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J Biol Chem, Vol. 275, Issue 17, 13082-13088, April 28, 2000

An NF-Y Binding Site Is Important for Basal, but Not Gonadotropin-releasing Hormone-stimulated, Expression of the Luteinizing Hormone  Subunit Gene^*

Ruth A. Keri, Daniel J. Bachmann, Alireza Behrooz, Brian D. Herr, Rebecca K. Ameduri, Christine C. Quirk, and John H. Nilson

From the Department of Pharmacology, Case Western Reserve University, Cleveland, Ohio 44106

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Regulated synthesis of luteinizing hormone (LH) requires coordinated transcriptional control of the  and LH subunits in pituitary gonadotropes. Several cis-acting elements and trans-acting factors have been defined for control of the LH promoter through heterologous cell culture models. In this report, we describe the identification of bipartite NF-Y (CBF/CP1) binding sites within the proximal bovine LH promoter. When multimerized, one of these sites activates the heterologous, minimal HSV thymidine kinase promoter in the gonadotrope-derived cell line T3-1. The functional role of the promoter-distal site in regulating the full-length bovine LH promoter was assessed in vivo using transgenic mice harboring a mutant promoter linked to the chloramphenicol acetyltransferase reporter gene. While this element is important for conferring high level activity of the LH promoter in pituitary, it does not appear to be essential for mediating gonadotropin-releasing hormone (GnRH) regulation. This is the first characterization of a cis-acting element within this GnRH-dependent promoter that is restricted to regulating basal expression and not GnRH-induced activity.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Stimulation of gametogenesis and synthesis of sex steroids by luteinizing hormone (LH)¹ is essential for reproduction in all mammalian species. Thus, understanding the regulated synthesis and secretion of this hormone from the gonadotropes in the pituitary is paramount to understanding the reproductive process.

LH is a member of the glycoprotein hormone family whose members are composed of a shared  subunit that combines with unique, hormone-defining  subunits. Synthesis and secretion of LH requires coordinated, gonadotrope-specific expression of the genes encoding both the  and LH subunits (1). Gonadotrope-specific expression of the  subunit gene requires a number of cis-acting elements located within the promoter-proximal 400 bp of the 5'-flanking region of the gene (2-7). Discernment of the essential cis-acting elements involved in expression of the LH subunit has lagged behind that made for the  subunit due to the lack of readily accessible, appropriate model systems.

Five models have been used to uncover regulatory elements within the LH promoter. These include cell lines corresponding to kidney fibroblasts (CV-1) (8, 9), modified somatotropes (GGH₃-1') (10, 11), primordial gonadotropes (T3-1) (12-14), and differentiated gonadotropes (LT2) (15, 16) as well as transgenic mice (17-21). Using these approaches, four transcription factors have been identified that regulate expression of the LH subunit gene through direct binding to its promoter (see Fig. 1A). These include the orphan nuclear receptor, SF-1 (8, 15, 21), an early growth response protein, Egr-1 (22, 23), a bicoid-related homeodomain protein, Pitx1 (9), and the ubiquitous transcription factor, Sp1 (11). There are two Egr-1 and SF-1 binding sites that exist in a pairwise conformation interrupted by a Pitx1 binding site within the promoter-proximal 150 bp (24). This configuration is conserved across many, if not all, mammalian LH promoters. Numerous recent studies have shown that SF-1 and Egr-1 functionally cooperate to activate transcription (16, 24, 25) of the LH subunit gene. In addition, Pitx1 further potentiates the activation mediated by these two proteins; however, this does not appear to require direct binding of Pitx1 to DNA (25). Rather, Pitx1 can interact directly with SF-1 and Egr-1 to induce expression (25). Sp1 regulates the rat LH promoter by binding two elements that reside further upstream in the promoter at (451/428) and (411/386) (11). This region is less conserved across species than the promoter-proximal sequences, and its importance in other species remains to be determined.

