(Received for publication, August 5, 1996, and in revised form, March 17, 1997)
From the Laboratory of Molecular Reproductive Biology and Medicine, Department of Obstetrics and Gynecology, University of Louisville, Health Sciences Center, Louisville, Kentucky 40292
We investigated the cis-acting
elements and trans-acting proteins required for the
transcriptional inhibition of the gonadotropin-releasing horomone
(GnRH) gene by human chorionic gonadotropin (hCG) in GT1-7 neurons.
Transient transfection of GT1-7 neurons with the 5-flanking region of
the rat GnRH gene-luciferase fusion constructs revealed that a 53-base
pair (bp) sequence between
126 and
73 bp is required for the hCG
inhibition. Nuclear extracts from GT1-7 neurons contained 110- and
95-kDa proteins that formed two complexes with the 53-bp sequence.
These proteins are not related to Fos, cAMP response element-binding
protein, Oct-1, or progesterone receptors, and hCG treatment
selectively increased the 95-kDa protein. DNase I footprinting with
GT1-7 cell nuclear extracts protected the
99 to
79-bp region,
which contained a so-called imperfect AP-1 site (
99 to
94 bp) and
two AT-rich palindromic sequences (
91 to
87 bp and
85 to
81
bp). The mutagenesis of the AT-rich regions, but not the AP-1 site,
resulted in a loss of DNA binding of the 95-kDa protein and the
inhibitory effect of hCG. In summary, our results are consistent with
hCG inducing a 95-kDa trans-acting protein, which binds to
91- to
81-bp AT-rich sequences in the 5
-flanking region to inhibit
the transcription of the GnRH gene.
The hypothalamic decapeptide, gonadotropin releasing hormone (GnRH),1 plays a central role in reproduction by controlling the synthesis and release of luteinizing hormone (LH) and follicle-stimulating hormone from the anterior pituitary (1, 2). The synthesis and release of GnRH itself is subjected to regulation by numerous agents (3, 4). The studies on the hypothalamic GnRH neurons are hampered by the fact that they are present in small numbers and harvesting them in quantities required for most studies is very difficult (5-7). The development of immortalized GnRH-containing GT1-7 neurons by targeted oncogenesis has allowed investigators to make rapid advances in understanding the regulatory mechanisms in the synthesis and release of GnRH (8, 9). One of these advances is that GT1-7 neurons have been shown to contain functional LH/human chorionic gonadotropin (hCG) receptors (10, 11), and these receptors are required for transcriptional inhibition of the GnRH gene by exogenous hCG in a dose- and time-dependent and hormone-specific manner (10). These findings supported the possible existence of a short loop feedback mechanism first proposed 30 years ago, in the synthesis and release of LH (12). The treatment of GT1-7 neurons with hCG under the conditions that it inhibits GnRH synthesis, activated protein kinase A, increased the synthesis of new proteins, increased the levels of phosphorylated cAMP response element-binding protein (CREB) and c-Fos and c-Jun proteins, and decreased the levels of GnRH receptors (13, 14). The present study focused on investigating the cis-acting elements and trans-acting proteins required for the transcriptional inhibition of the GnRH gene by hCG in GT1-7 neurons.
The following reagents were purchased from the
indicated commercial sources: the promoterless luciferase reporter
vector pGL2 basic DNA, luciferase, chloramphenicol acetyltransferase
(CAT), and -galactosidase assay systems, Klenow enzyme, restriction enzymes and T4 DNA ligase from Promega (Madison, WI);
[14C]chloramphenicol and [32P]dATP from
DuPont NEN; Dulbecco's modified Eagle's medium, fetal calf serum,
horse serum, antibiotic-antimycotic solution, Lipofectin reagent, and
Opti-MEM I medium from Life Technologies, Inc.; Sequenase Version 2.0 DNA sequencing kit from U. S. Biochemical Corp.; VCS-M13 helper phage
and pBluescript II KS+ vector from Stratagene Cloning Systems (LaJolla,
CA); kits for preparing single and double strand plasmid DNA from
QIAGEN Inc. (Chatsworth, CA); all reagents for synthesis of
oligonucleotides, SureTrack footprinting and BandShift kits from
Pharmacia Biotech Inc.; polyclonal antibodies to phosphorylated CREB,
c-Fos, and c-Jun from Upstate Biotechnology, Inc. (Lake Placid, NY);
polyclonal antibody to Oct-1 and consensus Oct-1 oligonucleotide from
Santa Cruz Biotechnology, Inc. (Santa Cruz, CA); monoclonal antibody to
progesterone receptor from Affinity Bioreagents (Neshanic Station, NJ).
