Transcription Factors Oct-1 and C/EBPß (CCAAT/Enhancer-Binding Protein-ß) Are Involved in the Glutamate/Nitric Oxide/cyclic-Guanosine 5'-Monophosphate-Mediated Repression of Gonadotropin-Releasing Hormone Gene Expression
Denise D. Belsham1 and
Pamela L. Mellon
Departments of Reproductive Medicine and Neurosciences The
Center for Molecular Genetics University of California, San
Diego La Jolla, California 92093
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ABSTRACT
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The physiological actions of nitric oxide (NO) as
a signaling molecule in endothelial and brain cells and as a toxic
molecule used by activated immune cells have been the focus of a wide
range of studies. Nevertheless, the downstream effector molecules of
this important neuromodulator are not well understood. We have
previously demonstrated that expression of the gene for the
reproductive neuropeptide, GnRH, is repressed by the
glutamate/NO/cyclic GMP (cGMP) signal transduction pathway through
cGMP-dependent protein kinase in the hypothalamic GnRH-secreting
neuronal cell line GT17. This repression localized within a
previously characterized 300-bp neuron-specific enhancer. Here, we find
that mutation of either of two adjacent elements within the enhancer
eliminates repression by this pathway. An AT-rich sequence located at
-1695 has homology to the octamer motif known to bind POU-homeodomain
proteins, while the adjacent element at -1676 has homology to the
C/EBP (CCAAT/enhancer-binding protein) protein family consensus
sequence. Antibody supershift assays reveal that one of the proteins
bound at the -1695 sequence is Oct-1, and one of the proteins bound to
the element at -1676 is C/EBPß. These two proteins can bind
simultaneously to the adjacent -1695 and -1676 binding sites in
vitro. In nuclear extracts of GT17 cells treated with an NO
donor, the intensity of the Oct-1 complex is increased. However,
although Western blot analysis indicates that neither Oct-1 nor
C/EBPß protein levels are increased, the relative binding affinity of
Oct-1 is increased. Dephosphorylation of the nuclear extracts decreases
binding of the Oct-1 complex to the -1695 site only in NO
donor-treated extracts. Thus, we conclude that Oct-1 and C/EBPß are
both downstream transcriptional regulators involved in the repression
of GnRH gene expression by the glutamate/NO/cGMP signal transduction
pathway.
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INTRODUCTION
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The signal transduction pathway utilizing nitric oxide (NO) as a
second messenger in the brain has been well characterized (1, 2). NO
mediates the actions of the excitatory amino acid glutamate in the
cerebellum and hippocampus, which may be linked to the phenomena of
long term potentiation and memory (3, 4, 5). NO has also been postulated
to be an important modulator of reproductive function. Acting on the
specific hypothalamic neurons responsible for the synthesis and
secretion of GnRH, NO has been shown to stimulate GnRH secretion
(6, 7, 8). Furthermore, NO serves as the downstream signaling intermediate
for induction of GnRH secretion by the glutamate receptor agonist
N-methyl-D-aspartic acid (NMDA) (9).
Since in vivo, GnRH is found in only a small number of
neurons scattered from the preoptic to the anterior hypothalamus (10),
the GnRH-secreting hypothalamic cell line, GT17, has been invaluable
in facilitating investigations of GnRH gene expression and function.
The GT17 cells were developed using targeted oncogenesis by
introduction of a hybrid gene containing the 5'-flanking region of the
rat GnRH gene linked to the coding region of the potent oncogene, SV40
T-antigen, into transgenic mice (11). The cells are morphologically and
physiologically neuronal (11, 12, 13) and secrete GnRH in a pulsatile
manner, very similar to GnRH neurons within the hypothalamus in
vivo (14, 15, 16). NMDA and NO also stimulate GnRH secretion from GT1
cells (6, 7, 8, 9). In fact, inhibitors of NO synthases block pulsatile
release of GnRH by GT17 cells (17), implicating NO synthesis as an
obligate intermediate in the pulsatile secretion of GnRH from the
hypothalamus. Thus, NO may serve as a potential modulator of the
synchronization of GnRH release, thereby implying a broader role in the
physiological control of the hypothalamic-pituitary-gonadal axis.
Previous attempts to study the molecular mechanisms involved in the
expression of genes within discrete populations of neurons, such as the
GnRH-secreting neurons of the hypothalamus, have been difficult.
Regulation of GnRH gene expression by second messengers has been the
focus of a number of studies in the GT17 cells. GnRH gene expression
is repressed by phorbol ester through the down-regulation of protein
kinase C (13, 18, 19, 20) and by glucocorticoids (21, 22) and PRL (23). We
have shown that GnRH gene expression is repressed by NMDA, acting
through a NO, cGMP signal transduction pathway that results in
activation of cGMP-dependent protein kinase. Further, we have found
that repression of GnRH gene expression is through a linear, obligate
pathway involving NO and requiring calcium (24). Upon binding the NMDA
receptor, glutamate (or NMDA) causes an influx of extracellular
calcium, which binds calmodulin and activates nitric oxide synthase,
thus producing NO. NO then binds guanylyl cyclase, thereby increasing
cyclic GMP (cGMP) levels and activating cGMP-dependent protein kinase.
We demonstrated that this pathway was acting at the transcriptional
level since in transfections, hybrid genes containing 3 kb of the
5'-regulatory region of the rat GnRH gene linked to a reporter gene
were also down-regulated after treatment of the GT17 cells with NMDA,
sodium nitroprusside (SNP, a NO donor), or 8Br-cGMP. The region
necessary for repression of GnRH gene expression was localized to 300
bp of DNA within the 5'-regulatory region known to contain a
neuron-specific enhancer that directs GnRH gene expression to the
GT17 neuron (24).
The GnRH neuron-specific enhancer is known to bind a number of nuclear
proteins from the GT17 cells (25). Some of these protein-binding
regions are required for basal GnRH gene activity. One of two GATA
transcription factor consensus sequences within the neuron-specific
enhancer binds GATA-4 and is essential for basal GnRH gene expression
(26). Further, two POU homeodomain transcription factor consensus
sequences, AT-a and AT-b, bind the transcription factor Oct-1, which is
also essential for GnRH gene expression (27).
The mechanisms by which the glutamate/NO/cGMP pathway regulates
transcription are not yet known. Developmental control of gene
expression has generally been shown to be due to the interactions of
multiple activator proteins bound to specific cis-regulatory
regions that together specify appropriate transcription (28). In
contrast, signal transduction pathways more often act by
posttranslational modification of an individual transcription factor.
Regulatory proteins occur in families that have common DNA recognition
properties (29). Members of the POU-homeodomain family bind an octamer
motif (30, 31, 32) and regulate transcription through differential
expression of the family members (30) or perhaps through interactions
with other transcription factors (33, 34, 35, 36). Similarly, members of the
CCAAT/enhancer-binding protein (C/EBP) family of transcription factors,
which are basic region-leucine zipper proteins, are differentially
expressed (37, 38) and are capable of protein-protein interactions with
members of this family or other transcription factor families
(38, 39, 40, 41, 42).
In this study, we show that two regions, AT-b (-1695 to -1702) and
-1676 (-1676 to -1684), within the 300-bp neuron-specific enhancer
of the GnRH gene are critical for repression of GnRH by NO. These two
sequences bind GT17 nuclear proteins, individually or as a continuous
sequence encompassing both sites, and contain DNA-consensus sequences
for the POU-homeodomain and C/EBP families of transcription factors. We
find that two of the proteins bound to these regions are Oct-1 and
C/EBPß. In addition, treatment of GT17 cells with an NO donor
increases the intensity of the DNA/protein complex formed on the
oligonucleotides representing the AT-b element, although overall Oct-1
or C/EBPß protein content in the GT17 cells does not appear to be
altered. Instead, Oct-1 in nuclear extracts from GT17 cells treated
with an NO donor demonstrates increased relative binding affinity for
the AT-b site indicating a potential mechanism for regulation. That
this mechanism is likely to involve increased phosphorylation of the
Oct-1 complex after activation of the glutamate/NO/cGMP signaling
pathway is supported by our finding that dephosphorylation reduces the
binding of the Oct-1 complex in NO-treated nuclear extracts. Thus, the
repression of GnRH gene expression by NO occurs at the transcriptional
level in the GT17 neuronal cells, and the downstream effector
molecules necessary for the effects of NO include the known
transcription factors, Oct-1 and C/EBPß.
