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Molecular Endocrinology 14 (8): 1246-1256
Copyright © 2000 by The Endocrine Society

The Otx2 Homeoprotein Regulates Expression from the Gonadotropin-Releasing Hormone Proximal Promoter

Carolyn G. Kelley1, Giovanni Lavorgna1, Melody E. Clark2, Edoardo Boncinelli and Pamela L. Mellon

Departments of Reproductive Medicine and Neuroscience and the Center for Molecular Genetics (C.G.K., M.E.C., P.L.M.) University of California, San Diego La Jolla, California 92093-0674
Dipartmento di Biologia e Tecnologia Istituto Scientifico H. S. Raffaele (G.L., E.B.) 20132 Milano, Italy
Centro per lo studio della Farmacologia Cellulare e Molecolare (E.B.) Consiglio Nazionale delle Ricerche 20129 Milano, Italy


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The GnRH gene is expressed exclusively in a highly restricted population of approximately 800 neurons in the mediobasal hypothalamus in the mouse. The Otx2 homeoprotein has been shown to colocalize with GnRH in embryonic mouse brain. We have identified a highly conserved bicoid-related Otx target sequence within the proximal promoter region of the GnRH gene from several species. This element from the rat GnRH promoter binds baculovirus-expressed Otx2 protein and Otx2 protein in nuclear extracts of a hypothalamic GnRH-expressing neuronal cell line, GT1–7. Transient transfection assays indicate that the GnRH promoter Otx/bicoid site is required for specific expression of the GnRH gene in GT1–7 cells and that it can confer specificity to a neutral Rous sarcoma virus (RSV) promoter in GT1–7 cells but not in NIH3T3 cells. Overexpression of mouse Otx2 in GT1–7 cells induces expression of a GnRH promoter plasmid, an effect that is dependent upon the Otx binding site. Thus, the GnRH proximal promoter is regulated by the Otx2 homeoprotein. Finally, we have now demonstrated the presence of Otx2 protein in the GnRH neurons of the adult mouse hypothalamus. These data suggest that Otx2 is important in the development of the GnRH neuron and/or in the maintenance of GnRH expression in the adult mouse hypothalamus.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Genetic and biochemical studies have shown that homeodomain proteins are sequence-specific transcriptional regulators often involved in developmental processes such as body segmentation, cell differentiation, and organogenesis. They act through transcriptional regulation of distinct sets of downstream target genes (1, 2, 3). Many different subclasses of homeodomain proteins have been shown to be involved in brain development (4, 5, 6).

Low-stringency hybridization cloning in mice has revealed a family of homeodomain proteins, termed Otx, closely related to D. melanogaster orthodenticle (otd) gene (7, 8). In Drosophila, expression patterns of the Otx proteins are restricted to the anterior head structures. In vertebrates, the most conserved Otx family member genes are Otx1 and Otx2 (9, 10, 11, 12, 13). Mouse knock-out experiments have shown that these genes are involved in anterior brain patterning; Otx2-/- mice lack forebrain and midbrain regions (14, 15, 16), while Otx1 -/- mice show several brain abnormalities (17). The early expression pattern of these genes, which has been studied in detail in Xenopus, suggests an even earlier role in development (10, 11, 12, 18).

The homeobox is a 180-bp DNA sequence segment that encodes a 60-amino acid residue domain, the homeodomain. The homeodomain of Otx and bicoid proteins is characterized by the presence of a conserved lysine at position 50; in other homeodomain-containing proteins, such as Antennapedia (Antp), it has been shown that the corresponding residue, a glutamine, directly contacts DNA bases (19). This amino acid difference between Otx and Antp homeodomains may be responsible for the differences between the Otx and Antp DNA recognition sequences. Previous studies from our group (13) have shown that Otx proteins can bind to the same functional target sequence motif recognized by bicoid on the hunchback promoter (20). We have also demonstrated that Otx1 and Otx2 proteins can transactivate a reporter gene, through a multimerized copy of the Otx/bicoid target sequence.

Using double immunohistochemistry, we have demonstrated previously that the cells that express Otx2 in the embryonic olfactory placode also express GnRH (21). GnRH is a decapeptide hormone that is released from a subset of hypothalamic neurons that controls many aspects of reproduction (22). GnRH-expressing neurons are found scattered throughout the basal forebrain and the rostral hypothalamus in the adult; however, they originate in the olfactory placode and migrate into the hypothalamus at E13 (23, 24, 25). Fewer than 800 GnRH neurons are present in the adult mouse hypothalamus (26), and these are not localized in a nucleus. Rather, they are scattered in several regions and can be found even along the original migratory route from the olfactory placode.

The low number and scattered distribution of GnRH neurons in the adult have made it difficult to study the molecular mechanisms underlying tissue-specific regulation of GnRH expression. We created a cultured cell model system to study GnRH gene regulation using targeted tumorigenesis in transgenic mice (27). This clonal GnRH-secreting hypothalamic cell line, GT1–7, is an excellent model system for the study of neuron-specific expression of the GnRH gene since these cells have retained many characteristics of GnRH neurons in vivo, including distinct neuronal morphology, expression of differentiated neuronal markers, and secretion of GnRH in response to appropriate signals (27, 28, 29, 30). The GT1–7 cells have been used to define a neuron-specific enhancer region (31) and demonstrate transcriptional activity of the GnRH proximal promoter (32, 33). Nuclear extracts from this cell line contain specific transcriptional regulatory proteins that bind both the enhancer and the promoter regions of the GnRH gene (31, 32, 33, 34, 35).

Recently, many new transcription factors have been isolated based on homology to known transcription factor families. Unfortunately, this approach does not provide any information on the target genes for these potential regulatory proteins. To find potential target genes for Otx, we developed a novel computer algorithm (36, 37) to search a sequence database for potential Otx binding sites. Interestingly, we found that the promoter of the GnRH gene from many species contains a previously undetected consensus Otx/bicoid target sequence. Herein we demonstrate that Otx2 regulates the rat GnRH gene through this Otx/bicoid site in the proximal promoter. To date, a few potential Otx target genes have been described in the sea urchin (38, 39, 40) and in the human tenascin-C promoter (41). The rat GnRH promoter Otx/bicoid site represents a natural functional binding site for Otx2 in the promoter of a brain-specific protein. These studies support the hypothesis that Otx2 participates in the development of the GnRH neuron and/or in the neuron-specific expression of the GnRH gene in the adult hypothalamus.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The GnRH Proximal Promoter Contains an Otx/Bicoid Consensus Site
The Otx family of homeoproteins is clearly associated with mammalian brain development (14, 15, 16, 17). However, since Otx2 was cloned by homology to other homeodomain proteins, it was not known to which target genes it binds nor had it been demonstrated to act as a transcriptional regulatory protein. We have recently developed computer software aimed at identifying potential target genes of transcription factors in sequence databases (36, 37). As Otx proteins have homology to bicoid within the homeodomain, we used a bicoid consensus binding element (TYTAATCC) for database screening (13, 20). Using this program, we found the presence of a well conserved Otx/bicoid target sequence upstream of the transcription start site in a number of genes (data not shown).

