, MD). Poly(dI-dC) was purchased from Roche Molecular Biochemicals (Indianapolis, IN).

Cell Culture and Transient Expression Assays
Transient transfection studies were performed in LßT2 cells, which were maintained in high-glucose DMEM containing 2 mM L-glutamine and supplemented with 10% FBS and antibiotics. The day before transfection, cells were plated at a density of approximately 2 x 106 cells per 35-mm well. Transfections were carried out using media lacking serum and antibiotics with 10 µl LipofectAMINE reagent (Life Technologies, Inc.), 2.0 µg each test vector, and 100 ng pRL-CMV (Promega Corp., Madison, WI), which was used to normalize data for transfection efficiency. Cell cultures were incubated with the transfection mixtures for approximately 18 h at 37 C in a humidified atmosphere with 5% CO2. After incubation, complete media were added to the cells, which, where indicated, were also supplemented with 100 nM GnRH. Twenty-four hours after the addition of fresh media and hormonal treatments, cells were lysed in passive lysis buffer (Promega Corp.), and a dual-luciferase assay was performed on each cellular lysate as per standard procedures. Transient transfections were performed a minimum of three times with at least two separate plasmid preparations for each construct that was tested. Luciferase activity was analyzed by two-way ANOVA (Fig. 2Go) or one-way ANOVA (Fig. 3Go), and differences among treatments were determined by the post-hoc test, Tukey’s Honestly Significant Difference, a very conservative pairwise comparison test.

Transgenic Mice
The µPitx1-LHßCAT insert was liberated from the vector using a SalI/BamHI digest. Insert DNA was purified by 0.7% agarose gel electrophoresis. Transgenic mice were generated as previously described (7). Mice were genotyped by PCR to amplify DNA found within the transgene. Primers that amplified a fragment of the endogenous murine ß-globin gene were also included in the PCR to confirm the integrity of genomic DNA within each reaction. Mice were bred to obtain single integration sites of the transgene, as determined by Mendelian inheritance patterns. All mice were housed in microisolator-plus units under pathogen-free conditions. Food and water were provided ad libitum, and animals were subjected to a 12-h light, 12-h dark cycle. Mice harboring the wt-LHßCAT transgene have been previously reported (16, 21).

Adult pituitaries were immersed in 200 µl 0.25 M Tris (pH 7.8). Pituitary lysates were obtained and CAT assays were performed as described previously (16, 21, 51). In all assays, 25 µg pituitary protein were used, and the assays were incubated for 2 h (wt-LHßCAT transgenic mice) or 18 h (µPitx1-LHßCAT and nontransgenic mice) and plotted as percent conversion/µg protein/h. The increased incubation for pituitary tissues extracted from both nontransgenic and µPitx1-LHßCAT mice has been shown to increase assay sensitivity in previous experiments (16), allowing for determination of low vs. no CAT activity. Radiolabeled chloramphenicol was obtained from NEN Life Science Products, and acetyl coenzyme A was purchased from Sigma (St. Louis, MO).

To examine GnRH responsiveness, sexually mature mice were ovariectomized under avertin anesthesia. Immediately after surgery, 300 µl antide (200 ng/ml; from Sigma) or vehicle (20% propylene glycol in normal saline) was injected subcutaneously (21). Additional injections were given every 48 h for a total of 10 days. On the tenth day, animals were killed and tissues and blood were collected. Serum LH concentrations were measured using a previously validated RIA (28). CAT activity was analyzed by two-way ANOVA. Differences among treatment groups were determined by Tukey’s Honestly Significant Difference.

Experimental Animals
All animal studies were conducted in accord with the principles and procedures approved by the Institutional Animal Care and Use Committee of Case Western Reserve University.


    ACKNOWLEDGMENTS
 
The authors wish to thank David Peck and Danielle Grove-Strawser for invaluable technical assistance as well as Dr. Joan Jorgensen for providing nuclear extracts from LßT2 cells for use in the EMSAs. We are also grateful to Xiaoming Sha and Dr. Terry Nett for performing LH RIAs; Dr. Pamela Mellon for providing the LßT2 cell line; and Drs. Paul MacDonald and Phillip Quirk for critical evaluation of this manuscript.


    FOOTNOTES
 
Address requests for reprints to: John H. Nilson, Ph.D., John H. Hord Professor and Chair, Department of Pharmacology, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106-4965. E-mail: jhn{at}po.cwru.edu

This work was supported by NIH Grant DK-28559 (to J.H.N.) and NIH National Research Service Award Fellowship DK-09843 (to C.C.Q.).

Received for publication August 7, 2000. Revision received December 12, 2000. Accepted for publication December 20, 2000.


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