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RESULTS
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Two Elements within the GnRH Neuron-Specific Enhancer Confer
Repression by the Glutamate/NO/cGMP Pathway
We have previously identified a neuron-specific enhancer 1.6 kb
upstream of the GnRH gene essential for GnRH gene expression in GT17
cells (25). This 300-bp neuron-specific enhancer (-1863 to -1571)
contains several protein binding regions as determined by DNase I
footprinting assays and electrophoretic mobility shift assay (EMSA) and
also confers repression by the glutamate/NO/cGMP pathway (24). To study
basal enhancer activity during the characterization of the 300-bp
region, block replacement mutants were previously prepared within the
major footprints located in the enhancer and placed upstream of the
truncated (-173) GnRH promoter and the chloramphenicol acetyl
transferase (CAT) reporter gene (25). We used these reporter plasmids,
as well as some point mutations in the two well characterized AT-rich
elements (27), to determine which region(s) of the GnRH enhancer
confers NO repression of GnRH gene expression by transfecting them into
GT17 neurons and treating with SNP, a NO donor, and ionomycin, a
calcium ionophore. Treatment with SNP and ionomycin repressed reporter
gene activity from most of the mutants, reproducing the repression
observed with the intact GnRH enhancer (EN on -173 vs. EN
on -173 with SNP+ Ca++; lanes 1 and 2, Fig. 1
). Although repression was slightly less
effective for most of the mutations (averaging
65% compared with
45% for wild type), only two elements abolished repression upon
treatment with NO, the block mutation from -1684 to -1676 and m AT-b,
a double-point mutation in one of the AT-rich elements (Fig. 1
).

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Figure 1. Two Elements within the 300-bp GnRH Enhancer
(-1863 to -1571) Confer Repression by NO
GT17 cells were transiently transfected with 15 µg of one of
the CAT expression vectors containing block replacement or point
mutations within the enhancer and then treated with SNP and ionomycin
in Opti-MEM or maintained in Opti-MEM alone. Treatments were for 4
h after previous transfection of the DNA for 12 h.
Bars represent the percent of CAT activity remaining
after SNP treatment as compared with control activity of each mutant
enhancer. EN on -173 represents the wild-type enhancer on the GnRH
promoter and is normalized to 100%. With SNP + Ca++, the
activity of EN on -173 is repressed to approximately 40%. All block
replacement mutants contain at least 7 of 9 bp changes within the
region indicated (25 ) and the AT-a, AT-a flank, and AT-b are
double-point mutations (see Fig. 2 and Ref. 27). RSV-luciferase was
included as an internal control for transfection efficiency. Each value
is an average of at least three independent measurements in duplicate
or triplicate ± SEM normalized to the internal
control.
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Specific Proteins Bind the Elements Necessary for Repression by the
Glutamate/NO/cGMP Signal Transduction Pathway
The two regions found to confer repression of GnRH gene expression
by NO both bind proteins, as determined by deoxyribonuclease I (DNaseI)
footprinting (25). To further study the proteins bound to these
regions, EMSAs were performed with oligonucleotide probes representing
the AT-b and -1684/-1676 regions (termed -1676 region) (Fig. 2A
). The two elements are adjacent within
the enhancer (Fig. 2
), but these oligonucleotides do not overlap.

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Figure 2. Sequences of the GnRH Enhancer at the AT-b and
-1676 Regions and the Oligonucleotides used for EMSA Studies
A, The boxed sequences represent the AT-b and -1676
elements DNase I footprinted by GT17 nuclear proteins (25 ). The
wild-type oligonucleotides used as probes in EMSA analysis are
represented under the appropriate regions. Some oligonucleotides have a
GATC linker sequence added to each 5'-end, which is not part of the
GnRH enhancer sequence. B, The mutations within the sequence of the
GnRH enhancer and the mutant oligonucleotides are represented. The
mutated bases within the sequences are changed accordingly in
bold and indicated by an X. C, The sequences of the
consensus C/EBP and Sp1 oligonucleotides are also shown.
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Nuclear extracts were prepared from GT17 cells untreated or
treated for 1 h with NMDA, NO, or cGMP (all with ionomycin) or
ionomycin alone, since in our initial study we found that calcium was
required for the repression of GnRH gene expression (24). The DNA-protein complexes formed on the AT-b region are
represented by three differentially migrating bands, as are the
complexes formed on the -1676 region (Fig. 3
). The slowest migrating complex formed on each of the two
regions appears to be unique, while the two lower complexes comigrate,
although the sequences of the two oligonucleotides are quite
different. Interestingly, the intensity of the upper band formed with
the AT-b oligonucleotide was consistently higher with the
NMDA/NO/cGMP-treated GT17 nuclear extract, but not with
untreated or treatment with ionomycin alone (Fig. 3
,
lanes 15; Fig. 4
, lanes 12; Fig. 5A
, lanes 1 and 6; Fig. 6
, lanes 1 and 3). The
upper band with the -1676 oligonucleotide is also increased by
the treatments in this figure, but this increase is not as distinct or
reproducible (Fig. 3
, lanes 610; Fig. 4
, lanes 5 and 6; Fig. 5B
, lanes 1 and 6;
Fig. 6
, lanes 6 and 9).

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Figure 3. GnRH Enhancer AT-b and -1676 Oligonucleotide
Probes Form Unique Complexes with GT17 Cell Nuclear Proteins, Some of
Which Are Induced by Treatment of the GT17 Cells with NMDA, SNP, or
8-Br-cGMP
GT17 cells were treated with NMDA, SNP, or 8-Br-cGMP (all with
calcium ionophore ionomycin), with ionomycin alone, or not treated
(none), as indicated. EMSAs were performed with 3 µg of treated or
control GT17 nuclear extract using the AT-b (lanes 15) or -1676
(lanes 610) oligonucleotides as probes, which are described in the
legend of Fig. 2 . The arrows indicate the specific
complexes formed on the AT-b or -1676 probes.
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Figure 4. AT-b and -1676 Mutant Oligonucleotides Fail to
Bind Most of the GT17 Nuclear Protein Complexes
GT17 cells were treated with SNP and ionomycin. Nuclear protein
extracts were prepared from treated and untreated cells as described.
EMSAs were performed using 3 µg of GT17 nuclear extract ±
SNP+Ca++ treatment and the wild-type (AT-b) or mutant AT-b
(m AT-b) oligonucleotides (lanes 14) or wild-type (1676) or mutant (m
1676) -1676 oligonucleotides (lanes 58) as probes. Figure 2
describes the specific mutations found within the oligonucleotides. The
arrows indicate the complexes formed on the probes.
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Figure 5. AT-b, -1676, Consensus C/EBP, and Consensus Sp1
Oligonucleotides Compete for Binding of the Protein Complexes Formed on
the AT-b, -1676, and Consensus Sp1 Probes
EMSAs were performed with a labeled oligonucleotide containing the AT-b
(panel A), -1676 (panel B), or consensus Sp1 sequences (panel C) and 3
µg of GT17 nuclear extract ± SNP + Ca++ treatment
as indicated. All competitor lanes represent 100-fold (AT-b or -1676)
or 10-fold (consensus Sp1 or consensus C/EBP) molar excess of unlabeled
oligonucleotide. Formation of the upper complex on the AT-b probe was
eliminated by the AT-b competitor alone, while the middle complex on
the AT-b probe was competed by the unlabeled -1676 or the consensus
Sp1 competitors. The upper complex on the -1676 probe was eliminated
by the unlabeled -1676 oligonucleotide or by the consensus C/EBP
competitor, while the middle complex was competed by both AT-b and
consensus Sp1 oligonucleotides. The single complex formed on the
consensus Sp1 probe was competed by the unlabeled consensus Sp1 or
-1676 oligonucleotides. None of the excess unlabeled oligonucleotides
were able to eliminate the lower complex formed on the AT-b or -1676
probes. Arrows indicate the specific complexes formed on
the probes.
