From the Division of Genetics, Department of
Medicine, Brigham and Women's Hospital and Harvard Medical School,
Boston, Massachusetts 02115, the § Department of Obstetrics
and Gynecology, Tufts University School of Medicine, Boston,
Massachusetts 02111, and the
Division of Endocrinology,
Metabolism, and Molecular Medicine, Northwestern University Medical
School, Chicago, Illinois 60611
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ABSTRACT |
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Recent in vivo and in
vitro studies have implicated the orphan nuclear receptor,
steroidogenic factor-1 (SF-1), and the early growth response protein 1 (Egr-1) in the transcriptional regulation of the luteinizing hormone
-subunit (LH
) gene. We have previously demonstrated the ability
of SF-1 to bind to and transactivate the rat LH
gene promoter acting
at a consensus gonadotrope-specific element (GSE) located at position
127. We have now identified a second functional GSE site at position
59. In addition, based on electrophoretic mobility shift assay,
in vitro translated Egr-1 is shown to bind to two putative
Egr-1 binding sites (positions
112 and
50), which appear to be
paired with the identified GSE sites. By transient transfection assay
in pituitary-derived GH3 cells, it was seen that Egr-1
increases promoter activity of region
207/+5 of the rat LH
gene
promoter through action at both Egr-1 sites. Furthermore, LH
gene
promoter activity is markedly augmented in the presence of both factors
together relative to activity in the presence of SF-1 or Egr-1 alone
(150-fold versus 14-fold and 12-fold, respectively). These
data define two composite SF-1-Egr-1 response-elements in the proximal
LH
gene promoter and suggest that SF-1 and Egr-1 act synergistically
to increase expression of the LH
gene in the gonadotrope.
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INTRODUCTION |
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Precise regulation of gonadotropin gene expression is required for
normal reproductive function. The pituitary gonadotropins, luteinizing
hormone and follicle-stimulating hormone, are composed of a common
-subunit linked to one of two unique
-subunits, LH
1 or FSH
. The common
-subunit can also associate with thyroid-stimulating hormone
-subunit in pituitary thyrotropes or, in humans, with placentally
derived chorionic gonadotropin
-subunit.
Studies of the -subunit gene promoter have identified a number of
transcription factors and cognate cis-acting DNA elements that provide
basal, tissue-specific, and hormonally mediated regulation of gene
expression. In particular, a gonadotrope-specific element (GSE) is
believed to be important for conferring gonadotrope-specific expression
of the
-subunit gene (1, 2).
The GSE, or Ad4 response element, regulates expression of a number of genes with a role in steroidogenesis, sexual differentiation, and adult reproductive function (3). The GSE/Ad4 site has been shown to interact with the transcription factor, steroidogenic factor-1 (SF-1), resulting in transcriptional activation of a variety of genes, including the steroidogenic P450, the Mullerian inhibiting substance, and the aromatase genes, among others (4-6).
SF-1 is a member of the nuclear hormone receptor superfamily. Although it was previously considered to be an orphan member of this family, it has recently been reported that SF-1-dependent transcriptional activity is increased in the presence of cholesterol metabolites, particularly 25-OH-cholesterol (7). It is currently unknown whether this putative ligand is important for SF-1 function in nonsteroidogenic tissues, such as the pituitary gland. SF-1 expression is restricted to the steroidogenic cells of the adrenal gland and gonads and to the gonadotrope subpopulation of the pituitary gland. Thus, the pattern of SF-1 expression suggests that SF-1 may contribute to tissue-specific gene expression.
In contrast with the -subunit, progress has only recently been made
in the identification of transcription factors that regulate the
gonadotropin
-subunit genes. Understanding
-subunit regulation at
the molecular level is critical because it is the
-subunits that
provide the functional specificity that distinguishes luteinizing hormone and follicle-stimulating hormone action.