The following items were obtained as gifts: immortalized GT1-7 neurons
from Dr. Pamela Mellon at the University of California (La Jolla, CA);
pGEM7 plasmid containing the
3026 to +116 bp of rat GnRH promoter
region from Dr. Margaret Wierman at the University of Colorado Health
Sciences Center (Denver, CO); promoter of cytomegalovirus (pCMV)-Fos
and pCMV-Jun expression plasmids from Dr. Tom Curran at Roche Institute
of Molecular Biology (Nutley, NJ); 5,12-O-tetradecanoyl
phorbol-13-acetate response element (TRE)/thymidine kinase (TK)-CAT
reporter vector from Dr. Inder Verma and CREB cDNA from Dr. Marc
Montminy, both at the Salk Institute for Biological Studies (La Jolla,
CA); TK-CAT and 3-cAMP response element (CRE)/TK-CAT reporter vectors
from Dr. Patrick Quinn at the Pennsylvania State University College of Medicine (Hershey, PA); dut nug mutant strain Escherichia
coli RZ 1032 from Dr. Mark Brennan and pCMV
-galactosidase
expression plasmid from Thomas Geoghean of our institution; highly
purified hCG (CR-127, 14,900 IU/mg) from the National Hormone and
Pituitary Program, supported by NIDDK, NICHHD, and USDA. The
oligonucleotides used for site-directed mutagenesis were synthesized in
our laboratory using a solid oligonucleotide synthesizer (Gene
Assembler Special, Pharmacia).
GT1-7 neurons were grown and maintained in Dulbecco's modified Eagle's medium supplemented with 5% fetal calf serum, 5% horse serum, 4.5 mg/ml glucose, and 1% antibiotic-antimycotic solution in 150-cm2 flasks. Cultures were incubated at 37 °C in a humidified atmosphere of 5% CO2 and 95% air. When neurons reached 90% confluence, they were replated in 150-cm2 flasks for the isolation of nuclear proteins and in six-well plates for transient transfection experiments.
Construction of CREB Expression VectorThe entire 1.2-kb
coding region of CREB cDNA, including the 150-bp untranslated
region at the 5 end and the 50-bp untranslated region at the 3
end in
PGEM-37f(
) plasmid (15), was released by digestion with
EcoRI and BamHI. The DNA fragment was then
subcloned into pCMV expression vector at the same sites.
Rat GnRH promoter
reporter fusion constructs were prepared as described previously (16).
Briefly, we excised a HindIII fragment of the GnRH promoter
region containing 3026 to +116 bp from pGEM7-GnRHP plasmid and
subcloned into the HindIII site of the promoterless luciferase reporter plasmid (pGL2-basic) containing the coding region
of luciferase gene. The sequential 5
deletion constructs were prepared
from the
3026- to +116-bp fragment using the convenient restriction
sites. All the constructs have the same 3
end at +116 bp. The sequence
of each deletion construct was confirmed by multiple endonuclease
restriction enzyme analyses.
GT1-7 neurons were plated at a density
of 5 × 105 cells/well in six-well cell culture plates
and were grown to 60% confluence, then transfected by a liposome-based
method. Cells were transfected with 30 µg of Lipofectin, 10 µg of
wild type or mutated pGL2-GnRH promoter constructs or 3CRE/TK-CAT or
5TRE/TK-CAT, 2 µg of pCMV -galactosidase expression plasmid, and 3 ml of Opti-MEM I per well. In some experiments, pCMV-CREB, pCMV-Fos,
and pCMV-Jun expression plasmids were co-transfected at the same
time.
For measurement of luciferase activity, the cells were lysed in 400 µl of reporter lysis buffer from the Promega luciferase assay kit. Twenty µl of cell lysates were mixed with 100 µl of luciferase assay mixture (270 µM coenzyme A, 470 µM luciferin, and 530 µM ATP in 20 mM Tricine, 1.07 mM (MgCO3)4Mg(OH)2·5H2O, 2.67 mM MgSO4, 0.1 mM EDTA disodium dihydrate, and 33.3 mM dithiothreitol (DTT), pH 7.8)), and luciferase activity was immediately measured using a luminometer (model 20E, Turner Designs, Sunnyvale, CA) at room temperature.