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Figure 6. Oct-1 and C/EBPß Antibodies Supershift the Upper
Complexes Formed on the AT-b and -1676 Oligonucleotide Probes,
Respectively
EMSAs were performed with labeled oligonucleotides representing the
AT-b (lanes 15) or -1676 (lanes 612) sequences and 3 µg of
nuclear extract from GT17 cells ± SNP + Ca++
treatment as indicated. Specific antibodies toward Oct-1 (lanes 2 and
4), C/EBPß (lanes 7 and 10), or Sp1 (lanes 8 and 11) were used, while
an equivalent amount of purified normal rabbit IgG was used as a
negative control (lanes 5 and 12). The uppermost arrow
indicates the supershifted complex with either probe.
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Oligonucleotides were also prepared containing the same mutations
used to map the regions necessary for NO-mediated repression of GnRH
gene expression (Fig. 2B
). The bottom strand of the AT-b region
contains an octamer motif (ATTAAAAT, matches 6 of 8 bases) that may
contain a putative POU-homeodomain transcription factor binding site
(43), while the upper strand of the -1676 region contains a putative
C/EBP consensus sequence (TGAAGCAAT, matches 9 of 9 bases) (44). The
mutant oligonucleotides incorporate base changes that specifically
eliminate the POU homeodomain and C/EBP consensus binding sequences.
Using these oligonucleotides as probes for EMSA analysis, we found that
no DNA/protein complexes were formed on the AT-b double point mutant,
while only the middle complex remained with the -1676 block
replacement mutant oligonucleotide (Fig. 4
). These results confirm that
the AT-b and -1676 regions bind proteins specifically and if mutated,
with the same base changes as the block replacement mutants, protein
binding is not tolerated with the exception of the middle band on the
-1676 probe.
To determine the specificity of protein binding, competition studies
were performed with each of the two regions. The upper and middle
complexes formed on the AT-b oligonucleotide are dramatically reduced
when a 100-fold molar excess of unlabeled AT-b oligonucleotide is added
to the EMSA reaction mixture, indicating that these two complexes
represent a specific interaction between protein and the AT-b sequence
(Fig. 5A
, lane 2). Similarly, the upper and middle complexes formed on
the -1676 oligonucleotide are competed by inclusion of a 100-fold
molar excess of unlabeled -1676 oligonucleotide (Fig. 5B
, lane 2). The
fastest migrating complex is not competed by any of the competitors
used with either of the oligonucleotides, indicating that it is a
nonspecific DNA/protein complex. Further, the upper complex formed with
the -1676 oligonucleotide is also eliminated by a 10-fold molar excess
of C/EBP consensus oligonucleotide (Fig. 2C
), indicating that the
protein(s) bound at this complex are also capable of binding the C/EBP
consensus sequence (Fig. 5B
, lane 5). When the C/EBP consensus sequence
is used as a labeled probe, the single band migrates to the same
position as the -1676 upper complex (data not shown).
The middle complex on AT-b is completely abolished with a
100-fold molar excess of the AT-boligonucleotide, while it is also
dramatically decreased by a 100-fold molar excess of the -1676 and
10-fold excess of Sp1 consensus oligonucleotides (Fig. 5A
, lanes 24).
Conversely, the middle complex formed on the -1676 oligonucleotide is
competed by a 100-fold molar excess of cold -1676, AT-b, and 10-fold
Sp1 consensus oligonucleotides (Fig. 5B
, lanes 24). The Sp1 consensus
oligonucleotide was originally used as an unrelated DNA sequence
control probe since there is no discernible homology to either element
(Fig. 2C
), until it was noted that the complex formed with GT17
nuclear extract migrated to the same level as the middle complexes of
the AT-b and -1676 oligonucleotides. When using the Sp1 consensus
sequence as the labeled oligonucleotide, one complex is formed and
migrates at the same position as the middle band in both the AT-b and
-1676 oligonucleotides (Fig. 5C
). Furthermore, a 10-fold molar excess
of unlabeled Sp1 consensus sequence and 100-fold -1676 oligonucleotide
abolish the appearance of this specific complex, but the complex is not
competed by 10-fold molar excess of C/EBP consensus sequence (Fig. 5C
, lanes 24). This indicates that the middle complex may be formed by
the binding of similar protein(s) on both the AT-b and the -1676
probes. This middle protein complex is not seen when using the
unrelated GATA binding motif of the human preproendothelin 1 (PPET)
gene as a labeled control probe (data not shown) (45).
The Transcription Factor, Oct-1, Binds the AT-b Element, While the
CCAAT/Enhancer Binding Protein (C/EBP) ß Binds to the -1676 Element
of the GnRH Neuron-Specific Enhancer
The identity of the proteins bound to the two elements required
for NO repression was determined by antibody supershift assays. The
consensus binding sites within the regions indicated which antibodies
might be used to determine the identity of the member(s) of the two
transcription factor families binding to these regions. Previous
studies in our laboratory indicated that Oct-1, a widely expressed
POU-homeodomain protein, bound to both the AT-b and AT-a regions (27).
AT-b and AT-a are similar elements within the GnRH enhancer containing
a POU-homeodomain consensus octamer sequence. When the Oct-1 antibody
was used with the SNP-treated GT17 nuclear extract, a supershifted
complex appeared, similar to that seen with the nuclear extract from
untreated GT17 cells (Fig. 6
, lanes 2 and 4). Changing the EMSA
conditions did not completely supershift the upper complex, indicating
that another protein may be present in the complex that cannot
supershift to the larger complex.
Analysis of the C/EBP consensus sequence present within the -1676
region of the GnRH enhancer revealed a 100% match to a C/EBPß
binding site. For this reason, we chose the C/EBPß antibody to use in
the supershift analysis of this complex. A supershifted complex was
seen with nuclear extract preparations from both SNP-treated and
untreated GT17 cells (Fig. 6
, lanes 7 and 10). Consistently, with
different nuclear extracts and incubation conditions, the untreated
cell nuclear extract preparation yielded a stronger supershift with the
C/EBPß antibody, indicating that the protein complexes formed on the
-1676 region with the nuclear extract from NO-treated and untreated
GT17 cells may differ.
Finally, a Sp1 antibody did not supershift the protein complexes formed
with any of the GT17 nuclear extracts (Fig. 6
, lanes 8 and 11),
although we did see a supershift using HeLa cell nuclear extract (data
not shown). These results imply that although a protein capable of
binding the Sp1 consensus oligonucleotide may be present in the middle
complex, Sp1 itself does not appear to be present in this complex, or
may not be capable of forming a supershift when it is part of the
middle complex, due to hindrance at the antigenic site. However, no Sp1
supershift was produced using the Sp1 consensus oligonucleotide as a
probe with the GT17 nuclear extract (data not shown), suggesting that
Sp1 may not be present in the GT17 cell nuclear extract. Rabbit IgG
was used as a nonspecific antibody control and showed no supershifts
with any of the nuclear extracts (Fig. 6
, lanes 5 and 12).
Oct-1 and C/EBPß Bind Simultaneously to the Sequence Encompassing
Their Adjacent Sites
A 34-bp oligonucleotide (AT-1676) that includes both the AT-b and
-1676 binding sites was used to determine whether the Oct-1 and
C/EBPß proteins can bind to the adjacent sites simultaneously (Fig. 2A
). EMSA analysis with the AT-1676 oligonucleotide produced a complex
that migrated more slowly than the three complexes previously detected
binding at either the AT-b or -1676 sites alone (Fig. 7
). All four complexes were eliminated by
competition with 100-fold molar excess of the AT-1676 oligonucleotide
(Fig. 7B
, lane 3). Interestingly, the intensity of the uppermost
complex formed on the AT-1676 oligonucleotide appears to be increased
with the SNP-treated nuclear extract (Figs. 7A
and 7B
, lane 2;
representing nuclear extracts from two separate experiments) when
compared with the control nuclear extract (Figs. 7A
and 7B
, lane
1).