The relative importance of Egr-1, SF-1, and Pitx1 in regulating LH gene expression has been confirmed with targeted disruption of each gene in mice. Mice deficient in Egr-1 exhibit a specific loss of LH gene expression, while the  and follicle-stimulating hormone  genes remain functional (22, 23). This suggests that Egr-1 is a selective transcriptional regulator of the LH gene and may mediate differential regulation of LH and follicle-stimulating hormone within the same cell. In contrast, mice deficient in SF-1 have a reduced gonadotrope population accompanied by attenuated expression of all three gonadotropin genes (26). Whether this is due solely to a defect within the gonadotrope (21) or to an additional impact in the hypothalamus (27) remains to be determined. Targeted disruption of the Pitx1 gene leads to gross defects in limb development (28, 29) as well as multiple pituitary effects (28). Although gonadotrope number and expression of the gonadotropin genes is significantly reduced in these animals, there is not an ablation of either (28). Thus, while Pitx1 is important for LH gene expression and gonadotrope development, it does not appear to be essential.

While earlier work focused on evaluating basal expression of the LH subunit gene, this rapidly evolved into analyses of the effects of gonadotropin-releasing hormone (GnRH). This reflects the exquisite dependence of transcriptional activity of the LH gene on pulsatile release of GnRH from the hypothalamus, exemplified by the hpg/hpg mouse (30) that harbors a spontaneous deletion of this gene (31). Mice harboring this mutation express the LH subunit gene at only 5-10% of normal levels (32), suggesting that "basal" activity of this gene requires activation of GnRH-induced signaling cascades. Indeed, all of the factors described to date as being important for LH gene expression also contribute to GnRH induction (11, 13, 14, 16, 21, 24, 25). In the current manuscript, we report the identification of an additional pairwise cis-acting element that binds NF-Y (CBF/CP1). In contrast to the previously described cis-acting elements and their cognate factors characterized for this gene, these sites are the first to be described that solely affect basal expression of this gene.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- Radiolabeled nucleotides and chloramphenicol were obtained from NEN Life Science Products. Acetyl coenzyme A and the GnRH antagonist, antide, were from Sigma. DNA-modifying enzymes were from either Roche Molecular Biochemicals or Life Technologies, Inc. Sequenase version 2.0 was from Amersham Pharmacia Biotech. Poly(dI-dC) was purchased from Roche Molecular Biochemicals. Sp1 antibody was obtained from Santa Cruz Biotechnology, Inc., and NF-Y (A subunit-specific) antibody was from Rockland Immunochemicals.

Plasmid Constructs-- The HSVtk-luc vector was constructed by inserting an XhoI/SalI fragment containing the HSVtk minimal promoter (105/+5) from BLCAT2 (33) into the XhoI site of pGL2-Basic (Promega). Kinased, double-stranded oligodeoxynucleotides corresponding to L1 or L2 (see below) were ligated into the SacI site 5' to the thymidine kinase promoter. The number and orientation of elements was then determined by dideoxynucleotide sequencing.

A transversion mutation of the L1 site within the context of the full-length (776/+10)bLH promoter was accomplished using an oligodeoxynucleotide replacement approach in the previously described (776/+10)bLHCAT vector (18). Oligodeoxynucleotides corresponding to the region to be mutated were 5'-CCGGGAACCGACAACGATTCCCCGGTTCAGAGGGGCGGTGCTGCA-3' and 5'-GCACCGCCCCTCTGAACCGGGGAATCGTTGTCGGTTCCCGG-3', with the mutant sequence indicated with boldface letters. The annealed oligodeoxynucleotide has a 5' blunt end and a 3' PstI overhang. The annealed, double-stranded oligodeoxynucleotide was inserted into the PstI sites at 397 and 355, relative to the start site of transcription, that flank the L1 consensus site. This required conversion of the 5' PstI site into a blunt end prior to ligation with the mutant oligonucleotide. The composition of the resultant clone was confirmed by sequencing the region containing the mutation and restriction digests confirming the remainder of the expression cassette. Isolation of the transgene was accomplished by restriction digestion with SalI and BamHI.

Cell Culture/Transient Transfections-- Transient transfection assays of T3-1 and MCF-7 cells were performed essentially as described (7) using 1.2 µg of test vector and 0.42 µg of RSV-gal per 35-mm² well containing 110,000-170,000 cells. Cells were harvested 48 h post-transfection in 150 µl of reporter lysis buffer (Promega). Luciferase and -galactosidase assays were performed as previously reported (7).