One of these genes, the GnRH gene, is of particular interest because the Otx/bicoid site is found in the proximal promoter of GnRH genes and is conserved across several vertebrate species, as shown in Fig. 1Go. In addition, the areas in which GnRH is expressed are areas dependent upon Otx2 for development. GnRH expression is limited to a subpopulation of hypothalamic neurons that are born in the olfactory placode, which is absent in the Otx2 knock-out mouse (14, 15, 16). In contrast, hypothalamic GnRH expression is unaltered in Otx1 -/- mice (42).



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  		Figure 1. Localization of an Otx/bicoid Site (boxed) in the Proximal Promoter of GnRH Genes from Several Vertebrate Species

The consensus sequence (TYTAATCC) is based on previous functional studies (13 20 ). The locations of the binding sites relative to the transcription start site are shown (32 55 56 57 58 ). A nucleotide of the human GnRH promoter, not matching the consensus sequence, is underlined. GenBank accession numbers for the GnRH sequences are as follows: M31670 (rat); M14872 (mouse); X15215 (human); X91408 (salmon).

 
Detection of Otx2 in GnRH Neurons and GT1–7 Cells
Using immunohistochemistry, we have shown previously that the GnRH-expressing neurons in the vomeronasal organ at e13 in the mouse also coexpress Otx2 (21). In Fig. 2AGo, we now show that this colocalization of Otx2 and GnRH is preserved in the adult mouse brain. Ten to 20% of neurons in the medial preoptic area and horizontal limb of the diagonal band have fibers stained brown by anti-GnRH antiserum/3,3'-diaminobenzidine tetrahydrochloride (DAB) reaction and nuclei stained black by the anti-Otx2 antiserum/Ni-DAB reaction.



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  		Figure 2. Otx2 is Found in GnRH Neurons in Adult Mouse Hypothalamus and in GT1–7 Cells

A, Immunohistochemical double-labeling shows colocalization of GnRH and Otx2 in hypothalamic neurons of the medial preoptic area (MPOA) and the organum vasculosum of the lamina terminalis (OVLT). For colocalization, 30-µm adult male mouse brain sections were stained with polyclonal rabbit anti-GnRH antiserum (52 53 ), visualized with a brown reaction product from a DAB reaction and a polyclonal rabbit anti-Otx2 antiserum (21 ) visualized with a blue-black product from a DAB-nickel mixture. Black arrows point to three cell bodies that show double label for Otx2 (black) with GnRH (brown). Approximately 10–20% of the GnRH cell nuclei stained for Otx2. B, Otx2 mRNA transcripts are present in GT1–7 cells. A Northern blot with 1 µg polyA+ RNA from GT1–7 (lane 1) and NIH3T3 (lane 2) cells was performed using a mouse Otx2 cDNA probe. A band of the appropriate size (2.6 kb) is present in the GT1–7 RNA.

 
The GT1–7 cell line is a clonal, immortalized cell line of hypothalamic GnRH neurons, created by targeted oncogenesis in mice (27). To determine whether GT1–7 cells express Otx2 transcripts as do their in vivo counterparts, we performed Northern hybridization analysis with polyA+ RNA from GT1–7 cells and NIH3T3 cells (as a negative control), using a mouse Otx2 cDNA probe. A 2.6-kb band, which is the expected size for Otx2 mRNA (13), is clearly detected in GT1–7 cells, whereas no signal was observed with NIH3T3 RNA (Fig. 2BGo). Otx1 was undetectable in GT1–7 cells by this method (data not shown).

Otx2 Binds to the GnRH Otx/Bicoid Site
To determine whether the GnRH Otx/bicoid site could bind Otx2 in vitro, we performed an electrophoretic mobility shift assay (EMSA) experiment in which we incubated recombinant mouse Otx2 protein with a radiolabeled, double-stranded oligonucleotide carrying the -163 to -133 portion of the rat GnRH gene (rGnRH Otx probe, Fig. 3AGo). This fragment contains a putative Otx/bicoid site starting at position -153. As shown in Fig. 3BGo, recombinant Otx2 protein binds to the rGnRH Otx probe (lane 1). This binding is competed by a 200-fold molar excess of self-competitor (lane 2) and by a 200-fold molar excess of the multimerized bicoid target sequence oligonucleotide (multi bicoid, lane 4) that has also been shown to recognize the Otx2 protein (13). The same Otx2 protein complex is not competed by a 200-fold molar excess of a mutated rGnRH Otx oligonucleotide (rGnRH mut, lane 3), in which four bases of the Otx/bicoid site had been changed to G residues (see underlined bases in Fig. 3AGo). Therefore, these EMSA experiments show that the rat GnRH gene promoter contains a Otx/bicoid site that can specifically bind to recombinant Otx2 protein in vitro.



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  		Figure 3. Binding of Otx2 Protein to Its Potential Target Sequence on the Rat GnRH (rGnRH) Promoter

A, Oligonucleotides used in the following EMSAs are shown. The rGnRH oligonucleotide (rGnRH Otx) encompasses positions -163 to -133 (from the transcription start site) of the GnRH promoter (32 ). In the rGnRH mut oligonucleotide, the core TAAT of the putative Otx2 consensus binding site is changed to GGGG. The singular bicoid oligonucleotide (bicoid) is comprised of the sequence from -77 to -47 of the Drosophila hunchback promoter containing a bicoid target sequence (20 59 ), and the multibicoid oligonucleotide is a synthetic trimer of the consensus bicoid-binding site that has been shown to specifically bind to mouse Otx2 protein (13 ). Bold bases indicate the Otx2 binding site and underlining indicates bases that have been mutagenized in the rGnRH mut oligonucleotide. B, Recombinant mouse Otx2 protein was incubated with rGnRH Otx, in the presence or absence of competitors; complexes were electrophoresed on a nondenaturing polyacrylamide gel. Competitions were as follows: lane 1, no competition; lane 2, 200-fold molar excess of rGnRH Otx; lane 3, 200-fold molar excess of rGnRH mut; lane 4, 200-fold molar excess of multibicoid oligonucleotide. C, Nuclear extract from either GT1–7 cells or NIH3T3 cells was incubated with the end-labeled oligonucleotide probe indicated and electrophoresed on a nondenaturing polyacrylamide gel. The reaction mixes were as follows: lanes 1–6, rGnRH Otx probe with GT1–7 nuclear extract; lane 1, no competitor; lane 2, 100-fold molar excess self-competition; lane 3, 100-fold molar excess bicoid oligonucleotide competitor; lane 4, 100-fold molar excess of rGnRH mut oligonucleotide competitor; lane 5, 1 µl Otx2 antibody; lane 6, 1 µl normal rabbit total IgG; lanes 7–9, rGnRH Otx probe with NIH3T3 nuclear extract; lane 7, no competitor; lane 8, 100-fold molar excess self-competition; lane 9, 1 µl Otx2 antibody; lane 10, rGnRH mut probe with GT1-7 nuclear extract; lanes 11–16, singular bicoid probe with GT1-7 nuclear extract; lane 11, no competition, lane 12, 100-fold molar excess self-competitor; lane 13, 100-fold molar excess rGnRH Otx competitor; lane 14, 100-fold molar excess rGnRH mut competitor; lane 15, 1 µl Otx2 antibody; lane 16, 1 µl normal rabbit total IgG; lanes 17–19, singular bicoid probe with NIH3T3 nuclear extract; lane 17, no competitor; lane 18, 100-fold molar excess self competition; lane 19, 1 µl Otx2 antibody.

