Characterization of the Promoter of SF-1, an Orphan Nuclear Receptor Required for Adrenal and Gonadal Development
Karen G. Woodson1,
Peter A. Crawford1,
Yoel Sadovsky1 and
Jeffrey Milbrandt
Division of Laboratory Medicine Departments of Pathology and
Internal Medicine (K.G.W., P.A.C., J.M.) Department of
Obstetrics and Gynecology (Y.S.) Washington University School of
Medicine St. Louis, Missouri 63110
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ABSTRACT
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Steroidogenic factor 1 (SF-1) is a transcription
factor shown to be critical for regulation of adrenal and gonadal
development and function. To dissect the mechanisms that direct
expression of this regulator, we have studied the promoter of the SF-1
gene and have identified cis-acting elements that recognize
a basic-helix-loop-helix transcription factor; the CAAT binding factor;
and Sp1. We demonstrate in Y1 adrenocortical cells that a 90-bp
proximal promoter fragment is sufficient to direct
steroidogenic-specific expression and that all three elements are
required for activity of the SF-1 promoter. Functional analysis of the
binding sites on a heterologous TATA box-containing promoter
demonstrates that the CAAT box and Sp1 site are not essential for
promoter activity when a TATA box is present, whereas the E box is
absolutely required for gene expression and is most likely the
steroidogenic cell-specific element. We also demonstrate that SF-1
itself does not significantly affect the transcription of its own gene,
and thus conclude that the E box, CAAT box, and Sp1 site of the
proximal promoter direct expression of the SF-1 gene.
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INTRODUCTION
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Steroidogenic factor 1 (SF-1) is a member of the nuclear hormone
receptor superfamily of transcription factors. Members of this
superfamily, which coordinate aspects of growth, development, and
metabolism, include receptors for steroid and thyroid hormones, vitamin
D, and retinoic acid. These receptors bind to specific
cis-acting DNA sequences called hormone response elements
and modulate gene expression, usually in the presence of their
respective ligands (1). Because no ligand has been isolated for SF-1,
it is termed an orphan nuclear receptor. SF-1 binds DNA as a monomer to
a sequence element comprised of an estrogen receptor half-site
(5'-AGGTCA) and three specific 5'-adjacent nucleotides (2). In the
adult mouse, SF-1 is constitutively expressed in all steroidogenic
tissues including the cortical cells of the adrenal gland, Leydig cells
of the testis, and thecal and granulosa cells of the ovary (3, 4). It
is thought to be a key regulator of steroid hormone biosynthesis as it
has been shown to bind and transactivate the promoters of cytochrome
P450 steroid hydroxylases that convert cholesterol to the various
steroids (2, 3, 5, 6).
SF-1 is the murine homolog of the Drosophila orphan nuclear
receptor FTZ-F1, a putative regulator of the fushi tarazu
homeobox gene during embryonic development (7, 8). Similarly, SF-1
expression is developmentally regulated in mammals. SF-1 is expressed
in the urogenital ridge before its differentiation into kidneys,
adrenal glands, and gonads (9), and it is also expressed in the
developing hypothalamus and in the gonadotropes of the anterior
pituitary, where it regulates gonadotropin synthesis (10, 11). The
developmental function of SF-1 was established when mice with a
targeted disruption of the SF-1 gene were found to lack adrenal glands
and gonads, resulting in neonatal lethality presumably due to
adrenocortical insufficiency (11, 12, 13). Moreover, analysis of staged
embryos of SF-1-deficient mice showed that gonadal precursors were
initially present, but later regressed by programmed cell death
(12).
Taken together, these observations extend SF-1s role beyond steroid
hormone biosynthesis to that of a key regulator of adrenal gland and
gonadal development. However, little is known regarding the molecular
mechanisms that control the tissue and developmental, stage-specific
expression of the SF-1 gene. Here we show that the promoter of SF-1
contains at least three elements that are essential for promoter
activity. The first element is an E box, which is recognized by factors
that belong to the basic-helix-loop-helix (bHLH) family of
transcription factors (14). These factors are thought to regulate many
developmental programs such as muscle differentiation, neurogenesis,
hematopoiesis, and sex determination (15, 16). The second element is a
CAAT box, a common proximal promoter element that is recognized by a
number of proteins (17, 18). The third is a GA-rich element, which is
shown to bind the transcription factor Sp1.
