The Basic Helix-Loop-Helix, Leucine Zipper Transcription Factor, USF (Upstream Stimulatory Factor), Is a Key Regulator of SF-1 (Steroidogenic Factor-1) Gene Expression in Pituitary Gonadotrope and Steroidogenic Cells
Adrienne N. Harris and
Pamela L. Mellon
Department of Reproductive Medicine (A.N.H., P.L.M.) Department
of Neuroscience and the Center for Molecular Genetics (P.L.M.)
University of California, San Diego La Jolla, California
92093-0674
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ABSTRACT
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Tissue-specific expression of the mammalian FTZ-F1
gene is essential for adrenal and gonadal development and sexual
differentiation. The FTZ-F1 gene encodes an orphan nuclear receptor,
termed SF-1 (steroidogenic factor-1) or Ad4BP, which is a primary
transcriptional regulator of several hormone and steroidogenic enzyme
genes that are critical for normal physiological function of the
hypothalamic-pituitary-gonadal axis in reproduction. The objective of
the current study is to understand the molecular mechanisms underlying
transcriptional regulation of SF-1 gene expression in the pituitary. We
have studied a series of deletion and point mutations in the SF-1
promoter region for transcriptional activity in
T31 and LßT2
(pituitary gonadotrope), CV-1, JEG-3, and Y1 (adrenocortical) cell
lines. Our results indicate that maximal expression of the SF-1
promoter in all cell types requires an E box element at -82/-77. This
E box sequence (CACGTG) is identical to the binding element for USF
(upstream stimulatory factor), a member of the helix-loop-helix family
of transcription factors. Studies of the SF-1 gene E box element using
gel mobility shift and antibody supershift assays indicate that USF may
be a key transcriptional regulator of SF-1 gene expression.
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INTRODUCTION
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The maintenance of physiological homeostasis and sexual
differentiation in mammals is predominantly controlled by the
appropriate temporal and spatial production of specific steroid
hormones. The biosynthesis of steroid hormones in specific tissues
requires the concerted action of a related group of cytochrome
P450 steroid hydroxylases (1), and considerable effort has
been directed toward defining the molecular mechanisms that regulate
expression of this family of biosynthetic enzymes. Functional analyses
of the promoter regions of the steroid hydroxylase genes revealed a
conserved element with homology to the estrogen receptor half-site that
was required for maximal gene expression (2). As a result of these
efforts, a developmentally regulated nuclear receptor, known as
steroidogenic factor-1 (SF-1) (3) or Ad4BP (4, 5), was identified that
interacts with this element to effect transcriptional regulation of
specific cytochrome P450 enzyme expression in gonadal and
adrenal tissues (2, 6).
SF-1 is one of two proteins encoded by the mammalian homologue of
fushi tarazu factor-1 (FTZ-F1) (3, 7, 8), a
Drosophila gene product that has been identified as a
regulator of the fushi tarazu homeobox gene (9). Isolation
and characterization of SF-1 cDNA clones (3, 7, 8, 10) revealed that
the factor is a member of the steroid/thyroid hormone receptor
superfamily (10), containing a zinc finger region and a
ligand-binding/dimerization domain. It was recently demonstrated that
endogenous oxysterols selectively enhance SF-1-mediated transcriptional
activity, suggesting that SF-1 is a ligand-activated receptor (11).
SF-1 is expressed in a highly tissue-restricted manner during embryonic
development (6, 12). SF-1 transcripts are first detected in the mouse
on embryonic day 9 (e9) in the urogenital ridge and localized to the
adrenal glands by e11. Interestingly, expression of SF-1 in the
embryonic gonad is sexually dimorphic by e14.5, with high levels of
SF-1 in testes and trace levels of detectable protein in ovaries. In
the adult rodent, SF-1 is expressed in all the primary steroidogenic
tissues, including the adrenal cortex, testicular Leydig cells, and the
theca and granulosa cells and corpus luteum of the ovary (6, 12). Since
SF-1 was thus strongly implicated in tissue-specific gene expression
and sexual differentiation, studies were initiated to analyze the gene
in intact mice. Targeted disruption of the mouse FTZ-F1 locus defined
SF-1 as the essential transcript of the gene (13) and as responsible
for the dramatic developmental abnormalities associated with
FTZ-F1-null mice (13, 14, 15). Adrenal glands in gene-disrupted mice are
absent from early stages in development, resulting in neonatal
lethality, presumably due to adrenocortical insufficiency (13, 14, 15). In
addition, FTZ-F1 null mice of both sexes lacked gonads but displayed
internal female genitalia. Thus SF-1 plays a pivotal role in the
formation and function of steroidogenic tissue in the developing mammal
and may represent one of the target genes of SRY in the cascade of gene
activation of sex determination genes (16, 17).
Importantly, control of reproductive function by the ventromedial
hypothalamus and pituitary is also selectively disrupted in
SF-1-knockout mice (15, 18). Of the five different endocrine cell types
present in the anterior pituitary, SF-1 is selectively expressed in the
gonadotrope (19, 20). In response to GnRH release from the
hypothalamus, the gonadotrope secretes the gonadotropin hormones LH and
FSH, resulting in increased steroid production in the target gonads. In
SF-1-knockout mice, ventromedial hypothalamus control of GnRH secretion
is impaired, which presumabably results in both decreased GnRH delivery
to the gonadotropes and decreased LH/FSH secretion (15, 18). Recently,
several targets of SF-1 transcriptional regulation through a consensus
DNA-binding site for SF-1 [or GSE (gonadotrope-specific element)
(20)] have been defined in the pituitary gonadotrope by analyzing the
5'-flanking sequences of gonadotrope-specific genes, including the GnRH
receptor (21), the
-subunit (20), and the LH-ß-subunit of the
glycoprotein hormones (22). Thus, SF-1 appears to play a global role at
the molecular and physiological level in the integrated interaction of
the hypothalamic-pituitary-gonadal axis in mammalian reproductive
function (23).