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Figure 7. Oct-1 and C/EBPß Bind Simultaneously to the
Adjacent AT-b and -1676 Elements
EMSAs were performed with a labeled oligonucleotide representing the
enhancer sequence encompassing both the AT-b and -1676 sequences
(AT-1676, panel A, lanes 1 and 2 and panel B, lanes 18), or
oligonucleotides with the AT-b site mutated (AT-1676MAT, panel B, lanes
9 and 10) or the -1676 site mutated (AT-1676M76, panel B, lanes 11 and
12), illustrated in Fig. 2 , with 3 µg of nuclear extract from GT17
cells ± SNP + Ca++ treatment. GT17 nuclear
extracts, from separate experiments (A and B) representing control (A
and B, lane 1) or SNP-treated (panel A, lane 2, and panel B, lanes
212) cells, were subjected to EMSA analysis. All competitor lanes
represent 100-fold molar excess of the unlabeled oligonucleotides,
AT-1676 (panel B, lane 3), AT-b (panel B, lane 4), or -1676 (panel B,
lane 5). Specific antibodies against Oct-1 (panel B, lane 6) or
C/EBPß (panel B, lane 7) were used to perform a supershift analysis
of the composite AT-1676 oligonucleotide, while an equivalent amount of
purified normal rabbit IgG was used as a negative control (panel B,
lane 8). Mutation of the AT-b site within the AT-1676 oligonucleotide
was used alone (panel B, lane 9) or with 100-fold molar excess of the
unlabeled -1676 oligonucleotide, and mutation of the -1676 site
within the AT-1676 oligonucleotide was used alone (panel B, lane 11) or
with 100-fold molar excess of the unlabeled AT-b oligonucleotide (panel
B, lane 12), in EMSA analysis. The uppermost band,
indicated with an arrow, represents the binding of both
the Oct-1 and C/EBPß protein complexes.
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The proteins bound at the uppermost complex were studied by EMSA
analysis, using the oligonucleotides representing the individual
binding sites and the antibodies against both Oct-1 and C/EBPß. Both
the AT-b and -1676 oligonucleotides, at 100-fold molar excess,
eliminated the uppermost complex (Fig. 7B
, lanes 4 and 5). The Oct-1
and C/EBPß antibodies supershifted the uppermost complex (Fig. 7B
, lanes 6 and 7, respectively), but IgG alone had no effect (Fig. 7B
, lane 8). The C/EBPß supershift was weak and required a longer
exposure of the EMSA gel to be visualized (data not shown). Mutation of
either the AT-b or the -1676 site within the AT-1676 oligonucleotide
(Fig. 2B
) abolished the appearance of the uppermost complex, although
binding at the remaining nonmutated element remained [Fig. 7B
, lanes 9 (AT-1676MAT) and 11 (AT-1676M76)]. Competition analysis
with 100-fold molar excess of the AT-b or -1676 oligonucleotide shows
that the lower three complexes are the same as those described for the
individual oligonucleotide probes (Fig. 7B
, lanes 10 and 12). These
results indicate that the protein complexes are also able to bind the
AT-1676 oligonucleotide individually.
Neither Oct-1 nor C/EBPß Protein Is Induced in the Nuclear
Protein Extracts from SNP-Treated GT17 Cells
The upper complex binding the AT-b oligonucleotide appears to be
increased in nuclear extract treated with NMDA, SNP, or cGMP (with
calcium) and similarly the upper complex binding the combined AT-1676
oligonucleotide appears induced, while induction of the binding on the
-1676 probe is variable. Therefore, we determined whether the
concentrations of these proteins were increased in the SNP-treated
nuclear extract by Western blot analysis. Utilizing the same antibodies
as for the antibody supershift analysis, we determined that the nuclear
extract from the GT17 cells treated with SNP and ionomycin did not
contain more Oct-1 or C/EBPß protein than did the untreated GT17
nuclear extract (Fig. 8
). Using Western
blot analysis, Oct-1 and C/EBPß antibodies produced bands of the
appropriate sizes (37). This finding indicates that although the band
intensity is increased in the upper bands with both the AT-b and -1676
oligonucleotides, the increase in band intensity cannot be accounted
for by an increased amount of Oct-1 or C/EBPß in the nuclear extract
from GT17 cells treated with SNP.

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Figure 8. Neither Oct-1 nor C/EBPß Protein Levels Are
Increased in Nuclear Extracts after NO Treatment of GT17 Cells
Nuclear extracts from GT17 cells ± SNP + Ca++
treatment were run on a 12% polyacrylamide gel and Western blotted
onto Immobilon-P membranes. Specific antibodies toward Oct-1 and
C/EBPß, the same as those used in EMSA supershift analysis, were used
as the primary antibody. The secondary antibody was
horseradish-peroxidase. The bands were visualized using enhanced
chemiluminescence. A specific band of approximately 100 kDa is seen
with the Oct-1 antibody (lanes 1 and 2), while a major band of
approximately 46 kDa and a 29-kDa minor band are seen with the C/EBPß
antibody (lanes 3 and 4).
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Oct-1 in Nuclear Extracts from GT17 Cells Treated with SNP Has
Increased Relative Binding Affinity for the AT-b Region in the GnRH
Neuron-Specific Enhancer Due to Increased Phosphorylation
Since neither Oct-1 nor C/EBPß protein levels are induced in
SNP-treated GT17 nuclear extracts by Western blot analysis, it is
possible that the binding affinity of the protein(s) for the
oligonucleotide has changed. To test this hypothesis, a competition
analysis was performed with increasing amount of unlabeled AT-b
oligonucleotide. The amounts of oligonucleotide competitor ranged from
30 pg to 10 ng DNA. The relative intensity of the uppermost band (which
represents the Oct-1 protein complex), was plotted vs. the
amount of competitor using a logarithmic scale. In three separate
nuclear extracts prepared from GT17 cells treated with SNP and
ionomycin, the IC50 for the AT-b oligonucleotide
changes by approximately 4- to 10-fold (Fig. 9
, representative graph) as determined by
the 50% displacement values. Because the increase in the upper complex
formed on the -1676 oligonucleotide was much less dramatic in EMSA
analysis, this same study was not possible using this
oligo-nucleotide.
To determine whether activation of the cGMP-dependent protein kinase
signaling pathway resulted in phosphorylation of proteins in the Oct-1
complex, we observed the DNA binding of Oct-1 after phosphatase
treatment of the nuclear extracts. Dephosphorylation of the SNP-treated
and control nuclear extracts with increasing concentrations of calf
intestinal alkaline phosphatase (CIP) was performed to determine
whether phosphorylation of nuclear proteins might be responsible for
the increased binding of the Oct-1 complex to the AT-b site. After
preincubation with CIP, but before the EMSA analysis, extracts were
treated with a phosphatase inhibitor cocktail to avoid
dephosphorylation of the labeled oligonucleotide probe. The efficiency
of the phosphatase inhibitor cocktail was determined by coincubation
with CIP before EMSA analysis. EMSAs using SNP-treated nuclear extract
demonstrated a decrease in DNA-binding activity to the AT-b element
upon increasing concentration of CIP, while binding to the AT-b
oligonucleotide by the CIP-treated control extract remained stable
(Fig. 10
). Thus, it is possible that
upon NO activation of the signaling pathway involving cGMP-dependent
protein kinase, the Oct-1 complex undergoes increased phosphorylation
resulting in increased DNA binding activity at the AT-b element.

View larger version (41K):
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|
Figure 10. Evidence that Phosphorylation Regulates the DNA
Binding Ability of the Oct-1 Complex at the AT-b Element from GT17
Cells Treated with SNP
EMSA was performed with labeled oligonucleotide probe representing the
AT-b element comparing 5 µg of nuclear extract from GT17 cells
± SNP + Ca++, treated with increasing concentrations of
CIP. Nuclear extracts were preincubated with CIP, followed by
incubation of the nuclear extracts with a phosphatase inhibitor
cocktail (50 mM sodium fluoride and 1 mM sodium
orthovanadate) to avoid dephosphorylation of the labeled probe. The
efficiency of the phosphatase inhibitor mix was determined by
coincubation with CIP (lane 6).
|
|
 |
DISCUSSION
|
---|
The molecular mechanisms involved in the physiological action of
NO on gene expression are not yet understood. Remarkably, only two
neuron-specific genes have been shown to be regulated by NO:
microtubule-associated protein 2 in hippocampal granule cells (46) and
GnRH in the GT17 hypothalamic neurons (24). In this study we have
defined two specific regions within the 5'-flanking region of the GnRH
gene that are critical for repression of GnRH gene expression, and we
find that the transcription factors binding these regions are members
of the POU-homeodomain and basic region-leucine zipper
transcription factor families.