Electrophoretic Mobility Shift Assays (EMSAs)-- Nuclear extracts from T3-1, MCF-7, and BeWo cells were prepared as described previously (7). EMSAs were performed essentially as described (21) except with 5-10 µg of nuclear protein. The following oligodeoxynucleotides were used (lowercase type indicates SacI sites added for cloning purposes): L1(+), 5'-cTGCAGCCAATCACCATCGGAAAATTGagct-3'; L1(), 5'-CAATTTTCCGATGGTGATTGGCTGCAgagct-3'; L1 µ610(+), 5'-cTGCAGTCAACACCATCGGAAAATTGagct-3'; L1 µ610(), 5'-CAATTTTCCGATGGTGTTGACTGCAgagct-3'; L110 µ18(+), 5'-cTGCAGCCAACACCATCTGAAAATTGagct-3'; L110 µ18(), 5'-CAATTTTCAGATGGTGTTGGCTGCAgagct-3'; L1 µ18/25(+), 5'-cTGCAGCCAATCACCATCTGAAAATGGagct-3'; L1 µ18/25(), 5'-CCATTTTCAGATGGTGATTGGCTGCAgagct-3'; L1 µ6(+), 5'-cTGCAGTCAATCACCATCGGAAAATTGagct-3'; L1 µ6(), 5'-CAATTTTCGATGGTGATTGACTGCAgagct-3'; L2(+), 5'-cTGTGAAGTCACCTTCTCCTGGGTGagct-3'; L2(), 5'-CACCCAAGGAGAAGGTGACTTCACAgagct-3'; B1(+), 5'-CTGCAGTCAACACCATCTGAAAATGGA-3'; B1(), 5'-CCATTTTCAGATGGTGTTGACTGCAGA-3' and an Sp1 consensus oligodeoxynucleotide obtained from Promega Corp. Double-stranded oligodeoxynucleotides were end-labeled with [-³²P]ATP using polynucleotide kinase. For the antibody supershift experiments, binding reactions were performed with 5-10 µg of T3-1 nuclear extract and 1 µg of poly(dI-dC) in 10 mM Tris-HCl (pH 7.4), 50 mM NaCl, 1 mM MgCl₂, 0.5 mM EDTA, 0.5 mM dithiothreitol, and 4% glycerol in a total volume of 15 µl. After a 30-min incubation on ice, 5 × 10⁴ cpm of labeled probe was added with an additional 30-min incubation on ice. For reactions containing antibody, 1 µl of anti-Sp1 or anti-NF-Y was added during the initial 30-min incubation.

Transgenic Mice-- All mice were housed in microisolator-plus units under pathogen-free conditions. Food and water were given ad libitum, and animals were subjected to a 12-h light/dark cycle. Mice harboring the (776/+10)bLHCAT construct have been previously described (18). For this work, we regenerated this transgenic strain to utilize mice that were as few generations from the founder animals as possible for direct comparison with mice harboring the µL1 construct.

To generate mice with the (776/+10)µL1bLHCAT construct, the 2.4-kilobase pair expression cassette was liberated with SalI plus BamHI. Transgenic mouse production, identification, and characterization of tissue-specific expression were done as reported (18). Chloramphenicol acetyltransferase (CAT) assays were performed as described (18) with 10-25 µg of cellular lysate for 1 or 4 h. Each assay contained control tissues from mice containing the wild type promoter. The amount of lysate used in each CAT assay was identical for each sample within that assay.

The ovariectomy/antide treatment paradigm was performed as follows. Female mice harboring either the wild type or mutant promoter were ovariectomized under avertin anesthesia. Following surgery, randomized mice were separated into two groups: those receiving vehicle (20% propylene glycol in saline) injections and those receiving antide injections. 60 µg of antide was given as a subcutaneous injection every 48 h for 10 days. On day 10, mice were killed by asphyxiation in a CO₂ chamber, and cardiac blood, pituitaries, and livers were collected. All animals used were adults and age-matched (within 1 month) within individual experiments. All animal studies were approved by the Case Western Reserve University Institutional Animal Care and Use Committee.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Alignment of the Bovine  and LH Promoters Reveals Three Conserved Regions-- Production of LH requires coordinated expression of both the  and LH genes. These genes must be expressed concurrently in a temporal and spatial pattern to form an intact heterodimer. In addition, regulation of their expression by GnRH and gonadal steroids may involve similar mechanisms for the two genes. Thus, we speculated that specific cis-acting elements may be shared between the two gonadotropin subunit genes as a means to confer similar signals for regulated expression. To begin to identify targets for functional analysis of the LH promoter in transgenic animals, we examined the bovine gonadotropin promoters for regions of apparent conservation. Three regions with greater than 80% homology were identified (Fig. 1B). One of these, the GSE, was previously shown to be important for activity of both the  (5) and LH (8, 21) genes. The other two elements located at positions 399/372 (element L1) and 228/204 (element L2) relative to the start site of transcription of the bovine LH gene, were subject to further analyses to determine their role, if any, in activating the LH promoter. A diagrammatic sketch of the positions of these elements as well as others identified for the bovine or rat genes is shown in Fig. 1A.