Progress toward defining the molecular mechanisms that control cell
type-specific expression of SF-1 in the pituitary gonadotrope has been
enhanced by the isolation of the promoter region of the gene (24, 25, 26).
In particular, recent reports have defined the functional importance of
an E box motif in the proximal promoter of the rat (24), mouse (26),
and human (26) SF-1 genes in tissue-specific expression, yet the
factors that interact with this motif have not been characterized. Here
we report that the E box motif in the proximal promoter of the rat SF-1
gene binds the transcription factor USF (upstream stimulatory factor),
a member of the helix-loop-helix (HLH) family of transcription factors.
In addition, we present evidence that nuclear proteins in the
T31
pituitary gonadotrope cell line exhibit different binding affinities
for distinct E box motifs that are important for gonadotrope-specific
gene regulation.
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RESULTS
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Promoter Activity of the Rat SF-1 Gene
To investigate the promoter activity of the SF-1 gene,
5'-flanking sequences were inserted upstream of the chloramphenicol
acetyltransferase (CAT) reporter gene in recombinant plasmids and used
in transient transfection assays. Previously, these SF-1 promoter
plasmids had been studied in the Y1 adrenocortical cell line (24).
Although SF-1 is also known to be expressed in the pituitary
gonadotrope (19) and
T31 pituitary gonadotrope cell line (20),
transcriptional regulation of the SF-1 gene has not been characterized
in these cell types. In this study, we assessed transcriptional
regulation of the SF-1 gene promoter in the
T31 and LßT2
pituitary gonadotrope cell lines.
Activity of the SF-1 gene directed by the proximal -4800 bp of
the SF-1 promoter was tested by transient transfection in
T31
(mouse pituitary gonadotrope), CV-1 (monkey kidney), JEG-3 (human
choriocarcinoma), and Y1 (mouse adrenocortical) cell lines (Fig. 1
). CAT activity of the SF-1 promoter plasmids
increased gradually when promoter sequences were deleted from -4800 to
-265 (CV-1 and Y1) or -92 (
T31 and JEG-3). However, a dramatic
reduction in CAT activity was observed when promoter sequences between
-92 and -60 were deleted (82% decrease in
T31 cells, 83% in
CV-1 cells, 90% in JEG-3 cells, and 75% in Y1 cells). Upstream
sequences between -4800 and -265 are regulated differently in
distinct cell types when normalized to CAT expression driven by the
constitutively active thymidine kinase (TK) promoter. Sequences between
-4800 and -265 appear to contain negative regulatory elements that
are active in steroidogenic Y1 adrenocortical cells and also in
T31 pituitary gonadotropes and CV-1 cells. However, overall
expression of the promoter is 2- to 4-fold higher in Y1 cells, a result
that correlates well with previously published studies (24).

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Figure 1. Fig. 1. Effect of SF-1 5' Flanking Sequence on CAT
Expression Transient transfection assays using plasmids carrying
5'-truncation mutations in the rat SF-1 promoter were performed in
T31 (pituitary gonadotrope), CV-1, JEG-3 (human choriocarcinoma),
and Y1 (adrenocortical) cells. Numbers below the bars
indicate base pairs included 5' from the transcription initiation site
of the SF-1 gene inserted upstream of a CAT reporter gene in pSV00CAT
(pSV00), a promoterless CAT expression vector (see Materials and
Methods). CAT enzyme activity values were corrected for
transfection efficiency by normalizing to a cotransfected
CMV-ß-galactosidase expression vector. Normalized CAT values are
expressed as a percentage of normalized TK-CAT activity in each cell
type. All values represent the result of a minimum of three separate
transfection experiments, with error bars representing the
SEM.
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The deletional analysis of the rat SF-1 gene promoter revealed
the presence of functional regulatory sequences between -92 bp and
-60 bp. Previous studies had established that a consensus E box motif
(CACGTG) at -82/-77 was required for maximal expression of the SF-1
gene in steroidogenic cell types (24). In this study, we have
established the importance of the E box for expression of SF-1 in the
T31 and LßT2 pituitary gonadotrope cell lines (Fig. 2
). An SF-1 promoter-CAT reporter gene
plasmid (-800) containing 800 bp of proximal 5'-flanking sequence was
tested by transient transfection assays in pituitary gonadotrope
(
T31, LßT2), steroidogenic (Y1), and nonsteroidogenic (JEG-3,
CV-1) cell lines. CAT activity of -800 in each cell type was set at
100% (black bars) and compared with CAT activity derived
from expression of the identical plasmid containing a specific
nucleotide substitution mutation in the E box at -82/-77 (E-mut)
generated by site-directed mutagenesis (24). This mutation converts the
wild-type sequence from CACGTG to CTGTAG. The presence of
this mutation has a direct functional effect on transcriptional
activation of the SF-1 promoter and results in a decrease in CAT
activity by 85% in
T31, LßT2, and CV-1 cells, 68% in JEG-3
cells, and 90% in Y1 cells (open bars). Results of these
studies further establish the importance of the E box as a
cis-regulatory element necessary for expression of the
proximal promoter of the SF-1 gene and, in particular, represents the
first transcriptional analysis of this element in the
T31 and
LßT2 pituitary gonadotrope cell lines. Unlike Woodson et
al. (26) we have not found that the SF-1 proximal E box imparts
tissue specificity to SF-1 gene expression in any cell type tested.

