Divergent and Composite Gonadotropin-Releasing Hormone-Responsive Elements in the Rat Luteinizing Hormone Subunit Genes
Jennifer Weck1,
Alice C. Anderson,
Shannon Jenkins,
Patricia C. Fallest and
Margaret A. Shupnik
Division of Endocrinology Department of Internal Medicine
(A.C.A., S.J., P.C.F., M.A.S.) Department of Molecular Physiology
and Biological Physics (J.W., M.A.S.), and The National
Science Foundation Center for Biological Timing University
of Virginia Charlottesville, Virginia 22903
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ABSTRACT
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GnRH pulses regulate gonadotropin subunit gene
transcription in a frequency-dependent, subunit-specific manner. The
-subunit gene is stimulated by constant GnRH and by rapid to
intermediate pulse frequencies, while stimulation of LHß subunit gene
transcription requires intermediate frequency pulses. We have defined
the GnRH-responsive elements of the rat LH subunit gene promoters by
deletion/mutation analysis and transfection studies in rat pituitary
cells and two clonal gonadotrope cell lines. The
-subunit gene
GnRH-responsive region lies between -411 and -375 bp. The region
contains two Ets-domain protein binding sites, and mutating either site
obliterates the response. DNA protein binding studies demonstrate the
two sites are not equivalent, and that Ets-1 does not mediate this
response. Studies of the LHß promoter reveal a major GnRH-responsive
region between -456 and -342 bp. Within this region, two Sp1
binding sites contribute to the GnRH response, and the 3'Sp1
site is also critical for basal expression. The 5'Sp1 site partially
overlaps a CArG box, and mutating the CArG element specifically
eliminates the response to pulsatile GnRH. DNA containing this
mutation cannot form intermediate mobility complexes with nuclear
proteins, but retains Sp1 binding. Mutation of the 3'Sp1 site and
either the 5'Sp1 or CArG element partially restores GnRH stimulation,
suggesting a downstream element contributes to the full GnRH response.
These studies demonstrate that unique composite elements and
transcription factors are responsible for GnRH stimulation of the LH
subunit genes and may contribute to their differential responses to
GnRH pulses.
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INTRODUCTION
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The gonadotropins LH and FSH act on the ovaries and testes to
stimulate steroidogenesis and gametogenesis. Synthesis and secretion of
the gonadotropins are dependent on appropriate secretion of the
hypothalamic peptide GnRH. GnRH is secreted in pulses that vary in
frequency and amplitude as a function of hormonal status and
reproductive cycle stage (1, 2, 3, 4, 5). The intermittent pattern of release is
critical for normal sexual development and gametogenesis, as
interruption of GnRH pulses or administration of long-acting GnRH
analogs and antagonists results in suppression of gonadotropins and sex
steroid production, thus resulting in infertility (1, 2, 3, 6). GnRH pulse
frequency differentially affects gonadotropin secretion, with higher
frequency pulses favoring LH and lower frequency pulses favoring FSH in
both humans and animal models (1, 2, 3, 4, 5). Frequency-dependent effects are
also noted at the level of steady-state mRNAs encoding the gonadotropin
subunits and the GnRH receptor (7, 8, 9, 10, 11). Administration of pulsatile
GnRH in vivo to male rats in which endogenous GnRH pulses
have been eliminated, and in vitro to cultured pituitary
cells, differentially stimulates subunit mRNA levels. In general, the
-subunit is stimulated by rapid to intermediate pulse intervals
(830 min) in vivo and by rapid pulses or constant GnRH
in vitro, the LHß subunit is favored by intermediate
pulses (3060 min), and the FSHß subunit and GnRH receptor are
favored by longer interval pulses (7, 8, 9). These results are mirrored at
the transcriptional level in vivo, and treatment of
perifused pituitary cells with GnRH emphasizes the requirement for
intermittent GnRH for stimulated transcription of the LHß and FSHß
genes (11, 12). Changes in the amplitude of the GnRH signal can
modulate the GnRH response but, in general, appear to have much less
impact than changes in frequency (9, 13).
The mechanisms by which GnRH differentially modulates gene
expression have been postulated to occur through at least two pathways,
including differential sensitivity of specific genes to the calcium
influx and protein kinase C (PKC) arms of the intracellular GnRH
signaling pathway (14, 15, 16, 17), and different gene targets for the GnRH
response. Several investigators have examined individual promoters of
the gonadotropin subunits or the GnRH receptor for sensitivity to GnRH
signaling cascades, and a few studies have compared the calcium and PKC
sensitivity of the gonadotropin subunit promoters to each other
(15, 16, 17, 18, 19, 20). These data suggest preferential sensitivity of different
genes to either calcium influx or stimulation through the
PKC/mitogen-activated protein kinase (MAPK) cascade (18, 19, 20, 21, 22). However,
at this time no clear consensus has emerged on either the subunit
specificity of the signaling responses or the mechanisms by which GnRH
pulses might preferentially activate individual pathways. Information
on the GnRH-responsive elements in the gonadotropin subunit and GnRH
receptor genes from several species indicates there is considerable
diversity in both the DNA sequence elements and the potential
transcription factors identified. Some GnRH-responsive regions for the
human (18, 23) and mouse (22)
-subunits have been identified and
include consensus binding sites for Ets-domain proteins. Several other
genes tested by transfection analysis, including those for the rat
LHß (24), ovine FSHß (25), and mouse GnRH receptor (26), have
composite GnRH-responsive regions containing several different
individual binding sites for multiple transcription factors. At
present, little is known about how GnRH pulses, as opposed to static
GnRH treatments of cultured cells, regulate gene transcription. The
majority of investigations have also concentrated on responsive areas
in individual genes, and several GnRH-responsive genes have not been
compared in the same experiments. Previously, such studies have been
hampered due to the lack of an appropriate clonal gonadotropin cell
line capable of expressing the promoters of multiple GnRH-responsive
genes.
We have used the LßT2 clonal gonadotrope cell line (27) to
analyze the GnRH responses of the rat LH subunit gene promoters to
static and pulsatile GnRH. We have found that the GnRH response of the
rat
-subunit gene with static GnRH treatment is modulated
through paired Ets-factor binding sites. In contrast, the rat LHß
gene is activated by pulsatile GnRH, acting in part through a large
upstream composite element containing binding sites for Sp1 and CArG
proteins. This element acts to modulate and integrate the response of
additional downstream regions containing binding sites for
steroidogenic factor-1 (SF-1) and early growth response protein-1
(Egr-1). Both upstream and downstream elements are required for the
maximal response to pulsatile GnRH.
