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Endocrinology Vol. 145, No. 11 4866-4879
Copyright © 2004 by The Endocrine Society

Regulation of the Follicle-Stimulating Hormone ß Gene by the LHX3 LIM-Homeodomain Transcription Factor

Brooke E. West, Gretchen E. Parker, Jesse J. Savage, Parinda Kiratipranon, Katherine S. Toomey, Lisa R. Beach, Stephanie C. Colvin, Kyle W. Sloop and Simon J. Rhodes

Department of Biology, Indiana University-Purdue University (B.E.W., G.E.P., J.J.S., P.K., K.S.T., L.R.B., S.C.C., S.J.R.); and Endocrine Discovery, Lilly Research Laboratories, Eli Lilly & Co. (K.W.S.), Indianapolis, Indiana 46202

Address all correspondence and requests for reprints to: Dr. Simon J. Rhodes, Department of Biology, Indiana University-Purdue University, 723 West Michigan Street, Indianapolis, Indiana 46202-5132. E-mail: srhodes{at}iupui.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
FSH is a critical hormone regulator of gonadal function that is secreted from the pituitary gonadotrope cell. Human patients and animal models with mutations in the LHX3 LIM-homeodomain transcription factor gene exhibit complex endocrine diseases, including reproductive disorders with loss of FSH. We demonstrate that in both heterologous and pituitary gonadotrope cells, specific LHX3 isoforms activate the FSH ß-subunit promoter, but not the proximal LHß promoter. The related LHX4 mammalian transcription factor can also induce FSHß promoter transcription, but the homologous Drosophila protein LIM3 cannot. The actions of LHX3 are specifically blocked by a dominant negative LHX3 protein containing a Krüppel-associated box domain. Six LHX3-binding sites were characterized within the FSHß promoter, including three within a proximal region that also mediates gene regulation by other transcription factors and activin. Mutations of the proximal binding sites demonstrate their importance for LHX3 induction of the FSHß promoter and basal promoter activity in gonadotrope cells. Using quantitative methods, we show that the responses of the FSHß promoter to activin do not require induction of the LHX3 gene. By comparative genomics using the human FSHß promoter, we demonstrate structural and functional conservation of promoter induction by LHX3. We conclude that the LHX3 LIM homeodomain transcription factor is involved in activation of the FSH ß-subunit gene in the pituitary gonadotrope cell.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE GONADOTROPIN HORMONES, FSH and LH, play important roles in mammalian reproductive development and function. FSH and LH are heterodimeric glycoproteins composed of a common subunit, {alpha}-glycoprotein ({alpha}GSU), and ß-subunits (FSHß or LHß) that confer biological specificity. In females, FSH stimulates the growth of ovarian follicles and, in the presence of LH, promotes estrogen secretion by the ovary. In males, FSH is important for spermatogenesis. LH induces ovulation and the formation of the corpus luteum in females; in males, it promotes testosterone synthesis by Leydig cells. FSH and LH are secreted in an episodic fashion from anterior pituitary gonadotrope cells in response to stimuli such as activins from the gonads and GnRH from the hypothalamus. The frequency and intensity of hypothalamic GnRH pulses can mediate differential regulation of FSH and LH production by the gonadotrope (reviewed in Refs. 1, 2, 3, 4).

The mechanisms controlling transcription of the LH and FSH ß-subunit genes are critical regulatory points in the production of the LH and FSH hormones. These mechanisms are complex, involving the actions of both tissue-specific and widely expressed transcription factors (reviewed in Refs. 1 and 3, 4, 5). It appears that the genes respond to external stimuli such as hypothalamic GnRH by different mechanisms. LHß transcription involves factors including p8, steroidogenic factor-1 (SF1), paired-like homeodomain transcription factor 1 (PITX1), specificity protein 1 (SP1), early growth response 1 (EGR1), nuclear factor Y (NFY), and orthodenticle-like (OTX) class homeodomain factors (6, 7, 8, 9, 10, 11, 12). GnRH may promote LHß transcription via SP1-, SF1-, and EGR1-dependent pathways (13, 14, 15). FSHß gene regulation appears to be mediated by some of the transcription factors that are required for LHß promoter activity, including SF1, NFY, and PITX class factors (6, 16, 17, 18). The activating protein 1 transcription factor also has been implicated in basal and GnRH-mediated FSHß transcription (reviewed in Ref. 5).

Studies of the regulation of the gonadotropin subunit genes have been enhanced by the generation of the LßT2 model of the mouse gonadotrope cell. This cell line was derived from a pituitary cell tumor expressing a rat LHß promoter-simian virus-40 T antigen transgene (19). LßT2 cells express the GnRH receptor, LHß, {alpha}GSU, steroid hormone receptors, activins, activin receptors, SF1, and produce FSH in response to activins and GnRH (16, 19, 20, 21, 22).

Activin, a signaling protein secreted by the gonads and the pituitary (23, 24), also has been implicated in the stimulation of the FSHß promoter (16, 21, 25, 26, 27, 28, 29). Recently, the transcription factors Sma/Mothers against decapentaplegic homolog (SMAD)3 and SMAD4 have been demonstrated to mediate activin stimulation of the rat FSHß promoter in LßT2 gonadotropes, an effect potentiated by the PITX2c homeodomain transcription factor (16). Although GnRH and activin stimulate FSHß expression, estrogen represses FSHß gene transcription through indirect mechanisms involving the proximal promoter (30).

LHX3 (also known as LIM3 or P-Lim) and LHX4 (LIM4/GSH4) are related LIM homeodomain (LIM-HD) transcription factors with demonstrated roles in vertebrate pituitary gland development (reviewed in Ref. 5). In rodents, the Lhx3 gene is expressed in the embryonic nervous system and the developing and mature pituitary gland (31, 32). LHX3 has been implicated in the transcriptional regulation of the promoters of pituitary genes, including those encoding {alpha}GSU, prolactin (PRL), TSHß, and the PIT1 transcription factor (31, 33, 34). For example, LHX3 (and other LIM-HD proteins, such as LHX2 and LHX4) can induce transcription from the {alpha}GSU gene by specifically binding to the pituitary glycoprotein basal element (PGBE) within the proximal region of the promoter (31, 33, 34, 35, 36), an element that is required to appropriately restrict expression of the gene to gonadotropes and thyrotropes in transgenic mice (37). Mammalian LHX3 genes produce two major mRNA transcripts that encode at least three protein isoforms: LHX3a, LHX3b, and M2-LHX3 (32, 33, 38). These LHX3 proteins possess different capacities to activate pituitary hormone gene promoters (33, 34).

In mice with targeted disruption of Lhx3, development of the gland is arrested after the formation of Rathke’s pouch (39). These animals die at or shortly after birth and lack gonadotropes, thyrotropes, somatotropes, lactotropes, and their hormone products. LHX3 mutations have been identified in human patients with combined pituitary hormone deficiency (40). These mutations reduce the ability of LHX3 to activate specific target genes in the pituitary (41, 42). Similar to the animal models, these patients display FSH, LH, GH, TSH, and PRL deficiencies (40), confirming the requirement for LHX3 in the development of four of the anterior pituitary cell types. During early pituitary development, LHX4 may act with other pituitary transcription factors, such as LHX3 and Prophet-of-Pit-1 (PROP1), to promote the initial formation and later expansion of Rathke’s pouch (43, 44). Unlike Lhx3 knockout mice, low numbers of all five of the differentiated cell types of the anterior pituitary are observed in mice lacking functional LHX4 (43).