We have localized two elements within the well characterized 300-bp
neuron-specific enhancer of the GnRH gene (25, 26, 47) that are each
required for the repression of GnRH gene expression through the
glutamate/NO/cGMP pathway. These regions lie adjacent within the
enhancer (the entire responsive region encompasses only 34 bp,
-1704/-1671), implying that protein-protein interactions may be
necessary for NO-mediated repression of gene regulation. Two specific
complexes are formed on each of the two important elements. One of
these protein complexes appears to be common to both the AT-b and
-1676 oligonucleotides, despite the fact that the only similarities
between the two sequences are the GATC linkers at the 5'-ends of the
oligonucleotides (Fig. 2
). The 34-bp sequence encompassing both the
AT-b and -1676 regions does not contain a GC-rich Sp1 consensus
sequence (42, 48), nevertheless a protein(s) capable of binding to both
of the oligonucleotides representing this region is also capable of
binding a GC-rich Sp1 consensus oligonucleotide in EMSA. This complex
is not Sp1 itself, however, since Sp1 cannot be identified in GT17
nuclear extracts. Interestingly, this complex is present in the EMSA
analysis utilizing the composite oligonucleotide AT-1676, which does
not contain any GATC linkers. Further studies will be undertaken to
determine the identity of the proteins in this complex. While one of
the mutations used in the functional analysis (m AT-b) did not bind
this common middle complex (although it contains the same GATC overhang
as its wild-type counterpart, AT-b), the block mutation at -1676 did
not eliminate this complex in EMSA even though it does prevent NO
repression. Thus, the binding of this protein is not likely to be
involved in NO repression since it remains bound in the presence of the
-1676 block mutation that eliminates repression.
The upper complex binding specifically to the AT-b oligonucleotide is
supershifted by antibodies specific for Oct-1. Oct-1 was previously
found to bind both the AT-a and AT-b regions of the GnRH
neuron-specific enhancer, but only the AT-a region was thought to be
important for GnRH enhancer activity since a block replacement mutant
in the AT-b region did not affect GnRH gene expression (27). In this
study, we prepared a mutation within the AT-b region which specifically
altered the critical two bases within the octamer consensus region
required for binding POU-homeodomain proteins such as Oct-1 (Fig. 2
).
This specific mutation prevented NO repression. Oct-1 is expressed in
several cell types (47, 49) and is known to interact with a number of
other DNA-binding proteins (33, 34, 35, 36, 50, 51), basal transcription
factors (52, 53), or with tissue-specific coactivators (54, 55).
Therefore, it has been postulated that Oct-1 may confer tissue-specific
gene expression through its interaction with other transcription
factors specifically expressed in a particular cell type or by complex
interactions with coactivators. The ability of Oct-1 to participate in
NO repression may also be due to its interaction with such
proteins.
The upper complex binding specifically to the -1676 oligonucleotide is
supershifted by antibodies specific for C/EBPß. Although published
C/EBP consensus sequences differ slightly (37, 42, 44), a comparison of
our binding site at -1676 shows a 100% match to a consensus sequence
generated by comparison of a number of C/EBP binding sites (44, 56).
The block replacement mutant -1684/-1676 changed the bases within the
C/EBP consensus sequence required to bind C/EBP proteins resulting in
the disappearance of the upper protein complex with the -1676
oligonucleotide in EMSA (Fig. 4
). C/EBPß is a member of the basic
region-leucine zipper family that controls transcription of a number of
genes through protein-protein interactions at the gene level. In
particular, C/EBPß can not only bind to its own family members such
as C/EBP proteins (38), fos, and jun (57), but also forms complexes
with other transcription factors such as nuclear factor-
B
(40, 41), estrogen receptor (41), and an Sp1 factor (42). Furthermore,
similar to Oct-1, C/EBPß may also alter transcription by complex
interactions with coactivators and basal transcription factors such as
TFIIB and p300 (58, 59).
C/EBPß has been previously shown to be induced by stimulation of the
glutamate pathway in rat cortical astrocytes in culture. This induction
is calcium and calmodulin dependent and is maximal (1.4-fold) after a
1-h treatment (60). However, the role of NO in this signaling was not
investigated.
The NO-stimulated repression of GnRH by Oct-1 and C/EBPß may
involve complex interactions between these two proteins, with other
transcription factors, the basal transcriptional machinery, and/or
coactivators. Our data indicate that the two protein complexes bind the
34-bp region of the enhancer simultaneously. The distance from the
center of the consensus site for Oct-1 to the center of the C/EBP
consensus site is 20 bp. Oct-1 is known to bind to the major groove
(61, 62), and indications are that C/EBP also binds to the major groove
(63). Therefore, the two proteins bind to the same side of the DNA
helix just one turn apart. In fact, the requirement for both binding
sites to be intact for repression to occur implies that they may
coordinate to bind a third protein together that is involved in the
response.
Our studies indicate that the binding of the GT17 nuclear
protein complex to the AT-b region, and perhaps to the C/EBP region, is
increased in extracts harvested after treatment of the GT17 cells
with SNP and ionomycin. Using Western blot analysis, we did not detect
an increase in the total protein levels of either Oct-1 or C/EBPß
using specific antibodies for these two proteins. Since the
concentration of protein is not changed within NO-stimulated cells,
then the affinity of the Oct-1 protein (and perhaps C/EBPß) for DNA
might be changed through a posttranslational modification, as has been
seen in other systems (64). Studies of GT17 nuclear extracts in EMSA
analysis, with or without SNP treatment, indicate that this is the case
for Oct-1. By increasing the amount of cold oligonucleotide in EMSA
competition studies (65), we demonstrate that the relative binding
affinity of Oct-1 to the AT-b region is increased 4- to 10-fold after
treatment with NO. Furthermore, dephosphorylation of the nuclear
extract shows that this increase is related to the phosphorylation
state of the proteins.
We chose to study the regions responsible for repression of GnRH gene
expression after treatment of the GT17 cells with NO. Nevertheless,
it appears that the protein binding at the AT-b and -1676 regions are
common to all three components of the glutamate/NO/cGMP signal
transduction pathway. After treatment with NMDA, SNP, or 8Br-cGMP (all
in the presence of the calcium ionophore ionomycin), we detect the same
protein complexes, and the Oct-1 complex appears to be increased by
each of the three treatments (see Fig. 3
).
One of the known effects of the elevation of cGMP is activation
of cGMP-dependent protein kinase (66). Thus, it is possible that
posttranslational modification of the proteins bound to these two
regions may be due to direct phosphorylation by cGMP-dependent protein
kinase, since repression of GnRH gene expression requires the action of
this kinase (24). Presently, very few substrates for this kinase have
been reported, and none of the known substrates to date are
transcription factors (66). However, a few as-yet-unidentified specific
substrates of cGMP-dependent protein kinase have been found in rat
brain (67). There are a number of examples in which the direct
phosphorylation of a protein alters its ability to bind DNA (68). Both
Oct-1 (68) and C/EBP family members (69) exhibit changes in DNA-binding
affinity, depending upon their phosphorylation state. Dephosphorylation
of C/EBP
severely inhibits its DNA binding activity on the serum
amyloid A promoter in liver (70). Interestingly it has also been
postulated that the DNA specificity of Oct-1 can be regulated by the
action of different kinases (71). As cells enter mitosis, Oct-1 is
hyperphosphorylated by protein kinase A, which results in the
inhibition of Oct-1 DNA binding activity (72). We can speculate that
depending upon the cell type and the available transcription factors,
Oct-1 phosphorylation may result in a differential effect on
DNA-binding activity. For instance, in GT17 cells, cGMP-dependent
protein kinase activation may result in increased phosphorylation of
Oct-1, causing the enhanced binding affinity of the Oct-1 complex to
the AT-b element. Alternatively, it is possible that another protein,
required as a corepressor, is recruited to the enhancer after a change
in phosphorylation due to activation of the signaling cascade. Whether
cGMP-dependent protein kinase or another protein kinase phosphorylates
either Oct-1 or C/EBPß in response to the activation of the
glutamate, NO, cGMP signal transduction pathway in the GT17
hypothalamic neurons is yet another question that remains to be
answered. Although at present we cannot say how an apparent increase in
DNA-binding activity might cause repression, this study demonstrates
that transcriptional repression of the GnRH gene by the NMDA/NO/cGMP
pathway depends on binding of the transcription factors Oct-1 and
C/EBPß and indicates a likely mechanism by which NO exerts its effect
on gene expression.
 |
MATERIALS AND METHODS
|
---|
Cell Culture and Reagents
GT17 cells were grown in DMEM (Life Technologies, Inc., Gaithersburg, MD) supplemented with 10% FBS, 4.5 mg/ml
glucose, and penicillin/streptomycin and maintained in an atmosphere
with 5% CO2 as described (11). Medium was
changed to Opti MEM (Life Technologies, Inc.), a
serum-free medium, before the treatments. NMDA (used at 500
µM), SNP (used at 50 µM), 8-bromo-cGMP
(8-Br-cGMP; used at 250 µM), and ionomycin, (used at 0.5
µM), were obtained from Sigma (St. Louis,
MO). Oligonucleotides were prepared by Operon Technologies, Inc. (Los Angeles, CA). Oct-1, C/EBPß, and Sp1
antibodies and consensus oligonucleotides were obtained from
Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).