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Figure 2. A Functional E box Is Required for Expression of
the SF-1 Gene
T31 and LßT2 (pituitary gonadotrope), JEG-3, CV-1, and Y1 cells
were transfected with either the -800 (wild-type) or E-mut [-800
containing a 4-bp substitution mutation (underlined) in
the E box at -82/-77] plasmids. Normalized CAT activity from the E
box mutant plasmid (open bars) is expressed as a
percentage of wild-type CAT activity of the -800 plasmid set to 100%
(black bars) in the corresponding cell type. CAT enzyme
activity values were corrected for transfection efficiency by
normalizing to a cotransfected CMV-ß-galactosidase expression vector.
All values represent the result of a minimum of three separate
transfection experiments (± SEM). The sequence and
location of the E box mutation relative to the transcription start site
are indicated.
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Mutation of the E Box, but Not the SF-1-Binding Site in the First
Intron, Has a Significant Effect on Transcriptional Activity
Previous studies had established the importance of a consensus DNA
recognition site for SF-1 [or GSE (20)] in the first intron as a
possible mechanism of transcriptional autoregulation by SF-1 in Y1
cells (25). In the current study we have used a series of plasmids
[previously described (25)] to assess the importance of this
SF-1-binding site as an autoregulatory mechanism in the pituitary
gonadotrope compared with steroidogenic and nonsteroidogenic cell types
(Fig. 3
). The -800Int plasmid
(representing 100% of CAT activity) includes the 3.4-kb first intron
of SF-1 in addition to -800 bp of 5'-flanking sequence linked upstream
of the CAT reporter gene. Nucleotide substitution mutations were
generated in the SF-1 binding site at +160/+167 in the first intron,
converting the SF-1 site from GAAGGCCG to GAATATCG
(S-mutant). Previous results indicated that this mutation in the SF-1
site significantly decreased transcriptional activation in Y1 cells
(25). Surprisingly, in our studies, this mutation resulted in a
moderate increase in transcriptional activity in all cell types tested,
including Y1 cells. However, inclusion of the 3.4-kb first intron,
which increases CAT activity by 400-2000% in these cells compared with
the CAT activity of -800 (data not shown), cannot compensate for loss
of the E box motif. CAT activity of the plasmid carrying the E box
mutation (Ebox-mutant) is dramatically reduced to 23% in
T31,
20% in LßT2, 10% in JEG-3, 22% in CV-1, and 42% in Y1 cells of
wild-type expression. CAT activity of the plasmid carrying both
mutations (S+Ebox-mutant) is not significantly different from the
Ebox-mutant alone, with activity reduced to 25% in
T31, 22% in
LßT2, 10% in JEG-3, 26% in CV-1, and 65% in Y1 cells of wild-type
expression. These data indicate that the E box motif in the proximal
promoter is essential for full transcriptional activity of the SF-1
gene in a variety of cell types, while the SF-1-binding site is
inconsequential.

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Figure 3. E Box, but Not SF-1-Binding Site, Mutations
Decrease SF-1 Promoter Activity
Recombinant plasmids were constructed from the -800Int (wild-type)
plasmid by nucleotide substitution mutations in either or both the E
box at -82/-77 and the SF-1 binding site located in the first intron
of the SF-1 gene (see Materials and Methods). -800Int
includes the 3.4-kb first intron linked upstream of the CAT reporter
gene in addition to -800 bp of 5' flanking sequence. Cells were
transfected with either -800Int or mutant plasmids, and results are
expressed in terms of normalized CAT activity of the mutant plasmid as
a percentage of CAT activity of -800Int (set to 100%). CAT enzyme
activity values were corrected for transfection efficiency by
normalizing to a cotransfected thymidine kinase promoter-luciferase
expression vector. All values represent the result of a minimum of
three separate transfection experiments (± SEM).
-800Int is represented schematically below the graph,
with mutations in the E box and SF-1-binding sites indicated by
crosses. The S mutant construct (open
bars) contains a mutation in the SF-1-binding site at +160/+167
in the first intron converting GAAGGCCG to GAATATCG. The
Ebox-mutant construct (black bars) contains a mutation
in the E box at -82/-77 converting CACGTG to CTGTAG, and
the S+Ebox-mutant construct (gray bars) contains both
mutations. The hatched bar represents Exon I (+1 to
+153), with the arrow indicating the transcription start
site.