For the CAT enzyme assay, 100 µl of cell lysates were preheated at
60 °C for 10 min and incubated with 25 µl of CAT enzyme assay
mixture (0.15 µCi of [14C]chloramphenicol, 25 µg of
n-butylryl coenzyme A) at 37 °C for 5 h. Ten µl of
ethyl acetate extract were spotted onto thin layer chromatography
plates and run for 1 h in a chloroform/methanol (97:3) mixture.
The plates were exposed to Kodak X-Omat film with intensifying screens
for 5 days at 80 °C. After autoradiography, the butyrylated
products were cut from the plates and counted in a liquid scintillation
counter for determination of CAT activity.
For measurement of -galactosidase activity, 10 µl of cell lysates
were incubated for 30 min at 37 °C with 150 µl of 2 ×
-galactosidase assay mixture (120 mM
Na2HPO4, 80 mM
NaH2PO4, 2 mM MgCl2,
100 mM
-mercaptoethanol, 1.33 mg/ml
o-nitrophenyl-
-D-galactopyranoside), and the
absorbance at 420 nm was measured using a multiplate reader. The
measurement of
-galactosidase activity served to monitor transfection efficiencies and also to normalize luciferase and CAT
enzyme data for
-galactosidase activity.
Nuclear extracts from
GT1-7 neurons were prepared essentially as described by Dignam
et al. (17). Briefly, cell pellets were suspended in
homogenization buffer (0.5 mM DTT, 0.5 mM
phenylmethanesulfonyl fluoride, 10 mM HEPES, 25 mM spermidine, 1 mM EDTA, 2 M
sucrose, and 10% glycerol, pH 7.6) and homogenized with a Dounce glass homogenizer. The homogenates were centrifuged to pellet nuclei. The
crude nuclear pellets were resuspended in lysis buffer (1 mM DTT, 0.1 mM phenylmethanesulfonyl fluoride,
10 mM HEPES, 100 mM KCl, 0.1 mM
MgCl2, 0.1 mM EDTA, and 10% glycerol, pH 7.6). After centrifugation, ammonium sulfate (0.3 g/ml) was added to precipitate nuclear proteins from the supernatant and then dialyzed against 100 volumes of dialysis buffer (0.5 mM DTT, 0.5 mM phenylmethanesulfonyl fluoride, 25 mM HEPES,
100 mM KCl, 0.1 mM EDTA, and 20% glycerol, pH
7.6). The nuclear extracts were stored in aliquots at 80 °C.
The wild type or
mutated DNA fragments from 126 to
73 bp of the GnRH promoter with
5
overhangs were labeled by a fill-in reaction using
[32P]dATP and Klenow enzyme. The labeled probes were
purified by polyacrylamide gel electrophoresis (PAGE). The
electrophoretic gel mobility shift assays were performed as described
in the BandShift kit from Pharmacia. Five-µg aliquots of the nuclear
extracts from untreated and hCG- treated GT1-7 neurons were incubated
with binding mixture (10 mM Tris-HCl, pH 7.5, 100 mM NaCl, 0.5 mM DTT, 10% glycerol, 0.05%
Nonidet P-40, 5 mM MgCl2, 2 µg of
poly(dI-dC), 0.1 mM EDTA, 0.5 ng of labeled probe (30,000 cpm/reaction)) for 20 min at room temperature. For competition studies,
a 100-fold excess of unlabeled probe was added to the binding mixture.
For the electrophoretic gel mobility supershift experiments, polyclonal antibodies to phosphorylated CREB, c-Fos, c-Jun, Oct-1, and monoclonal antibody to progesterone were added to the binding mixture and incubated for 30 min at 4 °C prior to adding the probes. After incubation, DNA-protein complexes were resolved by 4% native PAGE in
buffer containing 7 mM Tris-HCl, pH 7.5, 3 mM
sodium acetate, and 1 mM EDTA at 4 °C for 5 h. Gels
were dried and exposed overnight at
80 °C to Kodak X-Omat film
with intensifying screens.