View larger version (30K): [in this window] [in a new window]   Fig. 1.   Alignment of the bovine  and LH promoters reveals three conserved regions. A, a schematic diagram of the bovine LH promoter with the position of L1 and L2 is shown. Binding sites for SF-1 (GSE), Egr-1, and Pitx1 are indicated. The positions of Sp1 binding sites that are present within the rat LH promoter are also shown. L4 harbors a sequence that binds the same protein as L1. The Sp1 site that is 5' to the L1 site is outlined in gray to reflect the lack of an Sp1 consensus at this site in the bovine promoter. B, the bovine  and LH subunits were scanned for any regions of conservation between the two promoters. The sequences of three conserved elements are indicated. The GSE has previously been described (5, 8, 21). Element 1 and element 2 are designated L1 and L2 when referring to the elements within the LH promoter.

L1, but Not L2, Stimulates Transcriptional Activity of a Minimal Thymidine Kinase Promoter-- In the absence of GnRH, the bovine LH promoter has low activity in transiently transfected cells (18) that precludes functional analyses of the L1 and L2 elements in the context of the full-length promoter. Therefore, functional activity was initially assessed by determining whether multimerized L1 or L2 elements could stimulate expression of the HSVtk minimal promoter when linked to luciferase. The resulting constructs were transiently transfected into T3-1 cells. As shown in Fig. 2, the L1 element was capable of activating the HSVtk promoter 2-3-fold with inverted repeats of this sequence resulting in the greatest induction. In contrast, L2 was incapable of activating the heterologous promoter. Similar results were obtained in the breast cancer cell line, MCF-7 (data not shown). The ability of L1 to function independently of cell lineage suggested that it might bind a factor that is not gonadotrope-specific.

View larger version (42K): [in this window] [in a new window]   Fig. 2.   L1, but not L2, stimulates transcriptional activity of a minimal thymidine kinase promoter. Oligodeoxynucleotides corresponding to L1 or L2 were multimerized and ligated upstream of the HSVtk minimal promoter linked to luciferase. A shaded (L1) or black (L2) box indicates the presence and orientation (arrowheads) of each element. T3-1 cells were transiently transfected with each construct. Values are expressed relative to HSVtk-luc activity and represent the mean ± S.E. of three independent experiments each with three replicates.

L1, but Not L2, Binds a Nuclear Factor That Is Common to a Variety of Cell Lines-- To determine if L1 and L2 elements bind nuclear proteins, EMSAs were performed with T3-1 nuclear extracts. This assay revealed that L1 could bind a single complex with high specificity and affinity (Fig. 3A). While only a 50-100-fold molar excess of homologous competitor was necessary to eliminate binding of the factor to the probe, a 100-fold molar excess of heterologous competitor (LH GSE (21)) had no impact on protein binding. A monophasic Scatchard analysis revealed a high affinity interaction between the nuclear protein and the L1 site (K_D = 1.2 × 10¹⁰ M, data not shown) that was well within the known range for eukaryotic transcription factors that bind to DNA (34). In contrast to L1, incubation of the L2 probe with T3-1 nuclear extract failed to reveal any high affinity complexes (Fig. 3A). This supports data obtained from the transient transfection studies and suggests that L2 is nonfunctional in these cells. Given the transcriptional activity and protein binding function of L1, we chose to focus on further characterization of this element. Whether L2 has a transcriptional function in mature gonadotropes remains to be determined. Studies aimed at addressing this issue will probably involve complex experimental paradigms such as transgenic mice and preparation of nuclear extracts from mature gonadotrope cells.