A number of recent reports have suggested that SF-1 may play a role in
the regulation of LH gene expression in addition to its effects on
the common
-subunit. Targeted disruption of the Ftz-F1 gene encoding
SF-1 results in transgenic mice that lack transcripts for the
gonadotrope markers LH
, FSH
, and gonadotropin-releasing hormone
receptor and have greatly diminished levels of
-subunit mRNA
(3). Gonadotropin-releasing hormone replacement was able to restore
gonadotropin expression in four out of five of these animals,
suggesting that cells from the gonadotrope lineage are present but that
a specific defect in gonadotropin subunit gene expression exists (8).
By sequence homology, it has been shown that the LH gene promoter
contains a consensus GSE site at position
127 in the rat and
125 in
the cow. In vitro studies demonstrated the ability of SF-1
to bind to and transactivate the rat LH
gene promoter through
interaction with this putative GSE site (9). The physiologic significance of this site was confirmed using transgenic mice containing either the wild-type bovine LH
gene promoter or a promoter with a GSE site-specific mutation linked to a CAT reporter vector (10). Introduction of the GSE mutation substantially decreased
LH
gene promoter activity, indicating that the SF-1 binding site is
a critical regulator of LH
gene promoter activity in
vivo. Of note, however, in both the in vitro and
in vivo model systems, mutation of this GSE site failed to
eliminate fully LH
gene expression, suggesting the presence of a
second functional SF-1-response element.
Closer inspection of the rat LH gene promoter sequence identified a
second region with similarity to the consensus GSE site (Fig. 1). We
have termed this second putative site the 3'GSE and refer to the
previously defined site as the 5'GSE. The first aim of the current
study was to determine the functional significance of the putative
3'GSE site.
In vivo observations in transgenic mice have also suggested
a role for the transcription factor, early growth response protein 1 (Egr-1), in the modulation of LH gene expression. Egr-1, also known
as NGFI-A, zif/268, and Krox-24, is a member of the immediate early
gene family, the members of which contain a zinc finger domain with a
Cys2-His2 motif that recognizes the nucleotide
sequence CGCCC(C/A)CGC. Additional members of this family include Sp1
and the Wilms' tumor suppressor WT1 (11-13). Egr-1 is widely
expressed, and in a recent report, it has been shown to be expressed in
the gonadotrope, as well as in the somatotrope subpopulation of the pituitary (14). Of interest, two groups have demonstrated that targeted
disruption of the Egr-1 gene results in specific loss of LH
gene
expression, with maintenance of FSH
gene expression (14, 15).
Sequence analysis of the LH
gene promoter identifies two highly
conserved regions with homology to the consensus Egr-1 binding site
(Fig. 1). Lee et al. (15) have demonstrated that mutation of
the 3'Egr site correlates with loss of Egr-1-mediated transactivation.
However, the ability of Egr-1 to interact directly with the 3'Egr site
and the functional importance of the putative 5'Egr site have not been
determined.
In the study reported here, we have therefore verified the ability of
in vitro translated Egr-1 to bind to both of the putative Egr-1 binding sites and have shown that both sites confer Egr-1 responsiveness to the rat LH gene promoter. In addition, we have demonstrated the ability of SF-1 and Egr-1 to interact with each other
and have characterized the functional importance of this interaction to
the regulation of LH
gene expression.
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EXPERIMENTAL PROCEDURES |
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Oligonucleotides--
The oligonucleotides used for mutagenesis,
electrophoretic mobility shift assays (EMSAs), and polymerase chain
reactions are shown in Table I. The nucleotide sequence of the rat
LH gene promoter is based on newly obtained sequencing data
available at GenBankTM accession number AF020505, which
differ slightly from that of Jameson et al. (16). The 5'GSE
oligonucleotide contains additional 5'-BamHI and
3'-BglII restriction sites. The
207LH-S,
82LH-S, and
5'LH-S primers introduced BamHI restriction sites, whereas the +5LH-AS primer introduced a HindIII site, and the
3'LH-AS primer introduced BglII and XhoI
restriction sites (restriction sites not shown).