The importance of LHX3 in the development and function of the gonadotrope cell and the involvement of LIM-HD transcription factors in {alpha}GSU transcription prompted us to test the hypothesis that LHX3 and LHX4 are also regulators of the FSH and LH ß-subunit genes. We show that LHX3 activates transcription from the FSHß promoter, but not the proximal LHß promoter. In the FSHß promoter, LHX3 can bind to six conserved binding sites, including three that lie within an important proximal region that confers response to other pituitary transcription factors and to activins.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Plasmid construction and mutagenesis
The –5663 to +7 bp region of the FSHß gene and the –1158 to +8 bp region of the LHß gene were amplified from porcine genomic DNA by PCR using the following primers: 5'-gcgacgcgtgaattcaggaaagaggtcttctgttc-3', 5'-ccgctcgagaagctgtgggctgaatctagtctc-3' (pFSHß); and 5'-cgggatccagctgtgccctacccatcctgcc-3', 5'-cccaagcttccttggtgtctccaagccgtgtttac-3' (pLHß), based on reported sequences (45, 46). The –5663 to +7 bp region of the human FSHß (hFSHß); gene was amplified from human genomic DNA by PCR using primers 5'-cgcacgcgttgaggtggtgggaggtgctttaagcca-3' and 5'-cgcctcgaggagctgtagactgaatgaaatctcagtt-3', based on published sequences (47). These fragments were ligated into the pGL3 basic luciferase reporter vector (Promega Corp., Madison, WI). Nested deletions of the hFSHß or pFSHß promoters were constructed in the pGL3 basic vector by restriction enzyme digestion or using the PCR. Mutations were introduced into the –5663 bp pFSHß luciferase or the –398 bp pFSHß luciferase plasmids using the QuikChange site-directed mutagenesis protocol (Stratagene, La Jolla, CA). Mutagenic oligonucleotide sequences are available upon request. Mutagenesis reactions were repeated for sites A, B, and C to obtain plasmids containing double and triple mutations in these regions. The {alpha}GSU and PRL reporter genes and the LHX3a, LHX3b, M2-LHX3, LHX4, and PROP1 expression vectors have been previously described (33). The Krüppel-associated box (KRAB)-LHX3 expression vector was constructed by fusing cDNAs encoding the first 75 amino acids of human KOX1 (gift from Dr. Joseph Bidwell, Indiana University School of Medicine) and the first 230 amino acids of human LHX3a, then ligating the resulting DNA fragment into the pcDNA3.1 expression vector (Invitrogen Life Technologies, Carlsbad, CA). The mouse nescient helix loop helix factor 2 (NHLH2) cDNA was cloned from mouse pituitary cDNA into a pcDNA3 expression vector. The Drosophila LIM3 cDNA (a gift from Dr. Stephan Thor, Harvard Medical School, Boston, MA) was also cloned into the pcDNA3.1 expression vector. The PITX2c expression vector was a gift from Dr. Sally Camper (University of Michigan, Ann Arbor, MI), EGR1 was a gift from Dr. Eileen Adamson (Burnham Institute, La Jolla, CA), and SF1 was a gift from Dr. Holly Ingraham (University of California, San Francisco, CA). Plasmid integrity was confirmed by DNA sequencing (Indiana University School of Medicine).

Cell culture, transfection, and luciferase assays
Human embryonic kidney (HEK) 293T cells were cultured as previously described (33, 48). Mouse LßT2 pituitary gonadotrope cells (gift from Dr. Pamela Mellon, University of California, San Diego, CA) were cultured in DMEM (Cellgro, Mediatech, Inc., Herndon, VA). Typically, 1.5 x 105 cells 293T cells in a 35-mm well were transfected with 1–2 µg reporter gene plasmids and 0.5–1 µg expression vectors using the CalPhos system (BD Clontech, Palo Alto, CA). LßT2 cells (2.5 x 105/35-mm well) were transfected using FuGENE 6 (Roche, Indianapolis, IN) or Lipofectamine 2000 (Invitrogen Life Technologies) using the same amounts of plasmid DNA (or molar equivalents when appropriate). Luciferase activity in cell extracts was measured 48–72 h after transfection as previously described (49). Protein assays were performed by the Bradford method (Bio-Rad Laboratories, Hercules, CA), and luciferase activity was normalized to protein concentration. LßT2 cells were treated with recombinant human activin A (gift from Dr. Teri Belecky-Adams, Indiana University-Purdue University Indianapolis, or purchased from R&D Systems, Minneapolis, MN) at a concentration of 100 ng/ml in dimethylsulfoxide vehicle. Control cultures received equal volumes of vehicle alone.

RT-PCR and quantitative PCR analysis of gene expression
RNA extraction, cDNA synthesis, and PCR conditions to detect the murine LHX3 mRNA isoforms have been previously described (33). PCR products were analyzed on 11% acrylamide Tris-borate gels and detected by ethidium bromide staining. For quantitative PCR, RT reactions were performed in triplicate as described above using random hexamers and total RNA. Three RT reactions for each RNA sample were performed. Parallel negative control reactions lacking RT also were performed. Real-time quantitative PCR was performed using the 5' fluorogenic nuclease assay and an ABI 7900 PRISM (Applied Biosystems, Foster City, CA) to determine the relative abundance of the genes of interest. The 5' terminus of fluorogenic probes was labeled with 6-carboxy-fluorescein, and the 3' terminus contained the quenching dye 6-carboxytetramethylrhodamine. FSHß, {alpha}GSU, LHX3a, LHX3, and LHX4 primers and probes were synthesized by Biosearch Technologies, Inc. (Novato, CA). Primer and probe sequences were as follows: FSHß forward primer, 5'-cagaccatgatgaagttgatcca-3'; FSHß reverse primer, 5'-cagctatggcagcagattgc-3'; FSHß TaqMan probe, 5'-tgcatcttattctggtgctgga-3'; {alpha}GSU forward primer, 5'-ctgttgcttctccagggcata-3'; {alpha}GSU reverse primer, 5'-ttctttggaaccagcattgtctt-3'; {alpha}GSU TaqMan probe, 5'-cccactcccgccaggtccaa-3'; LHX3a forward primer, 5'-ggacaccccgcacgaa-3'; LHX3a reverse primer, 5'-tggcaatcgagttctgcttct-3'; LHX3a TaqMan probe, 5'-actggattagtgactgccatgctg-3'; LHX3 forward primer, 5'-aatgtcctggcagaaaacaaca-3'; LHX3 reverse primer, 5'-gtctcctaccaagccccatc-3'; LHX3 TaqMan probe, 5'-taacgccttgcttcatcgctt-3'; LHX4 forward primer, 5'-accaccatcacagcaaagca-3'; LHX4 reverse primer, 5'-catgccgggcaggtttt-3'; LHX4 TaqMan probe, 5'-tggagacactaaagaacgcatacaagaattccc-3'; 36B4 forward primer, 5'-ggcccgagaagacctcctt-3'; 36B4 reverse primer, 5'-tcaatggtgcctctggagatt-3'; and 36B4 TaqMan probe, 5'-ccaggctttgggcatcaccacg-3'. PCRs were run in triplicate 20-µl reactions that contained Universal Master Mix (Applied Biosystems), 4 pmol of each forward and reverse primer, 3 pmol probe, and 4 µl diluted cDNA from the RT reactions. Two-step PCR cycling was carried out as follows: 50 C 2 min for one cycle, 95 C 10 min for one cycle, and 95 C at 15 sec and 60 C at 1 min for 40 cycles. Data were normalized by determining the relative abundance of 36B4 mRNA.

EMSAs
EMSAs were performed as previously described (33). Results were visualized by autoradiography or using a Storm phosphorimager (Amersham Biosciences, Arlington Heights, IL; Molecular Dynamics, Sunnyvale, CA). Recombinant human LHX3a and M2-LHX3 were produced as glutathione-S-transferase fusions as previously described (33). Whole cell extracts of 293T cells transfected with LHX3a were prepared as previously described (49). Nuclear extracts from rat GH3 somatomammotrope cells were purchased from Active Motif, Inc. (Carlsbad, CA). Nuclear extracts of LßT2 cells were prepared by a modified Dignam method as previously described (50). EMSAs were performed using radiolabeled DNA probes representing the sequences listed in Table 1Go. The {alpha}GSU PGBE and LHX3 consensus site (LBC) probes have been described previously (36). The anti-LIM3/LHX3 antibody was purchased from Chemicon International (Temecula, CA).


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  		TABLE 1. Oligonucleotides used in EMSA experiments

