Multiple Orphan Nuclear Receptors Converge to Regulate Rat P450c17 Gene Transcription: Novel Mechanisms for Orphan Nuclear Receptor Action
Peilin Zhang and
Synthia H. Mellon
Department of Obstetrics, Gynecology, and Reproductive
Sciences (P.Z.)and The Metabolic Research Unit (S.H.M.),
University of California, San Francisco, California 94143-0556
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ABSTRACT
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The orphan nuclear receptor steroidogenic factor-1
(SF-1) plays a key role in regulating the expression of the rat P450c17
gene in testicular Leydig and in adrenocortical cells. Other DNA
sequences, not bound by SF-1, are also involved in transcriptional
regulation of the rat P450c17 gene in both cell types. The region from
-447/-399 or from -447/-419 increased both basal and cAMP-induced
transcription, and the region from -418/-399 increased basal
transcription to a greater extent than the intact -447/-399 DNA. The
-447/-399 DNA sequence contains three imperfect copies of the orphan
nuclear receptor-binding motif, AGGTCA, and at least three known orphan
nuclear receptors, chicken ovalbumin upstream promoter transcription
factor (COUP-TF), SF-1, and an early response gene induced by nerve
growth factor (NGFI-B), bind to -447/-399 DNA. The AGGTCA triad is
bound by one set of nuclear proteins when these three elements are
colinear and is bound by a different set of proteins when these
elements are separated. When the elements are separated, COUP-TF no
longer binds, and the region -418/-399 is bound by a protein that
greatly stimulates basal transcription. The region -447/-419 is bound
by two different proteins that mediate both basal and cAMP-stimulated
transcription. We call the protein binding to -418/-399 steroidogenic
factor inducer of transcription-1 (StF-IT-1), and one of the proteins
binding to -447/-419, StF-IT-2. SF-1 binds to a second AGGTCA element
in the -447/-419 region. StF-IT-1 and StF-IT-2 are both found in
Leydig and adrenal cells, and transcriptional regulation is similar in
both cell types. SF-1 and NGF-IB may increase transcription by
displacing COUP-TF (a transcriptional repressor) because these proteins
share DNA-binding domains. However, neither SF-1 nor NGF-IB alone,
binding as monomers, increases transcription. Rather, these proteins
must interact with another DNA-binding protein, e.g.
StF-IT-2, to increase transcription. StF-IT-2 also requires interaction
with SF-1 (or NGF-IB) bound to DNA and cannot increase transcription by
itself. This mechanism of action is different from the mechanism by
which SF-1 regulates transcription from the -84/-55 region of the rat
P450c17 gene. Thus, we have defined a novel mechanism of action for
orphan nuclear receptors that bind to DNA as monomers.
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INTRODUCTION
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Steroidogenesis is initiated by conversion of cholesterol to
pregnenolone by the mitochondrial cholesterol side chain cleavage
enzyme, P450scc. The cell type-specific synthesis of various steroids
in the gonads and adrenals is determined by cell-specific expression of
a variety of additional steroidogenic enzymes. The conversion of
pregnenolone and progesterone to their 17
-hydroxylated products and
then to dehydroepiandrosterone and androstenedione, respectively, is
catalyzed by a single protein, P450c17, encoded by a single gene (1, 2)
Thus, P450c17 mediates two enzymatic reactions, 17
-hydroxylation and
cleavage of the C17-C20 carbon bonds (lyase reaction) (3, 4, 5). The
expression of the P450c17 gene in steroidogenic tissues is species
specific: it is expressed in the rodent gonad (6, 7, 8) and placenta (9, 10), but not adrenal (11), and is expressed in the human adrenal and
gonad, but not placenta (11, 12, 13).
P450c17 gene expression is regulated in a tissue-specific and
species-specific fashion by trophic hormones via cAMP as a second
messenger. Bovine, but not human or rodent, adrenals lack P450c17 in
the absence of tropic stimulation (14, 15). Human adrenals (11, 12, 13) and
human and rodent gonads (6, 7, 8, 12) contain P450c17 mRNA in the absence
of tropic hormones or cAMP and hence exhibit basal transcription (7, 8, 13, 16, 17). Thus, the P450c17 gene is regulated by different
mechanisms in various species and in various tissues. Although the
P450c17 gene is not expressed in the rodent adrenal in vivo
(11), the human or rodent P450c17 promoter is readily expressible when
transfected into rodent adrenocortical cells, suggesting that the
transcription factors necessary for its expression exist in both rodent
adrenal and Leydig cells (2, 18, 19). One such factor is steroidogenic
factor-1 (SF-1), which regulates the expression of the rat P450c17 gene
in both adrenal and Leydig cells (2, 18). This regulatory diversity of
P450c17 in various species and tissues may reflect the involvement of
different nuclear transcriptional factors. Therefore, we sought to
determine whether nuclear transcription factors other than SF-1 bind to
the rat P450c17 promoter and are involved in regulation of rat P450c17
in mouse Leydig MA-10 cells. We now identify a segment of the
5'-flanking region of the rat P450c17 gene that is involved in both
basal and cAMP-mediated transcriptional regulation, identify the
multiple different transcription factors that bind to this region, and
characterize two previously undescribed transcription factors.