View larger version (57K): [in this window] [in a new window]   Fig. 3.   L1 binds a nuclear factor that is common to a variety of cell lines. A, EMSA were performed with either the L1 or L2 probes and T3-1 nuclear extracts. A single complex was observed with the L1 probe. To assess binding specificity, competition analyses were performed by adding 10-, 50-, or 100-fold molar excess of unlabeled, homologous L1 or L2 (as indicated) or heterologous GSE (21). Increasing concentrations of added oligodeoxynucleotides are indicated by triangles. B, EMSA were accomplished with nuclear extracts obtained from cell lines derived from gonadotrope (T3-1), choriocarcinoma (BeWo), or mammary epithelium (MCF-7). The L1 band is indicated by an arrow. Competition analyses were performed with unlabeled homologous L1 or an L1 mutant harboring a C/T transition at position 6 (see Fig. 4A for the L1 and mutant sequence).

To address whether the L1 protein-binding complex was unique to gonadotrope origin cell lines, we performed additional EMSA studies using nuclear extracts from MCF-7 and BeWo cells. Both extracts exhibited a complex with the same mobility and competition profile as that observed with T3-1 nuclear extracts (Fig. 3B). An additional complex displaying a faster migration pattern was also observed in both heterologous extracts. This interaction was considered nonspecific due to the increase in intensity that corresponded with increasing concentrations of unlabeled competitors. From these results, we conclude that the protein(s) that bind to LB1 are not cell-specific and are likely to be ubiquitous given their distribution in gonadotrope-, mammary-, and choriocarcinoma-derived cell lines.

The L1 Element Contains a CCAAT Motif That Is Essential for Protein Binding-- To begin to discern whether the L1 element was functionally conserved in the bovine  subunit promoter, we performed EMSA with its L1 homolog, designated B1. Interestingly, although homology analysis was utilized to originally identify L1, the B1 element did not bind any proteins with high affinity in EMSA with T3-1 nuclear extracts (data not shown). While function of the B1 element within the context of the full-length  subunit promoter was not addressed, lack of protein binding in gonadotrope-derived cells suggests that this region was not important for transcriptional function of the bovine  subunit gene promoter.

Only four nucleotide differences exist between the L1 and B1 elements. Thus, the lack of high affinity binding of a nuclear protein to B1 could be used to rapidly discern the specific nucleotides required for protein binding to L1. Mutations in the L1 element were made to mimic those nucleotide differences observed in B1. Fig. 4A outlines the mutant oligodeoxynucleotides utilized in EMSA. Two nucleotide changes were within a CCAAT motif, while two additional differences were external to this motif. The importance of these particular nucleotides in protein binding was addressed using pairwise mutations and competition analysis in EMSA utilizing the wild type L1 probe. As shown in Fig. 4B, mutation of nucleotides at positions 6 and 10 within L1 eliminated the ability of this oligonucleotide to compete for binding to the wild type L1 probe. Similar to the 6/10 mutation, mutation of nucleotides 10 and 18 also resulted in loss of L1 protein binding. In contrast, pairwise mutation of the two nucleotides outside the CCAAT motif at positions 18 and 24 resulted in no change in affinity. These results implicate the position 10 nucleotide (the T of CCAAT) in protein binding and minimized the likelihood that binding involved specific sequences eight nucleotides downstream of the CCAAT motif. To directly assess the importance of the C at position 6 of L1, an additional point mutant was used in EMSA. This mutant was incapable of competing with protein binding to the wild type L1 probe (Fig. 3B). Thus, both the initial C and the T of the CCAAT motif are essential for protein binding to L1.

View larger version (83K): [in this window] [in a new window]   Fig. 4.   The L1 element contains a CCAAT motif that is essential for protein binding. A, alignment of the element 1 sequences from the bovine  (B1) and LH (L1) promoters reveals only four nucleotide differences between the two that must dictate binding activity. These differences are indicated by boldface letters, and the position of each is denoted. B, EMSA with T3-1 nuclear extracts and L1 probe. Unlabeled L1 competitor (i.e. wild type) was added at 10- and 50-fold molar excess. Unlabeled mutant L1 oligonucleotide competitors were added at 50-, 100-, and 300-fold molar excess. The mutations within the L1 oligodeoxynucleotides are indicated by the position of the mutation. At each of these positions, the sequence was converted from the LH promoter sequence to that present within the  promoter.