 
Chromatin immunoprecipitation (ChIP)
ChIP was performed using a modification of the Upstate ChIP Assay Kit procedure (Upstate Cell Signaling Solutions, Charlottesville, VA) and that of Petz et al. (51). In brief, 1 million LßT2 cells were plated per 35 mm well in six-well dishes. Some groups were transfected with 5 µg Myc epitope-tagged hLHX3a for 24 h and/or treated with 100 ng/ml human activin A for 18 h as described above. Each group was set up in triplicate: one served as a control to normalize cell numbers, one served as a control to assay DNA fragmentation, and one served as the experimental group. After incubation, cells were washed twice with serum-free culture medium and then cross-linked in 1% formaldehyde in culture medium for 10 min at 37 C. Medium was then aspirated, and cells were washed with 1x PBS containing 1 mM phenylmethylsulfonylfluoride, 1 µg/ml aprotinin, 1 µg/ml pepstatin A followed by a wash with 1 x PBS. Cells were collected by scraping in 1 x PBS. Cells were pelleted by centrifugation then resuspended in 200 µl lysis SDS buffer (Upstate) supplemented with 1 mM phenylmethylsulfonylfluoride, 1 µg/ml aprotinin, and 1 µg/ml pepstatin A. After 10 min on ice, 800 µl ChIP dilution buffer (Upstate Cell Signaling Solutions) was added, and the mixture was sonicated on ice using three 10-sec bursts of a Branson sonifier cell disruptor microtip. For fragmentation controls, after cross-link reversal using 8 µl 5 M NaCl overnight at 65 C, DNA fragment length was checked by agarose gel electrophoresis. For experimental groups, soluble sonicated material was incubated with either specific antibody (or preimmune serum as a control) at a 1:100 dilution at 4 C overnight. Specific antibodies were rabbit polyclonal anti-LIM3/LHX3 (Chemicon International) or mouse anti-Myc monoclonal 4A6 (Upstate Cell Signaling Solutions). Samples then were incubated with 50 µl salmon sperm DNA/protein A agarose slurry (Upstate Cell Signaling Solutions) for 1 h at 4 C. The protein A beads were washed with ChIP dilution buffer, then with 1 ml low salt immune complex wash buffer, then with high salt immune complex wash buffer, then with LiCl immune complex wash buffer, and then with 1x TE [10 mM Tris-HCl and 1 mM EDTA (pH 8.0)]. Beads were then incubated in 500 µl elution buffer (1% sodium dodecyl sulfate and 0.1 M NaHCO3) for 30 min at room temperature. The supernatant then was reverse cross-linked by adding 70 µl 5 M NaCl, 10 µl 0.5 M EDTA, 20 µl 1 M Tris-Cl (pH 6.5), and 1 µl proteinase K (10 mg/ml) at 65 C for 1 h. DNA was extracted by phenol/chloroform extraction and ethanol precipitation. PCR was performed with 200 ng DNA with 5'-tgatagccctagaaatgagcaa-3' and 5'-agtttgaggctgggatttga-3' to amplify an approximately 370-bp region of the endogenous mouse FSHß gene encompassing the –509 bp/–139 bp region of the promoter. Touchdown cycling conditions began with annealing cycles at 63 to 59 C, followed by 35 cycles at 59 C.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
LHX3a activates the FSHß promoter, but not the proximal LHß promoter
To generate reporter genes with which to investigate transcriptional regulation of the FSHß and LHß genes, we amplified DNA fragments encompassing the –1158 to +8 bp region of the LHß gene and the –5663 to +7 bp region of the FSHß gene from porcine genomic DNA using the PCR. Similar or shorter regions of FSHß and LHß promoters have been demonstrated to confer pituitary gonadotrope-specific expression to reporter genes in transgenic mice (52, 53). We first cotransfected the LHß luciferase reporter gene with LHX3 isoform expression vectors into heterologous HEK 293T cells. Neither LHX3a nor LHX3b factors activated the LHß promoter (Fig. 1AGo). Consistent with previous studies (14, 15), EGR1 activated the LHß reporter gene in parallel positive controls (Fig. 1Go, A and C). Similar experiments were then performed using the mouse LßT2 model gonadotrope cell line (19, 20). This cell line expresses low levels of FSH (Ref. 21 and this study). We first tested whether the LßT2 cells produce the major transcripts from the murine Lhx3 gene. Low levels of the Lhx3a and Lhx3b RNAs were detected (Fig. 1BGo; see below). As observed in the heterologous cell experiments, LHX3 isoforms and the related LHX4 factor failed to activate the LHß promoter in LßT2 gonadotrope cells (Fig. 1CGo). We conclude that the –1158 bp region of the porcine LHß promoter is not regulated by LHX3 and LHX4 transcription factors.



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  		FIG. 1. LHX3 does not activate the proximal LHß promoter. HEK 293T cells (A) or LßT2 pituitary gonadotrope cells (C) were transiently transfected with a luciferase reporter gene containing –1158 bp of the pLHß promoter and the indicated cDNA expression vectors with EGR1 serving as a positive control. Negative controls (Control) received equivalent amounts of empty vector. Promoter activity was assayed by measuring luciferase activity 48 h after transfection. Activities are mean light units per 10 sec per microgram of total protein of triplicate assays ± SEM. A representative experiment of at least three experiments is depicted. B, RT-PCR was used to test for expression of the endogenous LHX3a (a, open arrow) and LHX3b (b, closed arrow) mRNAs in LßT2 cells. Amplified products were separated by PAGE and were detected by ethidium bromide staining. Parallel negative control reactions were performed omitting reverse transcriptase (no RT).

 
In contrast to the data obtained with the LHß promoter, we observed that LHX3a activates the –5663 bp FSHß promoter (Fig. 2AGo). In the same experiments, LHX3b caused only a moderate increase in FSHß promoter activity, and EGR1 inhibited transcription (Fig. 2AGo). To assay the requirements for distal regions of the FSHß promoter for the LHX3 response, we constructed a deletion series of reporter genes containing different lengths of the upstream region. The –4371, –1378, and –398 bp promoters had the highest basal activities in LßT2 gonadotrope cells, indicating that repressive elements probably lie between –5663 and –4371 bp, and also between –1378 and –398 bp (data not shown). Each of the FSHß reporter genes was activated by LHX3a in heterologous cells, with the –1378, –772, and –398 bp promoters displaying the most sensitivity to LHX3a induction (Fig. 2BGo). Similar results were obtained in experiments using LßT2 gonadotrope cells in which LHX3a, M2-LHX3, and LHX4 all activated FSHß reporter genes (Fig. 2CGo).



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  		FIG. 2. Specific isoforms of LHX3 and LHX4 activate the FSHß promoter. A, A luciferase reporter gene containing –5663 bp of the pFSHß promoter and the indicated cDNA expression vectors were transiently transfected into 293T cells. Controls received equivalent amounts of empty vector. Activities were measured as described in Fig. 1Go. B, A deletion series of the FSHß promoter was created containing the indicated fragments of the promoter. The responses of these reporter genes to LHX3a were determined as described in A. C, The responses of FSHß promoter reporter genes containing the indicated promoter fragments to LHX3a, M2-LHX3, and LHX4 in LßT2 gonadotrope cells were determined.

 
Regulation of the FSHß promoter by mammalian pituitary transcription factors
To compare the properties of LHX3 with those of other pituitary transcription factors, the –1378 bp FSHß reporter gene was cotransfected with expression vectors for various pituitary gene regulatory proteins. LHX3a, LHX4, PITX2c, and SF1 all activated the FSHß promoter (Fig. 3AGo). By contrast, PROP1, EGR1, nescient helix loop helix factor 2 (NHLH2), and combinations of transcription factors did not increase transcription (Fig. 3AGo and data not shown). LHX3b caused only a slight increase in activity (Fig. 3AGo). Similar results were obtained in experiments using the –5663 bp FSHß promoter reporter gene (data not shown). We also tested whether Drosophila LIM3 (DmLIM3) protein, an invertebrate homolog of LHX3 with roles in nervous system development, could activate the FSHß and other pituitary hormone gene promoters. In these experiments, mammalian LHX3a activated the {alpha}GSU promoter and the PRL promoter in synergy with PIT1, but DmLIM3 did not (data not shown). Similarly, LHX3a activated –5663 bp FSHß and –1378 bp FSHß, but DmLIM3 only slightly induced transcription from the FSHß promoter reporter genes (Fig. 3Go, B and C). We conclude that the FSHß promoter is activated by PITX2c, SF1, and specific isoforms of the LHX3/LHX4 subclass of LIM-HD proteins.



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  		FIG. 3. Activation of the FSHß promoter by the LHX3a, LHX4, SF1, and PITX2c pituitary transcription factors. A, Comparison of the ability of pituitary transcription factors to activate a reporter gene containing –1378 bp of the FSHß promoter. The reporter gene was cotransfected into 293T cells with the indicated expression vectors. Activities were measured as described in Fig. 1Go. The dashed line indicates the activity level of the control (empty vector). B and C, Comparison of the capacity of LHX3/LIM3 family members to activate the –5663 and –1378 bp FSHß reporter genes. Hs, Homo sapiens; Dm, Drosophila melanogaster.

 
Actions of LHX3 are blocked by a dominant negative protein derivative
To test whether an inhibitor of LHX3 actions could block the LHX3a-mediated induction of the FSHß promoter, a dominant negative derivative of the LHX3 protein containing the KRAB domain of KOX1 was constructed. The KRAB domain is a potent repressor of transcription that can physically interact with other factors, such as the KRAB-associated protein-1 and heterochromatin protein-1 (54, 55, 56, 57). The KRAB domain retains its repressive activity when fused to DNA binding domains, and this, and similar, dominant negative tools have been successfully used to probe the functions of other regulatory proteins (55, 56, 57, 58, 59, 60). In control experiments using established LHX3 target genes, a fusion protein containing a KRAB domain plus the amino-terminal LIM domains and HD of LHX3a (KRAB-LHX3) blocked both LHX3a induction of the {alpha}GSU promoter and synergistic activation of the PRL promoter by LHX3a and PIT1 (Fig. 4AGo and data not shown). A dominant negative LHX3 protein with KRAB domains fused at both the amino and carboxyl termini had similar properties (data not shown). In experiments using the –1378 bp FSHß promoter reporter gene, KRAB-LHX3 was similarly effective in blocking activation by LHX3a (Fig. 4BGo) and lowered FSHß promoter activity in LßT2 pituitary cells (Fig. 4CGo). By contrast, in parallel controls, a KRAB fusion protein containing the NMP4/CIZ zinc finger DNA binding domain (61) did not affect FSHß promoter transcription (Fig. 4CGo).