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RESULTS
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GnRH Responsiveness of the Rat
-Subunit Gene Requires Two
Ets-Domain Protein Consensus Binding Sites
We previously cloned the rat
-subunit promoter from -479
to +77 bp and showed it to be responsive to GnRH in transfected
T3
cells (20). Deletion/mutation analysis of transfected
promoter-luciferase constructs in LßT2 cells treated with static 10
nM GnRH (Fig. 1
) demonstrated
that the major GnRH-responsive region was located between -411 and
-287 bp. Examination of the rat
-subunit gene promoter sequence
between -411 and -375 bp (Fig. 2
),
corresponding to similar GnRH-responsive regions in the human and mouse
genes, revealed the existence of two putative Ets-domain protein
consensus binding sites, characterized by the core sequences GGAA,
underlined in Fig. 2
. Comparison with the human and mouse
gene sequences shows that these promoters have only one consensus Ets
site, and other groups have demonstrated that these sites are part of
the responsive regions for GnRH or second messengers such as PKC (18, 19). Transfer of a single copy of the -411/-375 bp region to the
heterologous thymidine kinase (tk) promoter (Fig. 3
) or the -106
-subunit minimal promoter, conferred GnRH responsiveness in both
transfected LßT2 cells (Fig. 1
) and
T3 cells (Table 1
). Mutation of either the distal
(tkmut5') or proximal (tkmut3') Ets sites, or both sites
(tkmut5',3') eliminated GnRH stimulation of the heterologous promoter,
suggesting that both sites are required for GnRH stimulation of the
-subunit gene. In agreement, mutation of either the distal site (411
mut5') or both sites (411 mut5',3') within the context of the entire
-411/+77 promoter completely abolished the GnRH response. These
mutations in the -411/+77
-promoter also suppressed basal
expression of the promoter 50% and 65%, respectively (Fig. 1
).
The data suggest that, in addition to playing a critical role in
the stimulated response, the sites contribute to basal expression.
Mutations in these regions would be expected to disrupt
binding of proteins important for the transcriptional
responses.

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Figure 2. DNA Sequences Representing the GnRH-Responsive
Regions of the Rat LH Subunit Genes
The rat -subunit responsive region is compared with similar gene
regions from the human and mouse genes. The putative Ets-domain protein
binding sites are shown in italics and
underlined in each gene. The LHß gene GnRH-responsive
region from -456 bp to -342 bp is shown. The 5'Sp1 and 3'Sp1 binding
regions are italicized and underlined.
The CArG box element is indicated by a double underline,
and a third putative Sp1 site is shown by a dotted
line.
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Figure 3. Binding of Nuclear Proteins to the -Subunit
GnRH-Responsive Region
Upper panel, The wild-type -411 to -375 bp -subunit
promoter region was labeled, and probe (50,000 cpm) was incubated with
5 µg of untreated LßT2 proteins and the indicated unlabeled
competitor oligonucleotides at 200-fold excess molar concentrations.
Competitor DNA included the homologous wild-type (WT) -subunit
promoter sequence, -subunit DNA mutated at the 5'Ets (M5'), 3'Ets
(M3') sites, or both Ets (M5', 3') sites, or consensus binding sites
for CREB, Sp1, or Ets-domain proteins. In the far right
portion of the panel, the consensus DNA sequence for Ets-domain
proteins was labeled, and the probe (50,000 cpm) was incubated with 5
µg of LßT2 proteins in the absence or presence of the Ets-1
antibody. Lower panel, Individual double-stranded
oligonucleotides corresponding to wild-type or mutant -subunit
probes as shown were labeled and incubated with 5 µg LßT2 proteins
in the absence or present of Ets-1 antibody before electrophoresis on a
nondenaturing gel. The positions of specific complexes lost upon
mutation of the Ets sites are shown by the arrowheads.
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Table 1. Basal and GnRH-Stimulated Activity of
Heterologous Promoter Luciferase Constructs Containing Portions of the
Rat LH Subunit Genes Transfected into T3
Cells
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Nuclear Protein Binding to the
-Subunit GnRH-Responsive
Region
Wild-type and mutated oligonucleotides corresponding to the
GnRH-responsive region of the
-subunit gene, from -411 to -375 bp,
were used in electrophoretic mobility shift assay (EMSA) studies to
characterize nuclear proteins bound to this region. Experiments using
nuclear proteins from LßT2 cells demonstrated four groups of
DNA-protein complexes with wild-type DNA (Fig. 3
, upper panel). Addition of
unlabeled competitor DNA representing wild-type or mutated
-subunit
promoter DNA or an Ets protein consensus binding site effectively
competed for the formation of these complexes, but addition of
unlabeled DNA representing binding sites for Sp1 and CREB had no
effect. Labeled DNA containing mutations corresponding to mut 5', mut
3' and mut 5',3' in Fig. 3
showed subtle differences in protein binding
(Fig. 3
, lower panel). Mutation of the distal Ets site (mut
5') inhibited formation of the most slowly migrating (upper) complex,
whereas mutation of the proximal Ets site (mut 3') inhibited formation
of the most rapidly migrating (lowest) complex. DNA with both mutated
sites (mut 5',3') could not form either the upper or lower complex, but
did form the two DNA-protein complexes of intermediate mobility.
Addition of antibody specific for Ets-1 did not interfere with the
formation or alter the mobility of any complex with any probe measured.
Addition of a pan-Ets antibody that binds both Ets-1 and Ets-2
likewise had no effect (not shown). However, treatment with the
Ets-1 antibody effectively eliminated the formation of protein
complexes with the consensus Ets-domain DNA binding element (Fig. 3
, upper panel), indicating that the antibody was effective.
Thus, it appears that the two putative Ets sites are not equivalent,
although both are critical for stimulated promoter activity, and form
slightly different protein complexes. Because unlabeled mutant DNA can
effectively compete with wild-type DNA for the formation of all four
complexes, they may permit binding of "core" proteins, while
prohibiting the formation of or destabilizing other heteromeric
complexes necessary for transcription. None of the binding proteins
appear to be Ets-1 or Ets-2. No significant differences were noted with
nuclear proteins from control or GnRH-treated cells (not shown).
Similar results were observed in studies with nuclear proteins from
T3 cells (not shown).
GnRH-Responsive Elements in the Rat LHß Subunit Gene
We performed initial studies in primary cultures of dispersed
female rat pituitary cells treated with either static or intermittent
10 nM GnRH (1 pulse/h). Under these conditions (Fig. 4
), the transfected LHß promoter was
stimulated 2- to 3-fold by pulsatile but not static GnRH. The data
suggest that in normal gonadotrope cells the nature of the GnRH signal
is critical in LHß gene regulation and agrees with previous
transcriptional run-off data of pituitary cells treated with pulsatile
and static GnRH (11). Deletions of the gene between -2.0 kb and -617
bp relative to the transcriptional start site slightly increased basal
activity, but subsequent deletions decreased basal activity. GnRH
responsiveness was eliminated when the region from -495 and -245 bp
was removed, indicating this area contained at least one
physiologically relevant GnRH response element. Transfer of either the
-495 to -342 bp region, or -450 to -342 bp region of the LHß
promoter to either the viral thymidine kinase promoter or the
-106
-subunit minimal promoter was sufficient to confer a GnRH
response to the heterologous promoters in
T3 cells (Table 1
). These
data are in agreement with results from other investigators who used a
heterologous sommatolactotrope pituitary cell line and identified a
major GnRH responsive element that resided in this region of the LHß
gene (24).

