From the Division of Genetics, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115
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ABSTRACT |
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The hypothalamic hormone gonadotropin-releasing
hormone (GnRH) plays a critical role in reproductive function by
regulating the biosynthesis and secretion of the pituitary
gonadotropins. Although it is known that GnRH induces luteinizing
hormone (LH
) gene transcription, the mechanisms by which this
occurs remain to be elucidated. We have shown previously that
GH3 cells transfected with the rat GnRH receptor
cDNA (GGH3-1' cells) support the expression of a
cotransfected fusion gene composed of 797 base pairs of rat LH
gene
5'-flanking sequence and the first 5 base pairs of the 5'-untranslated
region fused to a luciferase reporter (
797/+5LH
LUC) and respond to
a GnRH agonist with a 10-fold stimulation of activity. Furthermore, we
have shown that DNA sequences at
490/
352 confer GnRH responsiveness
to the rat LH
gene. We have now identified two putative binding
sites for Sp1, a three-zinc-finger transcription factor, within this
region. Using electrophoretic mobility shift assay, DNase I
footprinting, and methylation interference assays, we demonstrate that
Sp1 can bind to these sites and that Sp1 is responsible for DNA-protein
complexes formed using GGH3-1' and
T3-1 nuclear
extracts. Mutations of the Sp1 binding sites, which block binding of
Sp1, blunt the stimulation of the LH
gene promoter by GnRH. These
data define GnRH-responsive elements in the LH
5'-flanking sequence
and suggest that Sp1 plays an important role in conferring GnRH
responsiveness to the LH
subunit gene.
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INTRODUCTION |
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The pituitary gonadotropins luteinizing hormone
(LH)1 and
follicle-stimulating hormone (FSH) play integral roles in the
regulation of normal reproductive development and function. The
biosynthesis and secretion of these pituitary glycoproteins are
controlled by the complex interaction of multiple factors, among the
most important of which is gonadotropin-releasing hormone (GnRH).
Pulsatile GnRH stimulates the secretion of LH and FSH as well as
transcription, steady-state mRNA levels, and biosynthesis of the
gonadotropin subunits , LH
, and FSH
(1-4). This regulation is
dependent on GnRH pulse amplitude and frequency, which varies with
physiologic state, during puberty, during the rat estrous and human
menstrual cycles, and during menopause (5, 6). An understanding of the
mechanisms of regulation of LH
gene expression is an important first
step in elucidating the mechanisms of physiologic differential regulation of LH and FSH by GnRH.
Studies of the gonadotropin -subunit gene have identified a number
of DNA elements in the 5'-flanking region that mediate tissue-specific
and regulated expression and their cognate binding factors. Recently,
two transcription factors, steroidogenic factor-1 (SF-1) and early
growth response-1 (Egr-1), have been recognized to be involved in
expression of the LH
gene (7-9). Nevertheless, relatively little is
known about transcription factors that direct gonadotrope-specific or
hormonally regulated expression of the LH
and FSH
subunit genes.
A systematic approach to identifying mechanisms of hormonal regulation
of LH
and FSH
subunit gene expression has been hampered by the
lack of available cell lines that express either the endogenous or
transfected LH
and FSH
genes in a regulated manner. In our
studies, we have used GH3 cells, a well-characterized rat
pituitary somatolactotropic cell line, as a model for the analysis of
cis-regulatory elements in the rat LH
gene. We have demonstrated
previously that GH3 cells, when transfected with rat GnRHR
cDNA, bind and respond to GnRH (10-13). Cotransfection with the
5'-flanking region of the
, LH
, or FSH
subunit gene fused to a
luciferase reporter results in the expression of luciferase and a
stimulation of luciferase activity in response to GnRH (14).
Characterization of this cell model has demonstrated many similarities
in the GnRH response compared with that in primary pituitary cells,
including the specific intracellular signal transduction pathways
activated, the degree of stimulation of the gonadotropin subunit
promoter activities, and the presence of differential regulation of
LH
and FSH
gene promoter activities by GnRH. GH3
cells thus appear to be a useful model for the study of the regulation
of expression of the gonadotropin subunit genes by GnRH.