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  		FIG. 4. Inhibition of LHX3 induction of the FSHß promoter by a dominant negative LHX3 protein containing a KRAB domain. A, Activity of an {alpha}GSU luciferase reporter gene in the presence of the indicated LHX3 proteins in transfected 293T cells. LHX3a activation of transcription is blocked in the presence of KRAB-LHX3a. B, –1378 bp FSHß luciferase was cotransfected into 293T cells with the indicated expression vectors. C, Expression of KRAB-LHX3a in pituitary LßT2 cells reduces the activity of the FSHß promoter, but a nonspecific KRAB derivative containing a different DNA binding domain (KRAB-NMP4) does not.

 
LHX3 response elements in the FSHß promoter
Within pituitary hormone gene promoters, LHX3 binds to AT-rich DNA elements such as the PGBE element of the {alpha}GSU promoter (31, 33, 36, 49, 62, 63). In vitro, LHX3 selects a high affinity consensus DNA-binding site (the LHX3-binding consensus or LBC) containing ATTA core sequences (36). The –5663 bp pFSHß promoter contains multiple sequences with homology to the PGBE and LBC sites (Fig. 5AGo). Within the distal regions of the promoter, three DNA sequences were identified (sites D, E, and F; Table 1Go) that interacted with recombinant LHX3 proteins in EMSAs (Fig. 5BGo). Labeled PGBE and LBC sequences were used as positive controls (Fig. 5BGo). LHX3 proteins did not bind to mutated versions of the FSHß D, E, and F sites. Three additional LHX3-binding sites (FSHß sites A, B, and C; Fig. 5AGo) were identified within a proximal region of the promoter that has been demonstrated to contain response elements allowing regulation of FSHß promoters in multiple species by activins, PITX class factors, activating protein 1, and other factors (16, 18, 27, 64). In EMSA experiments, recombinant LHX3a and M2-LHX3 proteins bound to the A, B, and C sites, but not to mutant derivatives of the sequences (Fig. 5CGo). As expected, M2-LHX3 (which lacks the LIM domains that reduce DNA binding) (33) bound with higher affinity than LHX3a (Fig. 5CGo). Similarly, LHX3 proteins from LHX3-transfected 293T cells, but not mock-transfected cells, interacted with the FSHß B site and the LBC (Fig. 6AGo). Endogenous LHX3 proteins in nuclear extracts of GH3 pituitary mammosomatotrope and LßT2 pituitary gonadotrope cells bound to the FSHß B site (Fig. 6Go, A and B). LHX3/DNA complexes were disrupted by the addition of anti-LHX3 antibodies to the binding reactions, but not by preimmune serum (Fig. 6Go, A and B). In the case of the transfected cells, which express higher levels of LHX3 than GH3 or LßT2 cells, a supershifted complex could also be seen (Fig. 6AGo). To test LHX3 association with the endogenous mouse FSHß promoter of LßT2 pituitary gonadotrope cells, ChIP assays were performed. Anti-LHX3 antibodies precipitated chromatin containing the proximal region of the mouse FSHß promoter (that contains binding sites A, B, and C) from native LßT2 cells in the presence or absence of activin A (Fig. 6CGo). Similarly, anti-Myc antibodies precipitated DNA fragments containing the FSHß promoter from LßT2 cells transfected with LHX3-Myc (Fig. 6CGo). Association of LHX3 with the FSHß promoter was less robust in the absence of activin; this result may reflect a reduced ability of the ChIP assay using these antibodies to detect low levels of LHX3 associated with the less active FSHß promoter (see below) in untreated cells.



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  		FIG. 5. The FSHß promoter contains multiple LHX3 binding sites. A, Schematic diagram showing the location of six LHX3-binding sites in the pFSHß promoter. B, EMSA demonstrating interaction of recombinant M2-LHX3 protein with radiolabeled oligonucleotide probes representing the indicated FSHß regions [or mutated sequences (mut) as controls]. The LHX3 DNA binding consensus site (LBC) (36 ) and the {alpha}GSU promoter PGBE element were also included as positive controls. The DNA/protein complexes are shown. C, EMSA experiment testing the binding of recombinant LHX3a and M2-LHX3 protein isoforms to the indicated wild-type or mutated FSHß sequences. Reactions containing the LBC served as positive controls. F, Free probe; open arrow, LHX3a/DNA complexes; closed arrow, M2-LHX3/DNA complexes.

 


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  		FIG. 6. Interaction of pituitary cell LHX3 proteins with FSHß promoter elements. A, EMSA experiments testing binding of nuclear extract proteins from pituitary GH3 cells or from heterologous 293T cells transfected with an LHX3a expression vector or an empty vector (control). Reactions contained either radiolabeled pFSHß site B or the LBC. The presence of LHX3-containing complexes (white arrowhead) was confirmed by the addition of anti-LHX3 antibodies. Supershifted complexes are indicated (black arrowhead). Control reactions contained equivalent amounts of preimmune serum. B, EMSA experiments using nuclear extracts proteins from pituitary LßT2 gonadotrope cells. The probes were FSHß site B or the LBC site. LHX3-containing complexes (white arrowhead) were disrupted by the addition of anti-LHX3 antibodies. F, Free probe. C, ChIP assay of the mouse FSHß gene in LßT2 gonadotrope cells. Anti-LHX3 antibodies were used to precipitate chromatin in native LßT2 cells or anti-Myc antibodies were used to precipitate chromatin in LßT2 cells transfected with LHX3-myc. LßT2 cells were grown in the presence (upper panel) or absence (lower panel) of 100 ng/ml activin A. Preimmune serum was used in parallel controls. Precipitated DNA fragments were used in PCRs using primers representing the –509/–139 bp region of the mouse FSHß gene, and the products were separated by agarose gel electrophoresis. Mouse genomic DNA (Gen) served as a positive control for the PCR, and reactions lacking substrate (Neg) were used as negative controls.

 
The identified A, B, and C LHX3-binding sites lie within the proximal –398 bp of the FSHß promoter sequence that displays significant LHX3 response (Fig. 2Go, B and C). Others have observed that equivalent regions of rodent FSHß promoters are required for induction of the promoters by activin-stimulated SMAD pathways and other regulatory factors (16, 64). To test the roles of these LHX3-binding sites in FSHß promoter activity, site-directed mutations were introduced into the –398 bp reporter gene that altered the sites, both individually and in combination. Whereas the –398 bp wild-type promoter was induced by the expression of LHX3a or LHX4 in transfection experiments, the response to LHX3a was reduced after mutation of site A and was effectively abolished in double and triple mutants (Fig. 7AGo). Similarly, the mutations reduced the basal activity of the FSHß promoter by 39–86% in LßT2 gonadotrope cells (Fig. 7BGo).



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  		FIG. 7. Mutational analysis of FSHß LHX3-binding sites. A, Luciferase reporter genes containing the wild-type (WT) or mutated –398 bp pFSHß promoter were transfected into 293T cells, and their responses to cotransfection with LHX3a or LHX4 expression vectors were measured. Activities were measured as described in Fig. 1Go. B, Basal activities of the indicated –398 bp FSHß promoter reporter genes were measured after transfection into mouse LßT2 pituitary gonadotrope cells.

 
FSHß promoter responses to activin and LHX3
Activins are potent promoters of FSH secretion by pituitary gonadotropes (26, 29, 65, 66). To test whether the FSHß promoter reporter genes used in this study were responsive to activin, we introduced the –5663 and –1378 bp FSHß reporter genes into LßT2 pituitary gonadotrope cells and measured promoter activity in the presence of activin A. Activin A treatment resulted in a 20- to 32-fold increase in promoter activity compared with control cultures that received vehicle alone (Fig. 8AGo).