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Nuclear Proteins Form Distinct Complexes on the E Box Element in
the SF-1 Promoter
The effect on transcriptional activity of a site-specific mutation
of the E box at -82 in the SF-1 promoter suggests the possibility that
regulatory proteins interact with the E box to mediate full promoter
activity. To examine this possibility, we performed electrophoretic
mobility shift assays (EMSAs) with an oligonucleotide probe containing
SF-1 gene sequences from -90 to -68, which includes the E box element
at -82 (Fig. 4
). The sequences of all
double-stranded oligonucleotide probes and competitors are given in
Materials and Methods and Table 1
. The EMSA performed in the presence of
T31, CV-1, HeLa, JEG-3, and Y1 nuclear extracts revealed the
formation of distinct complexes with the wild-type E box probe. The
specificity of nuclear proteins for the E box is convincingly
demonstrated by comparing the pattern of binding complexes formed with
the E-mut probe to that obtained with the wild-type probe. The E-mut
oligonucleotide probe contains a site-specific mutation in the E box at
-82 (CACGTG to CTGTAG), identical in sequence to the
mutation created in the promoter plasmid used in transient
transfections (E-mut). Formation of the C1 and C2 complexes seen with
the wild-type E-box probe (left panel) is completely
eliminated in EMSAs employing the E-mut probe (right panel).
The variable complex above C1 is also partially eliminated in specific
extracts using the E-mut probe. The low-molecular weight complex
observed below C2 with CV-1, HeLa, and JEG-3 nuclear extracts may
represent non-E box-binding protein complexes formed elsewhere on the
probe. An equivalent mass of nuclear protein (10 µg) was used in each
EMSA; therefore the C1 and C2 complexes may represent ubiquitous
proteins present in a wide variety of cell types. However, the C2
complex is more variable in intensity and suggests the possibility that
a different cellular context may determine the expression pattern of E
box-binding proteins.

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Figure 4. An E box Mutation Reduces Nuclear Factor Binding as
Analyzed by EMSA
Wild-type SF-1 E box (E-box) and E box mutant (E-mut)
32P-labeled oligonucleotide probes were used in an EMSA
containing nuclear extracts (10 µg total protein in each lane) from
T31, CV-1, HeLa, JEG-3, or Y1 cells. Specific complexes (C1 and
C2) formed with the wild-type E box probe with all extracts are absent
when an identical oligonucleotide containing an E box mutation (E-mut)
is used as the labeled probe. -, No extract; Free, unbound
oligonucleotide probe. The size of the oligonucleotide probes and
sequence of the specific E box mutation are indicated
above the corresponding lanes. The full-length sequence
of the oligonucleotides is given in Materials and
Methods and Table 1.
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SF-1 E Box-Binding Proteins Migrate with Identical Mobility to
USF
USF is a ubiquitously expressed basic HLH-leucine zipper (bHLH-LZ)
transcription factor that is involved in transcriptional activation of
several promoters by forming functional heterodimers with ubiquitous
and cell-specific partners (27, 28, 29). In this capacity, basal
transcription is often facilitated by USF through a proximal E box
motif characterized by a core CACGTG sequence (30). The E box motif in
the SF-1 promoter is identical to the specific DNA sequence that is
recognized by USF and required for transcriptional activation (30).
To address the identity of the factors that form the specific C1 and C2
complexes and to test the hypothesis that one or both of these
complexes may contain USF, labeled SF-1 E box and USF probes were used
in EMSAs performed with nuclear extracts from multiple cell types (Fig. 5
). When the E box probe was incubated
with
T31, CV-1, HeLa, JEG-3, or Y1 nuclear extracts, the specific
C1 and C2 complexes were observed in all lanes (left panel).
Interestingly, when the USF probe was incubated with the identical
nuclear extracts, a very similar shift pattern emerged with the
appearance of the prominent C1 and C2 complexes migrating with the same
mobility as complexes formed on the E box probe (right
panel). HeLa cell nuclear extracts were included as a positive
control for USF binding (31). Due to the relatively long exposure time
required to obtain the E-mut binding complex signal seen in Fig. 4
(96
h exposure), the nonspecific binding complexes (below C2 and above C1)
are not observed in Fig. 5
(16 h exposure).

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Figure 5. Complexes with Identical Mobility to USF Are Formed
with the E box Oligonucleotide Probe
SF-1 E box and USF consensus 32P-labeled oligonucleotide
probes were used in an EMSA containing nuclear extracts (10 µg total
protein in each lane) from T31, CV-1, HeLa, JEG-3, or Y1 cells.
HeLa cell nuclear extracts have been included as a positive control for
USF binding. The specific C1 and C2 complexes formed with both the
wild-type E box and USF probes are present in all cell types tested.
-, No extract; Free, unbound oligonucleotide probe. The full-length
sequence of oligonucleotides is given in Materials and
Methods and Table 1.
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USF exhibits a heterogeneous pattern of expression in which proteins
with molecular masses of 43 kDa (USF-1) (31) and 44-kDa (USF-2) (29)
are responsible for USF activity, yet the two different forms of USF
show identical DNA-binding properties (29). Specific antibodies (29)
against these widely expressed proteins were used in EMSA supershift
experiments to further characterize the E box-binding complexes C1 and
C2 (Fig. 6
). Nuclear extracts from
T31, Y1, HeLa, CV-1, and JEG-3 cells were preincubated with 1 ng
of specific rabbit polyclonal antibody raised against USF-1 or USF-2
before the addition of the labeled E box oligonucleotide probe. One
nanogram of whole rabbit serum was included as a negative control for
the specificity of antibody reactions. Inclusion of control nonspecific
antibody [(-) lane] or whole mouse serum (IgG lane) has no effect on
the formation of the prominent C1 and C2 complexes. However, the
addition of specific
USF-1 antibody produces a supershifted complex
and a reduction in C1 and C2 complex formation in all cell types. The
addition of specific
USF-2 antibody has a weaker supershift effect
in all cell types, particularly in
T31 and JEG-3 extracts.