Using this cell model, we have recently identified two elements in the
rat LH gene promoter that contribute to the stimulation of LH
gene expression by GnRH (15). These two elements lie at positions
490/
352 (referred to as region A) and
207/
82 (region B),
relative to the transcriptional start site. A protein(s) present in
GH3 and
T3-1 nuclear extracts binds to DNA sequences within region A. In this study, we characterize the DNA sequences within region A to which protein binding occurs. These sequences have
homology to the consensus binding site for the three-zinc-finger transcription factor, Sp1, which binds to GC-rich sequences. We demonstrate that Sp1 binds to several sites within region A of the rat
LH
gene promoter. Mutations of these sequences abrogate Sp1 binding
and blunt the responsiveness of the LH
gene promoter to stimulation
by GnRH. These data suggest that Sp1 plays an important role in
conferring GnRH responsiveness to the LH
subunit gene.
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EXPERIMENTAL PROCEDURES |
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Materials-- The GnRH agonist des-Gly10, [D-Ala6]GnRH ethylamide (GnRHAg) was purchased from Sigma. Human Sp1 protein was purchased from Promega (Madison, WI). Anti-Sp1, anti-Sp3, and anti-Egr-1 polyclonal antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-follistatin polyclonal antibody was generated as described previously (16).
Reporter Plasmids and Expression Vectors--
An expression
vector encoding the rat GnRHR was prepared by subcloning the rat GnRHR
cDNA sequence into pcDNA1 (Invitrogen, San Diego, CA), as
described previously (10). An expression vector expressing
-galactosidase driven by the Rous sarcoma virus promoter
(RSV-
-galactosidase) was used as an internal standard and control
(17). The reporter constructs used had sequences of the rat LH
gene
promoter cloned into the pXP2 luciferase reporter vector (14, 18). The
rat LH
gene promoter was sequenced from rat genomic DNA by dideoxy
sequencing. The nucleotide sequence of the rat LH
gene promoter used
in these studies is based on our findings, with position
1 assigned
to the nucleotide immediately 5' of the transcriptional start site. The
GH50-pXP1 construct was prepared by subcloning the rat growth hormone
gene minimal promoter into BglII/SacI polylinker
restriction sites in pXP1 (7, 18, 19). All reporter constructs were
confirmed by dideoxy sequencing.
Cell Culture and Transfection--
GGH3-1' cells
were prepared by stably transfecting GH3 cells with the rat
GnRHR cDNA, as described previously (10). GGH3-1' cells
and T3-1 cells were maintained in monolayer culture in Dulbecco's
modified Eagle's medium supplemented with 10% (v/v) fetal bovine
serum at 37 °C in humidified 5% CO2/95% air. For transient transfection studies in GGH3-1' cells, cells were
cultured to 50-70% confluence and transfected by electroporation. In
each experiment, approximately 5 × 106 cells were
suspended in 0.4 ml of Dulbecco's phosphate-buffered saline plus 5 mM glucose containing the DNA to be transfected. The cells
received a single electrical pulse of 240 V from a total capacitance of
1000 microfarads, using an Invitrogen Electroporator II apparatus
(Invitrogen). After electroporation, cells were plated in
serum-containing medium. Medium was replaced 24 h after
transfection. Cells were treated with 100 nM GnRHAg or
vehicle for 6 h immediately prior to harvesting and analyzed
48 h after transfection. These conditions have been selected after
optimization analysis to give maximal levels of expression and GnRH
stimulation (10, 14). Cells were harvested in lysis buffer (125 mM Tris, pH 7.6, 0.5% (v/v) Triton X-100). Supernatant was
collected by centrifugation at 14,000 × g for 15 min
at 4 °C. Luciferase activity was measured using an LB 953 Autolumat
(EG & G Berthold, Nashua, NH) by standard protocols (20). Luciferase
activity was normalized for expression of RSV-
-galactosidase.
-Galactosidase activity was assayed colorimetrically by standard
protocols (17).
Preparation of Nuclear Extracts--
GGH3-1' and
T3-1 cells were grown to approximately 70% confluence and treated
with GnRHAg 100 nM or vehicle for varying time intervals;
then, cells were harvested, and nuclear extracts were prepared by the
method of Andrews and Faller (21).
Electrophoretic Mobility Shift Assay (EMSA)--
DNA sequences
encompassing LH region A (LHA) (
490/
334) were amplified by
polymerase chain reaction and subcloned into pBluescript KS+ (LHA-pBS)
as described previously (15). 32P-end-labeled LHA was
prepared by digesting LHA-pBS with XbaI or XhoI
and 3'-end labeling with [32P]dCTP and the Klenow
fragment of DNA polymerase I, followed by excision with a second
restriction enzyme, XhoI or XbaI. The
32P-labeled DNA fragment was purified on a 9%
polyacrylamide gel in 1× Tris borate-EDTA buffer (89 mM
Tris, 89 mM boric acid, 2 mM
ethylenediaminetetraacetic acid, pH 8.0). Oligonucleotides corresponding to the sense and antisense strands of subregions A1-A5
(Fig. 1A) were synthesized. The sense and antisense
oligonucleotides were then annealed and 5'-end-labeled with
[
-32P]ATP by T4 polynucleotide kinase and purified
over a Nick column (Amersham Pharmacia Biotech).