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  		FIG. 8. Regulation of the FSHß promoter by activin and LIM-HD transcription factors. A, The –5663 and –1378 bp pFSHß promoter reporter genes were transfected into LßT2 gonadotrope cells and parallel groups were treated with 100 ng/ml activin A or a vehicle control. Luciferase activity was assayed 48 h after transfection. Activities are mean light units per 10 sec per microgram of total protein of triplicate assays ± SEM. A representative experiment of at least four experiments is depicted. B, Activin promotes transcription from the endogenous FSHß gene of mouse LßT2 gonadotropes, but not from the {alpha}GSU, LHX3, or LHX4 genes. Using real-time PCR, quantitative fluorescent assays for the indicated mouse pituitary genes were developed. RNA was extracted from LßT2 cells that had been treated with activin A or a vehicle control as described above. cDNAs were generated using 3' gene-specific primers, and PCR was performed using gene-specific primers. Reactions were monitored in real time using internal fluorescent gene-specific TaqMan probes. Measured RNA levels were normalized to measurements of the acidic ribosomal phosphoprotein P0 (ARBP/36B4) mRNA level performed in parallel control reactions, and vehicle-treated values were set at 100. Results are the means of three assays ± SEM. Negative control reactions were performed lacking reverse transcriptase enzyme. C, –398 bp pFSHß promoter reporter genes with the indicated mutations in LHX3-binding sites were transfected into LßT2 gonadotrope cells, and their response to activin A treatment in comparison with that of the wild-type (WT) reporter gene was determined as described above.

 
We hypothesized that activin signaling pathways in the gonadotrope may act to promote FSHß transcription both directly by promoting SMAD transcription factor actions at the promoter and indirectly by increasing transcription of other regulators of FSHß transcription, such as LHX3 and LHX4. We therefore performed quantitative real-time measurements of the endogenous mouse FSHß, {alpha}GSU, LHX3a, LHX3 (i.e. both LHX3a and LHX3b mRNAs), and LHX4 mRNAs in LßT2 gonadotrope model cells after activin treatment. All of the examined genes were expressed at low levels in untreated cells (Fig. 8BGo). Activin A caused a 25-fold increase in endogenous FSHß mRNA levels (Fig. 8BGo), a response similar to that displayed by the transfected FSHß promoter reporter genes (Fig. 8AGo). By contrast, {alpha}GSU, LHX3, LHX3a, and LHX4 mRNA levels were not significantly altered by activin treatment (Fig. 8BGo). In transfection experiments using LßT2 cells, LHX3 and activin both increased the activity of FSHß promoter reporter genes, but did not act synergistically when used together (data not shown), either reflecting independent actions or perhaps that activin induction represents a near-maximal level of activity for the FSHß promoter. FSHß promoter genes with mutation of the proximal LHX3-binding sites retained activin inducibility in LßT2 gonadotrope cells (Fig. 8CGo). We conclude that activin regulation of the FSHß promoter does not require induction of the LHX3 gene and that the LHX3-binding sites are not required for the activin response.

Structural and functional conservation of FSHß promoter response to LHX3
Sequence analyses revealed that the three proximal LHX3-binding sites (sites A, B, and C) are well conserved in position and sequence in the porcine, rat, ovine, and human FSHß promoters (Fig. 9AGo). Other analyses revealed that the distal regions of these promoters all contained AT-rich sequences with strong homology to LHX3 binding sites D, E, and F (data not shown). In the human promoter, sites A and B are identical to those identified in the porcine promoter, and the core of site C is conserved (Fig. 9AGo). To test whether the human FSHß promoter is also inducible by LHX3, we generated luciferase reporter genes containing either –5663 or –1622 bp of the human promoter. Both reporter genes were induced by LHX3 in heterologous cells (Fig. 9BGo) and LßT2 gonadotrope cells (Fig. 9CGo). We conclude that the response to LHX3 induction is conserved in the human FSHß promoter.



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  		FIG. 9. Structural and functional conservation of the FSHß promoter response to LHX3. A, Sequence alignment of the indicated proximal regions of the porcine (P), ovine (O), rat (R), and human (H) FSHß promoters. The three identified LHX3-binding sites in this region of the FSHß gene are in bold. Recognition elements for SMAD and PITX2c transcription factors reported in the rat promoter (16 ) are also labeled. B, Activation of –5663 and –1622 bp fragments of the human FSHß promoter by LHX3a in transfected 293T cells. The porcine FSHß promoter was included as a positive control. C, Induction of –5663 and –1622 bp human FSHß promoters by LHX3a in LßT2 gonadotrope cells.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study we have demonstrated that LHX3a can activate the FSHß promoter, but not the –1158 bp LHß promoter. M2-LHX3 and LHX4 also increase FSHß promoter activity. Consistent with findings of others, we observed that SF1 and PITX2c also induce FSHß promoter transcription. The FSHß promoter contains multiple sites that can be bound by LHX3, including three within a proximal regulatory element that mediates responses to other transcription factors and to activins. Distinct pools of pituitary gonadotropes expressing various levels of the individual gonadotropins probably exist, and even in cells expressing both hormones, the production and release of FSH and LH may be distinctly controlled by agents such as GnRH and activins (reviewed in Refs. 1, 2, 3, 4). However, the precise mechanisms underlying the differential regulation of the gonadotropin genes remain to be elucidated. The observation that LHX3 activates the FSHß promoter, but not the proximal LHß promoter, suggests that LIM-HD proteins with known roles in pituitary gene regulation, such as LHX3, LHX4, and LHX2, may contribute to the selective regulation of the specific ß-subunit genes of the FSH and LH hormones. However, it is also possible that regions outside of the proximal promoter may mediate LHX3 regulation of LHß gene transcription, or LHX3 may act through indirect mechanisms. In these cases, LHX3 would be involved in the regulation of all three {alpha} and ß pituitary gonadotropin subunit genes.

Our data suggest that multiple positive and negative elements lie within the tested –5663 bp of the upstream FSHß promoter. The –1378 and –398 bp fragments of the promoters retain strong LHX3 inducibility and display high basal activities in gonadotrope cells, with the –1378 bp promoter having the most activity. To our knowledge, these are the first studies to functionally examine the activity of the porcine FSHß promoter, but Kato and colleagues (67) previously demonstrated that the –850 to –750 bp region (referred to as Fd2) of the pFSHß promoter is recognized by uncharacterized DNA-binding proteins extracted from anterior pituitary cells. We have now identified six LHX3-binding sites located at –209, –253, –284, –809, –1423, and –5030 bp. Four of the identified LHX3 sites are retained in the –1378 bp promoter reporter gene: one is located in the Fd2 region, and three are clustered in a proximal region in the –398 bp fragment. Similar elements are located within all sequenced mammalian FSHß promoters, either located at the same positions relative to the transcription start site or nearby. Homologous elements also are found within the corresponding regions of nonmammalian FSHß gene promoters, such as those of fish (68, 69). The –400 bp region of FSHß promoters from other species have been reported to contain critical regulatory elements recognized by many transcription factors and to mediate induction by GnRH and activin. The identified LHX3 recognition elements therefore lie within regions of the FSHß promoter that are important for multiple aspects of transcriptional regulation.

Transcriptional regulation by LHX3 is complex, involving DNA binding-dependent actions mediated by the HD and transcriptional activation mediated by a major activation domain located in the carboxyl terminus and weaker activation functions in other domains (33). In experiments using pituitary hormone reporter genes, LHX3 has been demonstrated to function both alone and in synergy with other transcription factors, such as PIT1 (31, 34, 49, 70). In addition, in the developing nervous system, LHX3 is proposed to participate in gene regulatory complexes that include other LIM-HD proteins, such as islet 1 (ISL1), and LIM-interacting partners, such as nuclear LIM interactor (NLI)/cofactor of LIM proteins (CLIM) (71). Observations that the LHX3 holoprotein and engineered derivatives lacking the LIM domains significantly bend their target DNA sites, and the association of LHX3 with the nuclear matrix, suggest that LHX3 also may play permissive architectural roles by altering local DNA topology to improve the actions of other transcription factors (36, 49). It is therefore likely that LHX3-mediated regulation of the FSHß promoter incorporates several mechanisms involving both direct and indirect actions at the FSHß promoter. In this study we demonstrate that LHX3 recognizes six binding sites in the FSHß promoter, but it is important to note that the FSHß gene contains additional, conserved, AT-rich elements that may also contribute to LIM-HD factor activation of the gene. The six LHX3-binding sites identified in the pFSHß promoter vary in their strength of binding with the protein; some are of similar affinity to the {alpha}GSU promoter PGBE element, whereas others are weaker. The mutagenesis experiments shown in Fig. 7Go demonstrate that these elements are important in combination for the LHX3 response. A component of LHX3 activation of the FSHß promoter may also include indirect actions involving induction of transcription factor genes that encode regulators of the FSHß gene, although our observations of LHX3 activation of FSHß promoter reporter genes in heterologous cells suggest that such indirect components do not require pituitary-specific factors.