However, specific supershifted complexes are seen using the
USF-2
antibody with Y1, HeLa, and CV-1 nuclear extracts.
Together, these data fit the hypothesis that the SF-1 promoter E
box is preferentially recognized by USF proteins (or proteins
antigenically related to USF). Previous studies have reported that
USF-1 and USF-2 readily form hetero- and homodimers in solution and
that these species represent the predominant DNA-binding complexes
(32). Our data suggest the possibility that C1 represents a USF-1:USF-2
heterodimer-DNA complex composed of 43- and 44-kDa proteins that
migrates with a relatively slow mobility. Thus, this complex is
recognized by both antibodies, as we have observed. Since the C2
complex is also preferentially supershifted by
USF-1 antibodies, C2
may be composed of a USF-1:USF-1 homodimer complex of 43-kDa proteins
that moves with a slightly faster mobility than C1. This pattern has
been observed using different ratios of bacterially produced USF-1 and
USF-2 proteins in EMSAs, wherein USF-1:USF-1>USF-1:USF-2>USF-2:USF-2
in terms of relative mobility (32). In addition, the protein charge and
cellular context may also play a role in determining the ratio and
binding affinity of USF-1 and USF-2 hetero- and homodimers for the SF-1
E box. The antigenic site required for recognition may be relatively
less accessible to the
USF-2 antibody in the heterodimer, possibly
leading to the appearance of a lower affinity of this antibody for the
C1 complex. If HeLa cell nuclear proteins may be used as a standard
from which to judge the formation of hetero- and homodimer complex
formation, USF-2:USF-2 homodimers are a relatively minor component of
an E box-binding complex (32), and such complexes have not been
observed under our EMSA conditions. Based on this observation, we can
conclude that the complex with higher electrophoretic mobility compared
with C1 is probably not a USF-2:USF-2 complex, but may represent a
complex with a relatively low affinity for the SF-1 E box compared with
USF.
Pituitary Gonadotrope Nuclear Proteins Exhibit Different Binding
Affinities for Distinct E Box Probes
A recent report provided evidence for E box-dependent
transcriptional activation of the human
-subunit of the glycoprotein
hormones in
T31 pituitary gonadotrope cells through two E box
motifs (
EB1 and
EB2) located at -51 and -21, respectively, from
the transcriptional start site (33). In this report it was determined
by supershift EMSA analysis using a cross-reactive polyclonal rabbit
anti-USF antibody that USF binds to
EB2, but not
EB1, E box
sequences. The
-subunit is expressed by cells committed to the
gonadotrope and thyrotrope cell lineage (34), and thus it was of
interest to determine whether
EB1 or
EB2 could compete for SF-1 E
box-binding proteins present in
T31 nuclear extracts.
To test this hypothesis, an SF-1 E box probe was used in an EMSA
containing
T31 nuclear extracts with the inclusion of a 100-fold
molar excess of unlabeled double-stranded competitor oligonucleotides
comprising the E box, E-mut, USF,
EB1, or
EB2 sequences (Fig. 7A
and Table 1). The specific C1 and C2
complexes are competed by an excess of both the wild-type SF-1 E box
and USF oligonucleotides. However,
EB1 and
EB2 were poor
competitors for SF-1 E box-binding proteins, demonstrating that the
proteins forming the C1 and C2 complexes may not have the equivalent
affinity for the
EB1 or
EB2 E box sequences. The sequence
differences in the E box motifs between oligonucleotide probes and
competitors are shown in Table 1.
The different affinity of the E box,
EB1, and
EB2 probes for E
box-binding proteins is further highlighted by the results presented in
Fig. 7B
. EMSAs were performed in which specific reactions using the
EB1 or
EB2 probes and
T31 nuclear extracts were additionally
incubated with a 100-fold molar excess of unlabeled double-stranded
competitor oligonucleotides as specified in Fig. 7A
. Two predominant
shifted complexes,
C1 and
C2, were formed with both the
EB1 or
EB2 probes. The
EB1 probe appears to have a higher affinity for
T31 nuclear proteins (left panel), as the
EB2
autoradiograph shows a weaker signal than that of
EB1 (right
panel). The
C1 complex migrates with equal mobility to the C1
complex on the SF-1 E box probe (data not shown), and it is likely that
C1 represents the same band previously described as C4 (33) that is
competed here by both the
EB1 (CAGGTG) and E box and USF (CACGTG)
oligonucleotides. However, the
C2 complex is competed by an excess
of all oligonucleotides including E-mut, and so most likely does not
represent a specific E box-binding complex. Finally, the C2 band
possibly representing a USF-1:USF-1 homodimer complex is completely
absent using either
EB1 or
EB2 probes. These results further
highlight the qualitative and quantitative differences in binding
patterns obtained with probes containing the CACGTG motif (E box and
USF) or CAGGTG motif (
EB1 and
EB2).
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DISCUSSION
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SF-1, also known as Ad4BP (5), was first identified as an orphan
nuclear receptor that regulates cytochrome P450 steroid
hydroxylase gene expression (2, 3, 5, 7, 10, 35, 36, 37, 38). An essential role
for SF-1 in the development of the adrenal glands and gonads was
established in mice by targeted disruption of the mammalian FTZ-F1
locus, which encodes the SF-1 and ELP transcripts (13, 14, 15, 18). These
studies extended the role of SF-1 in the control of reproductive
function by establishing that SF-1 knockout mice demonstrated defects
at all levels of the hypothalamic-pituitary-gonadal axis.