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Figure 4. Deletion Analysis of the Rat LHß Subunit Gene
Promoter Identifying the Region Responsive to Pulsatile GnRH
Luciferase constructs (15 µg) containing portions of the rat LHß
promoter and CMV-ß-galactosidase (1 µg) were transfected into
primary cultures of female rat pituitary cells for 4 h, followed
by a 15% glycerol shock. Constructs included promoter regions from the
5'-base indicated, to the common 3'-end at +41 bp. After incubation in
culture media for 16 h, transfected cells were treated with 10
nM GnRH for 8 h either continuously (static), or as a
series of 10-min pulses once per h (pulse) and compared with untreated
(control) cells. After treatment, luciferase activity was measured and
normalized for both protein content and ß-galactosidase activity.
Normalized luciferase activity (ALU/mg protein) is expressed as the
mean ± SEM for two experiments, with four culture
dishes per group. *, P < 0.05; **,
P < 0.01; ***, P < 0.001
vs. untreated controls.
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We next tested the response of several wild-type and mutated constructs
in LßT2 cells, which express the endogenous LHß gene. Because the
endogenous rat LHß gene (11) and LHß promoter constructs
transfected into primary cultures of rat pituitary cells require
pulsatile GnRH to stimulate promoter expression, we tested each
construct for 6 h with hourly GnRH pulses, each with matched
controls. Within this responsive region (Fig. 2
.) are several putative
binding sites for Sp1 at both the 5'- and 3'-ends. The 3'-region
contains up to three such Sp1 sites, with two sites almost completely
overlapping (shown by the underlined and italicized 3'-site
in Fig. 2
), while the 5' Sp1 site at -450 bp overlaps a CArG box at
-443 to -434 bp. CArG elements [CC(A/T)6GG]
are contained in many promoter enhancer regions, including the serum
response element (SRE) of the c-fos gene, and are often part
of composite elements involved in rapid and transient transcriptional
responses (29, 30, 31). In LßT2 cells, pulsatile GnRH treatments,
previously shown to be required for LHß mRNA stimulation (27), also
stimulated the intact LHß promoter constructs 5- to 6-fold (Fig. 5
). The degree of stimulation was greater
than that observed with GnRH treatment of
T3 cells transfected with
heterologous promoter constructs, and larger than responses observed
with the native promoter and pulsatile GnRH treatment of transfected
primary pituitary cells (Fig. 4
). Hourly GnRH pulses, while stimulating
the LHß gene promoter, were actually ineffective in stimulating
-subunit promoter activity (Control = 60,149 ± 5,948
vs. pulsed GnRH = 56, 908 ± 17, 444 arbitrary
luciferase units (ALU)/100 µg protein) compared with a 4-fold
stimulation with static GnRH in the same experiment (compare with Fig. 1
).

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Figure 5. Deletion/Mutational Analysis of the LHß Promoter
in LßT2 Cells with Pulsatile GnRH Treatment
Cells were transfected with the indicated promoter constructs (3 µg)
for 16 h, followed by 6 h of treatment of 100 nM
GnRH for 6 h with one 10-min pulse of GnRH per h. Each control
group had media changes identical to treatment groups. The sites of the
5'Sp1, CArG box, 3'Sp1, and Egr-1 sites are shown, as well as a
mutation in an A-T rich region at -340 bp. Mutated sites in the
upstream responsive region are indicated by an X in the bar
symbols along the Y axis. Normalized luciferase activity is
represented as the mean ± SEM for six experiments,
with three to four samples per group. *, P < 0.05;
**, P < 0.01; ***, P < 0.001
vs. untreated controls.
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Mutational analysis of the LHß gene suggested a complex
interrelationship between multiple elements in the -450 to -342 bp
responsive region. Although the GnRH response was significantly reduced
when the promoter region between -456 and -345 bp was removed, and
the upstream region from -450 to -342 bp conferred a GnRH response to
heterologous promoters (Table 1
), the -345 bp construct was still
stimulated by GnRH in LßT2 cells. This suggests that an additional
GnRH-responsive site or sites are present in the -345 bp promoter.
Mutation of an AT-rich site at -340 bp or deletion of the region
between -375 and -245 bp, which contains several additional Sp1
consensus binding sites, had little impact on either basal or
stimulated promoter activity. Individual mutations of either the CArG
box or the -365 bp 3'Sp1 site within the context of the entire
homologous promoter decreased basal stimulation, while mutation of the
5' Sp1 site actually increased basal expression. However, under
pulsatile GnRH treatment conditions, single mutations in either the
CArG box, the 5'Sp1, or 3'Sp1 sites effectively eliminated the GnRH
response (Fig. 5
), indicating that these mutations restrict the
activity of GnRH-responsive sites in the -345 bp promoter.
Additional mutational analysis suggested that the two Sp1 and CArG
sites interact in the intact promoter. Mutation of both the 3' and
5'Sp1 sites, or mutation of the 3'Sp1 site and the CArG box in the
CG/SP mutant, partially restored GnRH stimulation. Similarly, mutation
of the 5'Sp1 site in the presence of both the 3'Sp1 and CArG mutations
did not significantly change the response to GnRH compared with the
double mutant. These data demonstrate that stimulation of full-length
promoter activity by pulsatile GnRH treatment requires at least three
distinct upstream elements, including the two Sp1 sites that are
involved in the response to static GnRH (24), and an additional site in
the CArG box. Removal of this entire region, or mutation of sites at
both the 5' and 3' ends of this region, results in 2- to 3-fold GnRH
stimulation, equal to that observed with the -345 bp construct.
Recently, two binding sites for Egr-1, at approximately -112 and -50
bp, have been described in the rat LHß promoter and documented to be
responsive to pulsatile GnRH (41, 42, 43, 44, 45, 46). To test the possibility that the
Egr-1 sites contribute to the GnRH response, the -112 and -50 bp
sites were mutated individually within the context of the -456 to +44
promoter, with and without mutation of the upstream GnRH-responsive
element (Fig. 6
). Mutation of the -112
Egr-1 site had only a slight effect on either basal or GnRH-stimulated
expression, and inclusion of the -112 Egr-1 mutation in the CG/SP
double mutant had little effect on GnRH stimulation. Mutation of the
-50 Egr-1 site, however, reduced basal expression by 50% and
GnRH-stimulated promoter activity to 2.3-fold, compared with 5-fold for
the intact wild-type promoter. Furthermore, mutation of the -50 Egr-1
site within the context of the CG/SP mutant in which the CArG box and
the 3'Sp1 site are mutated effectively obliterates GnRH stimulation.