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RESULTS
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Sequence Analysis of the Mouse and Human SF-1 Promoters
To identify sequence elements important for the expression of
SF-1, genomic clones containing either the mouse or human SF-1 genes
were isolated. The nucleotide sequences of the human and mouse
5'-flanking regions, which are very similar, were analyzed for
potential transcription factor binding sites (Fig. 1
).
In particular, the sequence of the -93 to -38 region is almost
identical (54/55 bases). This region harbors a potential E box
(5'-CANNTG) (19), as well as an element that could function as an E box
or CAAT box, and a GA-rich region that contains potential sites for Ets
(20), GAGA (47), or the Sp1 (22) families of transcription factors.
Although no recognizable TATA element exists, there is a putative
transcription initiator element (23) (5'-PyPyA+1NT/APyPy),
which overlaps the transcription start site as previously determined
(24).

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Figure 1. Comparison of Nucleotide Sequences of SF-1
5'-Flanking Region between Human and Mouse
Nucleotides that are identical between the mouse and human 5'-flanking
sequences are indicated by vertical bars, and nucleotide
deletions (alignment gaps) are indicated by dashes. The
transcriptional start site (+1) (24) is indicated by the
arrow, and the nucleotides are numbered relative to this
position. Potential regulatory elements (an E box, an E and/or CAAT
box, and a GA-rich element) are boxed. The sequence
contains a potential initiator element spanning (+1) (23), but not a
recognizable TATA box.
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Deletion Analysis of the SF-1 Promoter
To localize the functional regions of the mouse SF-1 promoter, we
generated a series of luciferase reporter constructs that contained
deletions of the 5'-flanking regions with 5'-termini at nucleotide (nt)
-1885, -712, -190, -90, and -12 and a common 3'-terminus at +140.
The promoter activities of the 5'-deletion mutants were assessed by
luciferase assays after transient transfection into two separate
steroidogenic cell lines, Y1 (mouse adrenal cortex) and DC3 (rat
ovarian granulosa), both of which express SF-1 (our unpublished data).
A promoter fragment spanning nucleotide (nt) -90 to +140 (SF-1-90Luc)
was sufficient to drive transcription in Y1 and DC3 cell lines at
levels of
100-fold over the promoter-less luciferase reporter (Fig. 2
). Addition of further upstream flanking sequences up
to nt -1885 did not significantly increase the activity of the -90
fragment, whereas deletion of sequences to nt -12 abolished activity.
In contrast, when the promoter fragments were tested in NIH-3T3
fibroblasts, which are nonsteroidogenic and do not express SF-1, no
promoter activity was observed (Fig. 2
). These results indicate that
the region between nt -90 and -12 contains regulatory elements that
are required for steroidogenic cell-specific expression of SF-1.

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Figure 2. Deletion Analysis of the SF-1 Promoter
A schematic representation of the 5'-flanking region of the mouse SF-1
gene is shown. The locations of the relevant restriction sites used for
construction of the serial deletion mutants are indicated. The SF-1
promoter/luciferase reporter constructs were named according to the
nucleotide positions of their 5'-termini. All constructs were
transiently transfected into Y1 adrenocortical cells, DC3 granulosa
cells, and NIH-3T3 fibroblasts; luciferase assays were performed a
minimum of three times as described in Materials and
Methods. Mean luciferase activity (± SD) of each
construct in all three cell lines is given relative to the
promoter-less vector (pLuc).