 
To test whether this fragment of the rat GnRH promoter also binds to endogenous Otx2, we performed an EMSA using nuclear extract from GT1–7 cells. The EMSA in Fig. 3CGo shows that there are several complexes formed with GT1–7 nuclear extracts on the rGnRH Otx/bicoid site oligonucleotide (lane 1). One of these complexes, indicated by an arrow, appears to bind specifically to the probe. This protein complex is competed by a 100-fold molar excess of the rGnRH Otx oligonucleotide (lane 2) or by a 100-fold molar excess of the singular bicoid consensus site (bicoid) oligonucleotide (lane 3), whereas it is not competed by a 100-fold molar excess of rGnRH mut oligonucleotide (lane 4). To demonstrate that Otx2 binding participated in complex formation with the Otx/bicoid-containing oligonucleotide, we included Otx2 antibody in the binding reaction. Addition of 1 µl of Otx2 antibody to the binding reaction blocks this complex (lane 5), whereas incubation with 1 µl of normal rabbit total IgG does not perturb the complex (lane 6). Inclusion of 1 µl of a polyclonal antibody to another homeodomain protein (Emx1) also fails to perturb the complex (data not shown). Using the same GnRH Otx/bicoid site oligonucleotide as probe, no specific Otx2 binding activity was detected in NIH3T3 cells (lanes 7–9). As expected, the labeled rGnRH mut oligonucleotide does not detect any specific Otx2 binding activity in GT1–7 nuclear extracts (lane 10).

The bicoid oligonucleotide, which contains an Otx/bicoid site from the hunchback promoter (20), was also used as a probe in an EMSA with nuclear extracts from GT1–7 cells (lanes 11–16). This probe was incubated with extracts without competitor (lane 11), with 100-fold molar excess self-competition (lane 12), 100-fold molar excess rGnRH Otx competition (lane 13), 100-fold molar excess rGnRH mut competition (lane 14), 1 µl of Otx2 antibody (lane 15), or 1 µl of normal rabbit total IgG (lane 16). The bicoid probe forms a protein-DNA complex that comigrates with the specific band obtained using the labeled rGnRH oligonucleotide. This band is competed by itself and by the rGnRH oligonucleotide; incubation with Otx2 antibody also prevents the binding. The two bands that appear above the specific band are not affected by the Otx2 antibody, and we do not know their identity. The same two unidentified upper bands appear with NIH3T3 nuclear extracts on the singular bicoid consensus site oligonucleotide, whereas the specific Otx2 complex is not detected in NIH3T3 extracts (lanes 17–19).

This Otx2 antibody is thought to have low-level cross-reactivity with Otx1 (and the antibody for Otx1 also is thought to have low level cross-reactivity with Otx2); however, Otx1 and Otx2 are substantially different in size (355 amino acids vs. 289 amino acids, respectively), a difference that can easily by detected through EMSA. Although Otx1 was not detected in GT1–7 cells by Northern or Western blotting (data not shown), we further addressed this issue using nuclear extracts from NIH3T3 cells transfected with expression vectors for Otx1 or Otx2 in EMSA with the rGnRH Otx/bicoid probe or the singular bicoid consensus site probe. The band from the NIH3T3 cells transfected with Otx2 comigrated with that formed by the GT1–7 nuclear extract, while the band from the NIH3T3 cells transfected with Otx1 is retarded to a greater extent (data not shown). Although the Otx1 cDNA we used is human, the human and mouse Otx1 proteins differ in length by only one amino acid (with nine substitutions). Thus, the complex we have studied comigrates with Otx2 but not with Otx1 from transfected NIH3T3 cells. These data taken together demonstrate that GT1–7 cells contain Otx2 that is capable of binding specifically to the Otx/bicoid site in the rat GnRH promoter.

Activation of GnRH Transcription by Otx2
The functional relevance of Otx2 binding to the GnRH promoter region was assessed using transient transfection analyses with plasmids containing either the GnRH enhancer linked to the GnRH promoter or the GnRH promoter alone (see Materials and Methods). In the first experiment (Fig. 4Go), the effect of mutations in the Otx/bicoid site on the basal transcriptional activity of a GnRH promoter plasmid was assayed in GT1–7 cells. In all cases, mutagenized bases were the same as those that abolished Otx2 binding in vitro (Fig. 3Go). When four nucleotides in the center of the Otx/bicoid site were changed to G residues in the GnRH promoter, transcription measured by the luciferase reporter gene was reduced by more than 50% (Fig. 4Go, left panel). An even more dramatic effect was seen when using a reporter gene that contains the 300-bp GnRH neuron-specific enhancer upstream of the promoter (Fig. 4Go, right panel). In this case, the activity detected with the mutated plasmid is only about 20% of the wild-type activity. Taken together, these results show that the Otx2 binding element in the GnRH promoter is important for both full basal and enhancer-driven transcription of the GnRH gene in GT1–7 cells.



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  		Figure 4. Mutation of the Otx2 Site in the rGnRH Promoter Significantly Decreases Transcriptional Activity in GT1–7 Cells

Transient transfections were performed with 8 µg reporter plasmid DNA and 3 µg internal control TK-ß-galactosidase plasmid DNA. Values are normalized to the wild-type promoter plasmid, set equal to 1. The mean and SE bars result from nine separate experiments for all except the rGnRH promoter mutant where they result from seven. The star indicates statistical significance of P <0.0001, as determined by a one-way ANOVA.

 
Because the expression of Otx2 is restricted to neurons, we wanted to test the relative contribution of the Otx/bicoid site within the rGnRH promoter to the neuron-specific transcription of the GnRH gene. One method to investigate this issue involves transient transfections in two distinct cell types, one with highly specific transcription of GnRH, such as the GT1–7 cells, and one that has no endogenous expression of GnRH, such as the NIH3T3 cells. To control for differences in metabolic factors between distinct cell types, a heterologous Rous sarcoma virus (RSV) enhancer and promoter were linked to the luciferase gene (RSVe/RSVp-luc), and the same heterologous enhancer and promoter were linked to the ß-galactosidase gene (RSVe/RSVp-gal) to serve as the internal control. These plasmids were transfected into the cells in parallel with the expression vectors of interest (also cotransfected with the same internal control ß-galactosidase vector). The ratio of the RSVe/RSVp-luc value to the RSVe/RSVp-gal value was set to 1 within each cell type to allow comparison between the cell types.