Plasmid Constructions and Transfections
The plasmids containing the GnRH neuron-specific enhancer
upstream of the GnRH promoter (termed EN on -173) were created by
inserting the enhancer (-1863 to -1571 of the GnRH gene) in reverse
orientation into the polylinker upstream of the promoter in -173
GnRH-CAT as was previously described (25). The block replacement
mutants were constructed as previously described (25) or by designing
specific mutations using two homologous PCR primers each containing a
9-bp BamHI linker site (-1606/-1598, -1646/-1638,
-1659/-1651, -1684/-1676) or specific 2-bp changes (m AT-b, m AT-a,
and m AT-a flank). Briefly, two PCR products were generated using
flanking primers within the vector encompassing the GnRH
neuron-specific enhancer in combination with one of the homologous
mutation-containing primers, isolated, purified, and then used as
template to create a single PCR product with only the flanking vector
primers. This PCR product containing the GnRH enhancer block
replacement mutation was then cloned into the -173 GnRH-CAT plasmid
using the upstream polylinker. All plasmids were sequenced by the
dideoxy-chain termination method (73) in the presence of
[32P]dATP, 3000 Ci/mmol (New England
Nuclear, Boston, MA) and Sequenase (United States Biochemical Corp., Cleveland, OH). Transfections were performed using the
calcium phosphate precipitate method (74) containing 15 µg of plasmid
DNA and 5 µg of the internal control plasmid RSV (Rous sarcoma
virus)-luciferase (75). The cells were incubated for 1214 h
with DNA, followed by three PBS rinses, and then treated with SNP and
ionomycin in Opti MEM for 46 h before harvesting. Protein extracts
were prepared by freeze-thawing as described previously (76), and
protein concentrations were determined using the Coomasie blue
procedure (77). CAT (78) and luciferase assays (75) were done as
previously described.
Nuclear Extract Preparation and EMSA Analysis
Nuclear extracts were prepared following the method of Lee
et al. (79). After protein concentration was determined
using the Coomasie blue procedure (77), the extracts were stored frozen
at -75 C. EMSA and supershift assays were performed as previously
described (27), except EMSA reaction mixes were incubated 10 min at
room temperature with all reagents except the probe and then another 15
min or 30 min (AT-1676 oligonucleotides) at room temperature after
addition of 20,000 cpm of the labeled oligonucleotide. The
oligonucleotide sequences shown in Fig. 2
had a GATC linker added to
each 5'-end that were filled in using Klenow fragment both for labeled
probes, in the presence of
[32P]dATP, 3000
Ci/mmol (New England Nuclear, Boston, MA) or, in the case of competitor
oligonucleotides, with cold deoxynucleoside triphosphates.
Consensus C/EBP or Sp1 oligonucleotides were radiolabeled by incubation
of T4 polynucleotide kinase with
[32P]dATP
(6000 Ci/mmol; New England Nuclear, Boston, MA). All labeled probes
were gel purified by 6% polyacrylamide gel electrophoresis, crushed,
soaked overnight, and phenol-chloroform extracted, and the amount of
radioactivity was determined. Probes were diluted in 25
mM KCl. Competition studies were done by adding a
specified amount (10- to 100-fold, as indicated) of unlabeled
oligonucleotide to the reaction mix 15 min before the addition of
labeled oligonucleotide. Supershift assays were performed exactly as
the EMSA analysis except 1 µl of Oct-1, C/EBPß, or Sp1 antibody
(obtained from Santa Cruz Biotechnology, Inc.) was added
after the initial reaction and incubated for 45 min at room temperature
before loading onto the gel. The reactions were electrophoresed on a
30-min prerun 5% polyacrylamide gel in 0.25 x TBE for 3.5 h
at 175 V. After electrophoresis, the gels were dried and exposed to
Kodak XAR-5 film for autoradiography.
For the Oct-1 DNA binding analysis, the same nuclear protein extracts
prepared for the EMSA analysis described above were used. Three nuclear
extract preparations from treatments of GT17 cells in separate
experiments were used for the relative binding affinity analysis. EMSA
analysis was performed exactly as previously described except
increasing concentrations of unlabeled AT-b oligonucleotide were
incubated with the reaction mixture 15 min before the labeled AT-b
probe (20,000 cpm) was added. Concentrations of unlabeled AT-b
oligonucleotide ranged from 30 pg to 10 ng DNA. The polyacrylamide gels
were dried and exposed to an intensifying screen. Quantification of the
uppermost complex on the AT-b probe, corresponding to Oct-1 binding,
was done using a phosphor imaging system and the ImageQuant software
(Molecular Dynamics, Inc., Sunnyvale, CA). Relative band
intensity was plotted against the amount of oligonucleotide competitor
on a logarithmic scale. Change in relative binding affinity was
determined by comparing the concentration of unlabeled oligonucleotide
required to displace 50% of the Oct-1 protein bound to the AT-b probe.
Loading efficiency was controlled by normalization to the lower
nonspecific band on the EMSA gels.
Phosphorylation analysis was performed using a previously described
method (70), with modifications. Dephosphorylated nuclear extracts were
prepared by incubating 5 µg of SNP-treated and untreated nuclear
extracts with increasing concentrations of calf intestinal alkaline
phosphatase (0.010.5 U, CIP, Roche Molecular Biochemicals, Indianapolis, IN) at room temperature for
30 min. In some experiments, in addition to the phosphatase, nuclear
extracts were incubated in the presence of a combination of phosphatase
inhibitors (50 mM sodium fluoride and 1 mM
sodium orthovanadate). The nuclear extracts were then subjected to EMSA
analysis, as described above, after incubation with the phosphatase
inhibitor cocktail for 10 min at room temperature to avoid
dephosphorylation of the labeled probe.
Western Blotting
GT17 nuclear extract prepared as described above was also used
for Western analysis. Proteins (50 µg) were separated on an 12%
SDS-PAGE gel and transferred to Immobilon-P membranes (Millipore Corp., Bedford, MA). The membranes were incubated with antisera
to Oct-1 and C/EBPß (Santa Cruz Biotechnology, Inc.) in
1 x PBS with 5% nonfat dry milk and 0.001% sodium azide
overnight at room temperature with gentle shaking. Membranes were
washed sequentially with PBS, 5% non-fat dry milk, and 0.03% Tween-20
for 4 x 30 min without shaking. Membranes were then incubated
with horseradish peroxidase-labeled secondary antisera from rabbit for
36 h at room temperature and then washed with PBS containing 0.1%
Tween-20 as described above. The immunoreactive bands were visualized
by enhanced chemiluminescence (ECL, Amersham Pharmacia Biotech) as described by the manufacturer.
 |
ACKNOWLEDGMENTS
|
---|
We thank Teri Banks and Megan Houseweart for assistance in
cloning some block replacement mutant plasmids and Simon Lee and
Jocelyn Jovenal for excellent technical support. We are grateful to
Melody Clark, Mark Lawson, Shelley Nelson, and Jennifer Taylor for
critical reading of the manuscript. We also thank the members of the
Mellon laboratory for helpful discussions.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Pamela L. Mellon, Reproductive Medicine, 0674, University of California, San Diego, 9500 Gilman Drive, La Jolla, California 92093.
D.D.B. was supported by a fellowship from the Medical Research Council
of Canada. This research was supported by a grant from NIH to P.L.M.
(DK-44838).
1 Current address: Department of Physiology/Division of Reproductive
Science, University of Toronto, Toronto Hospital Research Institute,
200 Elizabeth Street,Toronto, Ontario, Canada M5G 2C4. 
Received for publication November 5, 1998.