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RESULTS
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Identification of cis-Active DNA Sequences in the Rat
P450c17 Gene
Previous studies showed that a DNA element located at -84/-55 of
the rat P450c17 gene mediated both basal and cAMP-inducible
transcription (2, 18). The nuclear protein bound to this element was
identified as the orphan nuclear receptor, SF-1 (20). Our transfection
studies (2) suggested that the region between -476/-267 was also
important for basal and cAMP-mediated transcription of this gene in
both mouse Leydig MA-10 and adrenal Y-1 cells. Therefore we studied the
sites and activities of protein-DNA interactions in the -476/-267
region.
DNase I footprinting of bases -476/-267 using cell extracts from
MA-10 cells identified a broad region from about -399 to -447
containing both DNase I-sensitive bands and newly created DNase
I-hypersensitive bands, suggesting the binding of several nuclear
proteins (Fig. 1
). The DNA sequence of the footprinted
region is shown beside the footprint. This footprinted region contains
multiple imperfect copies of the common DNA estrogen receptor half-site
motif, AGGTCA, which can be bound by a number of steroid/retinoid
intracellular receptors.

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Figure 1. DNase Footprint of the -447/-399 Region of the
Rat P450c17 Gene
P450c17 DNA from -476 to -267 was incubated with nothing (probe) or
with nuclear extracts from MA-10 cells (MA-10) and digested with DNase
I. One large region of DNA was bound by protein(s). The sequence to
which they bind are indicated beside the autoradiogram. Lane A + G
contains A + G chemical sequencing reactions.
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Gel mobility shift assays using bases -447/-399 and MA-10 cell
extracts identified three major protein-DNA complexes, called I, II,
and III (Fig. 2A
). Complex II may contain two different
protein-DNA complexes, but these complexes are not well resolved by gel
electrophoresis. All three complexes could be competed by adding
500-fold molar excess of unlabeled -447/-399 oligonucleotide (Fig. 2A
, lane 3). The DNA segment from -84 to -55 binds SF-1 (18). When
the DNA corresponding to -84/-55 was used as competitor, complex I
could be competed completely (lane 4), and complexes II and III were
also competed, but to a lesser extent. This suggests that SF-1 may be
involved in complex I and that SF-1 binding may affect binding of other
proteins in complexes II and III. SF-1 binds to the estrogen receptor
element half site, AGGTCA, but there is also a strong preference for
the sequence TCA at the 5'-end. Although the -447/-399 DNA contains
three AGGTCA-like motifs, there is no consensus SF-1 site (TCAAGGTCA)
in the -447/-399 DNA, suggesting that SF-1 may recognize a modified
estrogen response element (ERE) half-site and may thus recognize
different DNA sequences in different contexts.

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Figure 2. Gel Shift Analysis of Nuclear Extracts from MA-10
Cells Binding to a -447/-399 Oligonucleotide
A, Autoradiograph of an incubation of labeled oligonucleotide
containing a sequence from -447 to -399 bp of the rat P450c17 gene,
displayed on a 5% nondenaturing acrylamide gel (lane 1). Three
protein-DNA interactions, labeled I, II, and III, are observed when
incubated with MA-10 cell extract (lane 2). Each complex can be
competed by incubation with 500 x excess unlabeled
oligonucleotide (lane 3); competition by an oligonucleotide from
-84/-55 of the rat P450c17 gene, to which SF-1 binds (2, 18), is in
lane 4. B, Autoradiograph of an incubation of labeled oligonucleotide
containing a sequence from -447/-399 bp of the rat P450c17 gene,
displayed on a 5% nondenaturing acrylamide gel (lanes 13 and lanes
1012). Competition of complex formation by 500-fold concentration of
mutant oligonucleotides, lanes 4, 5, 6, 9, and 13. Competition of
complex formation by 500-fold concentration of wild type
oligonucleotides from -432/-399 (lane 7) and from -418/-399 (lane
8) of the rat P450c17 gene.