The heterologous RSV enhancer combined with the rGnRH promoter yields a plasmid that isolates the contribution of the rGnRH promoter to the cell type specificity. Transient transfections of this plasmid into GT1–7 and NIH3T3 cells showed a significantly lower level of transcription in NIH3T3 cells (Fig. 5AGo). When the rGnRH Otx/bicoid site has the core four bases mutated to GGGG, the statistically significant difference in transcription between the GT1–7 and NIH3T3 cells is eliminated (Fig. 5AGo). These results show that the Otx/bicoid site contributes to the cell type-specific transcription of the GnRH gene.



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  		Figure 5. The Otx2 Element Confers GT1–7 Neuronal Specificity to the GnRH Promoter

A, GT1–7 cells are 2.5-fold more efficient than NIH3T3 cells in transcribing from the RSV enhancer-rGnRH promoter, an effect that is lost when a 4-bp mutation is substituted into the rGnRH Otx2 binding element. Transient transfections were carried out with 1.5 µg reporter DNA and 0.5 µg internal control RSVe/RSVp-gal DNA. Values are expressed relative to RSVe/RSVp-luc (normalized to the internal control RSVe/RSVp-gal) set to 1 to allow direct comparisons between the two cell types. Each mean and SE bar represents the results from nine separate experiments. Statistical significance, indicated by a star, is equal to P <0.0001 as determined by a one-way ANOVA. B, The ratio of activity between GT1–7 and NIH3T3 cells is increased by insertion of the -163/-133 oligo containing the Otx2 site when the adjacent enhancer is the GnRH enhancer, but not with the RSV enhancer. Transient transfections are as in panel A and are the results of nine separate experiments. Diagrams to the left depict the arrangement of regulatory elements in the transfected plasmids. The ratio depicted is derived by dividing the luciferase activity in GT1–7 cells (already normalized to the internal control RSVe/RSVp-gal) by the luciferase activity in NIH3T3 cells for each reporter plasmid with the RSVe/RSVp-luc ratio set to 1 to simplify the comparison.

 
To further investigate the function of the Otx/bicoid site, we inserted the -163 to -133 oligonucleotide, which contains the Otx consensus binding site from the rGnRH promoter, between the rGnRH enhancer and RSV promoter (rGnRHe/163–133/RSVp-luc) and also between the RSV enhancer and RSV promoter (RSVe/163–133/RSVp-luc). In Fig. 5BGo we have plotted the ratio of activity in GT1–7 cells vs. NIH3T3 cells. Note that the plasmid containing the rGnRH enhancer and RSV promoter has a 4-fold higher level of transcription in GT1–7 cells vs. NIH3T3 cells (Fig. 5BGo, bar 3). By inserting the -163 to -133 rGnRH oligonucleotide (containing the Otx/bicoid site) between the GnRH enhancer and the RSV promoter, the relative level of transcription is increased 2.5-fold, to more than 10-fold higher in GT1–7 cells compared with NIH3T3 cells (Fig. 5BGo, bar 4). Interestingly, this effect cannot be seen in the absence of the GnRH enhancer when the -163 to -133 element is inserted between the RSV enhancer and the RSV promoter, perhaps indicating that this element interacts with the GnRH enhancer to provide neuronal specificity (Fig. 5BGo, bars 1 and 2). Nonetheless, insertion of this region of the rGnRH promoter containing the Otx/bicoid consensus binding site can confer neuron-specific expression in the GT1–7 cells in conjunction with the GnRH enhancer.

To determine whether overexpression of the Otx2 protein in GT1–7 cells could affect the level of GnRH expression, we cotransfected an Otx2 expression plasmid with the GnRH promoter reporter plasmid (rGnRHp-luc). This combination resulted in a statistically significant increase (~50%) in the activity of the reporter gene (Fig. 6Go). Since GT1–7 cells already contain Otx2 protein and the system may be nearly saturated, this degree of transactivation is appropriate. The effect of overexpression of Otx2 is dependent on the presence of a functional Otx/bicoid site, since it is not observed using the GnRH promoter reporter gene with a mutated Otx binding site (Fig. 6Go).



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  		Figure 6. Overexpression of Otx2 in GT1–7 Cells Increases GnRH Promoter Transcriptional Activity

Transient transfections were carried out with 8 µg reporter DNA, 3 µg internal control TK-ß-galactosidase DNA, and 4.88 µg of empty expression vector or 4.08 µg of Otx2 expression plasmid. Values are normalized to the internal control ß-galactosidase, and the bar for the wild-type promoter with the empty expression vector is set to 1 for ease of comparison. Each mean and SE bar represents the results from 15 separate experiments for all except the rGnRH promoter/empty vector, where it represents 13. Statistical significance, indicated by a star, is equal to P <0.0001, as determined by a one-way ANOVA. The P value for the mutant Otx/bicoid site transfected with the Otx2 expression vector compared with the empty vector is 0.7456.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
The Otx class of homeodomain proteins plays a critical role in central nervous system development. Knock-out mice homozygous for a null allele of Otx1 die in the first month after birth and have epilepsy as well as multiple abnormalities affecting mainly the telencephalic temporal and perirhinal areas, the hippocampus, the mesencephalon, and the cerebellum as well as the acoustic and visual organs (17). Knock-out mice homozygous for a null allele of Otx2 are characterized by an embryonic lethality and severe phenotypes, including complete absence of forebrain, midbrain, and anterior hindbrain regions (15, 16, 17). The most severe heterozygous Otx2 knock-out mice lack olfactory epithelium, olfactory bulb, and the vomeronasal organ (16). The GnRH neurons are born in the olfactory placode and migrate from the presumptive vomeronasal organ through the olfactory bulb to the hypothalamus (22). In fact, we have previously shown that Otx2 is expressed in the migratory neurons along this pathway and GnRH is coexpressed in some of the Otx2-positive cells (21). Here we show that this colocalization is preserved in the adult mouse hypothalamus. Thus, both the observation that these structures require Otx2 to form and the coexpression of Otx2 with GnRH support our findings that the GnRH gene is regulated by the Otx2 homeodomain protein. These data also imply a role for Otx2 in the development and/or migration of the GnRH neuron and perhaps in the maintenance of GnRH gene expression in the adult hypothalamus.

Previous studies have identified the importance of two DNA regions for correct expression of the GnRH gene in transfected GT1–7 cells (31). These two regions are the 300-bp GnRH enhancer, located 1.8 kb upstream of the transcription start site (31), and the 173-bp conserved GnRH proximal promoter region (32, 33). In this report, we have shown that the conserved GnRH proximal promoter contains an Otx2 binding site and that the GT1–7 hypothalamic cell line, which secretes GnRH, contains Otx2. Further we show that Otx2 binds to the GnRH promoter Otx/bicoid site, that this Otx2 binding site is necessary for both basal and enhancer-driven transcription of the GnRH gene in GT1–7 cells, and that it contributes to cell-type specific transcription of the GnRH gene. Lastly, cotransfection with an Otx2 expression vector activates the GnRH promoter through the Otx2 binding site, indicating that Otx2 plays a role in GnRH expression. Transgenic mouse experiments have shown that the GnRH promoter (as well as the enhancer) is important for specifying GnRH expression to the GnRH neurons in the hypothalamus. The combination of these two regulatory regions is sufficient to target appropriate transcription of a ß-galactosidase reporter gene in the GnRH neurons of transgenic mice, while the enhancer linked to a heterologous promoter (RSV) is not sufficient (M. Lawson and P. L. Mellon, personal communication). Thus, the GnRH promoter region contributes to neuron specificity of the GnRH gene in vivo. The Otx2 binding site is found from -153 to -146 of the rat GnRH gene within this crucial proximal promoter region.