In its role as a transcriptional regulator, SF-1 has been implicated in
the selective regulation of key markers of the pituitary gonadotrope
phenotype, including the human
-subunit (20), the LH ß-subunit in
multiple species (Ref. 22 and references therein), and the GnRH
receptor (21). We have focused primarily on the molecular mechanism for
directing expression of the rat SF-1 gene to the gonadotropes of the
anterior pituitary, and thus an analysis of the cis elements
and trans-acting factors governing expression of SF-1 is
critical to our understanding of the role of SF-1 in reproductive
function. In this study, we have demonstrated that optimal promoter
activity of the rat SF-1 gene in distinct cell types is primarily
regulated by a proximal E box at -82. We have defined members of a
specific subset of HLH transcription factors, USF-1 and USF-2, that may
bind the E box in dimer form and may be responsible for transactivation
of SF-1 in vivo. We have also demonstrated that nuclear
proteins obtained from the
T31 pituitary gonadotrope cell line
contain USF, and that these nuclear proteins exhibit distinct binding
affinities for different E box elements.
Previous studies have provided evidence that the E box regulates
expression of SF-1 in the Y1 and I-10 steroidogenic cell lines and not
in the nonsteroidogenic CV-1 cell line (24). We have presented data
demonstrating that regulation of SF-1 gene expression appears to occur
primarily through the E box in all cell types that were transiently
transfected, including the
T31 and LßT2 pituitary gonadotrope
cell lines, and nonsteroidogenic CV-1 and JEG-3 cells. Our data differ
from previously published reports (24, 25) with regard to expression of
the SF-1 promoter in nonsteroidogenic cells and the ability of SF-1 to
regulate its own promoter through a consensus DNA recognition site
(SF-1 site). We may have obtained different results based on our cell
culture and transfection technique (different media was used for Y1
cells). In addition, there appears to be variation in the
characteristics of Y1 clones in different laboratories (K. Morohashi,
personal communication). Cells transfected by the lipofection method
(24, 25) may yield significantly different quantitative and qualitative
results compared with the calcium phosphate method (39). The
sensitivity of transcriptional assays may be enhanced by the
calcium-phosphate method, allowing us either to transfect a larger
amount of plasmid or lower the threshold of detection for CAT
activity.
However, such results allow us to speculate on the mechanism by
which the consensus E box may regulate SF-1 gene expression in a wide
variety of cell types. We hypothesize that USF-1 and, to a lesser
degree, USF-2 or a heterodimer of USF-1:USF-2 form complexes on the
SF-1 proximal E box and are significantly involved in basal
transcription of the gene. This hypothesis is supported by several
recent observations of the function of USF proteins in transcriptional
regulation and the structure of the SF-1 gene. The SF-1 gene lacks a
recognizable TATA box, which is the hallmark of many eukaryotic gene
promoters that use RNA polymerase II for transcription initiation (40).
Although the mechanism of transcription initiation is less well
understood in TATA-less promoters, it has been shown that TFII-I, a
transcription initiation factor that activates core promoters through
an initiator motif (Inr) can cooperatively interact on binding with USF
at both the Inr and an upstream E box (41, 42). A potential Inr
sequence (43) is located in the rat (24), mouse (26), and human (26)
promoters at +1.
USF was first identified as a factor that bound to an upstream element
in the adenovirus major late promoter that stimulated transcription
(30, 41), and has subsequently been found to activate transcription of
the promoters of a number of cellular genes (27, 28). USF is a
ubiquitous nuclear protein that exists in two major biochemical forms
with apparent molecular masses of 43-kDa (USF-1) (31) and 44-kDa
(USF-2) (29). Sequence analysis of USF cDNA clones has demonstrated
that USF belongs to the myc family of regulatory proteins
characterized by a C-terminal basic region followed by a bHLH-LZ
structure responsible for dimerization and DNA binding (44). bHLH-LZ
proteins bind either as homodimers or heterodimers to a specific DNA
element, which contains the core sequence CANNTG or E box (45).
Dimerization between cell-specific [e.g. MyoD (46)] and
ubiquitously expressed HLH proteins [e.g. E12/E47 (47)] is
a critical step in controlling transcriptional activation and cell type
specificity during differentiation of diverse tissues (46, 48, 49).
The ubiquitous expression pattern of nuclear factors that was observed
to bind to the SF-1 E box and the specific architecture of the E box
led us to examine USF as a potential regulator of SF-1 gene expression.
USF binding is somewhat constrained at the first level by the central
core nucleotides at position -1 and +1
(C-3A-2N-1N+1T+2G+3),
preferring CG in these sequential positions (30, 50). In addition,
binding discrimination can be conferred by specific nucleotides 5' to
the E box core sequence, with USF exhibiting a pyrimidine selectivity
at -4 and a purine selectivity at -5 (50). The rat (24), mouse (26),
and human (26) SF-1 promoters contain a thymidine at -4 (relative to
the E box). Based upon these data, we tested the binding specificity of
pituitary nuclear factors for the USF E box from the SF-1 promoter
compared with the sequence of E box elements (
EB1 and
EB2) that
had been previously defined in the human
-subunit promoter using
T31 pituitary gonadotrope nuclear extracts (33). We observed that
only oligonucleotides containing the USF binding sequence (CACGTG)
could compete for the proteins bound to the SF-1 E box. However,
oligonucleotides containing the sequence CACGTG or CAGGTG could compete
for
EB1-binding proteins in the
C1 complex, a complex that
contributed a relatively low percentage to overall probe binding.