In Vitro Translated Proteins and Antisera Used in EMSA-- In vitro translated proteins were generated from plasmids containing 2.1 kilobase pairs of the mouse SF-1 cDNA (provided by Dr. K. L. Parker, Southwestern University School of Medicine) or 3.2 kilobase pairs of the mouse Egr-1 cDNA (provided by Dr. D. Nathans, Johns Hopkins University) using the TNT Coupled Reticulocyte Lysate System (Promega, Madison, WI) (11, 17). The resultant product was determined to be of appropriate size by comparison with [35S]methionine-labeled protein markers by SDS-polyacrylamide gel electrophoresis (PAGE).
The SF-1 polyclonal antibody, a generous gift of Dr. Parker, was generated in rabbits against a glutathione S-transferase-SF-1 fusion protein (18). The Egr-1 antiserum is a rabbit affinity-purified polyclonal antibody raised against the carboxyl terminus of mouse Egr-1 (Santa Cruz Biotechnology, Inc, Santa Cruz, CA). The Pit-1 antiserum, directed against amino acids 136-150 of rat growth hormone pituitary factor-1/Pit-1, was provided by C. Bancroft (Mt. Sinai School of Medicine) (19).Electrophoretic Mobility Shift Assays--
Region 141/
44 of
the rat LH
gene promoter (see Fig. 5B) was produced by
polymerase chain reaction using primers 5'LH-S and 3'LH-AS and
subcloned into the pGEM-T vector (Promega). BamHI and
BglII restriction enzymes were used to obtain the insert, which was agarose gel-purified and dephosphorylated. The remainder of
the double-stranded oligonucleotides used as probes and in cold
competition experiments were produced by annealing the sense oligonucleotide indicated in Table I with the corresponding antisense oligonucleotide (not shown). Probes were created by T4 polynucleotide kinase end-labeling with [
-32P]ATP followed by
purification over a NICK column (Amersham Pharmacia Biotech).
In Vitro Protein Interaction Assays--
Protein interaction
assays were performed as described previously (20). Briefly, a
His6-containing SF-1 fusion protein was obtained by
baculovirus transfection of insect cells. The fusion protein was
purified by affinity chromatography with nickel-nitrilotriacetic acid
(NTA) agarose (Qiagen, Hilden, Germany) followed by washing with 40 mM imidazole. In vitro translation of the Egr-1,
DAX-1, and RXR proteins was performed with the TNT Coupled
Reticulocyte Lysate System (Promega, Madison, WI) in the presence of
[35S]methionine. Labeled proteins were incubated with the
His6-SF-1 fusion protein bound to Ni-NTA agarose in buffer
containing 20 mM HEPES, pH 7.9, 10% glycerol, 50 mM KCl, 0.5% Nonidet P-40, 1 mM
phenylmethylsulfonyl fluoride, and 40 mM imidazole for
2 h at 4 °C. After extensive washing, bound proteins were
eluted by boiling in SDS sample buffer followed by 10% PAGE separation and autoradiography.
Plasmids Used in Transfection Studies--
Reporter constructs
used for these studies were created by subcloning polymerase chain
reaction products containing the LH gene promoter sequence into the
pXP2 vector using BamHI/HindIII restriction
sites, which were introduced by primers (18). The largest construct
used for these studies contained 207 base pairs of the 5'-flanking
sequence of the rat LH
gene and the first 5 base pairs of the
5'-untranslated region generated by primers
207LH-S and +5LH-AS. A
5'-truncated construct was obtained by subcloning the polymerase chain
reaction product obtained with primers
82LH-S and +5LH-AS.
Transfection Experiments-- Monkey kidney fibroblast (CV-1) cells or rat somatolactotrope (GH3) cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. CV-1 cells growing in 3.5-cm tissue culture wells (Flow Laboratories, McLean, VA) were transfected with expression (0.1 µg/well) and reporter (1.65 µg/well) plasmids using the calcium phosphate precipitation method (21). GH3 cells were cultured to 50-70% confluence and transfected by electroporation. Approximately 5 × 106 cells were suspended in 0.4 ml of Dulbecco's phosphate-buffered saline plus 5 mM glucose with the DNA to be transfected. The cells received a single electrical pulse of 240 V at a total capacitance of 1000 microfarads using an Invitrogen Electroporator II apparatus (Invitrogen, San Diego, CA).