DNase I Footprinting Assay-- DNase I footprinting assays were performed essentially according to the method of Hudson and Fried (22). Briefly, LHA-pBS was 3'-end-labeled on either the sense or antisense strand as described above. 50,000 cpm of end-labeled DNA fragments were incubated with 1 footprinting unit of Sp1 in a binding reaction mixture prepared as for EMSA. After 30 min at room temperature, samples were treated with DNase I (4 µl of 0.05-0.1 units/µl; Sigma) for 2 min. Reactions were stopped by the addition of 4 µl of formamide loading buffer to inhibit the DNase I. Samples were immediately transferred to a 95 °C water bath and incubated for 4-5 min. Denatured samples (4 µl) were subjected to electrophoresis on 6% denaturing polyacrylamide gels. The untreated end-labeled DNA fragments were also subjected to Maxam and Gilbert sequence reactions for G, A+G, C, and C+T to serve as markers on the gels (23). Gels were dried and subjected to autoradiography.
Methylation Interference Assay-- Methylation interference assays were performed essentially according to the method of Ikeda et al. (24). Briefly, LHA-pBS was 3'-end-labeled on either the sense or antisense strand as described above. 2 × 106 cpm of end-labeled DNA fragments were then partially methylated with dimethyl sulfate. Preparative EMSAs were performed as described above, using 25 µg of nuclear extract or 1-2 footprinting units of Sp1 and 250,000 cpm of methylated probe. Specifically complexed and free DNAs were visualized by autoradiography of the wet gel at 4 °C for 4 h and excised. The DNA was eluted, purified, cleaved with 1 M piperidine for 30 min at 90 °C, lyophilized, and resuspended in formamide loading buffer at a concentration of 1000 cpm/µl. The samples (6 µl) were subjected to electrophoresis on 6% denaturing polyacrylamide gels. Gels were dried and subjected to autoradiography.
Statistical Analysis--
Transfections were performed in
triplicate and repeated multiple times. Data in each experiment were
normalized to the basal levels of activity of 797/+5LH
LUC or
GH50-pXP1. Data were then combined across experiments to give a
mean ± S.E. for basal and GnRH-stimulated activities for each
construct, and fold stimulation in response to GnRH was calculated.
One-way analysis of variance, followed by post hoc
comparisons with Fisher's protected least significant difference test,
was used to assess whether changes in GnRH responsiveness among
different LH
promoter-luciferase reporter constructs were
significant. Significant differences were established as
p < 0.05.
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RESULTS |
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Protein Binding to Region A of the LH Gene Involves Subregions
A2 (
451/
428) and A4 (
411/
386)--
We have demonstrated
previously that proteins in GGH3-1' and
T3 nuclear
extracts bind specifically to region A of the rat LH
gene promoter
(15). We were interested in determining the DNA sequence(s) within
region A that was responsible for this protein binding activity. In
order to define more precisely the binding sequence, we divided region
A into five subregions, A1-A5 (Fig.
1A). Using oligonucleotides
corresponding to A1-A5, we performed EMSA to ascertain whether
GGH3-1' nuclear proteins were able to bind to these
sequences (Fig. 1B). A DNA fragment encompassing region A
(LHA;
490/
334) of the LH
5'-flanking sequence was 3'-end-labeled
and incubated with 5 µg of GGH3-1' nuclear extract. A1-A5 were used in 200-fold excess as unlabeled competitor DNAs. Subregions A2 and A4 were able to compete for binding to
GGH3-1' nuclear proteins, as indicated by the decreased
formation of a specific DNA-protein complex on the labeled LHA probe,
whereas subregions A1, A3, and A5 did not compete significantly. LHA, used as a positive control, was also able to compete successfully for
binding to the labeled LHA probe. A sequence containing the Pit-1
binding site, used as a negative control, was not able to prevent
binding of GGH3-1' nuclear proteins to LHA. For additional analysis of these five subregions, the A1-A5 oligonucleotides were
5'-end-labeled and used directly as probes in an EMSA (Fig. 1C). Complexes similar to those seen using LHA were seen
using sequences A2 (
451/
428) and A4 (
411/
386). A similar
complex was also formed using A3 (
427/
399) as the DNA probe, albeit with lower abundance, suggesting a lower affinity for the DNA-binding protein. The predominant DNA-protein complexes formed using A2 and A4
as probes migrated at similar rates in the EMSA, suggesting that the
same protein(s) may bind to both of these DNA elements. This was
confirmed by demonstrating that a 200-fold excess of unlabeled A2 could
prevent the formation of the major DNA-protein complex on A4 and,
conversely, that a 200-fold excess of unlabeled A4 could prevent the
formation of the major DNA-protein complex on A2 (data not shown). A
200-fold excess of unlabeled A3 (
427/
399) was also able to decrease
formation of protein-DNA complexes formed on either A2 or A4,
consistent with a weak affinity for the same DNA-binding protein(s).