DNase I footprint analysis of the rat GnRH proximal promoter region revealed seven distinct footprints bound by GT1–7 nuclear proteins (32). The Otx2 binding element falls near the 5'-end of footprint 6, which extends from -158 to -129 of the promoter region. Deletion of a larger region carrying this element (a -173 truncation as compared with a -126 truncation) demonstrated a 2-fold decrease in expression in GT1–7 cells (32, 33). Here, we show that a specific mutation of the Otx/bicoid site decreases expression by 80% in transiently transfected GT1–7 cells, indicating that the important element in this region is the Otx/bicoid site (the difference in degree of decrease due to deletion or internal mutation may be due to the use of luciferase in this study and chloramphenicol acetyl transferase as the reporter gene in the previous study). Further, cotransfection with an Otx2 expression vector activates through this element, demonstrating the capacity of Otx2 to regulate directly GnRH gene expression.

In addition to Otx2, other homeobox protein binding sites have been mapped both within the GnRH enhancer and the GnRH promoter. The Oct-1 POU homeodomain transcription factor is essential for activity of the rat GnRH enhancer (35) and promoter (33). POU homeodomain proteins are also known to interact specifically with a number of other DNA-binding proteins (43), some of which are other POU-domain transcription factors (44). Interestingly, a novel Otx-related homeodomain transcription factor, P-Otx, has been identified recently on the basis of its ability to interact with the transactivation domain of the pituitary-specific POU domain protein, Pit-1 (45). Hence the Otx and POU domain families of transcription factors can interact directly and might interact to produce neuron specificity of GnRH gene expression.

Recently, a functional SCIP/Oct-6/Tst-1 binding site has been mapped to the rat GnRH promoter at -151, in a position partially overlapping the Otx binding-site (46). SCIP/Oct-6/Tst-1 is a brain- and testis-specific member of the POU homeodomain transcription factor family (5, 47, 48, 49, 50) and is found in GT1–7 cells (35). When SCIP is overexpressed in GT1–7 cells, inhibition of rat GnRH transcription is seen (46). This finding suggests possible competition between the overexpressed SCIP and the endogenous Otx2 for binding to this region of the GnRH promoter. Further studies will be needed to elucidate the intriguing possibility of interaction between Otx and other brain factors, such as SCIP/Oct-6/Tst-1 and Oct-1, and their integration at the level of the GnRH promoter or other brain-specific promoters.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Database Screening
TargetFinder Web Service (http://hercules.tigem.it/TargetFinder.html) was used to search vertebrate sequences of release N. 107 of GenBank (in June 1998) for perfect matches to the consensus sequence TYTAATCC. The search was restricted by using capabilities built into TargetFinder, to the top strand of DNA and up to 1-kb upstream of the transcription start site. The search string was derived from the alignment of several bicoid target sites on hunchback promoter and from our previous studies (13, 20).

Immunohistochemistry
Immunohistochemistry was carried out according to MacConell et al. (51). For colocalization, 30-µm adult male mouse brain sections were stained with polyclonal rabbit LR1 antiserum [directed against GnRH (52, 53)], visualized with a brown reaction product from a DAB reaction and a polyclonal rabbit Otx2 antiserum (21) visualized with a blue-black product from a DAB-nickel mixture.

Plasmid Constructions and Block Replacement Mutagenesis
The reporter plasmid rGnRHe/rGnRHp-luc contains the rat GnRH enhancer (31) (positions -1571 to -1863 relative to the transcription start site) fused in reverse orientation to the GnRH minimal promoter (positions -173 to +112 relative to the transcription start site) upstream of the luciferase reporter gene. The reporter plasmid rGnRHp-luc contains only the rat GnRH minimal promoter upstream of the luciferase gene. The -173 (SmaI) to +112 (BglII) rat GnRH promoter (31) was cloned into pGL3 basic (Promega Corp., Madison, WI). The enhancer fragment (-1571 to -1863) from ENH-173 (54) was obtained by cutting with SmaI and SalI and filling it in with Klenow. It was then inserted in the reverse orientation upstream of the rGnRHp-luc plasmid, cut with the same enzyme.

rGnRHe/rGnRHp-luc was then used as the template DNA for a PCR-based mutagenesis method to make a mutation in the Otx2 binding site. The position of the Otx/bicoid site is at -146/-153. The block replacement mutation changed the core TAAT bases between -148 to -151 of the GnRH promoter to GGGG (Fig. 3AGo). PCR primers were designed for the sense and the antisense strands of the promoter, extending six bases beyond the 5'-end and 14 bases beyond the 3'- end of the mutagenized site. Two separate PCR reactions were assembled with the sense mutagenesis primer (5'-GGTTTGGGCCCTTAGAATGGTG) and the antisense vector primer (pGL3rev, Promega Corp.) combined in one reaction and the antisense mutagenesis primer (5'-TAAGGGCCCCAAAACCCAGACATG) and sense vector primer (RV primer 3, Promega Corp.) combined in another reaction. The primary products were electroeluted to purify them away from excess primers. The sense and antisense primary PCR products have 16 bases overlapping, including the mutagenized site. They were combined in equal amounts, denatured, and allowed to anneal, creating some heteroduplex species. Using both of the vector primers, a product equivalent to rGnRHe/rGnRHp-luc, except for the introduced mutation, was amplified. The secondary PCR product was digested with MluI and NcoI and inserted back into pGL3, digested with the same enzymes. The sequence of the fragment obtained by PCR was determined using dideoxy-chain termination reactions in the presence of [{alpha}32P]-dATP, 3000 Ci/mmol (NEN Life Science Products, Boston, MA) and Sequenase (Amersham Pharmacia Biotech, Arlington Heights, IL) under the conditions described by the manufacturer. The promoter alone mutant (rGnRHp-luc) was created by digesting the mutant rGnRHe/rGnRHp-luc construct with MluI and XmaI, filling in with Klenow, and cloning it back into pGL3.

The RSVe/RSVp-luc plasmid includes the RSV enhancer fused to the RSV promoter in the pGL3 basic plasmid (Promega Corp.). The coding region for luciferase was replaced with the coding region for ß-galactosidase to create the RSVe/RSVp-gal plasmid. The RSV enhancer-GnRH promoter-luciferase plasmid (RSVe/rGnRHp-luc) and the rGnRH enhancer-RSV promoter-luciferase (rGnRHe/RSVp-luc) plasmid each involve the replacement of one of the RSV regulatory regions with the corresponding GnRH regulatory region. The phosphorylated, double-stranded oligonucleotide corresponding to bases -163 to -133 of the rGnRH promoter was ligated into both the rGnRHe/RSVp-luc and RSVe/RSVp-luc plasmids after they were digested with SmaI, which is located at the junction of the specified enhancer and promoter to create rGnRHe/163–133/RSVp-luc and RSVe/163–133/RSVp-luc.