Thus, the upstream composite element cooperates with the Egr-1 binding
site at -50 bp to confer the full GnRH response to the LHß
promoter.

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Figure 6. Mutational Analysis of Egr-1 Sites with or without
Mutation of the Upstream GnRH-Responsive Region of the LHß Promoter
in LßT2 Cells Treated with Pulsatile GnRH
Cells were transfected with the indicated promoter constructs (3 µg)
for 16 h, followed by 6 h of treatment of 100 nM
GnRH for 6 h with one 10-min pulse of GnRH per h. Each control
group had media changes identical to treatment groups. Mutated sites in
each construct are designated by an X in the symbols
along the Y axis. Normalized luciferase activity is represented as the
mean ± SEM for four experiments with three samples
per group. *, P < 0.05; **, P
< 0.01 vs. untreated controls.
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Although pulsatile GnRH treatment is required for LHß gene
stimulation in vivo, treatment of LßT2 cells with acute
100 nM GnRH for 4 h can also stimulate LHß
promoter activity to some extent. Under these conditions, single
mutations of the 3'Sp1 and 5'Sp1 sites suppress the GnRH response
(5'SP1mut = 2.2-fold stimulation, and 3'SP1mut = 1.3-fold
stimulation vs. 4.8-fold stimulation for the wild-type
promoter), but mutation of the CArG box has no effect (CArGmut =
5.0-fold, and was no different from the wild-type construct in five of
five experiments). The loss of this sensitive interplay of gene
elements with static GnRH treatment suggests that proteins binding to
the GArG box are important for pulsatile GnRH responsiveness, which is
the relevant stimulating paradigm in vivo.
Nuclear Protein Binding to GnRH-Responsive Regions of the LHß
Gene
Oligonucleotides representing the portions of the upstream
composite GnRH- responsive region of the rat LHß gene formed several
DNA protein complexes with nuclear proteins from LßT2 gonadotrope
cells. Similar results were obtained with proteins from control or
GnRH-treated cells (not shown). An oligonucleotide representing the
3'Sp1 site at -365 bp binds nuclear proteins that include Sp1, based
on the mobility of the complex, the addition of pure Sp1 protein (Fig. 7
, top panel, Sp1 complex
shown by an arrowhead), and the inhibition of complex
formation by the addition of Sp1 antibody. The mutated 3'Sp1
oligonucleotide, corresponding to the transcriptionally inactive
construct (Fig. 5
), did not bind Sp1. Similar studies with the
wild-type 5'Sp1/CArG box oligonucleotide (Fig. 7
, bottom
panel) demonstrated that at least three to four complexes were
formed with nuclear proteins from gonadotrope cells. Individual
mutations of the 5'Sp1 site (5'SP1mut) or the CArG box (CarGmut) site
revealed that the two areas were functionally different when comparing
protein binding to these labeled probes. The 5'Sp1mut
probe does not form the upper complex corresponding to Sp1 from LßT2
nuclear proteins and could not bind pure Sp1 (not shown). In contrast,
the oligonucleotide corresponding to the mutated CArG box, which
effectively eliminated stimulation specifically by pulsatile GnRH,
still binds Sp1 effectively. These data suggest that Sp1 binding alone
is insufficient for pulsatile GnRH stimulation of transcription. Two
additional mutated oligonucleotides, representing our double mutation
of the 5'Sp1 and CArG box (5'SP1mCGm) and a previously described Sp1
mutation (24) with several widely spaced mutated bases (5'SP1m2CG),
were also tested. The double mutant 5'Sp1mCGm oligonucleotide does not
bind proteins effectively, while the previously described mutant
(5'Sp1m2CG) fails to bind Sp1 but retains some binding to the
intermediate mobility complexes, designated by a
bracket.

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Figure 7. Nuclear Protein Binding to Labeled Wild-Type and
Mutant DNA Oligonucleotides Representing Portions of the Upstream
GnRH-Responsive Region of the Rat LHß Gene
The position of the Sp1 complex is shown by an
arrowhead, and the positions of intermediate migrating
complexes are designated by brackets. Top
panel, Labeled DNA representing the wild-type and mutated 3'Sp1
LHß sequence was incubated with either 6 µg of untreated LßT2
cell nuclear protein or 1 footprinting unit of recombinant human Sp1.
In one set of reactions, nuclear proteins were incubated with wild-type
3'Sp1 probe in the absence or presence of antibody to Sp1.
Bottom panel, Labeled probes representing the wild-type
and mutated 5'Sp1CArG region of the LHß promoter, with individual or double mutations in the Sp1
or CArG region as shown, were incubated in the absence or presence of 6
µg of LßT2 nuclear proteins and run on a nondenaturing gel. In the
right three lanes of the panel, the 5'Sp1CGm (CArG mutation) probe is
shown next to a wild-type (WT) probe run on the same gel for direct
comparison.
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Competition studies performed with the wild-type labeled probe (Fig. 8
) were in essential agreement with the
above data. Addition of unlabeled wild-type oligonucleotide (WT
5'Sp1CG) inhibited binding to all protein complexes. In comparison,
addition of the previously described Sp1 mutation (5'Sp1m2CG) had no
effect on the formation of the Sp1 complex and moderately inhibited
formation of the intermediate complexes. Addition of the double mutant
5'Sp1mCGm oligonucleotide did not inhibit formation of any complexes.
Addition of unlabeled WT 3'Sp1 binding site inhibits formation of only
the Sp1-DNA complex, and similar results were obtained with an
unlabeled consensus Sp1 site. In contrast, addition of an unlabeled
oligonucleotide representing the CArG region, but not the entire 5'Sp1
site (WT CG), prevented formation of only the intermediate mobility
complexes. This result is similar to the binding profile observed with
competition by a full-length oligonucleotide mutated in only the CArG
region (5'Sp1CGm), which specifically inhibits transcriptional
stimulation by pulsatile GnRH. Addition of unlabeled consensus binding
sites for CREB or Ets-domain proteins had no effect on nuclear protein
binding (not shown). Thus, the functional interplay between the 5'Sp1
and CArG-box binding sites required for GnRH-stimulated transcription
via the upstream response region occurs by interaction between at least
two distinct transcription factors, one of which is Sp1. The second
factor or factors bind to the CArG box and are critical for maintaining
the transcriptional response to pulsatile GnRH signals. This complex is
required for optimal GnRH responsiveness and appears to cooperate with
downstream elements binding Egr-1.

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Figure 8. Competition Studies of Untreated LßT2 Nuclear
Protein Binding to the Labeled Wild-Type 5'Sp1CArG Region Probe in the
Presence of Unlabeled Competitor DNA
Nuclear proteins (4 µg) were preincubated with a 200-fold molar
excess of unlabeled DNA before incubation with labeled probe.