LHA could also prevent binding of GGH3-1' nuclear proteins
to both A2 and A4 when used as an unlabeled competitor DNA. The
formation of additional, specific DNA-protein complexes of lower
abundance was observed when A2 and A4 were used as probes. The
additional minor complexes formed using A3 and A5 were nonspecific (data not shown). Similar patterns of DNA-protein complexes were formed
on A1-A5 using
T3 nuclear extracts instead of GGH3-1' nuclear extracts (data not shown). This suggests that the protein(s) binding to these DNA sequences is common to both cell lines.
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DNA Sequences A2, A3, and A4 Confer GnRH Responsiveness to a
Heterologous Promoter--
We have demonstrated previously that region
A of the rat LH gene promoter can confer GnRH responsiveness to the
rat growth hormone (GH) minimal promoter (GH50), a heterologous gene
promoter (15). GH is normally expressed in the GH3 cell
line and is not GnRH-responsive or thyrotropin-releasing
hormone-responsive. We were interested in defining more precisely the
DNA sequences within region A that were responsible for this functional
GnRH response. In particular, we wished to correlate functional GnRH
responsiveness with the presence of DNA-protein interactions noted by
EMSA using A2 and A4, and to a lesser extent A3. We therefore subcloned
each subregion A1-A5, two copies in tandem, upstream of the rat GH50 minimal promoter in a luciferase reporter plasmid
(LHA12GH50LUC-LHA52GH50LUC). We tested these fusion constructs in a
transient transfection assay to determine whether they were
GnRH-responsive. Each of these constructs, or
797/+5LH
LUC or
LHAGH50LUC for comparison, or GH50-pXP1 or pXP1 as controls, was
transfected into GGH3-1' cells by electroporation. The
cells were harvested, and luciferase activity was measured 48 h
after transfection and after stimulation with 100 nM GnRHAg
or vehicle for 6 h immediately prior to harvesting (Fig.
2). Levels of expression of pXP1 and
GH50-pXP1 were very low, and a small but significant response to GnRHAg
was observed. All subsequent analyses of GnRH responsiveness were hence
made in comparison to GH50-pXP1. As noted in our previous studies (15), LHAGH50LUC increased basal luciferase activity substantially, in this
case by 25-fold, compared with GH50-pXP1, and LH
region A conferred
a 4.0 ± 0.6-fold GnRH response to the rat growth hormone minimal
promoter. This was somewhat less than the degree of stimulation by GnRH
of
797/+5LH
LUC (6.6 ± 0.4-fold), which contains the native
rat LH
gene minimal promoter and additional regions of the rat LH
gene 5'-flanking sequence. None of the five subregions of LHA were able
to confer full GnRH responsiveness to the level observed with the
entire LH
region A upstream of GH50 or to the level observed using
the native rat LH
gene promoter. However, LHA22GH50LUC and
LHA42GH50LUC increased basal luciferase activity and also conferred
partial GnRH responsiveness, to a significantly greater level than that
observed with GH50-pXP1. These reporter genes contain subregions A2 and
A4, respectively, corresponding to the oligonucleotides that bind
nuclear proteins present in GGH3-1' and
T3 cells. In
contrast, LHA12GH50LUC and LHA52GH50LUC had low basal activity and
little stimulation of activity in response to GnRH, and A1 and A5
oligonucleotides did not bind GGH3-1' and
T3 nuclear
proteins. LHA32GH50LUC increased basal activity but did not confer a
significant GnRH response compared with GH50-pXP1. Thus, there appears
to be a correlation between the presence of DNA-protein interactions
noted by EMSA and functional GnRH responsiveness for DNA sequences
within LH
region A. Furthermore, the sequences that confer GnRH
responsiveness are also able to enhance basal transcriptional
activity.