For both cell types, control wells received the appropriate "empty" expression vector. Cotransfection with an Rous sarcoma virus- ![]() |
RESULTS |
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SF-1 Binds Specifically to the Putative 3'GSE Site--
We have
previously demonstrated the ability of SF-1 to increase rat LH gene
promoter activity specifically through action at a functional GSE site
located at position
127/
119, now designated as the 5'GSE site (9).
These studies were primarily based on transient transfection of the
monkey kidney fibroblast cell line, CV-1, with preliminary confirmation
in the rat pituitary-derived somatolactotrope cell line,
GH3 (9). By Northern analysis, CV-1 cells lack the mRNA
that encodes the SF-1 homologue Ad4BP (24). We have also demonstrated
that both CV-1 and GH3 nuclear extracts fail to produce
specific protein-DNA interactions with the 5'GSE probe by EMSA (data
not shown).
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The 5'GSE and 3'GSE Sites Have Similar Affinity for in Vitro Translated SF-1-- We next wished to determine the relative affinities of the two GSE sites that we had identified for SF-1 binding. EMSA was performed using in vitro translated SF-1 and the 3'GSE oligonucleotide probe with an increasing molar excess of unlabeled 3'GSE or 5'GSE oligonucleotides (Fig. 2B). Based on this approach, the affinity of SF-1 for either of these sites is essentially identical within the sensitivity of this approach.
The Putative 3'GSE Site Contributes to SF-1-stimulated Increases in
LH Promoter Activity--
In order to determine the functional
significance of the interaction between SF-1 and the LH
gene
promoter sequences, transient transfection assays were performed in
CV-1 cells, the cell line used in characterization of the 5'GSE site
(9). Consistent with our previously published results, cotransfection
with the SF-1 expression vector resulted in an 80-fold increase in the luciferase activity of a wild-type reporter construct containing region
207 to +5 of the rat LH
gene promoter (Fig.
3A). Mutation of either the
5'GSE or 3'GSE sites eliminated over 90% of the SF-1-response, whereas
mutation of both sites decreased the SF-1-response to the level of the
empty reporter plasmid, pXP2.
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Egr-1 Binds to Both Putative Egr-1 Binding Sites in the LH Gene
Promoter--
In a recent report, specific loss of pituitary LH
mRNA expression was described in a transgenic mouse line deficient
in Egr-1 (15). In these studies, Lee et al. (15)
demonstrated that mutation of region
50 to
42 of the rat LH
gene
promoter sequence eliminated the ability of an Egr-1 expression plasmid
to increase promoter activity in both
T3-1 and CV-1 cells. On
further sequence analysis, we identified a second putative Egr-1 site
at position
112/
104 in the proximal rat LH
gene promoter with
homology to the consensus Egr-1 binding site. We have designated these two regions the putative 5'Egr and 3'Egr sites (Fig. 1). Interestingly, these putative Egr-1 elements are paired with the GSE sites, suggesting a functional interaction between the two associated transcription factors.
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EMSA Fails to Demonstrate an SF-1-Egr-1 Protein-Protein
Interaction--
Lee et al. (15) reported a synergistic
increase in LH gene promoter activity following transfection with
both SF-1 and Egr-1 expression vectors. We wished to determine whether
this observed functional cooperativity could be explained by direct protein-protein interaction between the two transcription factors, particularly in view of the noted pairing of their respective binding
sites. EMSA was performed using a probe that spanned the 3'GSE and
3'Egr sites (Fig. 5A) or a
probe containing base pairs
141 to
44 that spanned both GSE sites
and both putative Egr-1 binding sites (Fig. 5B). As seen in
Fig. 5A and confirmed by antibody treatment, the co-addition
of in vitro SF-1 and Egr-1 (lanes 3-6) resulted
in the production of two complexes that contained either SF-1 or Egr-1
but failed to demonstrate a higher order complex. Similarly, no
evidence of SF-1-Egr-1 heterodimerization was detected using a probe
that spanned the 5' and 3' GSE-Egr-1 pairs (oligonucleotide 5'GSE-3'Egr) (Fig. 5B, lane 5). Of note, the intensity of
the SF-1 and Egr-1 DNA complexes was the same whether they were
produced in the presence of one or both of the transcription factors.