The proteins in 100-µg aliquots of
nuclear extracts were separated by 8% discontinuous SDS-PAGE under
reducing and nonreducing conditions, then electrotransferred onto
Immobilon-P membranes. The proteins were renatured by placing the
membranes in Z buffer (25 mM HEPES-KOH, pH 7.6, 5 mM MgCl2, 20% glycerol, 0.1% Nonidet P-40,
0.1 M KCl, 1 mM DTT, 0.1 µM
ZnSO4, and 100 mM NaCl) containing 6 M guanidinium chloride. Nonspecific binding sites were
blocked by Z
buffer containing 3% nonfat dried milk (18). Finally, the membranes were incubated for 3 h at room temperature with binding buffer (Z
buffer plus 5 µg of poly(dI-dC), 30 µg of calf thymus DNA, and 1 × 106 cpm of
32P-labeled probe (
126- to
73-bp DNA fragment of the
GnRH promoter, wild type, or mutated
126- to +116-bp DNA fragment of
the GnRH promoter) per ml). In competition studies, a 100-fold excess
of unlabeled probe was added to the binding buffer. After washing three
times with Z
buffer, the membranes were exposed at
80 °C for 2 days to Kodak X-Omat film with intensifying screens.
The one end 32P-labeled
DNA fragments as probes were prepared as follows. For the coding
strand, the 1031 to +116 bp of the GnRH gene 5
-flanking region in
pGL2 vector was digested with PvuII and HindIII
to release a
126- to +116-bp DNA fragment with a 5
overhang only in
one end (HindIII site) for fill-in labeling; for the
noncoding strand, the same vector was first digested with EcoRI to generate a DNA fragment for fill-in labeling. Both
end-labeled DNA fragments were then digested with DraI. The
171- to
16-bp DNA fragment with only one end labeled was purified
by PAGE. DNase I footprinting assays were performed as described in the
SureTrack footprinting kit from Pharmacia. The nuclear extracts (0-60
µg of protein as indicated in the figure legends) were incubated at
room temperature for 30 min with a binding mixture (10% glycerol, 20 mM Tris-HCl, pH 7.5, 100 mM NaCl, 5 mM MgCl2, 1 mM DTT, 2 µg of
poly(dI-dC), 0.01% Nonidet P-40, and 20,000 cpm of labeled probe),
then 0.5 mM CaCl2 and 2 mM
MgCl2 were added, and the mixture was digested with 0.8 unit of DNase I for exactly 1 min. The reactions were terminated by
adding stop solution (192 mM sodium acetate, 32 mM EDTA, 0.14% SDS, and 64 µg/ml yeast RNA), extracted
with phenol/chloroform, and analyzed on an 8% polyacrylamide, 42%
urea sequencing gel. Then the gel was dried and exposed to Kodak X-Omat film with intensifying screens for 2 days at
80 °C. A Maxam and Gilbert G + A reaction was also performed on the same probes and run as
a marker for the footprint reactions.
The site-directed mutagenesis was
performed as described previously (19). Briefly, the 5-flanking region
of the GnRH gene was subcloned into pBluescript II KS+ vector and
transfected into a dut nug mutant E. coli RZ1032 to generate
single-stranded plasmid DNA. The single-stranded plasmid DNA was
annealed with oligonucleotides containing the mutated sequences
followed by in vitro DNA synthesis to produce
double-stranded plasmid DNA. The plasmid DNA was transfected into
competent cells, E. coli JM109. The mutants were identified by restriction enzyme analysis and confirmed by dideoxynucleotide sequencing with M13 universal primers. The mutated 5
-flanking regions
of the GnRH gene were subcloned into promoterless luciferase reporter
vector pGL2-basic for functional studies.
Each experiment was performed in duplicate. All the experiments were repeated at least three times on different occasions. The data presented are the means and S.E. of all the values. One-way analysis of variance and Duncan's multiple range test were used for statistical analyses of the data (20).
To determine
the transcriptional regulation of the GnRH gene by hCG and the common
transcription factors such as Fos, Jun, and CREB, we placed the
5-flanking region of the GnRH gene from
3026 to +116 bp in front of
the coding region of the luciferase gene and transiently transfected it
into GT1-7 neurons. Transfected GT1-7 neurons robustly expressed the
promoter activity of the GnRH gene. Treatment of these neurons under
optimal conditions (100 ng/ml hCG and 12 h) resulted in a
significant decrease in the promoter activity (Fig. 1).