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Identification of cis-Acting Functional Elements in the
SF-1 Promoter
Deletion analysis of the SF-1 promoter demonstrated that the
region between nt -90 and -12 was essential for promoter activity. To
functionally identify cis-acting regulatory elements, we
generated a series of substitution mutations spanning this region
within the context of the -90Luc construct (Fig. 3
). We
found that mutations in three different regions, at nt -84 to -75
(m1, a potential E box), nt -68 to -59 (m2, a potential E or CAAT
box), and nt -30 to -24 (m4, GA-rich region), reduced promoter
activity to 10% to 20% of wild type levels, whereas mutation of nt
-40 to -31 (m3) and nt -26 to -17 (m5) appeared to have no effect.
These mutations yielded a similar effect when incorporated into the
-1885-Luc construct (Fig. 3
).

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Figure 3. Mutational Analysis of the SF-1 Promoter
Substitution mutations of the -90 to -17 region were made in the
SF1-90Luc construct. The indicated mutants are identical to the wild
type sequence with the exception of the sequences shown. The effect of
these mutations in the context of the larger promoter fragment is also
shown. Constructs were transiently transfected into Y1 adrenocortical
cells, and luciferase activities were measured. Results shown are the
mean (± SD) of three independent experiments, calculated
as above.
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Because many promoters associated with steroidogenesis are regulated by
SF-1 in cultured cells, we assessed the ability of SF-1 to
transactivate two mouse SF-1 promoter fragments in CV-1 monkey kidney
cells and Y1 adrenocortical cells: -1885-Luc, which spans nt -1885 to
+140; and -3100-Luc, which spans nt -3100 to +3600. The latter
fragment traverses the genes first intron and extends to the natural
ATG of the mouse SF-1 gene, thus containing the SF-1-binding site
within the first intron (25). Although previous work on the rat SF-1
promoter has shown autoregulation of the SF-1 promoter by its own
product via this SF-1 binding site (25), neither of the SF-1 promoter
fragments tested here was activated by coexpression of SF-1 in CV-1
cells (Fig. 4
). Cotransfection of the SF-1 expression
vector with an SF-1 responsive luciferase reporter in CV-1 cells yields
more than 20-fold activation (data not shown), indicating the
functional integrity of our transfected SF-1. In addition, transfection
of these two SF-1 promoter constructs into Y1 cells, which
constitutively express SF-1, yielded equivalent results, despite the
presence of an SF-1-binding site in the -3100-Luc construct (Fig. 4
).
These data further indicate that SF-1 does not autoregulate its
expression.

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Figure 4. Effect of Additional 5'-Flanking Sequences,
Including an SF-1 Site, on Activity of the SF-1 Promoter
Diagrams schematically represent the promoter/luciferase reporter
construct used in transient transfection of CV-1 and Y1 cells. In CV-1
transfections, 50 ng of the nonrecombinant or CMV-SF-1 expression
vector were cotransfected with the reporter. In Y1 transfections,
reporters were transfected in the absence of any expression vector.
Results presented (mean ± SD) are representative of
two independent experiments, calculated as above.
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The Functional Element at nt -83 to -74 Is an E Box
To identify potential transcription factors that interact with the
identified elements, we performed gel mobility shift analysis.
Inspection of the functionally important regions reveals that the -83
to -74 region contains a putative E box; the -68 to -59 region
contains a palindromic (5'-CCAATTGG) sequence, which could potentially
be an E box, CAAT box, or both; and a GA-rich region at nt -30 to -24
(see below). Gel mobility shift assays using Y1 nuclear extracts and an
oligonucleotide containing nt -87 to -49 shows two specific complexes
(Fig. 5A
, lane 1, designated Complex I and II). Complex
I is formed by an interaction with the -83 to -74 region as a similar
complex was observed using an oligonucleotide probe spanning only nt
-87 to -69 (lane 5). In addition, formation of Complex I was
abolished when competed with the -87 to -69 region oligonucleotide
(lane 3). Complex II was formed by an interaction with the -68 to -59
region, as an oligonucleotide spanning nt -73 to -49 effectively
competed for the species (lane 2), and when used as a probe, a similar
complex was observed (lane 4).