Revision received October 18, 1999.
Accepted for publication November 8, 1999.
 |
REFERENCES
|
---|
-
Bredt DS, Snyder SH 1992 Nitric oxide, a novel neuronal
messenger. Neuron 8:311[Medline]
-
Garthwaite J 1991 Glutamate, nitric oxide and cell-cell
signalling in the nervous system. Trends Neurosci 14:6067[CrossRef][Medline]
-
Bliss TVP, Collingridge GL 1993 A synaptic model of memory:
long-term potentiation in the hippocampus. Nature 361:3139[CrossRef][Medline]
-
ODell TJ, Huang PL, Dawson TM, Dinerman JL, Snyder SH,
Kandel ER, Fishman MC 1994 Endothelial NOS and the blockade of LTP by
NOS inhibitors in mice lacking neuronal NOS. Science 265:542546[Medline]
-
Zhuo M, Hu Y, Schultz C, Kandel ER, Hawkins RD 1994 Role of
guanylyl cyclase and cGMP-dependent protein kinase in long-term
potentiation. Nature 368:635639[CrossRef][Medline]
-
Bonavera JJ, Sahu A, Kalra PS, Kalra SP 1993 Evidence that
nitric oxide may mediate the ovarian steroid-induced luteinizing
hormone surge: involvement of excitatory amino acids. Endocrinology 133:24812487[Abstract]
-
Moretto M, Lopez FJ, Negro-Vilar A 1993 Nitric oxide
regulates luteinizing hormone-releasing hormone secretion.
Endocrinology 133:23992402[Abstract]
-
Rettori V, Belova N, Dees WL, Nyberg CL, Gimeno M, McCann SM 1993 Role of nitric oxide in the control of luteinizing
hormone-releasing hormone release in vivo and in
vitro. Proc Natl Acad Sci USA 90:1013010134[Abstract]
-
Mahachoklertwattana P, Black SM, Kaplan SL, Bristow JD,
Grumbach MM 1994 Nitric oxide synthesized by gonadotropin-releasing
hormone neurons is a mediator of N-methyl-D-aspartate
(NMDA)-induced GnRH secretion. Endocrinology 135:17091712[Abstract]
-
Merchenthaler I, Setalo G, Csontos C, Petrusz P, Flerko B,
Negro-Vilar A 1989 Combined retrograde tracing and immunocytochemical
identification of luteinizing hormone-releasing hormone- and
somatostatin-containing neurons projecting to the median eminence of
the rat. Endocrinology 125:28122821[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]
-
Liposits Z, Merchenthaler I, Wetsel WC, Reid JJ, Mellon PL,
Weiner RI, Negro-Vilar A 1991 Morphological characterization of
immortalized hypothalamic neurons synthesizing luteinizing
hormone-releasing hormone. Endocrinology 129:15751583[Abstract]
-
Wetsel WC, Eraly SA, Whyte DB, Mellon PL 1993 Regulation of
gonadotropin-releasing hormone by protein kinases A and C in
immortalized hypothalamic neurons. Endocrinology 132:23602370[Abstract]
-
Wetsel WC, Valença MM, Merchenthaler I, Liposits Z,
L-pez 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]
-
Martinez de la Escalera G, Choi ALH, Weiner RI 1992 Generation
and synchronization of gonadotropin-releasing hormone (GnRH) pulses:
intrinsic properties of the GT11 GnRH neuronal cell line. Proc Natl
Acad Sci USA 89:18521855[Abstract]
-
Krsmanovic LZ, Stojilkovic SS, Merelli F, Dufour SM, Virmani
MA, Catt KJ 1992 Calcium signaling and episodic secretion of
gonadotropin-releasing hormone in hypothalamic neurons. Proc Natl Acad
Sci USA 89:84628466[Abstract]
-
López FJ, Moretto M, Merchenthaler I, Negro-Vilar A 1997 Nitric oxide is involved in the genesis of pulsatile LHRH secretion
from immortalized LHRH neurons. J Neuroendocrinol 9:647654[Medline]
-
Bruder JM, Drebs WD, Nett TM, Wierman ME 1992 Phorbol ester
activation of the protein kinase C pathway inhibits
gonadotropin-releasing hormone gene expression. Endocrinology 131:25522558[Abstract]
-
Bruder JM, Wierman ME 1994 Evidence for transcriptional
inhibition of GnRH gene expression by phorbol ester at a proximal
promoter region. Mol Cell Endocrinol 99:177182[CrossRef][Medline]
-
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]
-
Chandran UR, Attardi B, Friedman R, Dong K-W, Roberts JL,
DeFranco DB 1994 Glucocorticoid receptor-mediated repression of
gonadotropin-releasing hormone promoter activity in GT1 hypothalamic
cell lines. Endocrinology 134:14671474[Abstract]
-
Chandran UR, Attardi B, Friedman R, Zheng Z, Roberts JL,
DeFranco DB 1996 Glucocorticoid repression of the mouse
gonadotropin-releasing hormone gene is mediated by promoter elements
that are recognized by heteromeric complexes containing glucocorticoid
receptor. J Biol Chem 271:2041220420[Abstract/Free Full Text]
-
Milenkovic L, DAngelo G, Kelly PA, Weiner RI 1994 Inhibition
of gonadotropin hormone-releasing hormone release by prolactin from GT1
neuronal cell lines through prolactin receptors. Proc Natl Acad Sci USA 91:12441247[Abstract]
-
Belsham DD, Wetsel WC, Mellon PL 1996 NMDA and nitric oxide
act through the cGMP signal transduction pathway to repress
hypothalamic gonadotropin-releasing hormone gene expression. EMBO J 15:538547[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]
-
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]
-
Maniatis T, Weintraub H 1992 Gene expression and
differentiation. Curr Opin Genet Dev 2:197198[Medline]
-
Johnson P, McKnight S 1989 Eukaryotic transcriptional
regulatory proteins. Annu Rev Biochem 58:799839[CrossRef][Medline]
-
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]
-
Rosenfeld MG 1991 POU-domain transcription factors: pou-er-ful
developmental regulators. Genes Dev 5:897907[CrossRef][Medline]
-
Schöler HR, Hatzopoulos AK, Balling R, Suzuki N, Gruss P 1989 A family of octamer-specific proteins present during mouse
embryogenesis: evidence for germline-specific expression of an Oct
factor. EMBO J 8:25432550[Abstract]
-
Kutoh E, Stromstedt P, Poellinger L 1992 Functional
interference between the ubiquitous and constitutive octamer
transcription factor 1 (OTF1) and the glucocorticoid receptor by direct
protein-protein interaction involving the homeo subdomain of OTF-1. Mol
Cell Biol 12:49604969[Abstract]
-
OConnor M, Bernard H-U 1995 Oct-1 activates the
epithelial-specific enhancer of human papillomavirus type 16 via a
synergistic interaction with NF1 at a conserved composite regulatory
element. Virology 207:7788[CrossRef][Medline]
-
Tverberg LA, Russo AF 1993 Regulation of the
calcitonin/calcitonin gene-related peptide gene by cell-specific
synergy between helix-loop-helix and octamer-binding transcription
factors. J Biol Chem 268:1596515973[Abstract/Free Full Text]
-
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]
-
Wedel A, Ziegler-Heibrock HWL 1995 The C/EBP family of
transcription factors. Immunobiology 193:171185[Medline]
-
Williams SC, Cantwell CA, Johnson PF 1991 A family of
C/EBP-related proteins capable of forming covalently linked leucine
zipper dimers in vitro. Genes Dev 5:15531567[Abstract]
-
Williams SC, Baer M, Dillner AJ, Johnson PF 1995 CRP2
(C/EBPß) contains a bipartite regulatory domain that controls
transcriptional activation, DNA binding and cell specificity. EMBO J 14:31703183[Abstract]
-
Stein B, Cogswell PC, Baldwin ASJ 1993 Functional and physical
associations between NF-
B and C/EBP family members: a Rel
domain-bZIP interaction. Mol Cell Biol 13:39643974[Abstract]
-
Stein B, Yang MX 1995 Repression of the interleukin-6 promoter
by estrogen receptor is mediated by NF-
B and C/EBPß. Mol Cell
Biol 15:49714979[Abstract]
-
Lee Y-H, Yano M, Liu S-Y, Matsunaga E, Johnson PF,
Gonzalez FJ 1994 A novel cis-acting element controlling the rat
CYP2D5 gene and requiring cooperativity between C/EBPß and
an SP1 factor. Mol Cell Biol 14:13831394[Abstract]
-
Bendall AJ, Sturm RA, Danoy PA, Molloy PL 1993 Broad
binding-site specificity and affinity properties of octamer 1 and brain
octamer-binding proteins. Eur J Biochem 217:799811[Abstract]
-
Ryden TA, Beemon K 1989 Avian retroviral long terminal repeats
bind CCAAT/enhancer binding protein. Mol Cell Biol 9:11551164[Medline]
-
Wilson DB, Dorfman DM, Orkin SH 1990 A nonerythroid
GATA-binding protein is required for function of the human
preproendothelin-1 promoter in endothelial cells. Mol Cell Biol 10:48544862[Medline]
-
Johnston HM, Morris BJ 1994 NMDA and nitric oxide increase
microtubule-associated protein 2 gene expression in hippocampal granule
cells. J Neurochem 63:379382[Medline]
-
Clark ME, Dasgupta A 1990 A transcriptionally active form of
TFIIIC is modified in poliovirus-infected HeLa cells. Mol Cell Biol 10:51065113[Medline]
-
Kadonaga JT, Carner KR, Masiarz FR, Tjian R 1987 Isolation of
cDNA encoding transcription factor Sp1 and functional analysis of the
DNA binding domain. Cell 51:10791090[Medline]
-
Sturm R, Baumruker T, Franza BR, Herr W 1987 A 100-kD HeLa
cell octamer binding protein (OBF100) interacts differently with two
separate octamer-related sequences within the SV40 enhancer. Genes Dev 1:11471160[Abstract]
-
OReilly D, Hanscombe O, OHare P 1997 A single serine
residue at position 375 of VP16 is critical for complex assembly with
Oct-1 and HCF and is a target of phosphorylation by casein kinase II.