Co-transfections with increasing amounts of Fos or CREB expression
vectors also resulted in a dose-dependent significant
decrease in the promoter activity of the GnRH gene (Fig. 1). Similar
co-transfection with Jun expression vector, on the other hand, had no
effect on its own nor could it modify the inhibition conferred by Fos
or CREB (Fig. 1). The CREB was more effective than Fos, while Fos was
similar to hCG in inhibiting the promoter activity. There was no
synergism between CREB and Fos or between CREB, Fos, and Jun when they
were simultaneously co-transfected (Fig. 1).
We prepared next a series of deletion constructs to determine what
regions of the 5-flanking sequence are required for the inhibition of
promoter activity of the GnRH gene by hCG, CREB, and Fos. The basal
activity shown in Fig. 2 demonstrates that deletion of
the upstream sequence from
1031 bp resulted in a 71% decrease, and
further deletions to the
16-bp position resulted in a greater than
95% decrease in the promoter activity of the GnRH gene (Fig. 2). These
data are consistent with the presence of major activation and
neuron-specific enhancer regions at the upstream sequence from
1031
bp (16, 21-23).
After establishing the sequence requirements for basal promoter
activity, we treated the transfected GT1-7 neurons with hCG or
co-transfected them with Fos and CREB expression vectors and then
measured luciferase activity. Despite the decrease in basal activity,
the deletion of sequences up to the 126-bp position had no effect on
the ability of hCG, CREB, or Fos to inhibit the promoter activity of
the GnRH gene (Fig. 3). Deletion to the
73-bp position
resulted in a complete loss of hCG and Fos inhibition and partial loss
of CREB inhibition. Further deletion to the
16-bp position resulted
in an even greater loss of CREB effect (Fig. 3).
To determine whether inhibitory effects of hCG, Fos, and CREB are
specific to the GnRH promoter, we transfected GT1-7 neurons with
expression vectors containing three copies of the canonical CRE or five
copies of human metallothionein 11A TRE linked upstream of HSV TK
promoter and the CAT structural gene (24, 25). The cells were also
co-transfected with either pCMV-CREB, pCMV-Fos, or pCMV-Jun, or
combinations of them, or treated with 100 ng/ml hCG for 12 h. As
shown in Fig. 4, co-transfection with pCMV-CREB resulted
in an increase rather than a decrease in CRE/TK-CAT activity. Counting
the radioactivity in the butyrylated product revealed that CAT activity
was increased 5.8-fold by CREB. Co-transfection with pCMV-Fos or
pCMV-Jun resulted in a similar 4.8-fold increase in TRE/TK-CAT
activity.
Co-transfection with both pCMV-Fos and pCMV-Jun resulted in a 7.1-fold increase in TRE/TK-CAT activity. Treatment of CRE/TK-CAT and TRE/TK-CAT transfected GT1-7 neurons with hCG resulted in a 5.8- and 3.7-fold increase in CAT activity, respectively. These increases, plus the fact that Jun can increase TRE/TK-CAT activity in the absence of any effect on the promoter of the GnRH gene, would suggest that the inhibitory effects of hCG, Fos, and CREB are indeed specific to GnRH promoter.
Specific Binding of Nuclear Proteins to the Promoter Region of the GnRH GeneSince the inhibition of promoter activity of the GnRH
gene by hCG requires the 126- to
73-bp region of the 5
-flanking
sequence (Fig. 3), we used this DNA fragment for electrophoretic gel
mobility shift assays to identify and characterize the nuclear proteins from GT1-7 neurons. As shown in Fig. 5, nuclear
extracts from GT1-7 neurons formed three complexes with distinct
electrophoretic mobilities. These complexes were designated as C1, C2,
and C3 (Fig. 5). The formation of C1 and C2 but not the C3 complex was inhibited by the addition of 100-fold excess corresponding unlabeled DNA fragment, suggesting that the C3 complex is nonspecific (Fig. 5).