Competitor oligonucleotides included the 3'Sp1 site and wild-type and
three different mutated 5'Sp1CArG regions as shown in Fig. 6 . An
additional oligonucleotide that included the 5'CArG region but not the
entire 5'Sp1 site (WT CCG, 5'-cacccatttttggacccaatcc-3') was also
added. The middle lane shows binding of the wild-type
probe to recombinant human Sp1 for comparison.
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DISCUSSION
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Pulsatile hypothalamic release of GnRH plays an integral role in
regulating gonadotropin secretion and gene expression, and thus normal
sexual development and reproductive function. Specific pulse
frequencies of GnRH have differential effects on LH and FSH secretion,
as well as on the transcription of the three gonadotropin subunit genes
in vivo and in vitro. The common
-subunit gene
can be stimulated by either rapid to intermediate pulses or by static
GnRH, whereas the LHß gene requires intermittent GnRH stimulation
(9, 10, 11, 12, 13). Given that all three genes are expressed in the same cell type
and are stimulated by the same ligand and receptor, such
subunit-specific effects are potentially due to differential
sensitivity to GnRH-regulated signaling pathways and different nuclear
targets responsive to GnRH. Emerging evidence from several groups
suggest contributions from both possible mechanisms.
GnRH stimulates both calcium influx and phospholipase C/protein kinase
C signaling with PKC stimulation linked to subsequent activation of
MAPK activity (14, 15). No clear consensus on sensitivity to signaling
pathways has emerged, although genes from several species have been
tested in a variety of transfection systems. However, the relative
sensitivity of the LH subunit genes to the two arms of the signaling
pathways has differed in studies where the two mRNAs and promoters have
been tested simultaneously (16, 17, 20, 21). Transfection studies
performed in the heterologous GGH3 cell line suggest that the rat LHß
promoter is more sensitive to the PKC pathway, while the human
-subunit promoter is stimulated by both calcium and PKC (24).
Studies of GnRH stimulation of gonadotropin mRNA levels in primary rat
pituitary cultures have shown that GnRH-stimulated
-subunit, FSHß,
and GnRH-R mRNA levels are suppressed by inhibitors of MAPK activity,
but LHß mRNA stimulation is not (17). We have used nuclear run-off
assays in normal rat pituitary cells, transfection into
T3
gonadotrope cells, and transgenic animal studies and have found that
the rat
-subunit promoter is relatively more sensitive to MAPK
stimulation, while stimulation of the rat LHß gene is relatively more
sensitive to calcium influx (20). Such apparent discrepancies may be a
result of species differences in the gene promoters used, different
transfection systems and cell lines, or slightly different promoter
constructs used, including heterologous promoter/response element
constructs. It is also probable that cross-talk between signaling
pathways exists, and that the relative amount of signaling through each
pathway could vary under physiological conditions. Thus, although the
exact signaling pathways that are most critical for stimulation of each
responsive gene are not consistent in all systems, there is clear
divergence in the pathways importance among the GnRH-responsive
genes. Recent studies suggest that stimulation of the ovine FSHß
promoter (25) and the murine GnRH-R promoter by GnRH (26) occur via the
PKC/MAPK pathway acting through multiple promoter regions including
AP-1 sites. However, AP-1 sites do not appear to play a role in
stimulation of the rat
-subunit or LHß promoters by GnRH.
Studies with the mouse
-subunit gene promoter transfected into
clonal
T3 cells (19) or the human gene transfected into either rat
pituitary cells (23) or
T3 cells (18) have demonstrated that the
GnRH stimulation is tightly linked to PKC and MAPK kinase stimulation,
similar to our observations with the rat
-subunit gene in normal
pituitary or transfected
T3 cells (20). Stimulation of the human
promoter in the heterologous GGH3 line appears to occur primarily
through a calcium-mediated cascade (24). The mouse
-subunit promoter
requires two cooperating regions for complete GnRH stimulation (22).
One area from -337 to -330 bp is necessary for pituitary-specific
promoter expression and another between -416 to -385 bp (the GnRH-RE)
confers a GnRH response to heterologous promoters. This GnRH-RE region
is depicted in Fig. 2
and shares considerable sequence homology with
the GnRH-responsive region in the rat
-promoter, which lies between
-411 and -375 bp. The GnRH-responsive region of the human gene is
much less well defined and appears to require multiple interacting
regions, while second messenger specificity studies have been performed
with the -846 bp construct (19). Alignment of the human
-subunit
gene region from -428 to -397 bp with the rat GnRH-responsive region
from -411 to -375 bp, and the mouse gene from -416 to -385 bp (Fig. 2
) reveals significant DNA sequence homology and some important
differences. All three genes contain GGAA sequence motifs, which are
core consensus binding sites for Ets-domain proteins; however, the rat
gene contains two closely aligned sequences on opposite DNA strands,
and the mouse and human genes have only one (upstream) site. Ets
factors have been shown to be phosphorylated directly by or in response
to activated MAPK and thus could be direct mediators of GnRH
stimulation (32, 33). Studies with the transfected rat
-subunit
promoter show that inhibition of the MAPK pathway suppresses basal as
well as stimulated promoter activity (20), as is observed with mutation
of the putative Ets-factor binding sites, suggesting that these sites
modulate promoter activity through the MAPK pathway. Disruption of
either site in the rat gene suppresses basal and eliminates GnRH
stimulation of promoter activity.
A GAL4-Elk-1 fusion protein directly stimulated the mouse
-subunit
gene promoter, and both functional and DNA binding studies show that
Ets-2 can bind the mouse gene region; however, the gonadotrope
factor(s) binding directly to this region has not been identified (19).
Because the addition of an antibody to Ets1/Ets-2 that recognizes a
spectrum of Ets factor family members did not interfere with the
binding of LßT2 cell proteins to the rat
-subunit promoter, but
did recognize proteins binding to a consensus Ets site, a novel protein
or proteins may participate in the GnRH response. A consensus Ets
binding site oligonucleotide did compete for binding to this region,
suggesting that a transcription factor with similar DNA sequence
binding properties is involved. The unique positioning of two putative
Ets binding sites on the GnRH-responsive region of the rat
-subunit
gene, and the requirement for both intact sites for GnRH stimulation,
suggests functional cooperation between the sites. GABP, which confers
insulin sensitivity to the PRL gene promoter (34), is a heterodimeric
protein in which one subunit is an Ets-like protein that binds DNA, and
the other subunit contains a series of ankyrin repeats (35). Although
the
-subunit promoter does not contain a consensus site for GABP
binding, a similar type of protein or proteins, capable of forming
functional protein-protein interactions, may be involved in conferring
the GnRH response.