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Figure 5. Gel Shift Analysis of the E Box in the SF-1
Promoter
The DNA sequences of all oligonucleotides are shown in Table 1 . A,
32P-labeled oligonucleotides corresponding to SF-1
nucleotides (nt) -87 to -49 (lane 1), nt -73 to -49 (lane 4), and
nt -87 to -69 (lane 5) were incubated with nuclear extract prepared
from Y1 cells, as well as 100-fold molar excess of unlabeled SF-1 nt
-73 to -49 oligonucleotide (lane 2), or SF-1 nt -87 to -69 (lane 3)
oligonucleotide, as indicated. B, Gel shift analysis of the SF-1 -87
to -49 region probe using Y1 nuclear extracts incubated in the
presence of the 100-fold molar excess of the indicated E box mutant. C,
Gel shift analysis of the SF-1 -87 to -49 region probe using nuclear
extracts from Y1 and NIH-3T3 cells. The free probe did not remain on
the gel due to the longer electrophoresis time required to achieve
appropriate resolution.
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To confirm that Complex I interacts as an E box, we performed
competition analysis with mutant E box competitors (Fig. 5B
). Complex I
is effectively competed with the nt -87 to -69 mutant
oligonucleotides that contain mutations flanking immediately outside of
the E box (E box Em3, E box Em4) but not with those in which the E box
motif has been mutated (E box Em1, E box Em2) (Table 1
and Fig. 5B
). Furthermore, when the mutant E box oligonucleotides
were labeled and used in gel shift assays, no specific complex was
observed (data not shown).
To determine whether proteins that bind the SF-1 promoter are specific
to steroidogenic cells, we also used nuclear extracts from NIH-3T3
cells in mobility shift assays. Despite the complete inactivity of this
promoter fragment in NIH-3T3 cells (Fig. 2
), we found two species
corresponding in mobility to those obtained from Y1 nuclear extracts
(Fig. 5C
). Thus, the presence of proteins that bind these sequences
within the SF-1 promoter does not imply that the SF-1 gene is active in
these cells.
The Functional Element at nt -68 to -59 Binds the CAAT-Binding
Factor (CBF)
The region between nt -68 to -61 (5'-CCAATTGG) could
function as an E box, a CAAT box, or both. To distinguish these
possibilities, gel shift assays were performed using mutant
oligonucleotides that would distinguish between these two motifs (Fig. 6
). The first mutant oligonucleotide tested altered
5'-CCAATTG to 5'-ACAATTG, thus maintaining the E
box but destroying the CAAT box (E box +). A second mutant was tested
in which the 5'-CCAATTG was changed to
5'-CCAATCG, thus maintaining the CAAT box but mutating the E
box (CAAT+). Using the wild type nt -73 to -49 probe, Y1 nuclear
extracts, and these mutant oligonucleotides as competitors, it is clear
that complex II binds as a CAAT box rather than as an E box (Fig. 6
).
To determine whether the factor responsible for the shift of the CAAT
box was a previously characterized protein, we performed competition
analysis with oligonucleotides containing consensus binding sites for
the CAAT box-binding proteins C/EBP, NF-1/CTF, and CBF/CP1 (26, 27, 28). As
shown in Fig. 6
, the CBF site was an effective competitor that
abolishes formation of this complex, whereas the consensus CTF and
C/EBP oligonucleotide competitors did not compete even at levels of
100-fold molar excess (Fig. 6
). Finally, we performed a supershift
assay with an anti-CBF antibody, which completely supershifted complex
II, thus confirming that CBF (or a related protein) interacts with the
nt -68 to -59 element of the SF-1 promoter. The preimmune serum
control had no effect upon the complex, and the specificity of the
antiserum was demonstrated by its nonreactivity against an unrelated
complex (data not shown).