Neither the addition of endogenous SF-1 (present in
T3-1 nuclear
extracts) nor changes in the binding conditions of the EMSA incubation
reaction produced a detectable SF-1-Egr-1 complex (data not shown).
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SF-1 Interacts Directly with Egr-1 in a Protein-Protein Interaction Assay-- Because EMSA failed to demonstrate an interaction between SF-1 and Egr-1, we attempted to detect protein-protein binding using an alternative assay. A His6-SF-1 fusion protein linked to Ni-NTA agarose was tested for the ability to bind to Egr-1 that had been radiolabeled with [35S]methionine during in vitro translation. This assay has been used previously to detect an interaction between SF-1 and another gonadotrope-expressed transcription factor, DAX-1 (20). As shown in Fig. 6, Egr-1 bound to the SF-1 fusion protein, whereas no binding was observed to the Ni-NTA beads alone. Relative to the amount of radiolabeled input protein, approximately 6% of the Egr-1 bound to SF-1, similar in magnitude to the interaction between SF-1 and DAX-1 (7% of input). In contrast, no binding was detected between RXR and SF-1. Deletion of the carboxyl terminus of Egr-1 had no effect on, or even increased, binding (9% of input), whereas a more extensive deletion across the Egr-1 DNA binding domain eliminated the observed interaction. The lack of interaction between SF-1 and RXR and the loss of interaction following truncation of Egr-1 both strongly suggest that the binding of SF-1 to full-length Egr-1 is specific.
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Egr-1 Increases LH Gene Promoter Activity through Action at Both
Putative Egr-1 GSE Sites--
In order to verify the functional
significance of each of the putative Egr-1 binding sites, transient
transfection experiments were performed in GH3 cells. We
first tested the effect of introducing mutations into either of the
putative Egr-1 sites within the
207/+5 region of the LH
gene
promoter. These mutations corresponded to the mutations that eliminated
binding by in vitro translated Egr-1 on EMSA. In order to
optimize for detection of the response to the weaker 5'GSE site, cells
were cotransfected with relatively high amounts of Egr-1 expression
vector (5 µg/well), whereas lesser amounts were used for subsequent
experiments (1 µg/well). As seen in Fig.
7A, LH
gene promoter
activity increased by approximately 12-fold in the presence of Egr-1 in
the wild-type construct. Following mutation in the putative 3'Egr or
5'Egr sites, the Egr-1 response decreased to 1.7-fold or 3.5-fold,
respectively, but remained significantly greater than control
expression. Mutation in both putative Egr sites eliminated Egr-1
responsiveness.
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SF-1 and Egr-1 Act Synergistically to Increase LH Gene Promoter
Activity--
We next investigated whether the observed
protein-protein interaction between SF-1 and Egr-1 (Fig. 6) would
result in cooperative effects on LH
gene promoter function. As seen
in Fig. 7B, cotransfection with both SF-1 and Egr-1 resulted
in marked synergistic stimulation of LH
gene expression to over
150-fold, consistent with previous reports in CV-1 cells (15).
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SF-1 Interacts Functionally with Egr-1 through Both Adjacent and
Spaced Binding Sites--
As noted previously, the SF-1 and Egr-1
binding sites appear to be present as pairs in the LH gene promoter,
albeit with slightly different spacing between the paired sites. This
localization raised the possibility that the observed SF-1-Egr-1
functional interaction was dependent on the close proximity of the two
binding sites, perhaps promoting heterodimer formation. Alternatively, as suggested by the mutually exclusive binding observed on EMSA, the
proximity of the sites might prohibit simultaneous binding by the two
transcription factors. In this situation, SF-1 and Egr-1 would
presumably act through interaction between the two defined regions.
Mutation of the 5'GSE site allowed evaluation of the 3'GSE site with
the two Egr-1 binding sites. Additional mutation of either Egr-1 site
decreased, but did not eliminate, the SF-1-Egr-1 synergistic effect on
LH
gene promoter activity. These results suggest that both of these
mechanisms are functioning, i.e. the SF-1 response may be
augmented by Egr-1 acting on either the adjacent or the more distant
Egr-1 binding site.