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Because protein binding in complex I appears to affect the protein
binding in complexes II and III, we used various oligonucleotides
(Table 1
) to study these protein-DNA interactions in
greater detail. The -447/-399 DNA contains three variations of the
AGGTCA motif, listed in Table 1
as sites 1, 2, or 3, and hereafter
referred to as sites 1, 2, or 3. We created mutant oligonucleotides in
which we deleted a single ERE half-site at site 1 or 3 or mutated three
bases within the ERE half-sites (changing AGG to TTT) and used these as
competitors for the wild type -447/-399 sequence in gel shift assays
(Fig. 2B
).
Oligonucleotides containing a mutation at site 1 (-447/-399
1, lane
13) were able to compete with the wild type -447/-399 oligonucleotide
for formation of all three complexes. Similarly, oligonucleotides
containing a deletion of site 1 and mutation of either site 2 (Mut 2,
lane 4) or site 3 (Mut 3, lane 5) or a deletion of site 3 (WT
-432/-399, lane 7) were also able to compete with the wild type
-447/-399 oligonucleotide for the formation of all three complexes.
However, when sites 2 and 3 were mutated simultaneously (Mut 2/3, lane
6), the resulting oligonucleotide could no longer compete with the wild
type -447/-399 oligonucleotide. We observed competition with the
mutant oligonucleotides when only one ERE half-site at either site 2 or
3 remained intact. Oligonucleotides containing a single ERE half-site
at site 1 (WT -418/-399, lane 8) or a mutant ERE at site 1 (Mut 1,
lane 9) were unable to compete with the wild type -447/-399 DNA for
protein binding in any of the complexes, indicating that protein(s)
binding to the site 2 or 3, but not at site 1 alone, affect protein
binding at all three ERE half-sites. Because mutations at ERE
half-sites affected protein binding, the data further indicate that the
formation of complexes I, II, and III all require protein binding to
intact AGGTCA motifs.
-447/-399 DNA Contains Novel Basal- and cAMP-responsive
Transcriptional Elements
We determined whether the -447/-399 region of the P450c17 gene
was transcriptionally active by ligating the -447/-399
oligonucleotide into a luciferase expression vector containing the
herpes simplex virus thymidine kinase (TK) minimal promoter (TK32LUC)
and transfecting the resulting construct into mouse Leydig MA-10 and
adrenocortical Y-1 cells. As shown in Fig. 3A
, addition
of the -447/-399 sequences to the TK32LUC vector increased basal
luciferase activity 15-fold in MA-10 cells. Stimulation with cAMP for
6 h further increased luciferase activity approximately 12-fold.
Thus, DNA sequences between -447/-399 mediate both the basal and
cAMP-regulated transcription of the rat P450c17 gene. Qualitatively
similar results were obtained in Y-1 cells (Fig. 3B
), but the magnitude
of the responses was less than in MA-10 cells.

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Figure 3. Luciferase Assays of P450c17-TK32LUC Constructs
Luciferase reporter gene constructs containing the -447/-399 rP450c17
oligonucleotide (A and B) or mutants of this oligonucleotide (C) were
ligated to 32 bp DNA from the TK promoter (-447/-399 TKLUC). These
constructs, or the TK minimal promoter alone (TKLUC), were transfected
into cultured mouse Leydig MA-10 cells (A and C) and cultured mouse
adrenocortical Y-1 cells (B). Other wild type and mutant
oligonucleotides from -447/-419 or -418/-399 were similarly cloned
into the TK32LUC reporter plasmid and used to transfect A-10 (A) or Y-1
(B) cells. Cells were not treated (open bars) or were
treated with 1 mM 8-Br-cAMP for 6 h (black
bars) before luciferase activity was measured. Transfections
were performed in triplicate for each construct. This figure is one
representative of three separate transfection experiments with each
cell type. Error bars represent ±SD.