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Figure 6. Gel Shift Analysis of the CAAT Box in the SF-1
Promoter
The DNA sequences of all oligonucleotides are shown in Table 1 . A
32P-labeled oligonucleotide corresponding to SF-1 nt -73
to -49 was incubated with Y1 nuclear extract in the presence or
absence of either 10- or 50-fold molar excess of the indicated
competitor oligonucleotides: control without competitor; E box +, SF-1
nt -73 to -49 mutant in which the E box was maintained but the CAAT
box was destroyed; CAAT+, SF-1 nt -73 to -49 mutant in which the CAAT
box was maintained but the E box was destroyed; C/EBP consensus binding
site (27); CTF/NF-1 consensus binding site (26); CBF consensus binding
site (28). Supershift assay with anti-CBF: SF-1 nt -73 to -49 probe
in the absence and presence of anti-CBF polyclonal antibody; CBF
consensus site probe in the absence and presence of anti-CBF antibody.
The free probe did not remain on the gel due to the longer
electrophoresis time required to achieve appropriate resolution.
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The Functional Element at -30 to -24 Binds Sp1
As noted above, the GA-rich sequence that occupies nucleotides
-30 to -24 of the SF-1 promoter could potentially bind transcription
factors such as Ets (20), GAGA (47), and Sp1 (22). Gel mobility shift
assays using Y1 nuclear extracts and an oligonucleotide spanning nt
-40 to +12 reveals a single specific complex (Fig. 7
, lane 1). The complex is effectively competed by its homologous sequence
(lane 3) but not by an analogous mutant oligonucleotide in which nt
-30 to -24 region had been replaced (lane 2), confirming that the
complex was formed via interactions at nt -30 to -24. Gel shift
assays using consensus competitors for Sp1, Ets, and GAGA demonstrate
that this complex is effectively competed by the Sp1 site (lane 4) but
not the Ets (lane 5) and GAGA sites (lane 6), suggesting that the
complex is formed by an Sp1-like factor. To directly test whether Sp1
binds to the SF-1 promoter at this site, we performed a supershift
assay with an anti-Sp1 antibody, which diminished formation of the
complex (lane 7). The preimmune serum control had no effect upon the
complex, and the specificity of the antiserum against Sp1 was
demonstrated by its nonreactivity against an unrelated probe (data not
shown).

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Figure 7. Gel Shift Analysis of the GA-Rich Element in the
SF-1 Promoter
The DNA sequences of all oligonucleotides are shown in Table 1 . A
32P-labeled probe corresponding to SF-1 nt -40 to +12 was
incubated with Y1 nuclear extract in the absence (lane 1) or presence
of a 100-fold molar excess of the indicated unlabeled competitor [SF-1
nt -40 to +12, mutated within nt -30 to -24 (lane 2); wild type SF-1
nt -40 to +12 (lane 3); consensus site for Sp1 (22) (lane 4);
consensus site for Ets (20) (lane 5); consensus site for GAGA (47)
(lane 6)], or in the presence of anti-Sp1 antibody. Anti-Sp1 antibody
abolishes the complex yielded by the SF-1 nt -40 to +12 probe. A
supershift was achieved using an Sp1 consensus oligonucleotide and
anti-Sp1 antibody (lane 7). The free probe did not remain on the gel
due to the longer electrophoresis time required to achieve appropriate
resolution.
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The Identified Elements Confer Differential Activity to a
Heterologous Promoter
The E box, CAAT box, and Sp1 site are all required for SF-1
promoter activity (see Fig. 3
). To characterize the functional role of
each identified element, we tested their ability to activate the
heterologous minimal PRL promoter, which contains only a TATA box as an
identified promoter element. Transient transfection of Y1 cells
demonstrates that the -90 to -14 region of the SF-1 promoter (which
spans the E box, CAAT box, and Sp1 site) is sufficient for
100-fold
activation of this heterologous promoter (Fig. 8
, construct 1). In this context, mutation of either the Sp1 site
(construct 2) or both the Sp1 site and CAAT box (construct 3) does not
appear to have a significant effect over promoter activity, indicating
that they are not required in the presence of a TATA box. In contrast,
mutation of the putative E box (construct 4) significantly reduced
promoter activity. Furthermore, when a construct containing promoter E
box element in the incorrect orientation 5' to the TATA-box was tested,
full promoter activity was observed (data not shown). Therefore, on a
TATA-containing promoter, the E box appears to serve as an enhancer,
whereas the CAAT box and Sp1 site do not. Moreover, the wild type -90
to -14 fragment in this heterologous context was inactive in NIH-3T3
cells, further indicating that the E box serves as a steroidogenic
cell-specific element on the SF-1 promoter (data not shown).