Previously, USF proteins had been characterized in
T31 nuclear
extracts using the
EB2 probe in supershift analyses employing an
anti-USF antibody that was cross-reactive between USF-1 and USF-2 (33).
In this same report, functional studies demonstrated that
overexpression of the Id protein compromised the activity of the
-subunit promoter, possibly through interference with USF binding at
the
EB2 site. However, HLH proteins that contain a leucine zipper
motif (e.g. USF-1 and USF-2) resist inactivation by Id (51),
and so it is likely that the effect of Id in reducing
-subunit gene
expression as described is indirect. Therefore, whether the
C1
complex contains specific USF proteins is inconclusive, although in our
studies the
EB1 and
EB2 probes encourage binding by proteins
comprising the
C1 complex that are specifically competed by the
CACGTG E box.
Results from previous Southwestern and far-Western experiments
demonstrated that factors sharing the conserved properties of bHLH
proteins are present in pituitary nuclear cell extracts (52). Further,
in gel mobility shift experiments using nuclear extracts from mature
adult pituitary tissue, complexes distinct from those produced using
the
T31 pituitary extracts are observed. These results may have
significant implications for our understanding of developmental
regulation of the pituitary gonadotrope lineage. We may use pituitary
cell lines immortalized at discrete stages of development, which
express differentiated markers of the pituitary gonadotrope cell (34),
to determine the temporal and spatial expression pattern of HLH
proteins that may be required for the differentiation process to occur.
USF-1 and USF-2 proteins exhibit a heterogeneous pattern of expression
in both human and mouse, resulting from differential splicing and
alternative poly(A) site usage (32). Thus it is possible that the
abundance of functional USF is regulated under physiological conditions
by the predominance or ratio of encoded proteins in different cell
types. Our studies represent a first step in the identification of a
subset of HLH proteins that appear to be required for expression of a
marker gene in the pituitary gonadotrope. Results from these studies
may allow us to develop a model with which to understand the mechanism
by which the ubiquitous USF bHLH-LZ proteins may interact with a
tissue-specific partner to dictate expression of SF-1 to the
appropriate tissues, as has been demonstrated in other systems
utilizing HLH transcriptional determinants of gene expression.
 |
MATERIALS AND METHODS
|
---|
Plasmid Construction
Construction of rat SF-1 gene promoter-CAT reporter gene
plasmids has been previously described (24). Briefly, a 4.8-kb fragment
containing the 5'-flanking region upstream from the EcoRI
site located in the first exon of the SF-1 gene was ligated upstream of
the CAT structural gene in pSV00CAT to produce Ad4CAT4.8K. The deletion
mutant constructs including, and created from, Ad4CAT4.8K have been
designated here as -4800, -2000, -1200, -800, -265, -92, and -60
(numbers indicate base pairs included 5' from the transcription
initiation site) and correspond to Ad4CAT4.8K, Ad4CAT2.0K, Ad4CAT1.2K,
Ad4CAT0.8K, Ad4CAT265, Ad4CAT92, and Ad4CAT60, respectively, as
described previously (24). The E-mut plasmid (generated from
Ad4CAT0.8K) contains a site-specific mutation in the SF-1 E box that
converts the E box at -82/-77 from CACTGT to
CTGTAG. E-mut corresponds to the Ad4CATM3
plasmid as previously described (24).
Construction of SF-1 gene promoter/intron reporter gene plasmids
containing mutations in either or both the E box at -82 or the SF-1
binding site in the first intron has been previously described (25).
The -800Int plasmid (corresponding to plasmid Ad4ECAT0.8K) contains
the 4.4-kb DNA fragment located between the BamHI site at
0.8-kb upstream from the transcription initiation site and a
SmaI site generated by site-directed mutagenesis in the
second exon at 5 bp upstream from the initiation methionine. Mutations
in either the E box (Ebox-mutant), SF-1 binding site (S-mutant), or
both (S+Ebox-mutant) were generated from the wild-type -800Int plasmid
by site-directed mutagenesis. These constructs correspond to
Ad4ECAT0.8KM, Ad4ECAT0.8KA, and Ad4ECAT0.8KMA, respectively (25).
Cell Culture and Transfection
T31 (53) and LßT2 (54) (mouse pituitary gonadotrope),
CV-1 (monkey kidney), JEG-3 (human choriocarcinoma), and HeLa (human
cervical carcinoma) cells were maintained in DMEM (GIBCO BRL)
supplemented with 10% FBS (HyClone. Logan, UT), 4.5 mg glucose per ml,
100 U of penicillin per ml, and 0.1 mg of streptomycin per ml in a
humidified atmosphere of 5% CO2. Y1 (mouse adrenocortical)
cells were cultured in Hams F-10 medium (GIBCO BRL) supplemented with
15% horse serum (GIBCO BRL), 5% FBS, and additional supplements and
atmosphere as indicated above.