Calf thymus DNA had no effect on the formation of any of the three
complexes (Fig. 5). Using equal amounts of nuclear extracts, the GT1-7
neurons treated with 100 ng/ml hCG showed an increase in the C2 complex
at 12 h followed by a decline to the control level by 24 h
(Fig. 5).
To determine whether the proteins in C1 and C2 complexes were related to CREB, c-Fos, c-Jun, Oct-1, or progesterone receptors, we examined supershifts in electrophoretic gel mobility assays. In this assay, nuclear extracts from hCG-treated GT1-7 neurons were preincubated with the polyclonal antibodies to the above transcription factors, and the assays were performed. The results showed that none of the antibodies was able to induce supershifts (data not shown). To determine whether the assay is sensitive enough for detection of supershifts, we performed a procedural control in which Oct-1 antibody was added to the nuclear extracts from GT1-7 neurons and then performed the assay with the 32P-labeled oligonucleotide containing the consensus ectomere sequence. The results showed that Oct-1 antibody had indeed caused a supershift (data not shown).
To further characterize the nuclear proteins from GT1-7 neurons that
bound to the 126- to
73-bp region of the 5
-flanking sequence of
the GnRH gene, we performed Southwestern blotting. In this assay, the
proteins in nuclear extracts from GT1-7 neurons were resolved by PAGE
under reducing conditions, then transferred to membranes and probed
with 32P-labeled
126- to
73-bp DNA fragment. Fig.
6 shows that 110- and 95-kDa proteins bind to the DNA
fragment (lane 1). The addition of excess unlabeled
corresponding DNA fragment resulted in an inhibition of binding of both
the proteins (lane 5). Using equal amounts of nuclear
extracts, GT1-7 neurons treated with 100 ng/ml hCG showed an increase
of the 95-kDa, but not the 110-kDa protein, at 9 h (lane
2) and 12 h (lane 3) followed by a decline to the control level by 24 h (lane 4). Densitometric analysis
revealed that there was a 5-7-fold increase in the 95-kDa protein at
12 h after hCG treatment.
Identification of DNA-Protein Contact Sites
To identify the
location and nucleotide sequence of the binding site within the 126-
to
73-bp region, we performed DNase I footprinting with the probes
extending from
171 and +116 bp of GnRH promoter. In this assay, we
preincubated the DNA fragment with the nuclear extracts from GT1-7
neurons and then treated it with DNase I. As shown in Fig.
7, nuclear extracts protected, from DNase I, a
99- to
79-bp region within the coding strand and also in the corresponding
region in the noncoding strand. This protection in both strands
increased as the increasing amounts of nuclear extracts from GT1-7
neurons were added. Using equal amounts of nuclear extracts showed that
treatment of GT1-7 neurons with hCG resulted in a greater protection
as compared with the control (data not shown). Nucleotide sequencing
revealed that the
99- to
79-bp region contained a so-called
imperfect AP-1 site at the
99- to
94-bp position (5
-TGACCA-3
) and
a palindromic AT-rich sequence at the
91- to
87-bp position
(5
-TTTAA-3
) and at the
85- to
81-bp position (5
-AAAAT-3
) (Fig.
7). The palindromic sequence at the
91- to
87-bp position somewhat
differed from the consensus TATAA sequence.
Effect of Mutations at Positions
To test the importance of the 21-bp region in the transcriptional inhibition of the GnRH gene by hCG, we prepared block replacement or internal deletion mutants using site-directed mutagenesis.
The wild type construct (RGPLW) showed a robust luciferase
activity and the formation of two protein DNA complexes designated as
C1 and C2 (Fig. 8, panels A and
B). The block replacement of the imperfect AP-1 site with
AGATCT (RGPLM1) resulted in a significant decrease in basal
promoter activity of the GnRH gene and disappearance of the C1 complex
(Fig. 8, panels A and B). The block replacement of the AT-rich sequence at the 85- to
81-bp position with AGGCGT (RGPLM2) resulted in a significant increase in basal
promoter activity and disappearance of the C2 complex (Fig. 8,
panels A and B). The block replacement of the
AT-rich sequence in the
91- to
87-bp position with GCGATGC
(RGPLM3) or internal deletion of the entire 21-bp region
(RGPLM4) resulted in a significant decrease of the basal
promoter activity of the GnRH gene and disappearance of both the C1 and
C2 complexes (Fig. 8, panels A and B).