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Sp1 Can Bind to Subregions A2 and A4 and Is Responsible for the
Major DNA-Protein Complexes Observed Using These DNA Sequences with
GGH3-1' Nuclear Extracts--
Analysis of the DNA sequences within
subregions A2 and A4 revealed the presence of GC-rich sequences within
both of these elements. These sequences have homology with the
consensus binding site for Sp1, a three-zinc-finger transcription
factor (Fig. 3). We therefore sought to
test the hypothesis that Sp1 was able to bind to both A2 and A4 and
that the DNA-protein complexes observed by EMSA on A2, A4, and LHA
using GGH3-1' nuclear extracts contained Sp1 protein. EMSA
studies demonstrated that a 100-fold excess of oligonucleotide
containing the consensus Sp1 binding site was able to compete
successfully for GGH3-1' nuclear extract binding to A2, A4,
or LHA (Fig. 4A).
Oligonucleotides containing the consensus binding sites for AP1 or AP2
were also tested because these transcription factors are both activated
by protein kinase C, which is, in turn, activated by GnRH. Hence, these
are also putative mediators of transcriptional responses to GnRH.
However, in contrast to the results observed using the Sp1
oligonucleotide, oligonucleotides containing the consensus binding
sites for AP1 or AP2 did not compete for binding to A2, A4, or LHA when
present in 100-fold molar excess. Purified human Sp1 is able to bind to
LHA, giving a binding pattern similar to that observed using
GGH3-1' nuclear extracts (Fig. 4B). Similarly,
Sp1 is able to bind to A2 and A4 (data not shown). Co-incubation of Sp1
antibody with GGH3-1' nuclear extract and LHA, A2, or A4
DNA probe in an antibody supershift assay resulted in the formation of
a more slowly migrating "supershifted" complex (Fig.
4B), whereas no supershift was observed using an anti-follistatin antibody as a negative control (Fig. 4B) or
using an anti-Sp3 antibody or an anti-Egr-1 antibody (data not shown). Similarly, the Sp1 antibody was also able to supershift protein-DNA complexes observed using T3 nuclear extracts and LHA, A2, or A4
probes (data not shown). Taken together, these studies indicate that
Sp1 or an antigenically related protein is present in
GGH3-1' and
T3 nuclear extracts and binds to regions A2
(
451/
428) and A4 (
411/
386) of the LH
gene promoter.
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DNase I Footprinting Studies Demonstrate That Sp1 Binds to GC-rich
Sequences in Subregion A2--
Our EMSA studies demonstrated that Sp1
could bind to DNA sequences in A2 and A4 as well as to LH region A. Based on homology to the Sp1 consensus DNA binding sequence, we
hypothesized that Sp1 binding occurred to GC-rich sequences within
these elements. In order to test this hypothesis and define the Sp1
binding sites more precisely, we performed DNase I footprinting
experiments, using human Sp1 and 3'-end-labeled LHA (Fig.
5). These studies demonstrated that Sp1
could bind to
456/
442 of the rat LH
gene promoter, using either
the sense or antisense labeled DNA fragment as the probe, resulting in
protection from DNase I digestion. Binding was reduced using a 100-fold
excess of Sp1 oligonucleotide, LHA, A2, or A4 as an unlabeled DNA
competitor but not using an oligonucleotide containing the Pit-1
binding sequence, used as a negative control (25), confirming the
specificity of the Sp1-DNA interaction. Bovine serum albumin was not
able to protect the DNA fragment from DNase I digestion and therefore
did not generate a footprint. We were unable to observe a footprint on
the GC-rich DNA element corresponding to A4 (
411/
386); however,
this sequence was able to compete for Sp1 binding to
456/
442 (Fig.
5B). Therefore, the lack of a footprint on this region
likely represents a limitation of the assay rather than an absence of
Sp1 binding to this sequence. Due to the high GC content of this
sequence, secondary structure may have interfered with resolution of
this region on denaturing polyacrylamide gels, possibly obscuring the
presence of a footprint.
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Methylation Interference Studies Demonstrate That Sp1 and a
Protein(s) in GGH3-1' and T3-1 Nuclear Extracts Bind to
GC-rich Sequences in Subregion A2--
In order to define more
precisely the base pairs contacted by Sp1 and to compare the pattern of
binding of Sp1 with that observed using GGH3-1' or
T3
nuclear extracts, we performed methylation interference studies with
gel retardation, using 3'-end-labeled LHA. As shown in Fig.