NF-Y Is the L1-binding Protein-- The requirement for sequences corresponding to a CCAAT motif suggested that the L1 element might bind to a known CCAAT box-binding factor. To begin to determine the nature of the CCAAT-binding protein that interacts with L1, we performed a variety of competition EMSA studies with consensus oligodeoxynucleotides and antisera to known CCAAT box-binding factors. Antiserum directed against NF-Y was capable of "supershifting" the L1 protein band (Fig. 5A). NF-Y is a ubiquitous transcription factor that binds to CCAAT motifs. Thus, the protein that interacts with L1 is either NF-Y or a highly related protein. Within the rat LH promoter, an Sp1 site that resides just 5' to the NF-Y binding site was shown to be important for mediating GnRH regulation. No Sp1 consensus binding sequence exists in the bovine promoter at this site; however, to determine if Sp1 could also bind to the L1 element, we performed additional EMSA. Antiserum to the transcription factor Sp1 did not affect binding of nuclear factors to the L1 element but did retard the mobility of a complex formed with an oligodeoxynucleotide corresponding to a consensus Sp1 element (Fig. 5A). Thus, we conclude that NF-Y, but not Sp1, binds to this site in the bovine LH promoter.

View larger version (63K): [in this window] [in a new window]   Fig. 5.   NF-Y is the L1- and L4-binding protein. A, EMSA were performed with T3-1 nuclear extracts and either L1-, L4-, or Sp1-labeled probes. Supershift assays were performed with antisera directed against NF-Y or Sp1 to assess the nature of the protein complexes associated with these oligodeoxynucleotide probes. Supershifted complexes containing either NF-Y or Sp1 are indicated with arrows. B, the L1 and L2 core CCAAT elements are aligned with the NF-Y consensus sequence (36). The L1 site harbors an exact consensus sequence for NF-Y. Lowercase letters indicates mismatches from the consensus. The full sequences of the probes used in the EMSA are delineated under "Experimental Procedures."

The L1 Element Exists as an Inverted Repeat in the Bovine LH Promoter-- Subsequent analysis of the bovine LH promoter for additional CCAAT motifs surprisingly indicated that no motif was present in the region surrounding 80 bp relative to the start site of transcription. In contrast, as shown in Fig. 5B, an additional imperfect motif (CCTAT) was observed in the opposing orientation of the LH promoter at positions 332 to 328. This sequence, within the context of a larger oligodeoxynucleotide (designated L4), was analyzed for its ability to bind a nuclear protein in EMSA (Fig. 5A). Interestingly, L4 bound the same protein as L1 as shown by complete cross-competition and antibody supershift experiments. However, L4 bound this protein with significantly lower affinity. While only a 100-fold molar excess of unlabeled L1 is required to compete for binding to the L1 probe, over 500-fold molar excess of unlabeled L4 is required for a similar level of competition (data not shown). It is intriguing that the inverted orientation of the L4 and L1 elements within the LH promoter is identical to the most active multimerized L1 elements in transient transfection (Fig. 2). Inverted repeats of NF-Y sites have been identified in multiple genes, suggesting that these sequences may cooperate to lead to full expression of the LH promoter (35).

The L1 Element Regulates Pituitary-specific Expression of the Bovine LH Promoter-- To determine the functional importance of the L1 element in the context of a full-length promoter in an appropriate physiological context, transgenic mice were made. These mice harbored the 776-bp bovine LH promoter containing a transversion mutation throughout the entire 26-bp region of the L1 element linked to CAT. Pituitary CAT activity in these mice was compared with that observed in mice harboring the wild type promoter linked to CAT. Previous studies have shown that the full-length, wild type promoter is highly active only in gonadotropes of transgenic mice (18). In addition, the promoter responds appropriately to GnRH and gonadal steroids (18). Pituitary CAT activity from six independent lines of mice harboring the mutant LH promoter was compared with activity observed in three independent lines of mice harboring the wild type promoter. Mutation of the L1 element reduced activity of the LH promoter to approximately 14% of the wild type promoter's activity (Fig. 6). While this reduction was substantial, it is important to note that the L1 mutant promoter had detectable activity in a number of mice. In particular, mutant lines 1, 3, and 4 displayed higher activity than the other lines of mice harboring the mutation. This suggests that while L1 is important for high level expression of the LH promoter, some integration sites permit low level expression from this attenuated promoter. Thus, the remaining cis-acting elements must compensate to some degree for the loss of the distal NF-Y binding site.