Southwestern blot analysis was performed to determine the molecular
size of proteins that formed C1 and C2 DNA complexes and also to
confirm the protein binding to wild type and mutated constructs through
competition experiments. As shown in Fig. 9, the protein in the C1 complex has a molecular mass of 110 kDa, and the one in the
C2 complex has a molecular mass of 95 kDa. As expected from the direct
binding studies in Fig. 8, both 110- and 95-kDa proteins can bind to
32P-labeled wild type construct, and this binding can be
inhibited by unlabeled RGPLW DNA fragment. Again as expected from the
data in Fig. 8, while RGPLM1 construct competed for the binding of 95-kDa protein and RGPLM2 competed for the binding of 110-kDa protein,
RGPLM3 and RGPLM4 could not compete for binding of either of the
proteins to the RGPLW construct.
Fig. 10 shows that block replacement of the imperfect
AP-1 site had no effect on the inhibition of promoter activity of the GnRH gene by hCG, Fos, or CREB. However, block replacement of the
AT-rich regions or internal deletion of the entire 21-bp region resulted in the reversal of hCG and Fos inhibition without affecting CREB inhibition of promoter activity of the GnRH gene.
The 5-flanking sequence of the rat GnRH gene from
3026 to +116
bp, which has been cloned and characterized (16, 21-23, 26, 27) was
used in the present study to determine cis-acting elements
and trans-acting proteins required for the transcriptional inhibition of the GnRH gene by hCG. The results showed that treatment of transiently transfected GT1-7 neurons with hCG resulted in a
decrease in the promoter activity of the GnRH gene. This decrease was
similar in magnitude with the decreases in steady state GnRH mRNA
levels and transcription rate of the gene (10). Although truncations of
the 5
-flanking region greatly diminished basal promoter activity, hCG
was able to inhibit the promoter activity until the 53-bp sequence
between
126 and
73 bp had been deleted.
Our previous study demonstrating that the treatment of GT1-7 neurons
with hCG resulted in an increase of phosphorylated CREB, Fos, and Jun
before the decrease in the expression of the GnRH gene suggested that
these transcription factors might be involved in the hCG action (13).
This led us to investigate the effect of overexpression of Fos, Jun,
and CREB on the promoter activity of the GnRH gene. The results showed
that Fos and CREB, but not Jun, inhibited the promoter activity in a
dose-dependent manner in the 3026- to +116-bp construct.
Fos, but not Jun, inhibiting basal proximal promoter activity of the
GnRH gene is in agreement with a previous finding (26). CREB was more
effective than Fos, and Fos was similar to hCG in inhibiting the
promoter activity. There was no synergism between any of these three
transcription factors, suggesting that heterodimerization, which is a
known feature in the control of gene activity (28), may not play a role
in regulating the promoter activity of the GnRH gene in the
3026- to
+116-bp construct.
Squelching of common transcription factors by overexpressed immediate
early genes has been reported as a mechanism for down-regulation of
target genes (29-31). However, this could not explain the present findings, because while overexpressed pCMV-Jun had no effect, overexpressed pCMV-Fos and pCMV-CREB could inhibit the GnRH promoter activity. If the CMV promoter was squelching transcription factors that
otherwise would bind to the GnRH promoter, then overexpressed pCMV-Jun
should also have inhibited the GnRH promoter activity. The experiments
performed with GT1-7 neurons transfected with three copies of
canonical CRE or five copies of human metallothionein 11A TRE linked
upstream of HSV TK promoter, and the CAT structural gene revealed that
the inhibitory effects of Fos and CREB are specific to GnRH promoter.
The finding that hCG increased both CRE and TRE/TK-CAT activities
indicates that the hCG effect is also promoter-specific to the GnRH
gene and may not be mediated by CRE or TRE. In fact, the site of CREB
inhibition is downstream from the site of hCG inhibition in the
5-flanking region of the GnRH gene.