6, methylation of the guanine nucleotides at positions
450/
443 on the noncoding strand interferes with human
Sp1 binding, as well as with GGH3-1' nuclear extract
binding. The identical pattern was observed using
T3-1 nuclear
extract (data not shown). No methylation interference pattern was
observed using the coding strand of LHA (data not shown); however,
there are no guanine residues in this region of the coding strand.
These methylation interference assay data, taken together with the
DNase I footprinting studies and EMSA analyses, suggest that Sp1 can bind to sequences at positions
456/
442 and
411/
386 and that the
binding observed using GGH3-1' and
T3 nuclear extracts
is due to Sp1 or a protein antigenically related to Sp1 and able to
recognize and bind to the same sequences as Sp1.
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Point Mutations in Subregions A2 and A4 Abolish Binding of Sp1 and
GGH3-1' Nuclear Proteins--
In order to study the
functional role of Sp1 binding to sequences in LH region A in
mediating the stimulation of LH
gene promoter activity by GnRH, we
wanted to abolish binding of Sp1 to this region. Oligonucleotides
containing point mutations in the putative Sp1 binding sites were
generated and are referred to as M1 and M2 (Fig.
7A). When these two mutant
oligonucleotides were 5'-end-labeled and used in EMSA studies,
GGH3-1' nuclear extract and Sp1 were no longer able to bind
to these DNA sequences (Fig. 7B). Furthermore, these
oligonucleotides were unable to prevent DNA-protein interactions
between the A2 or A4 oligonucleotides and GGH3-1' nuclear
extracts, when used in 200-fold excess as unlabeled competitors,
whereas the wild-type sequences were able to reduce such DNA-protein
interactions (Fig. 7B). These studies confirm that Sp1
binding occurred to the GC-rich sequences identified in LH
region A
as having homology to the Sp1 consensus binding site and that the point
mutations abolished Sp1 binding.
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Mutations That Abrogate Sp1 Binding to Sequences in Region A Blunt
the Stimulation of the LH Gene Promoter by GnRH--
The EMSA,
DNase I protection, and methylation interference studies defined
two DNA elements within the LH
gene promoter that bind Sp1 or an
Sp1-like protein and that lie within a GnRH-response element. We
therefore hypothesized that this protein-DNA interaction was necessary
to mediate stimulation of the LH
promoter activity by GnRH. In order
to test this hypothesis, we used site-directed mutagenesis to introduce
point mutations into these two Sp1 binding sites in
797/+5LH
LUC,
which would abolish Sp1 binding as demonstrated by EMSA analysis
(LH
Sp1M1LUC, LH
Sp1M2LUC, and LH
Sp1M1M2LUC). We then used these
mutant constructs in transient transfection assays in order to compare
the stimulation of luciferase activity by GnRH of the wild-type
797/+5LH
LUC to that using the Sp1 binding site mutants (Fig.
8). Mutation of either of the Sp1 binding
sequences decreased the stimulation of the LH
gene promoter by GnRH
(wild-type, 5.4 ± 0.3-fold compared with vehicle-treated
controls; mutation of 5' Sp1 site (M1), 3.7 ± 0.4-fold response
to GnRH (p < 0.005); mutation of 3' Sp1 site (M2),
3.1 ± 0.5-fold response to GnRH (p < 0.005)).
Mutation of both Sp1 sites causes a slight but not statistically
significant further decrease in the GnRH response (2.7 ± 0.5-fold; p = NS). Mutations in either or both of the
Sp1 binding sites also decreased basal luciferase activity to 45-50% of the wild-type promoter. Thus, Sp1 both binds to and mediates GnRH
stimulation of the rat LH
gene promoter. These data suggest that Sp1
or an Sp1-like protein not only binds to the LH
gene promoter at
positions
456/
442 and
411/
386 but also plays an important role
in conferring GnRH responsiveness to the LH
subunit gene.
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DISCUSSION |
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The gonadotropins play an integral role in the regulation of
reproductive development and function, responding to GnRH and other
modulatory influences with changes in production and secretion that, in
turn, act on the gonads to regulate gametogenesis and gonadal hormone
production. LH and FSH secretion are known to be tightly regulated
throughout development, at puberty, menopause, and particularly during
the menstrual or estrous cycle. LH and FSH production are also highly
regulated, and much of this regulation occurs at the level of gene
transcription. Although studies of the -subunit gene have led to
some understanding of the molecular mechanisms involved in this
transcriptional regulation, little is known about the mechanisms
involved in the regulation of the LH
and FSH
subunit genes. In
this study, we have demonstrated that two GC-rich DNA elements, which
have homology to the consensus binding site for Sp1 and are able to
bind Sp1, are necessary and sufficient to mediate stimulation of LH
gene expression by GnRH. Mutations in these elements that prevent Sp1
binding also decrease the stimulation of LH
gene transcription by
GnRH.