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Figure 8. Functional Analysis of the Identified Promoter
Elements
Schematic representations of luciferase reporter constructs containing
a TATA box from the minimal PRL promoter and various SF-1 mutant
promoter fragments are shown. All constructs were transiently
transfected into Y1 cells, and luciferase activities were measured. The
graph shows luciferase activities averaged (±SD) from
three separate experiments, relative to the minimal PRL promoter
construct, and controlled by measurement of ß-gal activity as
directed by the plasmid RSV-ß-gal.
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DISCUSSION
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SF-1 is a transcription factor that regulates steroidogenic enzyme
expression and is a critical developmental regulator for the adrenal
gland and gonads (11, 12, 13). Because SF-1 expression is restricted to the
adrenal gland, gonads, and the neural and pituitary elements of the
hypothalamic-pituitary-adrenal axis, the factors that regulate its
temporal and spatial expression are crucial to the proper development
and function of these organs. Our analysis of the SF-1 promoter has
revealed several elements important for expression in steroidogenic
cells. These include a bHLH transcription factor site at -83 (E box),
a CAAT box protein-binding site at -68, and a GA-rich Sp1 site at
-30.
The bHLH family of transcription factors includes both cell
type-restricted and ubiquitously expressed members. This family of
proteins includes master regulators of cell fate such as MyoD and
myogenin, which have been shown to be instrumental for initiation of
muscle cell differentiation (30). It is generally considered that the
cell-restricted bHLH proteins heterodimerize with the ubiquitously
expressed bHLH proteins and these dimers bind to E box elements (31, 32). The E box of the SF-1 promoter, which was also identified in a
previous study (24), may determine cell-specific expression of SF-1 as
the other factors, CBF and Sp1, are ubiquitously expressed. Moreover,
the SF-1 promoter E box may bestow steroidogenic-specific expression as
it alone can promote activation on the minimal PRL promoter in Y1
adrenocortical cells, but not in NIH-3T3 fibroblasts. While no
steroidogenic cell-specific bHLH factors have yet been cloned, USF,
which is a class B, E box-binding protein that is ubiquitously
expressed, has been shown to bind to the E box within the SF-1 promoter
(33) and may contribute to the regulation of the SF-1 gene in
steroidogenic tissues. Because of its ubiquitous expression, however,
USF is clearly not sufficient to direct tissue-specific expression of
SF-1.
A second functional element identified in this study is recognized by
the ubiquitous transcription factor, CBF. The CAAT box is a proximal
promoter element that is found between nucleotides -80 and -60 in the
promoters of many genes (17, 18). Although multiple factors, such as
C/EBP, CTF/NF-1, and CBF, interact with the CAAT box, only CBF binds to
the SF-1 CAAT element. CBF is a heteromeric factor that consists of
three subunits, CBF-A, CBF-B, and CBF-C, and is highly conserved
throughout evolution (21, 28, 34, 36). CBF has been identified as a
critical factor in the regulation of many genes including albumin (35)
and major histocompatibility complex class II genes (37). Although
several lines of evidence suggest that CBF facilitates the formation of
stable preinitation complexes, as CAAT elements are present in
promoters with or without a TATA box, the role of CBF in transcription
is unclear. It is possible that within the SF-1 promoter, CBF works in
concert with Sp1 to direct and/or initiate site-specific initiation
(38, 39). This is consistent with evidence gathered from other studies
of TATA-less promoters, which show that Sp1 directs site-specific
initiation. Indeed, our data reveal that Sp1 is not required for SF-1
promoter activity when a TATA element is present.