The full-length mouse Otx2 cDNA sequence was cloned into the mammalian expression vector pSG-5, under the control of the SV40 early promoter (pSG-mOtx2). This pSG-mOTX2 construct was generated by cloning into the EcoRI site of pSG-5 a 900-bp fragment containing the full-length cDNA of mouse Otx2 (a gift from Antonello Mallamaci).

Cell Culture and Transfections
GT1–7 cells (27) and NIH3T3 cells were used in transient transfections. Cells were maintained in DMEM with 10% FBS, penicillin (100 U/ml), streptomycin (0.1 mg/ml), and 4.5% glucose in an atmosphere with 5% CO2. Transient transfections (for Figs. 4Go and 6Go) were performed using calcium phosphate precipitates containing 8 µg plasmid DNA, 4.88 µg of Otx2 or 4.08 µg of empty vector control (molar equivalents), and 3 µg internal control plasmid [herpes virus thymidine kinase (TK) promoter-ß-galactosidase] in 10-cm petri dishes. Cells were incubated for 16 h with the DNA precipitates, followed by washing twice with PBS and then adding new medium. The cells were incubated 30 h more before harvesting with a rubber policeman into 0.15 M NaCl, 1 mM EDTA, and 40 mM Tris, pH 7.4. Cell pellets were obtained by centrifugation. Resuspension in lysis buffer (100 mM potassium phosphate, pH 7.8, 0.2% Triton X-100) yielded cellular proteins. ß-Galactosidase assays were performed as directed by the manufacturer (Tropix, Bedford, MA) after heat inactivation of the cellular extracts. Luciferase assays were performed as previously described (35). Luciferase activity was divided by the internal control ß-galactosidase values to control for transfection efficiencies between plates. Values were normalized to wild-type GnRH regulatory region with empty vector control.

The comparison of transcription in GT1–7 and NIH3T3 cells (shown in Fig. 5Go, A and B) involved transient transfections in 6-cm petri dishes with the FuGENE 6 reagent (Roche Molecular Biochemicals, Indianapolis, IN). Cells were maintained as above. Reporter plasmid DNA (1.5 µg) and 0.5 µg of internal control DNA plasmid were mixed with 3 µl of FuGENE. FuGENE was used according to the manufacturer’s directions. The mixture was incubated with the cells for 24 h. The cells were then washed and harvested as above. The ratio of RSVe/RSVp-luc to RSVe/RSVp-gal in the three control plates was set to one in both GT1–7 and NIH3T3 cells and used for normalization of the rest of the plates.

Nuclear Extracts and EMSA
The recombinant mouse Otx2 protein used in the EMSAs has been previously described (21). The method for Fig. 3BGo came from Clark and Mellon (35). The procedure for Fig. 3CGo follows: nuclear extracts used for the experiment in Fig. 3CGo were prepared according to Clark and Mellon (35). Annealed oligonucleotides (1 pmol, Operon Technologies, Alameda, CA) were phosphorylated with [{gamma}-32P]ATP (6000 Ci/mmol; NEN Life Science Products) and T4 polynucleotide kinase. Then, probes were purified over a MicroSpin G-50 column (Pharmacia Biotech, Piscataway, NJ). The 10-µl reaction mixes contained 1 µg of protein and 1 fmol probe in 100 mM KCl, 37 mM HEPES (pH 7.9), 2.5 mMM EDTA, 1.25 mM dithiothreitol, 12.5% glycerol, and 4 mM phenylmethylsulfonyl fluoride. In the appropriate reactions, 100 fmol (100x labeled probe) of competitor or 1 µl antibody (or control rabbit IgG) were added 10 min before the addition of probe. The probe was added and incubated for 5 min before loading onto a 5% polyacrylamide nondenaturing gel that had prerun for 30 min. After 2 h at 200 V, gels were dried under vacuum and then exposed to film at room temperature for 1–3 days.

Northern Blot Analysis
RNA for Northern hybridization analysis was prepared according to the protocol provided with the Trizol Reagent (Life Technologies, Inc., Gaithersburg, MD). To prepare the polyA+ RNA, the PolyATtract mRNA Isolation System IV (Promega Corp.) was used according to the manufacturer’s instructions. One microgram of this polyA+ RNA (either from GT1–7 or NIH3T3 cells) was loaded onto a 1% agarose gel and transferred to Hybond-N+ membrane (Amersham Pharmacia Biotech) by capillary blotting. The filter was prehybridized in 25% formamide buffer at 55 C and then hybridized with 1,000,000 cpm of Otx2 probe for 16 h. The probe itself was made by incubating the 900-bp EcoRI fragment of pSG-mOTX2 with Klenow and [{alpha}-32P]ATP (6000 Ci/mmol, NEN Life Science Products) and then purifying it over an S-200 HR column (Pharmacia Biotech). The filter was washed in 0.1% SDS at 65 C for 40 min, dried, and then exposed to film at -70 C with an intensifying screen for 5 days.


   ACKNOWLEDGMENTS
 
We are indebted to J. Wijnholds, P. Gruss, and A. Mallamaci for the generous gift of plasmids and G. Corte for Otx2 recombinant protein and antibody. We thank K. Huang, M. Lawson, J. Jovenal, and S. Nelson for plasmids. We thank M. Sottocorno and A. Rosete for excellent secretarial assistance, L. MacConell for neuroanatomical advice, Sally B. Hall for performing immunocytochemistry, L. Pintonello for technical support, and K. Friggi for performing some experiments referred to in the text.


   FOOTNOTES
 
Address requests for reprints to: Pamela L. Mellon, Department of Reproductive Medicine 0674, University of California, San Diego, 9500 Gilman Drive, La Jolla, California 92093-0674. E-mail: pmellon{at}ucsd.edu

This work was supported by the NIH Grant R01 DK-44838 to P.L.M. and by grants from the EC BIOTECH (Nos. 0378 and 0042) and BIOMED (No. 0777) Programmes, the Telethon-Italia Programme (No. D78), and the Italian Association for Cancer Research (AIRC) to E.B. C.G.K. was supported by a predoctoral fellowship from the Howard Hughes Medical Institute, and M.E.C. was supported by a fellowship from the American Cancer Society and the National Alliance for Research on Schizophrenia and Depression Foundation.