In comparative studies we show that mammalian LHX3 and LHX4 transcription factors, but not their homolog, Drosophila LIM3, can activate specific mammalian pituitary hormone gene promoters, including FSHß. This observation is in accord with previous structure-function assays of the properties of LHX3 protein domains. The major trans-activation domain of mammalian LHX3 proteins lies within a central region of the carboxyl terminus of the protein (33). Sequence alignments reveal that this region of the protein is poorly conserved between Drosophila and mammals (72). By contrast, the LIM domains and HD display high sequence identity. In Drosophila, LIM3 functions have mostly been associated with motor neuron pathway selection in the nervous system (72). Mammalian LHX3 proteins also serve critical functions in spinal cord neuron development (71). However, LIM3 may also be expressed in the developing Drosophila ring gland (72), a compound endocrine organ that secretes hormones such as ecdysone, and it is therefore intriguing to speculate that LIM3 may nevertheless play some role in gene regulation in the insect endocrine system.

We show that both the FSHß promoter reporter gene and the endogenous FSHß gene within the LßT2 model gonadotrope cell are strongly induced by the addition of recombinant activin A. Our quantitative measurement of induction of the LßT2 FSHß gene is in accord with observations made by others that levels of FSHß mRNAs increase upon treatment of primary pituitary cells or gonadotrope model cells with activin (21, 25, 26, 28, 29). The LßT2 cell line expresses activin, follistatin, inhibin, and activin receptor mRNAs (21). Our data therefore represent induction of the LßT2 FSHß gene above the low basal levels that occur in the presence of endogenously produced activins. Indeed, it has been postulated that activin may be a necessary component of basal FSHß gene activity (3, 73).

In comparison with the dramatic induction of endogenous FSHß mRNA levels in LßT2 cells, treatment with recombinant activin did not cause notable changes in the levels of all LHX3 transcripts or the LHX3a, LHX4, and {alpha}GSU mRNAs. We conclude that the effects of activin on the FSHß gene are probably, for the most part, directly mediated by SMAD and other signaling pathways and do not involve transcriptional induction of the LHX3 and LHX4 genes. The demonstrated SMAD-binding sites in the rat FSHß promoter (16) are not perfectly conserved at the equivalent positions in the human and porcine promoters, but it is likely that similar sequences confer a SMAD-mediated activin response to the promoters of other species, such as the promoters examined in this study.

There are slight discrepancies in the literature regarding the transcriptional effects of activins on the {alpha}GSU gene. Experiments using reporter genes in LßT2 model cells have suggested that activins can increase transcription by 40% from the {alpha}GSU proximal promoter (21). Other studies have reported repression of {alpha}GSU reporter gene activities in the {alpha}T3–1 pregonadotrope model cell line (74). Assays of the {alpha}GSU mRNA by blotting methods suggest that activin causes either modest increases or no significant changes after treatment of primary rat pituitary cells (28, 29). Our quantitative measurements suggest that activin does not cause significant changes in the expression of the endogenous {alpha}GSU gene of LßT2 cells.

In future experiments it will be interesting to test whether the response of the FSHß promoter to GnRH involves LIM-HD proteins such as LHX3. The PGBE element of the {alpha}GSU gene (that is bound by LIM-HD proteins such as LHX2, LHX3, and LHX4) (31, 33, 35) has been implicated in some aspects of the activation of {alpha}GSU by GnRH (35, 62, 74). Clearly, transcriptional regulation of FSHß by GnRH and activins is complex. GnRH induction of the FSHß promoter in LßT2 cells is reduced by treatment with follistatin, an activin blocker (21), and activins increase GnRH-mediated FSH release in primates (65). Preliminary experiments indicate that the pFSHß promoter is GnRH responsive in LßT2 cells (West, B. E., and S. J. Rhodes, unpublished observations).

In conclusion, we have shown that the LHX3 LIM-HD protein activates the FSHß promoter by recognizing multiple elements within the promoter. This observation, in accord with previous studies from our laboratory and others, is consistent with the idea that LHX3 plays important roles both in the early stages of pituitary development and in differentiation and cell-specific gene expression in four of the five anterior pituitary cell types. These studies therefore are relevant to future investigations examining the role of LIM-HD factors in pituitary-associated diseases, such as combined pituitary hormone deficiency, precocious puberty, delayed puberty, and pituitary tumor formation.


   Acknowledgments
 
We are grateful to E. Adamson, T. Belecky-Adams, J. Bidwell, S. Camper, B. P. Herring. H. Ingraham, K. Marrs, P. Mellon, A. Nardulli, S. Thor, B. Yaden, and Y. Ziegler for reagents and advice. We also thank J. Cao and A. Moberly for assistance.


   Footnotes
 
This work was supported by grants (to S.J.R.) from the National Institutes of Health (HD-42024) and the National Science Foundation (IBN-0131702).

B.E.W. and G.E.P. contributed equally to this study.

Abbreviations: ChIP, Chromatin immunoprecipitation; EGR1, early growth response 1; {alpha}GSU, {alpha}-glycoprotein; HD, homeodomain; HEK, human embryonic kidney; KRAB, Krüppel-associated box; PGBE, pituitary glycoprotein basal element; PITX1, paired-like homeodomain transcription factor 1; PRL, prolactin; PROP1, Prophet-of-Pit-1; SF1, steroidogenic factor-1; SMAD, Sma/Mothers against decapentaplegic homolog; SP1, specificity protein 1.

Received May 11, 2004.