EMBO J 16:24202430[Abstract/Free Full Text]
-
Lai JS, Herr W 1997 Interdigitated residues within a small
region of VP16 interact with Oct-1, HCF, and DNA. Mol Cell Biol 17:39373946[Abstract]
-
Wong MW, Henry RW, Ma B, Kobayashi R, Klages N, Matthias P,
Strubin M, Hernandez N 1998 The large subunit of basal transcription
factor SNAPc is a Myb domain protein that interacts with Oct-1. Mol
Cell Biol 18:368377[Abstract/Free Full Text]
-
Inamoto S, Segil N, Pan ZQ, Kimura M, Roeder RG 1997 The
cyclin-dependent kinase-activating kinase (CAK) assembly factor, MAT1,
targets and enhances CAK activity on the POU domains of octamer
transcription factors. J Biol Chem 272:2985229858[Abstract/Free Full Text]
-
Luo Y, Fujii H, Gerster T, Roeder RG 1992 A novel B
cell-derived coactivator potentiates the activation of immunoglobulin
promoters by octamer-binding transcription factors. Cell 71:231241[Medline]
-
Strubin M, Newell JW, Matthias P 1995 OBF-1, a novel B
cell-specific coactivator that stimulates immunoglobulin promoter
activity through association with octamer-binding proteins. Cell 80:497506[Medline]
-
Lu M, Seufert J, Habener JF 1997 Pancreatic beta-cell-specific
repression of insulin gene transcription by CCAAT/enhancer-binding
protein ß. Inhibitory interactions with basic helix-loop-helix
transcription factor E47. J Biol Chem 272:2834928359[Abstract/Free Full Text]
-
Hsu W, Kerppola TK, Chen PL, Chen-Kiang S 1994 Fos and Jun
repress transcription activation by NF-IL6 through association at the
basic zipper region. Mol Cell Biol 14:268276[Abstract]
-
Mink S, Haenig B, Klempnauer KH 1997 Interaction and
functional collaboration of p300 and C/EBPß. Mol Cell Biol 17:66096617[Abstract]
-
Miau LH, Chang CJ, Tsai WH, Lee SC 1997 Identification and
characterization of a nucleolar phosphoprotein, Nopp140, as a
transcription factor. Mol Cell Biol 17:230239[Abstract]
-
Yano S, Fukunaga K, Takiguchi M, Ushio Y, Mori M, Miyamoto E 1996 Regulation of CCAAT/Enhancer-binding protein family members by
stimulation of glutamate receptors in cultured rat cortical astrocytes.
J Biol Chem 271:2352023527[Abstract/Free Full Text]
-
Dekker N, Cox M, Boelens R, Verrijzer CP, van der Vliet PC,
Kaptein R 1993 Solution structure of the POU-specific DNA-binding
domain of Oct-1. Nature 362:852855[CrossRef][Medline]
-
Pomerantz JL, Sharp PA 1994 Homeodomain determinants of major
groove recognition. Biochemistry 33:1085110858[Medline]
-
Avitahl N, Calame K 1994 The C/EBP family of proteins distorts
DNA upon binding but does not introduce a large directed bend. J
Biol Chem 269:2355323562[Abstract/Free Full Text]
-
Calkhoven CF, Ab G 1996 Multiple steps in the regulation of
transcription-factor level and activity. Biochem J 317:329342[Medline]
-
Ingraham HA, Flynn SE, Voss JW, Albert VR, Kapiloff MS, Wilson
L, Rosenfeld MG 1990 The POU-specific domain of Pit-1 is essential for
sequence-specific, high affinity DNA binding and DNA-dependent
Pit-1-Pit-1 interactions. Cell 61:102133[Medline]
-
Butt E, Geiger J, Jarchau T, Lohmann SM, Walter U 1993 The
cGMP-dependent protein kinase - gene, protein, and function. Neurochem
Res 18:2742[Medline]
-
Wang X, Robinson PJ 1995 Cyclic GMP-dependent protein kinase
substrates in rat brain. J Neurochem 65:595604[Medline]
-
Boulikas T 1995 Phosphorylation of transcription factors and
control of the cell cycle. Crit Rev Eukaryot Gene Expr 5:177[Medline]
-
Osada S, Yamamoto H, Nishihara T, Imagawa M 1996 DNA binding
specificity of the CCAAT/enhancer-binding protein transcription factor
family. J Biol Chem 16:38913896[CrossRef]
-
Ray A, Ray BK 1994 Serum amyloid A gene expression under
acute-phase conditions involves participation of inducible
C/EBP-ß and C/EBP-
and their activation by phosphorylation.
Mol Cell Biol 14:43244332[Abstract]
-
Grenfell SJ, Latchman DS, Thomas NS 1996 Oct-1 and Oct-2
DNA-binding site specificity is regulated in vitro by
different kinases. Biochem J 315:889893[Medline]
-
Segil N, Roberts SB, Heintz N 1991 Mitotic phosphorylation of
the Oct-1 homeodomain and regulation of Oct-1 DNA binding activity.
Science 254:18141816[Medline]
-
Sanger F, Nicklen S, Coulson AR 1977 DNA sequencing with
chain-terminating inhibitors. Proc Natl Acad Sci USA 74:54635467[Abstract]
-
Mellon PL, Parker V, Gluzman Y, Maniatis T 1981 Identification
of DNA sequences required for transcription of the human
1 globin gene using a new SV40 host-vector
system. Cell 27:279288[Medline]
-
Mellon PL, Clegg CH, Correll LA, McKnight GS 1989 Regulation
of transcription by cyclic AMP-dependent protein kinase. Proc Natl Acad
Sci USA 86:48874891[Abstract]
-
Gorman C 1985 High efficiency gene transfer into mammalian
cells. In: Glover DM (ed) DNA Cloning: A Practical Approach. IRL Press,
Oxford, UK, vol 2:143190
-
Bradford M 1976 A rapid and sensitive method for the
quantitation of microgram quantities of protein utilizing the
principles of protein-dye binding. Anal Biochem 72:248254[CrossRef][Medline]
-
Seed B, Sheen JY 1988 A simple phase-extraction assay for
chloramphenicol acyltransferase activity. Gene 67:271277[CrossRef][Medline]
-
Lee KAW, Bindereif A, Green MR 1988 A small-scale procedure
for preparation of nuclear extracts that support efficient
transcription and pre-mRNA splicing. Gene Anal Technol 5:2231[CrossRef]