The GnRH-responsive region for the rat LHß gene is highly divergent
from that of the
-subunit promoter, in keeping with the differential
sensitivity of the two genes in vivo to different GnRH pulse
regimens, and different responses of the transfected subunit promoters
to various signaling pathways. Instead of relying on Ets-like binding
sites, the LHß gene upstream GnRH-responsive region contains a number
of interacting transcription factor binding sites, including two Sp1
binding regions and a CArG box element. In these studies, we have
demonstrated the importance of the Sp1 binding sites in both
GnRH-stimulated and basal expression in gonadotrope cells, as others
have previously demonstrated in heterologous cell lines (24, 36).
However, Sp1 binding alone is insufficient for the GnRH response, as
mutations in the newly defined CArG element permit Sp1 binding, yet
abolish the response to pulsatile GnRH (Figs. 5
and 7
). Proteins that
bind to CArG boxes, such as serum response factor (SRF) and related
proteins, often form multiprotein complexes and are involved in rapid
and transient transcriptional responses (29, 30, 31). The smooth muscle
myosin heavy chain gene promoter contains a negative-acting GC-rich
region located between two stimulatory CArG elements that bind SRF (30, 31). The GC-rich region in the myosin promoter binds both Sp1 and Sp3
and modifies the gene response to SRF without interfering with SRF
binding. In the LHß gene, the 5'-Sp1 binding site and CArG element
overlap, but Sp1 and CArG box protein binding can be differentially
inhibited by mutation analysis. Protein binding to both sites appears
to be required for complete GnRH stimulation. The role of SRF and
related proteins in rapid and transient transcriptional responses is
intriguing in light of the preferential response of LHß to GnRH
pulses, and the unique role of the CArG element in that response. It is
unlikely that SRF, a muscle protein, is contained in gonadotropes, but
a similar type of protein may be involved.
There is an interesting and complex relationship between the composite
5'Sp1/CArG element and the 3'Sp1 binding site. Although mutations in
any of these three sites completely suppressed the GnRH response,
mutation of the 3'Sp1 site in combination with the 5'Sp1 and/or the
CArG box restores responsiveness to the level observed with the -345
bp construct. These data suggest that interactions between the two Sp1
sites occur and restrict the responsiveness of the gene promoter. The
-345 bp construct contains two composite binding sites for two
transcription factors found to have a critical role in gonadotropin
expression, SF-1 and Egr-1 (37, 38, 39, 40, 41), and several groups have now
described cooperative functional interactions between these two
proteins on the rat and equine LHß promoters (42, 43, 44, 45). Our studies
used a larger promoter region than the constructs used in other
studies, and the data demonstrate that Egr-1 binding sites can
cooperate with other upstream GnRH-responsive regions as well. In the
context of the larger LHß promoter, both the upstream Sp1/CArG
regions and the Egr-1 sites are required for the full response to
pulsatile GnRH.
Disruption of either the SF-1 (40) or Egr-1 (41) genes in mice severely
impairs LHß gene expression, but the relationships are not completely
straightforward. For example, SF-1 gene disruption eliminates
expression of all gonadotropin subunit genes, but injection of the
animals with GnRH partially restores expression (40). Disruption of the
Egr-1 gene completely eliminates Egr-1 protein expression in both
males and females, but only in females is LHß gene expression
completely eliminated with resulting infertility (41). In a separate
study, target disruption of the Egr-1 gene at a different position
completely eliminated LHß protein expression and resulted in
infertility in both sexes (46). Both SF-1 (39) and Egr-1 mRNA (43, 44, 45)
can be stimulated by GnRH treatment, and Egr-1 may also be stimulated
by PKC. Particularly dramatic increases in Egr-1 mRNA have been
observed in LßT2 cells with pulsatile GnRH (43), although increased
Egr-1 expression has also been noted in
T3 cells (43, 44). The
resulting increase in Egr-1 protein levels thus stimulates expression
of the LHß promoter, and mutations in Egr-1 binding sites have a
larger effect on basal and GnRH-stimulated promoter activity than do
mutations in SF-1 sites. Our studies, and previous studies in a
heterologous cell line, suggest that GnRH stimulation of the rat LHß
promoter requires an integrated response of multiple transcription
factors binding to several discrete promoter elements, distinct from
and in addition to the promoter regions binding SF-1 and Egr-1 (19, 20). Studies with the isolated LHß response elements on two
heterologous promoters (Table 1
and Ref. 20) demonstrate that the
region between -456 to -342 bp is capable of conferring a GnRH
response separate from that conferred by the SF-1/Egr-1 regions and may
help explain some differences in cell signaling transfection studies.
The GnRH response of the promoter may thus be governed partly by Egr-1
levels that are modulated by GnRH, and partly by other GnRH-mediated
pathways that stimulate the Sp1/CArG containing upstream region.
Relatively high or static Egr-1 levels may permit the upstream region
of the gene to play a larger role in GnRH regulation and might explain
the very low response of the smallest LHß promoter construct in
primary cultures. Overall, these studies indicate that the
transcription factors involved are distinct from those conferring GnRH
stimulation to the rat
-subunit gene and represent the first direct
comparison of these two subunits from the same species. There may be
physical as well as functional interactions between these factors, in
addition to modification of transcription factors by calcium and
PKC-activated kinases as well as MAPK. Direct interaction between Sp1
and Egr-1 has been demonstrated in osteoclast cells, with Egr-1
limiting the amount of free Sp1 available to activate gene
transcription. (46). Functional interactions between Egr-1 and Sp1
could occur in the gonadotrope cell, either in solution or bound to DNA
and help explain the restrictive influence of Sp1 mutations on the GnRH
stimulation of the LHß promoter.
 |
MATERIALS AND METHODS
|
---|
Vector Preparation
The rat
-subunit gene promoter from -479 to +77 relative to
the transcriptional start site was cloned from rat genomic DNA and
inserted into the luciferase reporter vector as previously described
(18). Deletion constructs corresponding to 5'-ends of -411, -354,
-245, and -106 bp were obtained from this construct by PCR
amplification and cloning into the PCRII vector
(Invitrogen, San Diego, CA). After DNA sequence
verification of both strands, each promoter was inserted into the LUCII
expression vector (18). Primers used for constructing promoter regions
included intronic primer from +77 to +59 bp
(5'-ttccccaaatgatttcatc-3'), and the 5'flanking sequence primers at
-411 bp (5'-atggatccacttttctgtttcctgttc-3'), -354 bp
(5'-ccgaaaacatcaggcac-3'), -245 bp (5'-cccttttctggccaag-3'), -179 bp
(5'-ccccaaaatgtctaaaagc-3'), and -106 bp
(5'-ccataccaagtaccatc-3').