Accepted for publication July 15, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Brown P, McNeilly AS 1999 Transcriptional regulation of pituitary gonadotrophin subunit genes. Rev Reprod 4:117–124[Abstract]
  2. Shupnik MA 1996 Gonadotropin gene modulation by steroids and gonadotropin-releasing hormone. Biol Reprod 54:279–286[Abstract]
  3. Schwartz NB 1995 The 1994 Stevenson Award Lecture. Follicle-stimulating hormone and luteinizing hormone: a tale of two gonadotropins. Can J Physiol Pharmacol 73:675–684[Medline]
  4. Miller WL, Shafiee-Kermani F, Strahl BD, Huang HJ 2002 The nature of FSH induction by GnRH. Trends Endocrinol Metab 13:257–263[CrossRef][Medline]
  5. Savage JJ, Yaden BC, Kiratipranon P, Rhodes SJ 2003 Transcriptional control during mammalian anterior pituitary development. Gene 319:1–19[CrossRef][Medline]
  6. Tremblay JJ, Lanctot C, Drouin J 1998 The pan-pituitary activator of transcription, Ptx1 (pituitary homeobox 1), acts in synergy with SF-1 and Pit1 and is an upstream regulator of the Lim-homeodomain gene Lim3/Lhx3. Mol Endocrinol 12:428–441[Abstract/Free Full Text]
  7. Halvorson LM, Ito M, Jameson JL, Chin WW 1998 Steroidogenic factor-1 and early growth response protein 1 act through two composite DNA binding sites to regulate luteinizing hormone ß-subunit gene expression. J Biol Chem 273:14712–14720[Abstract/Free Full Text]
  8. Rosenberg SB, Mellon PL 2002 An Otx-related homeodomain protein binds an LHß promoter element important for activation during gonadotrope maturation. Mol Endocrinol 16:1280–1298[Abstract/Free Full Text]
  9. Keri RA, Bachmann DJ, Behrooz A, Herr BD, Ameduri RK, Quirk CC, Nilson JH 2000 An NF-Y binding site is important for basal, but not gonadotropin-releasing hormone-stimulated, expression of the luteinizing hormone ß subunit gene. J Biol Chem 275:13082–13088[Abstract/Free Full Text]
  10. Keri RA, Nilson JH 1996 A steroidogenic factor-1 binding site is required for activity of the luteinizing hormone ß subunit promoter in gonadotropes of transgenic mice. J Biol Chem 271:10782–10785[Abstract/Free Full Text]
  11. Quirk CC, Lozada KL, Keri RA, Nilson JH 2001 A single Pitx1 binding site is essential for activity of the LHß promoter in transgenic mice. Mol Endocrinol 15:734–746[Abstract/Free Full Text]
  12. Quirk CC, Seachrist DD, Nilson JH 2003 Embryonic expression of the luteinizing hormone ß gene appears to be coupled to the transient appearance of p8, a high mobility group-related transcription factor. J Biol Chem 278:1680–1685[Abstract/Free Full Text]
  13. Weck J, Anderson AC, Jenkins S, Fallest PC, Shupnik MA 2000 Divergent and composite gonadotropin-releasing hormone-responsive elements in the rat luteinizing hormone subunit genes. Mol Endocrinol 14:472–485[Abstract/Free Full Text]
  14. Tremblay JJ, Drouin J 1999 Egr-1 is a downstream effector of GnRH and synergizes by direct interaction with Ptx1 and SF-1 to enhance luteinizing hormone ß gene transcription. Mol Cell Biol 19:2567–2576[Abstract/Free Full Text]
  15. Kaiser UB, Halvorson LM, Chen MT 2000 Sp1, steroidogenic factor 1 (SF-1), and early growth response protein 1 (egr-1) binding sites form a tripartite gonadotropin-releasing hormone response element in the rat luteinizing hormone-ß gene promoter: an integral role for SF-1. Mol Endocrinol 14:1235–1245[Abstract/Free Full Text]
  16. Suszko MI, Lo DJ, Suh H, Camper SA, Woodruff TK 2003 Regulation of the rat follicle-stimulating hormone ß-subunit promoter by activin. Mol Endocrinol 17:318–332[Abstract/Free Full Text]
  17. Jacobs SB, Coss D, McGillivray SM, Mellon PL 2003 Nuclear factor-Y and steroidogenic factor-1 physically and functionally interact to contribute to cell-specific expression of the mouse FSHß gene. Mol Endocrinol 17:1470–1483[Abstract/Free Full Text]
  18. Zakaria MM, Jeong KH, Lacza C, Kaiser UB 2002 Pituitary homeobox 1 activates the rat FSHß (rFSHß) gene through both direct and indirect interactions with the rFSHß gene promoter. Mol Endocrinol 16:1840–1852[Abstract/Free Full Text]
  19. Alarid ET, Windle JJ, Whyte DB, Mellon PL 1996 Immortalization of pituitary cells at discrete stages of development by directed oncogenesis in transgenic mice. Development 122:3319–3329[Abstract]
  20. Turgeon JL, Kimura Y, Waring DW, Mellon PL 1996 Steroid and pulsatile gonadotropin-releasing hormone (GnRH) regulation of luteinizing hormone and GnRH receptor in a novel gonadotrope cell line. Mol Endocrinol 10:439–450[Abstract/Free Full Text]
  21. Pernasetti F, Vasilyev VV, Rosenberg SB, Bailey JS, Huang HJ, Miller WL, Mellon PL 2001 Cell-specific transcriptional regulation of follicle-stimulating hormone-ß by activin and gonadotropin-releasing hormone in the LßT2 pituitary gonadotrope cell model. Endocrinology 142:2284–2295[Abstract/Free Full Text]
  22. Vasilyev VV, Pernasetti F, Rosenberg SB, Barsoum MJ, Austin DA, Webster NJ, Mellon PL 2002 Transcriptional activation of the ovine follicle-stimulating hormone-ß gene by gonadotropin-releasing hormone involves multiple signal transduction pathways. Endocrinology 143:1651–1659[Abstract/Free Full Text]
  23. Gaddy-Kurten D, Tsuchida K, Vale W 1995 Activins and the receptor serine kinase superfamily. Recent Prog Horm Res 50:109–129
  24. Welt C, Sidis Y, Keutmann H, Schneyer A 2002 Activins, inhibins, and follistatins: from endocrinology to signaling. A paradigm for the new millennium. Exp Biol Med 227:724–752[Abstract/Free Full Text]
  25. Weiss J, Guendner MJ, Halvorson LM, Jameson JL 1995 Transcriptional activation of the follicle-stimulating hormone ß-subunit gene by activin. Endocrinology 136:1885–1891[Abstract]
  26. Graham KE, Nusser KD, Low MJ 1999 LßT2 gonadotroph cells secrete follicle stimulating hormone (FSH) in response to active A. J Endocrinol 162:R1–R5
  27. Huang HJ, Sebastian J, Strahl BD, Wu JC, Miller WL 2001 Transcriptional regulation of the ovine follicle-stimulating hormone-ß gene by activin and gonadotropin-releasing hormone (GnRH): involvement of two proximal activator protein-1 sites for GnRH stimulation. Endocrinology 142:2267–2274[Abstract/Free Full Text]
  28. Carroll RS, Corrigan AZ, Gharib SD, Vale W, Chin WW 1989 Inhibin, activin, and follistatin: regulation of follicle-stimulating hormone messenger ribonucleic acid levels. Mol Endocrinol 3:1969–1976[Abstract/Free Full Text]
  29. Attardi B, Miklos J 1990 Rapid stimulatory effect of activin-A on messenger RNA encoding the follicle-stimulating hormone ß-subunit in rat pituitary cell cultures. Mol Endocrinol 4:721–726[Abstract/Free Full Text]
  30. Miller CD, Miller WL 1996 Transcriptional repression of the ovine follicle-stimulating hormone-ß gene by 17ß-estradiol. Endocrinology 137:3437–3446[Abstract]
  31. Bach I, Rhodes SJ, Pearse RV II, Heinzel T, Gloss B, Scully KM, Sawchenko PE, Rosenfeld MG 1995 P-Lim, a LIM homeodomain factor, is expressed during pituitary organ and cell commitment and synergizes with Pit-1. Proc Natl Acad Sci USA 92:2720–2724[Abstract/Free Full Text]
  32. Zhadanov AB, Copeland NG, Gilbert DJ, Jenkins NA, Westphal H 1995 Genomic structure and chromosomal localization of the mouse LIM/homeobox gene Lhx3. Genomics 27:27–32[CrossRef][Medline]
  33. Sloop KW, Dwyer CJ, Rhodes SJ 2001 An isoform-specific inhibitory domain regulates the LHX3 LIM homeodomain factor holoprotein and the production of a functional alternate translation form. J Biol Chem 276:36311–36319[Abstract/Free Full Text]
  34. Sloop KW, Meier BC, Bridwell JL, Parker GE, Schiller AM, Rhodes SJ 1999 Differential activation of pituitary hormone genes by human Lhx3 isoforms with distinct DNA binding properties. Mol Endocrinol 13:2212–2225[Abstract/Free Full Text]
  35. Roberson MS, Schoderbek WE, Tremml G, Maurer RA 1994 Activation of the glycoprotein hormone {alpha}-subunit promoter by a LIM-homeodomain transcription factor. Mol Cell Biol 14:2985–2993[Abstract/Free Full Text]
  36. Bridwell JA, Price JR, Parker GE, McCutchan Schiller A, Sloop KW, Rhodes SJ 2001 Role of the LIM domains in DNA recognition by the Lhx3 neuroendocrine transcription factor. Gene 277:239–250[CrossRef][Medline]
  37. Brinkmeier ML, Gordon DF, Dowding JM, Saunders TL, Kendall SK, Sarapura VD, Wood WM, Ridgway EC, Camper SA 1998 Cell-specific expression of the mouse glycoprotein hormone {alpha}-subunit gene requires multiple interacting DNA elements in transgenic mice and cultured cells. Mol Endocrinol 12:622–633[Abstract/Free Full Text]
  38. Sloop KW, Showalter AD, Von Kap-Herr C, Pettenati MJ, Rhodes SJ 2000 Analysis of the human LHX3 neuroendocrine transcription factor gene and mapping to the subtelomeric region of chromosome 9. Gene 245:237–243[CrossRef][Medline]