Sp1 is a member of a family of three-zinc-finger transcription factors, binding to GC-rich elements found in a wide variety of cellular and viral promoters (26, 27). Transcriptional activation is thought to occur through interaction with co-activator proteins that, in turn, interact with the basal transcriptional machinery (28, 29). Sp1 is posttranslationally modified by multiple N- and O-linked glycosylations as well as by phosphorylation by a DNA-dependent protein kinase (30). Sp1 is widely expressed and is often involved in transcriptional activation of genes in a non-hormonally regulated manner. In recent years, it has also been shown that Sp1 is involved in the hormonal regulation of gene expression for a variety of genes by a number of different mechanisms. Two Sp1 binding sites mediate cAMP-induced transcription of the CYP11A gene by adrenocorticotropic hormone (31). Regulation of the low density lipoprotein receptor gene promoter by cholesterol requires a sterol regulatory element (SRE) and an adjacent binding site for Sp1 (32). The SRE functions as a conditionally positive element, binding its cognate factor, SRE-binding protein and activating expression only when sterol levels are low. It cannot function efficiently by itself but requires binding of Sp1 to an adjacent site. It is hypothesized that the concerted action of Sp1- and SRE-binding protein results in synergistic activation of the low density lipoprotein receptor gene by coupling two different co-activator pathways together. Thyroid hormone suppression of the epidermal growth factor receptor promoter is mediated by overlapping Sp1 and thyroid hormone receptor-retinoid X receptor complex binding sites (33). In contrast, when a thyroid hormone response element is separated from Sp1 binding sites, synergism is observed. The induction of the cathepsin D and heat shock protein 27 genes by estrogen depends on the presence of both estrogen receptor and Sp1 binding sites in the gene promoters; mutation of either binding site prevents induction by estradiol (34, 35).
The DNA binding of Sp1 to the LH gene promoter is not regulated by
GnRH at either of the two binding sites that we have identified. Sp1
binding patterns on A2 and A4 by EMSA were the same using nuclear
extracts prepared from GGH3-1' cells that had been treated with GnRH for varying time intervals ranging from 10 min to 6 h,
as they were for untreated or vehicle-treated cells (data not shown).
The mechanism by which Sp1 binding to the LH
gene promoter activates
transcription in response to GnRH is unclear. The stimulation of cells
by GnRH may lead to posttranslational modification of Sp1 that results
not in changes in DNA binding but in changes in transcriptional
activity by altering the interaction of Sp1 with the basal
transcriptional machinery, either directly or indirectly through a
coactivator(s). Another possible mechanism is by interaction with a
protein(s) binding to the LH
gene promoter at sites adjacent to the
Sp1 binding sites. These proteins may be tissue-specific and/or
specific to the LH
gene promoter, and their DNA binding or
transcriptional activation functions may be regulated by GnRH but
require interaction with Sp1 for full DNA binding or transcriptional activity. This would be analogous to the cathepsin D and heat shock
protein genes, which are dependent on Sp1-estrogen receptor interactions, or to the sterol-dependent regulation of the
low density lipoprotein receptor gene by SRE-binding protein and Sp1 (32, 34, 35). Alternatively, Sp1 bound to subregions A2 and A4 may
interact with proteins bound to other regions of the LH
gene
promoter to regulate gene expression. We have shown previously that
full GnRH responsiveness of the LH
gene promoter requires sequences
at positions
207/
82 (region B) in addition to the DNA sequences in
region A (15). Region B includes sequences with homology to the SF-1
consensus binding site and has been shown to bind SF-1 and activate
expression of the rat LH
gene (7). It is possible that Sp1 bound to
region A interacts with SF-1 bound to region B to mediate stimulation
by GnRH. Indeed, it has been shown that Sp1 and SF-1 can associate
in vivo in a mammalian two-hybrid system and function
cooperatively in the transactivation of the bovine CYP11A gene promoter
(36).