View larger version (26K): [in this window] [in a new window]   Fig. 6.   The L1 element regulates pituitary-specific expression of the bovine LH promoter. Transgenic mice harboring either the wild type (wt, gray bars) or an L1 mutant (µ, black bars) LH promoter linked to CAT were made. Pituitary CAT activity was measured in three lines of mice with the wild type promoter and six lines of mice harboring the mutant promoter. Values are the mean ± S.E. The following numbers of animals were used: 29 (wt1), 44 (wt2), 11 (wt3), 12 (µ1), 5 (µ2), 8 (µ3), 6 (µ4), 1 (µ5), and 1 (µ6). The µ5 and µ6 animals were founder transgenics. In all animals in which liver activity was examined, it was below detection and similar to the levels of activity observed in µ3, µ5, and µ6 animals. Expression of the wild type transgene versus the mutant transgene was significantly different (p  0.0001, two-tailed Student's t test assuming unequal variances).

L1 Is Unnecessary for GnRH Regulation of the LH Promoter-- Numerous cis-acting elements have been described for the LH promoter, and most, if not all, mediate GnRH stimulation (11, 14, 16, 21, 24, 25). To determine the role of the L1 element in conveying GnRH regulation, female transgenic mice harboring either the wild type promoter or the L1 mutant promoter were subjected to an ovariectomy/antide treatment paradigm. Ovariectomy removes negative feedback from ovarian steroids and causes an increase in GnRH synthesis and secretion from the hypothalamus that further results in increased LH gene expression (1). Treatment with the GnRH-specific antagonist, antide, can block this increase (18). Thus, the degree of repression of transgene activity following antide administration is an indicator of the degree of promoter responsiveness to GnRH. To assess the impact of the L1 mutation, mice were ovariectomized and treated with either vehicle or antide for 10 days. As previously shown (18, 21), antide treatment lead to a 38-63% reduction in wild type promoter activity in two independent lines of mice (Fig. 7). Similarly, antide treatment of ovariectomized mice harboring the L1 mutation resulted in 50% reduction in promoter function. This indicates that while the basal function of the LH promoter is significantly attenuated by mutation of the L1 element, this element is not necessary for mediating GnRH-regulated expression. Whether NF-Y can mediate GnRH regulation through the intact L4 site or additional, uncharacterized, binding sites is unknown and warrants further study.

View larger version (33K): [in this window] [in a new window]   Fig. 7.   L1 is unnecessary for GnRH regulation of the LH promoter. Transgenic mice harboring either the wild type (wt) or mutant (µL1) (776/+10) LH promoter were subjected to a GnRH regulation assay. The mutant line used in this study corresponds to µ1 in Fig. 6. Mice were ovariectomized and treated with either vehicle (OVX, gray bars) or 60 µg antide (OVX + antide, black bars) every 48 h. for 10 days. Pituitaries were collected, and CAT enzymatic activity was measured. Values are expressed relative to the OVX control for each line of mice and represent the mean ± S.E., n = 5 animals per group. In each case, antide treatment resulted in CAT activity that was significantly different from OVX animals (p  0.05, one-tailed Student's t test assuming unequal variances).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Expression of luteinizing hormone in the gonadotrope of the pituitary requires the coordinated expression of the  and LH subunits. Both subunit genes respond to GnRH and sex steroids to promote synthesis and secretion of appropriate levels of the intact heterodimer (1). Several elements have been defined for transcriptional control of both the  and LH subunit genes. However, a complete picture of LH gene regulation has not been resolved. This has largely been due to the lack of appropriate cell culture models to study expression of this gene.

Using transgenic mice, we and others have shown that the LH promoter-proximal region confers high level expression that is confined to gonadotropes and responds appropriately to GnRH and gonadal steroids (17-20). In this report, we describe an inverted repeat element that binds to NF-Y and is important for full activity of the LH promoter in transgenic mice. In contrast to the other elements that have been described thus far for this promoter, the distal NF-Y binding site is not required for mediating GnRH responsiveness. Thus, two different classes of cis-active elements exist for the LH promoter. These include basal and GnRH-responsive sites, both of which contribute to transcriptional tone. Since expression of the LH gene is very low in the absence of GnRH (32), the so-called basal elements may play a role in amplifying a signal that must be initiated by GnRH acting through the promoter-proximal SF-1, Pitx1, and Egr-1 binding sites, as well as potential Sp1 sites.