With regard to the ability of SF-1 to regulate its own promoter, our
data differ from a previous report (25), which showed SF-1 binding to a
divergent SF-1 site within the first intron of the gene, and 2- to
3-fold transactivation of the promoter through this element in CV-1
cells. While our results do not rule out SF-1s ability to bind to
this element, it would appear that this element plays little role in
the regulation of SF-1 expression in steroidogenic tissues, as its
incorporation into a promoter fragment offers no additional activity to
a luciferase reporter in Y1 (SF-1 expressing) adrenocortical cells.
Moreover, coexpression of SF-1 in CV-1 cells does not enhance the
activity of a promoter fragment that includes this site. Therefore,
whether SF-1 autoregulates the SF-1 gene remains unclear.
Our gel mobility shift assays using nuclear extracts from NIH-3T3 cells
demonstrate that nuclear proteins from this cell line bind to the E box
and CAAT box. However, in contrast to their function in steroidogenic
cells, they are not sufficient to activate expression of the SF-1 gene
in NIH-3T3 cells. There are four plausible reasons for this finding.
First, the Y1 bHLH transcription factor(s) could be distinct from those
found in nonsteroidogenic tissues (and yet yield similar migration on a
mobility shift assay). Second, a steroidogenic cell-specific
coactivating molecule may be required for transcriptional activity of
the bHLH transcription factor but not for its ability to bind DNA.
Third, a repressive molecule may be present in nonsteroidogenic tissues
that prevents transcriptional activation through E boxes in certain
contexts. Finally, steroidogenic cells may posttranslationally modify
and thereby activate the bHLH transcription factor. Given the numerous
bHLH transcription factors expressed in a tissue-specific fashion that
bind as heterodimers, these possibilities are by no means mutually
exclusive. The activities of bHLH transcription factors are often
tissue-specific by virtue of the presence of refined interactive
(activating or repressive) factors characteristic of that tissue
(40, 41, 42, 43). Therefore, it is likely that tissue-specific expression of
the SF-1 gene is guided by a bHLH transcription factor and associated
tissue-specific coactivators and repressors.
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MATERIALS AND METHODS
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Cloning of SF-1 Genomic Sequences
Isolation of the mouse SF-1 genomic clone (strain 129Sv) has
been reported (13). A genomic P1 clone of human SF-1 was obtained from
Genome Systems, Inc. (St. Louis, MO). Both the mouse and human genomic
clones were mapped, subcloned, and partially sequenced by the
dideoxynucleotide method on a DNA sequencer (Applied Biosystems, Foster
City, CA; model 373A). Sequence construction and nucleic acid homology
searches were performed by the Lasergene sequence analysis program
(DNAStar, Madison, WI).
Plasmids
For construction of the SF-1 promoter-luciferase reporter
plasmids, appropriate restriction sites were used to insert fragments
of the mouse SF-1 gene in a 5' to 3' orientation into the polylinker of
the promoterless plasmid pLuc. The -1885, -712, -190, -90, and -12
-bp constructs were generated using genomic fragments spanning from
the KpnI (nt -1885), XbaI (nt -712),
PstI (nt -90), and PvuI (nt -12) sites,
respectively, to the EcoRI site located at +140 (see Fig. 2
). For site-directed mutagenesis of the SF-1 promoter, four 71-mer
oligonucleotide sets comprising DNA corresponding to the -90 to -17
region with the indicated nucleotide substitution mutations were
synthesized (see Fig. 3
). The -90-Luc reporter constructs were cloned
by replacement of the PstI (nt -90)/PvuI (nt
-12) fragment of -90-Luc or -1885-Luc with mutant oligonucleotides.