We have demonstrated that DNA sequences with homology to the Sp1
consensus binding site function as GnRH response elements in the rat
LH gene promoter and that purified human Sp1 can bind to these
sites. Using anti-Sp1 antibodies, we show that Sp1 is present in the
complexes formed between A2 or A4 and nuclear extracts from
GGH3-1' or
T3 cells. The possibility remains that a
protein antigenically related to Sp1, rather than Sp1 itself, binds to these elements to mediate the GnRH effect. Using antibodies, we have
shown that Egr-1 and Sp3 are not responsible for the DNA binding we
observed (data not shown). However, we have not ruled out all members
of this family of transcription factors. Overexpression of Sp1 in
GGH3-1' cells does not augment the GnRH response (data not
shown), but this is most likely explained by the presence of adequate
levels of Sp1 occurring endogenously in the cells. Other investigators
have used the Drosophila Schneider cell line for such
studies because these cells lack Sp1 (28), but these cells would not be
expected to be GnRH-responsive and therefore would not be suitable for
our studies.
Anti-Sp1 antibody failed to supershift all of the protein-DNA complexes
observed using LH region A or A2 as the DNA probe, as indicated in
Fig. 4B. This suggests that an additional protein(s) present
in GGH3-1' nuclear extracts (and
T3-1 nuclear extracts, not shown) binds to these DNA sequences. The identity of these additional proteins has not yet been determined. It is possible that
these proteins may bind to the LH
gene promoter adjacent to the Sp1
binding sites and interact with Sp1 to mediate the GnRH response, as
outlined above.
Sp1 binding sites close to the transcriptional start site are
known to result in high levels of transcriptional activity (37). This
likely accounts for the high levels of basal activity observed using
LHA22GH50LUC, LHA32GH50LUC, and LHA42GH50LUC, in which the Sp1 binding
sites have been moved immediately upstream of the transcriptional start
site. Mutation of the Sp1 binding sites in the context of the native
rat LH gene, relative to the transcriptional start site, resulted in
a 50% decrease in basal activity of the promoter in addition to
decreasing stimulation by GnRH, suggesting that Sp1 binding to LH
region A also contributes to basal activity of the rat LH
gene.
Although the GH3 cell line is pituitary in origin, it is
not gonadotrope-derived and does not express the LH gene. Therefore, at best, it can be used as a model for the study of LH
gene
expression and regulation, and results need to be confirmed in other,
more physiologic systems. Nevertheless, we have shown previously that the regulation of the gonadotropin subunit promoter activities by GnRH
in this cell line, when transfected with the GnRHR, closely reflects
the regulation observed in primary pituitary cells (14). Furthermore,
the DNA-protein complexes observed using GH3 nuclear extracts are identical to those observed using nuclear extracts from
the gonadotrope-derived
T3 cell line. The role of Sp1 in the
regulation of LH
gene expression by GnRH provides some insight into
this effect. Sp1 is widely expressed in all mammalian cells. Therefore,
the stimulation of LH
promoter activity by GnRH could be predicted
to occur in any cell expressing the GnRHR on its cell surface, because
Sp1 would be present. However, full activity of the LH
gene likely
requires additional factors. Indeed, other studies have demonstrated
roles for SF-1 and Egr-1 in basal and possibly GnRH-stimulated activity
of the LH
gene (7-9, 38, 39). These two factors are also not
gonadotrope-specific, and it is likely that additional,
gonadotrope-specific factors are necessary for full LH
promoter
activity. The recent development of a gonadotrope-derived cell line,
L
T2 cells, may be useful for such studies (40, 41).
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ACKNOWLEDGEMENT |
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We thank Dr. Masato Ikeda for assistance with the DNase I footprinting and methylation interference assays.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grants HD-33001 (to U. B. K.) and HD-19938 (to W. W. C.) and an American Society for Reproductive Medicine-Serono Research Grant (to U. B. K.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF020505.
To whom correspondence should be addressed: G. W. Thorn Research
Bldg., Rm. 1009, Brigham and Women's Hospital, 20 Shattuck St., Boston, MA 02115. Tel.: 617-732-5856; Fax: 617-732-5123; E-mail: kaiser{at}rascal.med.harvard.edu.
1
The abbreviations used are: LH, luteinizing
hormone; LHA, LH region A; FSH, follicle-stimulating hormone; GnRH,
gonadotropin-releasing hormone; GnRHR, GnRH receptor; SF-1,
steroidogenic factor-1; GnRHAg, GnRH agonist; EMSA, electrophoretic
mobility shift assay; GH, growth hormone; SRE, sterol regulatory
element; Egr-1, early growth response-1; RSV, Rous sarcoma virus.
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REFERENCES |
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