Transient transfections were performed by the calcium phosphate
precipitation method without glycerol shock (55). Cells were plated at
a density of 4060% confluency on 10-cm plates 18 h before
transfection. Five micrograms of SF-1 gene promoter-CAT reporter
expression plasmids were cotransfected with 5 µg of TK
promoter-luciferase plasmid or ß-galactosidase under the control of
the human cytomegalovirus promoter (CMV-ß-gal) as an internal
transfection control. Additional plates were transfected with a TK-CAT
reporter gene plasmid as a control for transfection in each different
cell type. Cells were incubated with precipitates for 5 h in a
humidified atmosphere of 5% CO2 and washed once each in
PBS and culture medium, followed by a change of medium. Cells were
harvested after 36 h by scraping into a buffer of 150
mM NaCl, 1 mM EDTA, and 40 mM
Tris-Cl (pH 7.4) at 4 C. Extracts of harvested cells were made by brief
centrifugation and resuspension of cell pellets in 100 µl of 250
mM Tris-Cl (pH 7.8) at 4 C followed by three cycles of
freeze-thawing. Extracts were clarified by centrifugation for 5 min in
an Eppendorf 5415C centrifuge. Supernatants were assayed for luciferase
activity (56) with an AutoLumat 953 or MicroLumat 96P luminometer (EG&G
Berthold, Bad Wildbad, Germany), ß-galactosidase activity (57), and
CAT activity by the organic phase extraction method (58) with minor
modifications (59). Results are reported as the mean CAT activity from
at least three separate transfection experiments corrected for the
activity of the cotransfected internal control. Error is reported as
SEM.
EMSAs
Nuclear extracts from
T31, CV-1, JEG-3, Y1, and HeLa cells
were prepared as previously described (60). Shift reactions were
performed at a final volume of 15 µl in a solution containing 10
mM HEPES, pH 7.9, at 25 C, 10% (vol/vol) glycerol, 50
mM KCl, 5 mM MgCl2, 5
mM dithiothreitol, 0.2 mg BSA per ml, 0.5 mM
phenylmethylsulfonyl fluoride, 1 mM benzamidine, 0.1%
Nonidet P-40, and 15 ng/µl poly(dI-dC). One to two microliters of
nuclear extract were included at 510 µg/µl as determined relative
to a BSA standard by the method of Bradford (61). Reaction mixtures
with extract were preincubated for 10 min on ice followed by the
addition of 2050 fmol of radiolabeled, double-stranded
oligonucleotide probe either with or without the addition of unlabeled
competitor oligonucleotide at a 100-fold molar excess. Reaction
mixtures were further incubated for 5 min at 25 C, and DNA-protein
complexes were resolved by electrophoresis on 5%
acrylamide-N,N'-bisacrylamide (30:1) gels at 9 V/cm. An
autoradiogram of the gels was made by exposing dried gels to XAR-5 film
(Kodak, Rochester, NY) using DuPont Cronex intensifying screens at -80
C. Antibody supershift assays were performed identically with the
inclusion at the preincubation step of 1 ng of purified, reconstituted
rabbit anti-USF-1 or anti-USF-2 antibody (a generous gift of Michele
Sawadogo), or normal rabbit IgG (Vector Laboratories, Inc., Burlingame,
CA).
The self-complementary oligonucleotide representing the SF-1 E box (E
box) contains the sequence (5'-tTGCAGAGTCACGTGGGGGCAGAG-3')
and comprises the nucleotide sequence from -90 to -68 of the rat SF-1
gene (24). The self-complementary E-mut oligonucleotide is identical to
the E box oligonucleotide sequence, with the exception that CACGTG was
converted to CTGTAT, creating a site-specific mutation from
-81/-78 in the E box. The self-complementary oligonucleotide
representing the consensus USF binding site (USF) contains the sequence
5'-tCTGAATTCCTGGTCACGTGACCGCAGCTGT-3' (62). The
self-complementary oligonucleotides representing two different E boxes
in the human
-subunit promoter (33) contain the sequences
5'-tGCTTAGATGCAGGTGGAAACACT-3' (
EB1) and
5'-tGTATAAAAGCAGGTGAGGACTTC-3' (
EB2). One thymidine
nucleotide (t) was included at the 5'-end of each synthetic
oligonucleotide to facilitate end-labeling reactions (57). Ten to 20
pmol of each double-stranded oligonucleotide were used in fill in
labeling reactions with Klenow fragment and 100 µCi of
[
32P]dATP (3000 Ci/mmol, DuPont NEN, Boston, MA) to
generate oligonucleotide probes. Probe reactions were stopped with the
addition of 5 mM EDTA, extracted with phenol-chloroform
(1:1), and purified over a Probe-Quant G-50 microcolumn (Pharmacia
Biotech, Piscataway, NJ). The probe concentration was adjusted to
2050 nM in 10 mM Tris-Cl (pH 7.4), 1
mM EDTA, and 50 mM NaCl and stored at -20
C.
 |
ACKNOWLEDGMENTS
|
---|
We thank Ken-Ichirou Morohashi and Masatoshi Nomura for
generously providing the rat SF-1 promoter-CAT reporter plasmids. We
also thank Michele Sawadogo for providing anti-USF-1 and USF-2
antibodies, and Sandra Holley and Flavia Pernasetti for critical review
of the manuscript.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Pamela L. Mellon, Departments of Reproductive Medicine-0674, University of California, San Diego, School of Medicine, Cellular and Molecular Medicine Building, Room 2057, 9500 Gilman Drive, La Jolla, California 92093-0674. E-mail:
pmellon{at}ucsd.edu
This work was supported by NRSA Grant DK-09468 from the NIDDK (to
A.N.H.) and NIH Grants RO1DK-20377 and HD-12303 (to P.L.M.).
Received for publication September 10, 1997.
Revision received December 12, 1997.
Accepted for publication January 14, 1998.
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