The rat LHß gene promoter region from the BamHI site at
-2 kb to the KpnI site at +41 bp relative to the
transcriptional start site was obtained from a rat genomic DNA clone
and inserted into the Luciferase expression vector as described (20, 48). Deletion constructs of this region were made by using the 3'-end
KpnI site at +44 bp and restriction enzyme sites
corresponding to 5'-ends at -617 (EcoRI) and -245 bp
(PstI). Additional deletion constructs were generated by PCR
amplification of the genomic clone using primers corresponding to the
3'-KpnI site and 5'ends at -456 bp
(5'-gctgaaaccacacccatttttggaccc-3'), and -345 bp
(5'-gaggatccgaacaccgaagctgtgc-3'). All amplified promoter regions were
first cloned into the PCRII vector, and then inserted into the
luciferase expression vector at the BamHI polylinker site.
All constructs were sequenced to ensure fidelity. A construct
containing the promoter region from -617 to +44 bp, but with a -370
to -246 bp deletion (
370/246), was made from a -617 to +44 bp
LHß promoter clone in a pGEM3Z plasmid in which the PstI
site was eliminated. This construct was cut with PstI and
KpnI to remove the -617 to -245 bp region. This gene
fragment was then cut with DraI, and the resultant 250 bp
fragment from -617 to -371 bp was isolated and inserted by blunt end
ligation into the pGEM3Z vector containing the remaining gene region,
before cloning into the luciferase reporter.
Isolated putative GnRH-responsive regions were fused to two
heterologous promoters that were not responsive to GnRH, including the
-106
-subunit luciferase construct, and the Herpes simplex virus
thymidine kinase (tk) promoter construct, as previously described. For
the
-subunit gene, the region between -411 and -375 was generated
by PCR amplification. The amplified fragment was excised from the PCRII
vector with BamHI and inserted into the BamHI
site at the 5'-end of the -106
-subunit promoter region, or in the
polylinker region of the tkLUC vector. A similar strategy was followed
for the LHß gene, in which the gene regions between -495 and -342,
and between -450 and -342 bp, were amplified and fused to the
heterologous promoters.
Mutagenesis
Two separate mutagenesis strategies were used, depending on the
location of the altered region. For internal mutations within the
context of the homologous promoter, the Altered Sites II in
vitro mutagenesis kit (Promega Corp., Madison, WI)
was used. Briefly, a template was prepared in which the -617 to +44 bp
region of the rat LHß gene was inserted at the KpnI and
BamHI sites into the pAlter vector, which contains potential
ampicillin and tetracycline resistance regions. This construct was
denatured to anneal with mutagenic oligonucleotides, including
mutations for the identified 3'Sp1 site at -365 bp
(5'-ggggctgggcgagggTTTgcgcccacctctgg-3'), the CArG-box region at -436
bp (5'-aaccacacccattttCCgacccaatccaggcatc-3'), an AT-rich region at
-340 bp (5'-cacctctggttgtatCCCaagcaaatttggaggc-3'), the Egr-1 site at
-112 bp (5'-ctgaccttgtctgtctCgCCCcaaagattagtgtc-3''), and the Egr-1
site at -50 bp (5'-gtggccttgccacccCCaCaaccCgcaggtataaagcc-3'').
Simultaneously, ampicillin repair and tetracycline knock-out
oligonucleotides were added to change the antibiotic resistance as a
marker for successful mutagenesis. Each of these single mutations was
introduced, and the resultant constructs were sequenced and inserted
into the luciferase expression vector. The mutated 3'Sp1 site plasmid
(3'SP1 mut) was used as a template to introduce the CarG box mutation
(CArGm) and to form a double mutant (CGm3'SP1m). Alternatively, for
mutations in the LHß promoter -456 to +44 bp constructs, PCR
amplification was performed with wild-type or mutated -617 to +44 bp
pAlter templates to introduce mutations at the 5'Sp1 site (5'SP1mut
with oligonucleotide 5'-taggtaccacacAAatttttggacccaatcc-3'), alone or
in combination with other mutations. The oligonucleotide 5'SP1mCGm
(5'-taggtaccacacAAattttCCgacccaatcc-3') was used with the mutated 3'Sp1
site template to produce a triple mutant construct (5'SP1mCGm3'SP1m).
Triple mutants containing single Egr-1 mutations in combination
with the 5'CArG site and 3'SP1 site were similarly created. All mutated
bases are shown in capital letters.
A similar strategy was used to introduce mutations into the potential
Ets-domain binding sites of the rat
-subunit promoter. A mutation in
the 5'-Ets site (
Mut5', with the primer 5'-
atccatcttttctgttGActgttggaata-3') was made by PCR amplification from
the -479 to +71 bp construct, and the 3'-genomic primer. The
Mut5' construct was then used with the
Mut5',3' primer (5'-
atccatcttttctgttGActgttgTGataacgtaga-3') to prepare a construct with
mutations in both Ets sites. Mutant
-subunit GnRH-responsive
elements obtained by chemical synthesis of appropriate complementary
oligonucleotides were annealed and inserted into tkLUC or -
106LUC
vectors as described. Single mutations were incorporated into each Ets
site with the following oligonucleotide sequences that contained
5'KpnI and 3'BamHI sites: MUT5'
(5'-cttttctgttGActgttggaataacgtagag-3') and MUT3'
(5'-tttctgtttcctgttgTGataacgtagag-3') and both mutations.
Cell Cultures and Transfections
For
-subunit gene constructs, and studies of both LH subunit
gene isolated elements in heterologous promoter constructs,
transfection studies were first performed in the
T3 cell line, and
subsequently in the LßT2 cell line. The
T3 precursor gonadotrope
cell line (28) contains functional, well characterized GnRH receptors
and expresses the
-subunit gene, but neither the endogenous nor
exogenous transfected LHß promoter. Cells were grown in DMEM
containing 10% FBS, 2 mM glutamine, 100 U/ml penicillin,
and 100 mg/ml streptomycin. Cells were plated at a density of
0.50.7 x 106/60 mm well 36 h before
a 16 h CaPO4 transfection with 5 µg DNA
per well. After transfection, cells were washed and treated for 6
h with 10-7 M GnRH or the des-gly
agonist (GnRHa). For all transfection studies, cells were then washed
and collected in 250 µl lysis buffer (Promega Corp.),
vortexed, spun for 1 min, and assayed in a Turner 20e luminometer
(Turner Designs, Mountain View CA). The same lysates were used for
protein determination, using the colorimetric protein assay system
(Bio-Rad Laboratories, Inc., Hercules, CA). Luciferase
activity was expressed as ALU per 100 µg protein. In each experiment,
treatment groups contained three to four wells, and values represent
the results of three to six experiments. Control and GnRH treatment
groups were compared for each construct, and statistical significance
was assigned by Students t test. In some mutational
analysis studies, values are expressed relative to untreated controls
for each construct, set at 100% or 1.0.