1 These authors contributed equally. Back

2 Present address: Neurocrine Biosciences, 10555 Science Center Drive, San Diego, California, 92121. Back

Received for publication October 6, 1999. Revision received May 5, 2000. Accepted for publication May 9, 2000.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Gehring WJ, Affolter M, Burglin T 1994 Homeodomain proteins. Annu Rev Biochem 63:487–526
  2. Gehring WJ, Yan YQ, Billeter M, Forukubo-Tokunaga K, Schier AF, Resendez-Perez D, Affolter M, Otting G, Wuthrich K 1994 Homeodomain-DNA recognition. Cell 78:211–223
  3. Scott MP, Tamkun JW, Hartzell GWI 1989 The structure and function of the homeodomain. Biochim Biophys Acta 989:25–48
  4. Fujii H, Hamada H 1993 A CNS specific POU transcription factor, Brn-2, is required for establishing mammalian neural lineages. Neuron 11:1197–1206
  5. Hara Y, Rovescalli A, Kim Y, Nirenberg M 1992 Structure and evolution of four POU domain genes expressed in mouse brain. Proc Natl Acad Sci USA 89:3280–3284
  6. He X, Treacy MN, Simmons DM, Ingraham HA, Swanson LW, Rosenfeld MG 1989 Expression of a large family of POU-domain regulatory genes in mammalian brain development. Nature 340:35–42
  7. Boncinelli E, Mallamaci A 1995 Homeobox genes in vertebrate gastrulation. Curr Opin Genet Dev 5:619–627
  8. Finkelstein R, Boncinelli E 1994 From fly head to mammalian forebrain: the story of otd and Otx. Trends Genet 10:310–315
  9. Bally-Cuif LM, Gulisano M, Broccoli V, Boncinelli E 1994 c-Otx2 is expressed in two different phases of gastrulation and is sensitive to retinoic acid treatment in chick embryo. Mech Dev 49:49–63
  10. Kablar R, Vignali R, Menotti L, Pannese P, Andreazzoli M, Polo C, Giribaldi MG, Boncinelli E, Barsacchi G 1996 Xotx genes in the developing brain of Xenopus laevis. Mech Dev 55:145–158
  11. Li Y, Allende ML, Finkelstein R, Weinberg ES 1994 Expression of two zebrafish orthodenticle-related genes in embryonic brain. Mech Dev 48:229–244
  12. Pannese M, Polo C, Andreazzoli M, Vignali R, Kablar B, Barsacchi G, Boncinelli E 1995 The Xenopus homologue of Otx2 is a maternal homeobox gene that demarcates and specifies anterior body regions. Development 121:702–720
  13. Simeone A, Acampora D, Mallamaci A, Stornaiuolo A, D’Apice R, Nigro V, Boncinelli E 1993 A vertebrate gene related to orthodenticle contains a homeodomain of the bicoid class and demarcates anterior neuroectoderm of the gastrulating mouse embryo. EMBO J 12:2735–2774
  14. Acampora D, Mazan S, Lallemand Y, Avantaggiato V, Maury M, Simeone A, Brulet P 1995 Forebrain and midbrain regions are deleted in OTX2-/- mutants due to a defective anterior neuroectoderm specification during gastrulation. Development 121:3279–3290
  15. Ang S-L, Jin O, Rhinn M, Daigle N, Stevenson L, Rossant J 1996 A targeted mouse OTX2 mutation leads to severe defects in gastrulation and formation of axial mesoderm and to deletion of rostral brain. Development 122:243–252
  16. Matsuo I, Kuratani S, Kimura C, Takeda N, Aizawa S 1995 Mouse OTX2 functions in the formation and patterning of rostral head. Genes Dev 9:2646–2658
  17. Acampora D, Mazan S, Avvantaggiato V, Barone P, Tuorto F, Lallemand Y, Brulet P, Simeone A 1996 Epilepsy and brain abnormalities in mice lacking the Otx1 gene. Nat Genet 14:218–222
  18. Blitz IL, Cho KWY 1995 Anterior neuroectoderm is progressively induced during gastrulation: the role of the Xenopus homeobox gene orthodenticle. Development 121:993–1004
  19. Otting G, Quian YQ, Billeter M, Mueller M, Affolter M, Gehring WJ, Wuthrich K 1990 Protein contacts in the structure of a homeodomain-DNA complex determined by nuclear magnetic resonance spectroscopy in solution. EMBO J 9:3085–3092
  20. Driever W, Nuesslein-Volhard C 1989 The bicoid protein is a positive regulator of hunchback transcription in the early Drosophila embryos. Nature 337:138–143
  21. Mallamaci A, DiBlas E, Briata P, Boncinelli E, Corte G 1996 OTX2 homeoprotein in the developing central nervous system and migratory cells of the olfactory area. Mech Dev 58:165–178
  22. Schwanzel-Fukuda M, Jorgenson KL, Bergen HT, Weesner GD, Pfaff DW 1992 Biology of normal luteinizing hormone-releasing hormone neurons during and after their migration from olfactory placode. Endocr Rev 13:623–634
  23. Wray S, Nieburgs A, Elkabes S 1989 Spatiotemporal cell expression of luteinizing hormone-releasing hormone in the prenatal mouse: evidence for an embryonic origin in the olfactory placode. Dev Brain Res 46:309–318
  24. Seeburg PH, Mason AJ, Stewart TA, Nikolics K 1987 The mammalian GnRH gene and its pivotal role in reproduction. Recent Prog Horm Res 43:69–98
  25. Silverman A 1988 The gonadotropin-releasing hormone (GnRH) neuronal systems: immunocytochemistry. In: Knobil E, Neill JD (eds) The Physiology of Reproduction. Raven Press, New York, vol 1:1283–1304
  26. Wray S, Grant P, Gainer H 1989 Evidence that cells expressing luteinizing hormone-releasing hormone mRNA in the mouse are derived from progenitor cells in the olfactory placode. Proc Natl Acad Sci USA 86:8132–8136
  27. Mellon PL, Windle JJ, Goldsmith P, Pedula C, Roberts J, Weiner RI 1990 Immortalization of hypothalamic GnRH neurons by genetically targeted tumorigenesis. Neuron 5:1–10
  28. Stojilkovic SS, Krsmanovic LZ, Spergel DJ, Catt KJ 1994 Gonadotropin-releasing hormone neurons: intrinsic pulsatility and receptor-mediated regulation. Trends Endocrinol Metab 5:201–209
  29. Wetsel WC, Valença MM, Merchenthaler I, Liposits Z, López FJ, Weiner RI, Mellon PL, Negro-Vilar A 1992 Intrinsic pulsatile secretory activity of immortalized LHRH secreting neurons. Proc Natl Acad Sci USA 89:4149–4153
  30. Wetsel W, Culler M, Johnston C, Negro-Vilar A 1988 Processing of the luteinizing hormone-releasing hormone precursor in the preoptic area and hypothalamus of the rat. Mol Endocrinol 2:22–31
  31. Whyte DB, Lawson MA, Belsham DD, Eraly SA, Bond CT, Adelman JP, Mellon PL 1995 A neuron-specific enhancer targets expression of the gonadotropin-releasing hormone gene to hypothalamic neurosecretory neurons. Mol Endocrinol 9:467–477