NF-Y is a ubiquitous CCAAT box-binding factor that is composed of three subunits (36). It regulates both TATA-containing and TATA-less promoters (35). Functional NF-Y sites within promoters are usually closely associated with the start site of transcription, occurring within the promoter-proximal 100 bp (36). However, there are several genes that contain NF-Y binding sites that are over 1 kilobase pairs 5' to the promoter (35). Both the L1 and L4 sites are located more distally than the typical NF-Y site. In addition, this factor displays an almost strict requirement for the pentanucleotide CCAAT motif that can occur on either the coding or noncoding strand (35, 36). While L1 harbors an exact consensus for NF-Y, the L4 site has a CCTAT motif on the opposite strand from L1. Although the L4 site also binds NF-Y, it does so with lower affinity than L1 (data not shown). The L4 motif has been observed in the cdc25 gene (37) as well as the MHC Class II DPA gene (38). Interestingly, these sequences, like L4, all occurred in the reverse orientation on the coding strand. Unlike the LH subunit gene, however, both of these genes are TATA-less.

There are numerous examples of NF-Y cooperativity with other transcription factors (39-41). It is particularly interesting to note that NF-Y can synergize with the transcription factor Sp1 (39, 41). An Sp1 site has been characterized within the rat LH promoter that is immediately adjacent to a conserved NF-Y sequence (11). Whether these two proteins synergize to activate the rat promoter remains to be determined. With regard to LH promoters from other species, the Sp1 sequence is not conserved at this site, although the adjacent NF-Y sequence is. Within the bovine LH promoter, we have shown that an oligodeoxynucleotide that encompasses the NF-Y site fails to bind Sp1. Thus, the transcriptional regulation that occurs through this cis-acting element cannot be attributed to Sp1. It will be important to determine if Sp1 consensus sequences located within other regions of this promoter can bind to Sp1 and potentially synergize with the NF-Y binding sites to activate transcription.

Sp1 and the other factors that define transcriptional activity of the LH promoter have largely been characterized using heterologous cell culture systems. These include the use of kidney fibroblasts (CV-1) (8, 9, 22), somatotropes (GH3 and GGH3-1') (10, 11, 24), and immature gonadotropes (T3-1) (13-15). None of these cell lines express the endogenous LH subunit gene, and they are probably devoid of essential factors for activating its transcription. While these models have allowed ascertainment of the ability of specific transcription factors to induce the LH promoter, most depended upon overexpression of those factors. Thus, the relative importance of these factors in defining the transcriptional tone of the LH promoter within the mature gonadotrope is unknown. The recently derived LT2 cell line, which does express the endogenous LH subunit gene (42), will probably present an ideal avenue to confirm studies previously performed in these alternative models. Prior to development of this cell line, however, the only mature gonadotrope model effectively used for analyses of the LH promoter was the pituitary of transgenic mice. Even with this cell line, the transgenic mouse presents the most physiologically relevant system to assess important factors in regulating expression of the LH gene. In this regard, we have characterized a new site that is important for basal expression of this gene but not GnRH induction. The function of this element contrasts with the previously defined GSE that was shown to be essential for pituitary activity as well as GnRH activation of this promoter in transgenic mice (21). While previous in vivo and in vitro studies have focused specifically on the promoter-proximal 150 bp, the studies presented herein indicate that additional sequences involved in regulation of promoter activity reside upstream of this region. Thus, any attempts to study the proximal promoter in isolation will probably result in a limited picture of the transcription control of this gene.

	ACKNOWLEDGEMENTS

We acknowledge the Pharmacological Sciences Consortium Core Transgenic Facility at Case Western Reserve University for production of transgenic mice. We also thank David Peck for excellent technical assistance and Paul MacDonald for helpful discussions during the preparation of this manuscript.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grants DK28559 (to J. H. N.) and DK09843 (to C. C. Q.) and an Endocrine Society fellowship (to B. D. H.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Pharmacology, Case Western Reserve University School of Medicine, 2109 Adelbert Rd., Cleveland, OH 44106. Tel.: 216-368-4497; Fax: 216-368-3395; E-mail: jhn@po.cwru.edu.

	ABBREVIATIONS

The abbreviations used are: LH, luteinizing hormone; SF-1, steroidogenic factor-1: GnRH, gonadotropin-releasing hormone; CAT, chloramphenicol acetyltransferase; HSVtk, herpes simplex virus thymidine kinase; EMSA, electrophoretic mobility shift assay; GSE, gonadotrope-specific element; bp, base pair(s).

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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