The construct -3100-Luc contains a 6.7-kb 5'-flanking fragment that
was cloned as four smaller fragments from the mouse genomic clone
(NotI-KpnI, nt -3100 to -1885;
KpnI-XbaI, nt -1885 to -712;
XbaI-EcoRI, nt -712 to +140;
EcoRI-SacII, +140 to +3600) into pBluescript KS
(Stratagene, La Jolla, CA) and subsequently the promoter-less pLuc. The
expression vector for SF-1 (CMV-SF-1) was generated by sequential
subcloning of the SF-1 cDNA (a gift from K. Parker) using
linker-generated EcoRI sites to clone into the
EcoRI site of pBluescript KS, and then the flanking
KpnI and XbaI sites from the bluescript
polylinker to clone into pCMVneo. To generate constructs for the
heterologous promoter experiments, the individual SF-1 promoter
fragments mutated as above were cloned into the BamHI site
immediately upstream of the minimal PRL promoter that contains
nucleotides -26 to +1, which directs expression of the luciferase
reporter from the plasmid pPrl-Luc (a gift from Stuart Adler).
Cell Culture and Transient Transfections
Y1 adrenocortical cells were grown in 50% DMEM/50% Hams F-12
medium supplemented with 10% FBS and antibiotics. CV-1 monkey kidney
and NIH-3T3 fibroblast cells were grown in DMEM supplemented with 10%
FCS and antibiotics. DC3 ovarian granulosa cells (44) were grown in
Iscovess modified DMEM supplemented with 20% FBS and antibiotics.
All plasmid DNA samples tested were prepared over Qiagen columns
(QIAGEN Inc., Chatsworth, CA), and at least three preparations of each
plasmid were tested. Cells were plated at
6 x 104
cells per 35-mm well and transfected 24 h later with 0.5 µg of
reporter and a total of 2 µg plasmid DNA per well. Transient
transfection of DC3 cells was performed using LipofectAMINE reagent
(GIBCO BRL, Gaithersburg, MD) as described by the manufacturer.
Transient transfection of NIH-3T3, CV-1, and Y1 cells was performed by
the calcium phosphate coprecipitation method (45). Luciferase assays
were performed 48 h after transfection and normalized to
ß-galactosidase activity from the cotransfected plasmid
RSV-ß-galactosidase.
Electrophoretic Mobility Shift Assays
Nuclear extracts were prepared as described (46). Probes (refer
to Table 1
) were prepared by end-labeling 100 ng of the primary strand
oligonucleotide by 20 µCi of [
-32P]ATP, using T4
polynucleotide kinase, and annealing to the complementary
oligonucleotide. Five micrograms of nuclear extract were mixed with 1
µg of poly (deoxyinosinic-deoxycytidylic) acid in a binding buffer
consisting of 10 mM Tris, pH 7.5, 50 mM NaCl, 1
mM EDTA, 1 mM dithiothreitol, and 5% glycerol.
After a 5-min preincubation, 1.0 ng of labeled probe was added, and the
mixture was incubated for 20 min at 25 C. The binding reactions (50
µl total volume) were loaded onto 4% polyacrylamide gels and run at
150 V for 4 h in 0.5 x Tris-borate-EDTA at 4 C. The gels
were dried and exposed to film or a PhosphorImager screen (Molecular
Dynamics, Sunnyvale, CA). For competition experiments, the competing
unlabeled oligonucleotide was added at the indicated concentration and
preincubated at 25 C for 5 min before addition of
32P-labeled oligonucleotide. For antibody supershift
experiments, the reaction mixture was incubated with the
32P-labeled oligonucleotide for 5 min, after which 1 µl
of antibody [anti-CBF (36), a gift from Benoit de Crombrugghe, or
anti-Sp1 (Promega, Madison, WI)] was added and the mixture was
incubated for an additional 15 min at 25 C.
 |
ACKNOWLEDGMENTS
|
---|
We thank Shelly Audrain for excellent technical assistance, and
Robert Heuckeroth and John Svaren for critical comments on the
manuscript.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Jeffrey Milbrandt, M.D., Ph.D., Departments of Pathology and Medicine, Washington University School of Medicine, 660 South Euclid Avenue, Box 8118, St. Louis, Missouri 63110.
This work was supported by Grant P01 CA49712-06A1 from the Nation
Cancer Institute. J.M. is an Established Investigator of the American
Heart Association.
1 The first three authors contributed equally to this study. 
Received for publication August 16, 1996.
Revision received October 25, 1996.
Accepted for publication November 4, 1996.
 |
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