LHß promoter constructs were first tested in primary cultures of rat
pituitary cells. Primary cultures of dispersed randomly cycling female
rat pituitary cells were prepared from mascerated glands as previously
described (49) by digestion with 0.125% trypsin at 37 C for 1.5
h, and collagenase (2 mg/ml, Worthington Biochemical Corp., Freehold NJ) for 3045 min at 37 C. Cells were plated in
60-mm wells (1.52.0 x 106 cells per well)
in DMEM/10% FBS media. After 2024 h, cells were transfected by the
CaPO4 method for 4 h, followed by a 5-min
incubation with 15% glycerol in PBS. Each well received 15 µg LHß
construct and 1.0 µg cytomegalovirus (CMV)-ß-galactosidase. After
transfection, wells were washed and incubated for 16 h in culture
media before treatment with 10- 8 M
GnRH either continuously for 8 h, or in a regimen of 1 pulse of
10-8 M GnRH for 10 min per h.
Luciferase activity was measured and normalized for protein and
ß-galactosidase activity. Data are represented as the mean ±
SEM for six wells per group, compared with untreated
controls.
All wild-type and mutated LHß constructs were subsequently analyzed
in the murine LßT2 gonadotrope cell line (27). These cells express
both endogenous mouse and transfected rat LH subunit genes, secrete LH,
and contain functional GnRH receptors that respond to pulsatile GnRH
stimulation. Cells were grown in DMEM with 10% FBS and antibiotics and
were plated in 35-mm wells at a density of 1.5 x
106 cells per well 1624 h before
CaPO4 transfection. Each well was transfected
with 3 µg of vector for 16 h, washed, and treated with
10-7 M GnRH for either 6 h
continuously (acute static treatment), or with
10-7 M GnRH for 10 min each hour for
6 h (pulsatile treatment). When acute and pulsatile treatment
regimens were compared, separate controls (no GnRH) were used for the
acute and pulsed treatment groups. The controls for the pulsatile GnRH
treatment were handled identically to the treatment groups with respect
to time out of the incubator and media changes with control (no
treatment) media, to ensure that the observed responses were not due to
experimental artifacts. In several experiments, a CMV-ßgalactosidase
reporter was included (0.3 µg/well) to normalize luciferase activity.
After treatment, cells were collected and assayed as above. Each
experiment includes three to four wells per treatment group. Data are
represented as the mean ± SEM and represents data
from three to six experiments as indicated.
Nuclear Proteins and Electrophoretic Mobility Shift
Assays
Nuclear proteins were isolated from LßT2 cell nuclei by the
method of Dignam et al. (50). The extraction buffer [20
mM HEPES pH 7.3, 0.6 M KCl,
20 µg/ml ZnCl2, 0.2 mM
EGTA, 0.5 mM dithiothreitol (DTT)] contained
protease inhibitors (10 µg/ml each of aprotonin, antipain,
chymostatin, leupeptin, and 1 µg/ml pepstatin). After
ultracentrifugation at 100,000 x g, supernatant
proteins were subjected to chromatography on a Sephadex G column with
buffer (20 mM HEPES, pH 7.3, 1.5
mM MgCl2, 0.15
M KCl, 0.5 mM DTT, and
protease inhibitors). For electrophoretic mobility shift (gel shift)
assays, nuclear protein (46 µg) was incubated with labeled DNA
50100(50100,000 cpm) and buffer containing final concentrations of 10
mM Tris-HCl, pH 7.5, 4% glycerol, 1
mM MgCl2, 0.5
mM EDTA, 0.5 mM DTT, 50
mM NaCl, and 1 µg poly(dI-dC). Final volumes
were 1520 µl, and final salt concentrations were adjusted to
100125 mM KCl. Samples were incubated on ice
for 45 min. For competition experiments, protein and buffer were
incubated with unlabeled oligonucleotides, at 200-fold excess molar
concentrations, on ice for 30 min before addition of labeled DNA. A
similar preincubation step was included in studies with antibody
addition. After incubation, reactions were subjected to electrophoresis
on 5% acrylamide (19:1 acrylamide-bisacrylamide) 1 x
Tris-borate-EDTA gel. Gels were run for 1 h at 100 V at 4
C, before sample loading, and samples were subjected to electrophoresis
at 100120 V for an additional 34 h.
Complimentary oligonucleotides representing
-subunit gene sequences
in EMSA included the wild-type sequence
WT
(5'-cttttctgtttcctgttggaataacgtacc-3') and mutated sequences
M5'
(5'-cttttctgttGActgttggaataacgtacc-3'),
M3'
(5'-cttttctgtttcctgttgTGataacgtacc-3') and
M5',3' with both mutated
sites. Mutated bases are designated by capital letters. In
some experiments, competitor DNA representing consensus binding sites
for Ets factors (5'-cggctgcttgaggaagtataagag-3'), CREB, and SP1 were
also included. Antibodies specific for Ets-1, or for Ets-1/Ets-2 were
obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz,
CA).
Complimentary oligonucleotides representing rat LHß gene sequences
for EMSA included wild-type and several mutant sequences representing
the 5'Sp1/CArG box region. The wild-type sense strand sequences include
the entire region as WT 5'Sp1CG
(5'-gctgaaaccacacccatttttggacccaatccaggcatcc-3') and the CArG region
without the entire 5'Sp1 site as WT CG (5'-cacccatttttggacccaatcc-3').
Mutated sequences include a previously described 5'Sp1 binding mutation
with two separate mutated sites 5'Sp1m2CG
(5'-gctgaaaccacacAAatttttggaTccaatccaggcatcc-3'); a mutation in the
CArG box only as 5'Sp1CGm
(5'-gctgaaaccacacccattttCCgacccaatccaggcatcc-3'); and a mutation in
both Sp1 and CArG sites as 5'Sp1mCGm
(5'-gctgaaaccacacAAattttCCgacccaatccaggcatcc-3'). The oligonucleotide
representing the wild-type 3'Sp1 site was 3'Sp1
(5'-gctgggcgaggggcggcgcccacctc-3') and the oligonucleotide representing
the mutated site was 3'Sp1m (5'-gctgggcgagggTTTgcgcccacctc-3'). Pure
human recombinant Sp1 was obtained from Promega Corp..
 |
ACKNOWLEDGMENTS
|
---|
The authors would also like to thank Drs. Gary Owens and Chris
Mack for many helpful discussions during presentations of this work and
Dr. Pamela Mellon for the cell lines.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dr. Margaret A. Shupnik, Box 578 HSC, 7141 Multistory Building, University of Virginia Medical Center, Charlottesville, Virginia 22903.
This work was supported by NIH Grant RO1-HD25719 (to M.A.S.), the
National Science Foundation Center for Biological Timing (Grant DIR
890162), and as part of the Specialized Cooperative Centers Program in
Reproductive Research (Grant NIH/NICHHD U54-HD-28934).
1 Current Address: Department of Biochemistry, Molecular Biology, and
Cell Biology, Northwestern University, Evanston, Illinois. 
Received for publication June 14, 1999.
Revision received December 15, 1999.
Accepted for publication January 3, 2000.
 |
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