Electrophoretic gel mobility shift assays have indicated that nuclear
extracts from GT1-7 neurons contained proteins that formed two
specific complexes with the 126- to
73-bp region of the 5
-flanking
sequence of tbe GnRH gene. The molecular masses of these proteins as
determined by Southwestern blotting were 110 and 95 kDa. Of these, only
the 95-kDa protein responded to hCG treatment by about a 5-7-fold
increase at 9 to 12 h followed by a decline at 24 h. This
change preceded or coincided with a decrease in the expression of the
GnRH gene (10). Even though the molecular mass of the 95-kDa protein is
higher than those of Fos, CREB, and Oct -1, we nevertheless tested, by
supershift experiments, whether they might somehow be related to the
95-kDa protein. The results showed that the antibodies to these
transcription factors failed to induce supershifts, thereby eliminating
this possibility. The size of the 95-kDa protein is similar to that of
progesterone receptor A. The anti-progesterone receptor antibody, which
recognizes both A and B forms, failed to induce supershift, suggesting
that the 95-kDa protein is not a progesterone receptor. Even though
GT1-7 neurons do not contain progesterone receptors, co-transfection
with progesterone receptor has been shown to inhibit promoter activity
via binding to non-consensus sequences in the
171- to
126-bp region
of the 5
-flanking sequence of the GnRH gene (32). This region is
obviously different from the one required by hCG (
91 to
81 bp) to
inhibit the promoter activity of the GnRH gene.
DNase I footprinting showed that 110- and 95-kDa proteins bind to a
21-bp region within the 99- to
79-bp sequence of the 5
-flanking
region of the GnRH gene. Examination of the sequence revealed that it
contained a so-called imperfect AP-1 site at the
99- to
94-bp
position and an AT-rich region at the
91- to
81-bp position. The
block replacement of the imperfect AP-1 site eliminated DNA binding of
the 110-kDa protein and decreased the basal activity. Although hCG,
Fos, and CREB were able to inhibit GnRH promoter activity in the
mutated imperfect AP-1 construct, they were less effective compared
with their inhibitory effects in the
126- to
73-bp construct. This
may suggest that even though imperfect AP-1 is not absolutely required,
it may still play a role in determining the extent of inhibition of
GnRH promoter activity by hCG, Fos, and CREB. Nevertheless, this
finding is in keeping with the results which showed that hCG treatment
does not increase the 110-kDa protein, implying that it is not
absolutely required for hCG action. The block replacement of the
85-
to
81-bp region resulted in a complete loss of DNA binding of the 95-kDa protein, moderate loss of basal activity, and more importantly, the complete loss of the inhibitory effect of hCG. This suggests that
the 95-kDa protein is required for the hCG effect, which is in keeping
with hCG treatment increasing the level of this protein. The block
replacement of the
91- to
87-bp region and internal deletion
resulted in elimination of DNA binding of both the 95- and 110-kDa
proteins, moderate loss of basal activity, and the disappearance of hCG
effect.
How the 95-kDa protein actually exerts its repressive activity is not known. Previous studies have suggested that competition, quenching, direct inhibition, and squelching may explain transcriptional repression of the genes (31). It is possible that the 95-kDa protein induced by hCG may use one or more of these mechanisms to repress the transcription of the GnRH gene. hCG may also induce labile protein(s) or non-DNA-binding adapter protein(s) that may facilitate the repression of the GnRH gene by the 95-kDa protein (33).
In this report, we identified the cis-acting elements and
trans-acting protein that mediate the transcriptional
inhibition of the GnRH gene by hCG. From the previous and present
results, it is reasonable to propose a model in which LH/hCG binding to receptors activates a cAMP/protein kinase A pathway, which increases the phosphorylation of CREB, and the phosphorylated CREB induces c-Fos,
c-Jun, and 95-kDa proteins. The 95-kDa protein could be a downstream
target of not only hCG but also of Fos action on the GnRH gene. Even
though this protein does not appear to bind Fos, the action of Fos may
depend on both this protein as well as on its ability to bind to
AT-rich sequences in the 91- to
81-bp region in the 5
-flanking
sequence of the GnRH gene. The CREB, however, must work through a
completely different mechanism because the mutation of the AT-rich site
does not compromise its inhibitory effect. The identity of the 95-kDa
protein is not known. We have not been able to place it in any group of
the known transcription factors, either by our experiments or by size
comparison.
In summary, transcriptional inhibition of the GnRH gene by hCG is
mediated by a 95-kDa trans-acting protein, which binds to AT-rich cis-acting elements in the 91- to
81-bp region
of the 5
-flanking sequence of the GnRH gene.