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
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ABSTRACT
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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, GT17. Transient transfection assays indicate that
the GnRH promoter Otx/bicoid site is required for specific expression
of the GnRH gene in GT17 cells and that it can confer specificity to
a neutral Rous sarcoma virus (RSV) promoter in GT17 cells but
not in NIH3T3 cells. Overexpression of mouse Otx2 in GT17 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.
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INTRODUCTION
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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, GT17, 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 GT17 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.
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RESULTS
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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. 1
. 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).
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Detection of Otx2 in GnRH Neurons and GT17 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. 2A
, 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 GT17 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 1020% of the GnRH cell nuclei stained for Otx2. B,
Otx2 mRNA transcripts are present in GT17 cells. A Northern blot with
1 µg polyA+ RNA from GT17 (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 GT17 RNA.
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The GT17 cell line is a clonal, immortalized cell line of
hypothalamic GnRH neurons, created by targeted oncogenesis in mice
(27). To determine whether GT17 cells express Otx2 transcripts as do
their in vivo counterparts, we performed Northern
hybridization analysis with polyA+ RNA from GT17 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 GT17 cells, whereas no signal was observed with NIH3T3
RNA (Fig. 2B
). Otx1 was undetectable in GT17 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. 3A
). This fragment contains a putative
Otx/bicoid site starting at position -153. As shown in Fig. 3B
, 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. 3A
). 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 GT17 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 16, rGnRH Otx probe with GT17 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 79, 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 1116, 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 1719, 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.
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To test whether this fragment of the rat GnRH promoter also binds to
endogenous Otx2, we performed an EMSA using nuclear extract from GT17
cells. The EMSA in Fig. 3C
shows that there are several complexes
formed with GT17 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 79). As expected, the labeled rGnRH mut oligonucleotide
does not detect any specific Otx2 binding activity in GT17 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 GT17 cells (lanes 1116). 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
1719).
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 GT17 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 GT17 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 GT17 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. 4
), the effect of
mutations in the Otx/bicoid site on the basal transcriptional activity
of a GnRH promoter plasmid was assayed in GT17 cells. In all cases,
mutagenized bases were the same as those that abolished Otx2 binding
in vitro (Fig. 3
). 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. 4
, 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. 4
, 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 GT17 cells.

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Figure 4. Mutation of the Otx2 Site in the rGnRH Promoter
Significantly Decreases Transcriptional Activity in GT17 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.
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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 GT17 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
GT17 and NIH3T3 cells showed a significantly lower level of
transcription in NIH3T3 cells (Fig. 5A
).
When the rGnRH Otx/bicoid site has the core four bases mutated to GGGG,
the statistically significant difference in transcription between the
GT17 and NIH3T3 cells is eliminated (Fig. 5A
). 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 GT17 Neuronal
Specificity to the GnRH Promoter
A, GT17 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 GT17
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 GT17 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.
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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/163133/RSVp-luc) and also between the RSV
enhancer and RSV promoter (RSVe/163133/RSVp-luc). In Fig. 5B
we have
plotted the ratio of activity in GT17 cells vs. NIH3T3
cells. Note that the plasmid containing the rGnRH enhancer and RSV
promoter has a 4-fold higher level of transcription in GT17 cells
vs. NIH3T3 cells (Fig. 5B
, 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 GT17 cells compared with NIH3T3 cells (Fig. 5B
, 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. 5B
, 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
GT17 cells in conjunction with the GnRH enhancer.
To determine whether overexpression of the Otx2 protein in GT17 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. 6
). Since GT17 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. 6
).

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Figure 6. Overexpression of Otx2 in GT17 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.
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DISCUSSION
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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 GT17 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 GT17 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 GT17
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 GT17 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 GT17 cells (32, 33). Here, we show that a specific mutation of the
Otx/bicoid site decreases expression by 80% in transiently transfected
GT17 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 GT17 cells (35). When SCIP is overexpressed in GT17
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
|
---|
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. 3A
). 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
[
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/163133/RSVp-luc and
RSVe/163133/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
GT17 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. 4
and 6
) 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 GT17 and NIH3T3 cells (shown in
Fig. 5
, 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 manufacturers
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 GT17 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. 3B
came from Clark and
Mellon (35). The procedure for Fig. 3C
follows: nuclear extracts used
for the experiment in Fig. 3C
were prepared according to Clark and
Mellon (35). Annealed oligonucleotides (1 pmol, Operon Technologies, Alameda, CA) were phosphorylated with
[
-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 13 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 manufacturers instructions.
One microgram of this polyA+ RNA (either from GT17 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 [
-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. 
2 Present address: Neurocrine Biosciences, 10555 Science Center Drive,
San Diego, California, 92121. 
Received for publication October 6, 1999.
Revision received May 5, 2000.
Accepted for publication May 9, 2000.
 |
REFERENCES
|
---|
-
Gehring WJ, Affolter M, Burglin T 1994 Homeodomain
proteins. Annu Rev Biochem 63:487526[CrossRef][Medline]
-
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 78:211223[Medline]
-
Scott MP, Tamkun JW, Hartzell GWI 1989 The structure and
function of the homeodomain. Biochim Biophys Acta 989:2548[CrossRef][Medline]
-
Fujii H, Hamada H 1993 A CNS specific POU transcription
factor, Brn-2, is required for establishing mammalian neural lineages.