  39. Sheng HZ, Zhadanov AB, Mosinger Jr B, Fujii T, Bertuzzi S, Grinberg A, Lee EJ, Huang SP, Mahon KA, Westphal H 1996 Specification of pituitary cell lineages by the LIM homeobox gene Lhx3. Science 272:1004–1007[Abstract]
  40. Netchine I, Sobrier ML, Krude H, Schnabel D, Maghnie M, Marcos E, Duriez B, Cacheux V, Moers A, Goossens M, Gruters A, Amselem S 2000 Mutations in LHX3 result in a new syndrome revealed by combined pituitary hormone deficiency. Nat Genet 25:182–186[CrossRef][Medline]
  41. Sloop KW, Parker GE, Hanna KR, Wright HA, Rhodes SJ 2001 LHX3 transcription factor mutations associated with combined pituitary hormone deficiency impair the activation of pituitary target genes. Gene 265:61–69[CrossRef][Medline]
  42. Howard PW, Maurer RA 2001 A point mutation in the LIM domain of Lhx3 reduces activation of the glycoprotein hormone {alpha}-subunit promoter. J Biol Chem 276:19020–19026[Abstract/Free Full Text]
  43. Sheng HZ, Moriyama K, Yamashita T, Li H, Potter SS, Mahon KA, Westphal H 1997 Multistep control of pituitary organogenesis. Science 278:1809–1812[Abstract/Free Full Text]
  44. Raetzman LT, Ward R, Camper SA 2002 Lhx4 and Prop1 are required for cell survival and expansion of the pituitary primordia. Development 129:4229–4239
  45. Hirai T, Takikawa H, Kato Y 1990 The gene for the ß subunit of porcine FSH: absence of consensus oestrogen-responsive element and presence of retroposons. J Mol Endocrinol 5:147–158[Abstract/Free Full Text]
  46. Ezashi T, Hirai T, Kato T, Wakabayashi K, Kato Y 1990 The gene for the ß subunit of porcine LH: clusters of GC boxes and CACCC elements. J Mol Endocrinol 5:137–146[Abstract/Free Full Text]
  47. Jameson JL, Becker CB, Lindell CM, Habener JF 1988 Human follicle-stimulating hormone ß-subunit gene encodes multiple messenger ribonucleic acids. Mol Endocrinol 2:806–815[Abstract/Free Full Text]
  48. Feister HA, Torrungruang K, Thunyakitpisal P, Parker GE, Rhodes SJ, Bidwell JP 2000 NP/NMP4 transcription factors have distinct osteoblast nuclear matrix subdomains. J Cell Biochem 79:506–517[CrossRef][Medline]
  49. Parker GE, Sandoval RM, Feister HA, Bidwell JP, Rhodes SJ 2000 The homeodomain coordinates nuclear entry of the Lhx3 neuroendocrine transcription factor and association with the nuclear matrix. J Biol Chem 275:23891–23898[Abstract/Free Full Text]
  50. Torrungruang K, Shah R, Alvarez M, Bowen DK, Gerard R, Pavalko FM, Elmendorf JS, Charoonpatrapong K, Hock J, Rhodes SJ, Bidwell JP 2002 Osteoblast intracellular localization of Nmp4 proteins. Bone 30:931–936[Medline]
  51. Petz LN, Ziegler YS, Schultz JR, Kim H, Kemper JK, Nardulli AM 2004 Differential regulation of the human progesterone receptor gene through an estrogen response element half site and Sp1 sites. J Steroid Biochem Mol Biol 88:113–122[CrossRef][Medline]
  52. Keri RA, Wolfe MW, Saunders TL, Anderson I, Kendall SK, Wagner T, Yeung J, Gorski J, Nett TM, Camper SA, Nilson JH 1994 The proximal promoter of the bovine luteinizing hormone ß-subunit gene confers gonadotrope-specific expression and regulation by gonadotropin-releasing hormone, testosterone, and 17ß-estradiol in transgenic mice. Mol Endocrinol 8:1807–1816[Abstract/Free Full Text]
  53. Huang HJ, Sebastian J, Strahl BD, Wu JC, Miller WL 2001 The promoter for the ovine follicle-stimulating hormone-ß gene (FSHß) confers FSHß-like expression on luciferase in transgenic mice: regulatory studies in vivo and in vitro. Endocrinology 142:2260–2266[Abstract/Free Full Text]
  54. Bellefroid EJ, Poncelet DA, Lecocq PJ, Revelant O, Martial JA 1991 The evolutionarily conserved Kruppel-associated box domain defines a subfamily of eukaryotic multifingered proteins. Proc Natl Acad Sci USA 88:3608–3612[Abstract/Free Full Text]
  55. Ayyanathan K, Lechner MS, Bell P, Maul GG, Schultz DC, Yamada Y, Tanaka K, Torigoe K, Rauscher III FJ 2003 Regulated recruitment of HP1 to a euchromatic gene induces mitotically heritable, epigenetic gene silencing: a mammalian cell culture model of gene variegation. Genes Dev 17:1855–1869[Abstract/Free Full Text]
  56. Margolin JF, Friedman JR, Meyer WK, Vissing H, Thiesen HJ, Rauscher III FJ 1994 Kruppel-associated boxes are potent transcriptional repression domains. Proc Natl Acad Sci USA 91:4509–4513[Abstract/Free Full Text]
  57. Witzgall R, O’Leary E, Leaf A, Onaldi D, Bonventre JV 1994 The Kruppel-associated box-A (KRAB-A) domain of zinc finger proteins mediates transcriptional repression. Proc Natl Acad Sci USA 91:4514–4518[Abstract/Free Full Text]
  58. Herchenroder O, Hahne JC, Meyer WK, Thiesen HJ, Schneider J 1999 Repression of the human immunodeficiency virus type 1 promoter by the human KRAB domain results in inhibition of virus production. Biochim Biophys Acta 1445:216–223[Medline]
  59. Reynolds L, Ullman C, Moore M, Isalan M, West MJ, Clapham P, Klug K, Choo Y 2003 Repression of the HIV-1 5' LTR promoter and inhibition of HIV-1 replication by using engineered zinc-finger transcription factors. Proc Natl Acad Sci USA 100:1615–1620[Abstract/Free Full Text]
  60. Bradford AP, Wasylyk C, Wasylyk B, Gutierrez-Hartmann A 1997 Interaction of Ets-1 and the POU-homeodomain protein GHF-1/Pit-1 reconstitutes pituitary-specific gene expression. Mol Cell Biol 17:1065–1074[Abstract]
  61. Thunyakitpisal P, Alvarez M, Tokunaga K, Onyia JE, Hock J, Ohashi N, Feister H, Rhodes SJ, Bidwell JP 2001 Cloning and functional analysis of a family of nuclear matrix transcription factors (NP/NMP4) that regulate type I collagen expression in osteoblasts. J Bone Miner Res 16:10–23[CrossRef][Medline]
  62. Schoderbek WE, Kim KE, Ridgway EC, Mellon PL, Maurer RA 1992 Analysis of DNA sequences required for pituitary-specific expression of the glycoprotein hormone {alpha}-subunit gene. Mol Endocrinol 6:893–903[Abstract/Free Full Text]
  63. Sloop KW, Walvoord EC, Showalter AD, Pescovitz OH, Rhodes SJ 2000 Molecular analysis of LHX3 and PROP-1 in pituitary hormone deficiency patients with posterior pituitary ectopia. J Clin Endocrinol Metab 85:2701–2708[Abstract/Free Full Text]
  64. Coss D, Jacobs SB, Bender CE, Mellon PL 2003 A novel AP-1 site is critical for maximal induction of the FSHß gene by GnRH. J Biol Chem 279:152–162
  65. McLachlan RI, Dahl KD, Bremner WJ, Schwall R, Schmelzer CH, Mason AJ, Steiner RA 1989 Recombinant human activin-A stimulates basal FSH and GnRH-stimulated FSH and LH release in the adult male macaque, Macaca fascicularis. Endocrinology 125:2787–2789[Abstract/Free Full Text]
  66. Schwall RH, Nikolics K, Szonyi E, Gorman C, Mason AJ 1988 Recombinant expression and characterization of human activin A. Mol Endocrinol 2:1237–1242[Abstract/Free Full Text]
  67. Kato Y, Tomizawa K, Kato T 1999 Multiple binding sites for nuclear proteins of the anterior pituitary are located in the 5'-flanking region of the porcine follicle-stimulating hormone (FSH) ß-subunit gene. Mol Cell Endocrinol 158:69–78[CrossRef][Medline]
  68. Vischer HF, Teves AC, Ackermans JC, van Dijk W, Schulz RW, Bogerd J 2003 Cloning and spatiotemporal expression of the follicle-stimulating hormone ß subunit complementary DNA in the African catfish (Clarias gariepinus). Biol Reprod 68:1324–1332[Abstract/Free Full Text]
  69. Rosenfeld H, Levavi-Sivan B, Gur G, Melamed P, Meiri I, Yaron Z, Elizur A 2001 Characterization of tilapia FSHß gene and analysis of its 5' flanking region. Comp Biochem Physiol B Biochem Mol Biol 129:389–398[CrossRef][Medline]
  70. Bach I, Carriere C, Ostendorff HP, Andersen B, Rosenfeld MG 1997 A family of LIM domain-associated cofactors confer transcriptional synergism between LIM and Otx homeodomain proteins. Genes Dev 11:1370–1380[Abstract/Free Full Text]
  71. Thaler JP, Lee SK, Jurata LW, Gill GN, Pfaff SL 2002 LIM factor Lhx3 contributes to the specification of motor neuron and interneuron identity through cell-type-specific protein-protein interactions. Cell 110:237–249[CrossRef][Medline]
  72. Thor S, Andersson SG, Tomlinson A, Thomas JB 1999 A LIM-homeodomain combinatorial code for motor-neuron pathway selection. Nature 397:76–80[CrossRef][Medline]
  73. Corrigan AZ, Bilezikjian LM, Carroll RS, Bald LN, Schmelzer CH, Fendly BM, Mason AJ, Chin WW, Schwall RH, Vale W 1991 Evidence for an autocrine role of activin B within rat anterior pituitary cultures. Endocrinology 128:1682–1684[Abstract/Free Full Text]
  74. Attardi B, Klatt B, Little G 1995 Repression of glycoprotein hormone {alpha}-subunit gene expression and secretion by activin in {alpha}T3–1 cells. Mol Endocrinol 9:1737–1749[Abstract/Free Full Text]



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