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DISCUSSION |
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The gonadotropins are critical modulators of reproductive
development and function, acting on the gonads to stimulate both steroidogenesis and gametogenesis. A wide variety of studies suggest that gonadotropin biosynthesis is tightly controlled and depends, to a
large degree, on regulation at the level of gene transcription. Although a number of transcription factors have been identified that
modulate expression of the -subunit gene, only recently have studies
begun to identify the molecular mechanisms that regulate
-subunit
gene expression.
The results reported here substantially extend our understanding of the
transcription factors and DNA response elements that stimulate activity
of the LH gene promoter. These studies clearly demonstrate the
ability of SF-1 to bind to a second region of the rat LH
gene
promoter with homology to the consensus GSE site. This site, located at
position
59, acts in conjunction with the previously identified GSE
site at position
127 to confer marked SF-1 responsiveness to the
LH
gene. Our studies also demonstrate the ability of in
vitro translated Egr-1 to bind to and transactivate the LH
promoter through action at two sites located at positions
112 and
50. In addition, these experiments confirm the previously reported
synergistic induction of LH
gene expression in the presence of both
transcription factors, SF-1 and Egr-1 (15). As we now demonstrate, this
functionally cooperativity appears to be provided by both Egr-1 binding
sites and, at least in the presence of an intact 3'GSE site, occurs
through interactions between either proximal or spaced SF-1 and Egr-1
regulatory elements.
In the functional studies reported here, we utilized a heterologous
system in which expression vector(s) for SF-1 and/or Egr-1 and a
reporter construct containing LH gene promoter sequences were
transiently transfected into a pituitary-derived somatolactotrope cell
line, GH3. Although these studies ideally would have been performed in a gonadotrope-derived cell line, there is precedent for
the study of the LH
gene in GH3 cells. The
GH3 cell line has been shown to support transcription
initiation from the authentic start site of the LH
gene and to allow
cAMP-mediated increases in LH
promoter activity (28). These cells
have also been utilized to identify an estrogen-responsive element in
the LH
gene promoter (29). When transfected with the
gonadotropin-releasing hormone receptor, GH3 cells have
been shown to support gonadotropin-releasing hormone-induced regulation
of gonadotropin subunit promoter activity, which closely parallels the
regulation observed in primary pituitary cells (30). As indicated under
"Results," preliminary studies in the newly available,
gonadotrope-derived L
T2 cell line have supported the results
obtained in the GH3 cell line.
SF-1 is a member of the nuclear hormone receptor superfamily, which
includes the thyroid hormone, estrogen, progesterone, and retinoic acid
receptors. Interestingly, the consensus GSE sequence resembles a
nuclear receptor binding half-site. Although members of the nuclear
hormone receptor family are best known for binding to pairs of
recognition half-sites, monomer binding to a single 5'-extended
half-site has been described for both SF-1 and another orphan nuclear
receptor, NGFI-B (31). Although we have identified two GSE sites in the
proximal LH promoter, many aspects of our data argue against the
need for SF-1 multimer binding to two DNA half-sites. Although DNA
bending could allow for close approximation of the two GSE sites,
interaction between nuclear hormone binding sites with separations of
over 70 base pairs has not been previously described. From a functional
standpoint, we have demonstrated that SF-1-mediated increases in LH
promoter activity can be observed in the presence of a single intact
GSE site (Fig. 3).
Our data demonstrate functional cooperativity between SF-1 and Egr-1 on
the rat LH gene promoter activity and suggest that this effect may
be due to direct interaction between these two transcription factors,
as demonstrated by use of a His-tagged SF-1 fusion protein. Based on
preliminary data using Egr-1 truncation mutants, formation of
SF-1-Egr-1 heterodimers requires the presence of the Egr-1 DNA binding
domain. Further studies are under way to characterize fully the SF-1
and Egr-1 protein domains that are required for protein-protein
interactions and/or functional synergy.