Neuron 11:11971206[Medline]
-
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 89:32803284[Abstract]
-
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 340:3542[CrossRef][Medline]
-
Boncinelli E, Mallamaci A 1995 Homeobox genes in vertebrate
gastrulation. Curr Opin Genet Dev 5:619627[CrossRef][Medline]
-
Finkelstein R, Boncinelli E 1994 From fly head to mammalian
forebrain: the story of otd and Otx. Trends Genet 10:310315[CrossRef][Medline]
-
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 49:4963[CrossRef]
-
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 55:145158[CrossRef][Medline]
-
Li Y, Allende ML, Finkelstein R, Weinberg ES 1994 Expression
of two zebrafish orthodenticle-related genes in embryonic brain. Mech
Dev 48:229244[CrossRef][Medline]
-
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 121:702720
-
Simeone A, Acampora D, Mallamaci A, Stornaiuolo A, DApice 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 12:27352774[Abstract]
-
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 121:32793290[Abstract/Free Full Text]
-
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 122:243252[Abstract/Free Full Text]
-
Matsuo I, Kuratani S, Kimura C, Takeda N, Aizawa S 1995 Mouse
OTX2 functions in the formation and patterning of rostral head. Genes
Dev 9:26462658[Abstract]
-
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 14:218222[Medline]
-
Blitz IL, Cho KWY 1995 Anterior neuroectoderm is progressively
induced during gastrulation: the role of the Xenopus
homeobox gene orthodenticle. Development 121:9931004[Abstract/Free Full Text]
-
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 9:30853092[Abstract]
-
Driever W, Nuesslein-Volhard C 1989 The bicoid protein is a
positive regulator of hunchback transcription in the early
Drosophila embryos. Nature 337:138143[CrossRef][Medline]
-
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 58:165178[CrossRef][Medline]
-
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 13:623634[Medline]
-
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 46:309318[Medline]
-
Seeburg PH, Mason AJ, Stewart TA, Nikolics K 1987 The
mammalian GnRH gene and its pivotal role in reproduction. Recent Prog
Horm Res 43:6998[Medline]
-
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 1:12831304
-
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 86:81328136[Abstract]
-
Mellon PL, Windle JJ, Goldsmith P, Pedula C, Roberts J, Weiner
RI 1990 Immortalization of hypothalamic GnRH neurons by genetically
targeted tumorigenesis. Neuron 5:110[Medline]
-
Stojilkovic SS, Krsmanovic LZ, Spergel DJ, Catt KJ 1994 Gonadotropin-releasing hormone neurons: intrinsic pulsatility and
receptor-mediated regulation. Trends Endocrinol Metab 5:201209
-
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 89:41494153[Abstract]
-
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 2:2231[Abstract]
-
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 9:467477[Abstract]
-
Eraly SA, Mellon PL 1995 Regulation of GnRH transcription by
protein kinase C is mediated by evolutionarily conserved,
promoter-proximal elements. Mol Endocrinol 9:848859[Abstract]
-
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 12:469481[Abstract/Free Full Text]
-
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 16:35963605[Abstract]
-
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 15:61696177[Abstract]
-
Lavorgna G, Boncinelli E, Wagner A, Werner T 1998 Detection of
potential target genes in silico? Trends Genet 14:375376[CrossRef][Medline]
-
Lavorgna G, Guffanti A, Borsani G, Ballabio A, Boncinelli E 1999 TargetFinder: searching annotated sequence databases for
target genes of transcription factors. Bioinformatics 15:172173[Abstract/Free Full Text]
-
Mao CA, Gan L, Klein WH 1994 Multiple Otx binding sites
required for expression of the Strongylocentrotus purpuratus Spec2a
gene. Dev Biol 165:229242[CrossRef][Medline]
-
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 181:284295[CrossRef][Medline]
-
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 171:195211[CrossRef][Medline]
-
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 16:559567
-
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 125:12291239[Abstract/Free Full Text]
-
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 337:573576[CrossRef][Medline]
-
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 5:13091320[Abstract]
-
Szeto DP, Ryan AK, OConnell SM, Rosenfeld MG 1996 P-OTX: a
PIT-1-interacting homeodomain factor expressed during anterior
pituitary gland development. Proc Natl Acad Sci USA 93:77067710[Abstract/Free Full Text]
-
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 17:16521665[Abstract]
-
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 18:73577365[Abstract]
-
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[Medline]
-
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 11:17391744[Medline]
-
Monuki ES, Kuhn R, Weinmaster G, Trapp BD, Lemke G 1990 Expression and activity of the POU transcription factor SCIP. Science 249:13001303[Medline]
-
MacConell LA, Widger AE, Barth-Hall S, Roberts VJ 1998 Expression of activin and follistatin in the rat hypothalamus.
Endocrine 9:233241[Medline]
-
Shivers BD, Harlan RE, Morrell JI, Pfaff DW 1983 Absence of
oestradiol concentration in cell nuclei of LHRH-immunoreactive
neurones. Nature 304:345347[Medline]
-
Witkin JW, Silverman A 1985 Synaptology of luteinizing
hormone-releasing hormone neurons in rat preoptic area. Peptides 6:263271[CrossRef][Medline]
-
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 12:364377[Abstract/Free Full Text]
-
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 GT17
neuronal cells. Nucleic Acids Res 24:36143620[Abstract/Free Full Text]
-
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 115:113122[CrossRef][Medline]
-
Hayflick JS, Adelman JP, Seeburg PH 1989 The complete
nucleotide sequence of the human gonadotropin-releasing hormone gene.
Nucleic Acids Res 17:64036404[Medline]
-
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 234:13661371[Medline]
-
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 327:383389[CrossRef]