  32. Eraly SA, Mellon PL 1995 Regulation of GnRH transcription by protein kinase C is mediated by evolutionarily conserved, promoter-proximal elements. Mol Endocrinol 9:848–859
  33. Eraly SA, Nelson SB, Huang KM, Mellon PL 1998 Oct-1 binds promoter elements required for transcription of the gonadotropin-releasing hormone gene. Mol Endocrinol 12:469–481
  34. Lawson MA, Whyte DB, Mellon PL 1996 GATA factors are essential for activity of the neuron-specific enhancer of the gonadotropin-releasing hormone gene. Mol Cell Biol 16:3596–3605
  35. Clark ME, Mellon PL 1995 The POU homeodomain transcription factor Oct-1 is essential for activity of the gonadotropin-releasing hormone neuron-specific enhancer. Mol Cell Biol 15:6169–6177
  36. Lavorgna G, Boncinelli E, Wagner A, Werner T 1998 Detection of potential target genes in silico? Trends Genet 14:375–376
  37. Lavorgna G, Guffanti A, Borsani G, Ballabio A, Boncinelli E 1999 TargetFinder: searching annotated sequence databases for target genes of transcription factors. Bioinformatics 15:172–173
  38. Mao CA, Gan L, Klein WH 1994 Multiple Otx binding sites required for expression of the Strongylocentrotus purpuratus Spec2a gene. Dev Biol 165:229–242
  39. Sakamoto N, Akasaka K, Mitsunaga-Nakatsubo K, Takata K, Nishitani T, Shimada H 1997 Two isoforms of orthodenticle-related proteins (HpOtx) bind to the enhancer element of sea urchin arylsulfatase gene. Dev Biol 181:284–295
  40. Wei Z, Angerer LM, Gagnon ML, Angerer RC 1995 Characterization of the SpHE promoter that is spatially regulated along the animal-vegetal axis of the sea urchin embryo. Dev Biol 171:195–211
  41. Gherzi R, Briata P, Boncinelli E, Ponassi M, Querze G, Viti F, Corte G, Zardi L 1997 The human homeodomain protein OTX2 binds to the human tenascin-C promoter and trans-represses its activity in transfected cells DNA. Cell Biol 16:559–567
  42. Acampora D, Mazan S, Tuorto F, Avantaggiato V, Tremblay JJ, Lazzaro D, di Carlo A, Mariano A, Macchia PE, Corte G, Macchia V, Drouin J, Brûlet P, Simeone A 1998 Transient dwarfism and hypogonadism in mice lacking Otx1 reveal prepubescent stage-specific control of pituitary levels of GH, FSH and LH. Development 125:1229–1239
  43. Poellinger L, Yoza BK, Roeder RG 1989 Functional cooperativity between protein molecules bound at two distinct sequence elements of the immunoglobulin heavy-chain promoter. Nature 337:573–576
  44. Voss JW, Wilson L, Rosenfeld MG 1991 POU-domain proteins Pit-1 and Oct-1 interact to form a heteromeric complex and can cooperate to induce expression of the prolactin promoter. Genes Dev 5:1309–1320
  45. Szeto DP, Ryan AK, O’Connell SM, Rosenfeld MG 1996 P-OTX: a PIT-1-interacting homeodomain factor expressed during anterior pituitary gland development. Proc Natl Acad Sci USA 93:7706–7710
  46. Wierman ME, Xiong X, Kepa JK, Spaulding AJ, Jacobsen BM, Fang Z, Nilaver G, Ojeda SR 1997 Repression of gonadotropin-releasing hormone promoter activity by the POU homeodomain transcription factor SCIP/Oct-6/Tst-1: a regulatory mechanism of phenotype expression? Mol Cell Biol 17:1652–1665
  47. Meijer D, Graus A, Kraay R, Langeveld A, Mulder MP, Grosveld G 1990 The octamer binding factor Oct6: cDNA cloning and expression in early embryonic cells. Nucleic Acids Res 18:7357–7365
  48. Zimmermann EC, Jones CM, Fet V, Hogan BLM, Magnuson MA 1991 Nucleotide sequence of mouse SCIP cDNA a POU domain transcription factor. Nucleic Acids Res 19:956
  49. He X, Gerrero R, Simmons DM, Park RE, Lin CR, Swanson LW, Rosenfeld MG 1991 Tst-1 a member of the POU domain gene family binds to the promoter of the gene encoding the cell surface adhesion molecule Po. Mol Cell Biol 11:1739–1744
  50. Monuki ES, Kuhn R, Weinmaster G, Trapp BD, Lemke G 1990 Expression and activity of the POU transcription factor SCIP. Science 249:1300–1303
  51. MacConell LA, Widger AE, Barth-Hall S, Roberts VJ 1998 Expression of activin and follistatin in the rat hypothalamus. Endocrine 9:233–241
  52. Shivers BD, Harlan RE, Morrell JI, Pfaff DW 1983 Absence of oestradiol concentration in cell nuclei of LHRH-immunoreactive neurones. Nature 304:345–347
  53. Witkin JW, Silverman A 1985 Synaptology of luteinizing hormone-releasing hormone neurons in rat preoptic area. Peptides 6:263–271
  54. Lawson MA, Buhain AR, Jovenal JC, Mellon PL 1998 Multiple factors interacting at the GATA sites of the gonadotropin-releasing hormone neuron-specific enhancer regulate gene expression. Mol Endocrinol 12:364–377
  55. Kepa JK, Spaulding AJ, Jacobsen BM, Fang Z, Xiong X, Radovick S, Wierman ME 1996 Structure of the distal human gonadotropin releasing hormone (hGnRH) gene promoter and functional analysis in GT1–7 neuronal cells. Nucleic Acids Res 24:3614–3620
  56. Coe IR, van Schalburg K, Sherwood NM 1995 Characterization of the Pacific salmon gonadotropin-releasing hormone gene, copy number and transcription start site. Mol Cell Endocrinol 115:113–122
  57. Hayflick JS, Adelman JP, Seeburg PH 1989 The complete nucleotide sequence of the human gonadotropin-releasing hormone gene. Nucleic Acids Res 17:6403–6404
  58. Mason AJ, Hayflick JS, Zoeller RT, Young WS, Phillips HS, Nikolics K, Seeburg PH 1986 A deletion truncating the gonadotropin-releasing hormone gene is responsible for hypogonadism in the hpg mouse. Science 234:1366–1371
  59. Tautz D, Lehmann R, Schnurch H, Schuh R, Seifert E, Kienlin A, Jones K, Jackle H 1987 Finger protein of novel structure encoded by hunchback, a second member of the gap class of Drosophila segmentation genes. Nature 327:383–389



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H. H. Kim, A. Wolfe, G. R. Smith, S. A. Tobet, and S. Radovick
Promoter Sequences Targeting Tissue-specific Gene Expression of Hypothalamic and Ovarian Gonadotropin-releasing Hormone in Vivo
J. Biol. Chem., February 8, 2002; 277(7): 5194 - 5202.
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D. Roy, N. L. Angelini, H. Fujieda, G. M. Brown, and D. D. Belsham
Cyclical Regulation of GnRH Gene Expression in GT1-7 GnRH-Secreting Neurons by Melatonin
Endocrinology, November 1, 2001; 142(11): 4711 - 4720.
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