For reasons that remain unclear, SF-1-Egr-1 protein interactions could
not be detected on EMSA despite multiple attempts to optimize binding
conditions. Although this result could be due to a weak interaction
between the two proteins, the amount of Egr-1 bound as a percentage of
input was similar to the interactions of RXR and thyroid hormone
receptor
in analogous assays (data not shown). Precedent exists for
difficulty in detecting interactions between SF-1 and other
transcription factors by EMSA. For example, despite clear functional
interactions, the ability of SF-1 to bind to DAX-1 required the use of
the His6-SF-1 approach utilized for these studies (20).
SF-1 has previously been shown to bind to DAX-1, another orphan member
of the nuclear hormone receptor superfamily with effects on gonadotrope
function. Mutations in DAX-1 are believed to cause hypogonadotropic
hypogonadism in conjunction with X-linked adrenal hypoplasia congenita
(20, 32). Ito et al. (20) have demonstrated that DAX-1
blunts the ability of SF-1 to increase expression of a heterologous
reporter construct containing a consensus GSE site. This DAX-1 effect
did not require DNA binding by DAX-1. In contrast, Egr-1 augments
SF-1-mediated increases in LH gene promoter activity, and this
action depends upon the presence of two Egr-1 DNA binding sites in the
LH
gene promoter. Interestingly, in studies of the CYP11A gene
promoter, SF-1 has been shown to interact with Sp1, a member of the
zinc finger transcription family, which includes Egr-1 (33).
It must be noted that the observed Egr-1 effects on LH promoter
activity are physiologically relevant only if gonadotropes express
endogenous Egr-1. Topilko et al. (14) recently demonstrated that Egr-1 is, in fact, expressed in gonadotropes based on
co-localization of LH
-subunit protein and X-gal staining, which is
conferred by a lacZ transgene inserted 3' to the endogenous
Egr-1(Krox-24) promoter (14). Interestingly, we have been unable to
detect Egr-1 binding to the 3'Egr site using untreated nuclear extracts from the gonadotrope-derived cell lines
T3-1 and L
T2 (data not shown).
In studies of nonreproductive systems, it has been observed that levels
of Egr-1 mRNA and protein, although low under basal conditions, are
rapidly and markedly induced by a number of stimuli, including serum,
phorbol 12-myristate 13-acetate, nerve growth factor, and fibroblast
growth factor (34). As is well known, gonadotropin gene expression is
regulated by a wide variety of hormonal factors arising from the
gonads, the hypothalamus, and the pituitary itself, factors that have
been postulated to increase Egr-1 levels in the gonadotrope.
Experiments are currently underway to define the physiologic stimuli
that regulate Egr-1 levels in the gonadotrope and thereby increase
expression of the LH gene.
In summary, this study has defined two composite GSE/Egr-1
elements in the proximal rat LH gene promoter, and it suggests that
SF-1 and Egr-1 can act both alone and synergistically to increase
expression of the LH
gene by the gonadotrope.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grants R03-HD34692 (to L. M. H.), R01-HD19938 (to W. W. C.), and U54-HD29164 (to M. I. and J. L. J.) and by an American Society for Reproductive Medicine-Ortho Pharmaceutical Research Grant in Reproduction (to L. M. H.).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.
¶ To whom correspondence should be addressed: G. W. Thorn Research Bldg., Rm. 1013, Brigham and Women's Hospital, 20 Shattuck St., Boston, MA 02115. Tel.: 617-732-5856; Fax: 617-732-5123; E-mail: Halvorson{at}rascal.med.harvard.edu.
1
The abbreviations used are: LH, luteinizing
hormone
-subunit; FSH
, follicle-stimulating hormone
-subunit;
SF-1, steroidogenic factor-1; Egr-1, early growth response protein 1;
EMSA, electrophoretic mobility shift assay; RXR, 9-cis-retinoic acid
receptor; DAX-1, dosage-sensitive sex reversal-adrenal hypoplasia
congenita critical region on the X chromosome, gene 1; NTA,
nitrilotriacetic acid; GSE, gonadotrope-specific element.
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REFERENCES |
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