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We removed site 1 by deleting sequences from -418 to -399 and
ligated the remaining -447/-419 oligonucleotide, containing sites 2
and 3 to the TK32LUC vector. This construct, -447/-419TK32LUC, was
transfected into MA-10 and Y-1 cells to determine its basal and
cAMP-induced transcriptional activity. In MA-10 cells, basal luciferase
activity of the -447/-419TK32LUC plasmid was less than that of the
intact -447/-399TK32LUC plasmid, but was still 6-fold greater than
the vector alone. This truncated plasmid still showed a 7-fold
increased response to stimulation with cAMP. By contrast, this
construct showed only a 2-fold response to cAMP in Y-1 cells. We
mutated sites 2 and 3 individually (mutants 2 and 3) (Table 1
) or
together (mutant 4), ligated each to the TK32LUC plasmid, and
transfected these plasmids into Y-1 and MA-10 cells. All three of these
mutants completely lost both basal and cAMP-induced transcription.
Mutant 4 demonstrated some cAMP stimulation in MA-10 cells, although
neither mutant 2- or 3-TK32LUC showed activity. Similar results were
obtained when the constructs were transfected into Y-1 cells (Fig. 3B
),
suggesting that the-447/-399 sequence is functional in both Leydig
and adrenocortical cells, and that the same transcription factors may
be functioning in both types of steroidogenic cells.
We also analyzed the activity of the -418/-399 DNA, after ligation to
the TK32LUC plasmid, in transfected MA-10 and Y-1 cells (Fig. 3
, A and
B). Basal luciferase activity of the -418/-399TK32LUC plasmid in
MA-10 cells was 4 times greater than the activity of the intact
-447/-399TK32LUC and was 45 times greater than the vector alone.
However, cAMP had a minimal effect on -418/-399TK32LUC activity. When
this construct was transfected into Y-1 cells, the results were
similar, but again, the magnitude of the responses was less than in
MA-10 cells (Fig. 3B
). When site 1 in -418/-399TK32LUC was mutated to
TTTAGA, (called Mut 1 -418/-399 TK32LUC), it had no basal or
cAMP-induced luciferase activity in MA-10 or Y-1 cells. These data
indicate that an intact AGGTCA-like motif is required for
transcriptional activation, and that the sequence between -418 and
-399 contains a strong basal transcription activator whose activity is
attenuated by sequences between -447 and -419.
Transcriptional activities were also assessed in the intact
-447/-399 DNA, but in which site 1 was individually mutated (called
-447/-399
1). This oligonucleotide was ligated to TK32LUC and
transfected into MA-10 cells (Fig. 3C
). The data demonstrate that basal
activity of the -447/-399
1TK32LUC plasmid was slightly greater
than the intact -447/-399TK32LUC plasmid. The -447/-399
1TK32LUC
plasmid showed a 12-fold response to cAMP stimulation, similar to the
response seen with the -447/-419TK32LUC construct (Fig. 3A
).
Finally, we analyzed the activity of the -432/-399 DNA after
ligation to the TK32LUC plasmid (Fig. 3
, A and B). This DNA contains
sites 1 and 2, but lacks site 3. This plasmid had neither basal nor
cAMP-stimulated activity in either MA-10 or Y-1 cells. These data,
together with the data from the other constructs, indicate that
transcription from -432/-399 DNA is repressed. Thus, the basal
transcription and cAMP induction from -447/-399 P450c17 DNA is due to
the combination of activating and repressing interactions, and this
transcriptional activation requires intact AGGTCA-like motifs.
Chicken Ovalbumin Upstream Promoter-Transcription Factor (COUP-TF)
Binds to the -447/-399 Region of the Rat P450c17 Gene
Our functional data (Fig. 3
) suggest that the intact -447/-399
sequence has less transcriptional activity than the truncated
-418/-399 sequence, and that the -432/-399 sequence had no
activity. This suggests that a repressor may be involved in attenuating
transcriptional activity in the intact fragment. COUP-TF is a factor
that can bind to AGGTCA-like sequences and repress transcription (21, 22) and the DNA sequence between -447/-399 contains a potential
COUP-TF binding site. To determine whether COUP-TF is involved in rat
P450c17 gene transcription, we performed gel mobility shift assays with
MA-10 cell extracts and several oligonucleotides encompassing the
-447/-399 region, in the absence and presence of a COUP-TF antibody.
As shown previously in Fig. 2A
, the entire -447/-399 element formed
three protein-DNA complexes with MA-10 cell extracts (Fig. 4
, lane 2). Addition of COUP-TF antibody decreased the
formation of complexes II and III, increased the amount of complex I,
and generated an additional band (lane 4), possibly a supershift of
either complex II or III. As the antibody binds to both COUP-TF I and
COUP-TF II, the data suggest that one or both of these forms of COUP-TF
binds to the 5'-flanking DNA of the rat P450c17 gene. COUP-TF not only
bound to the -447/-399 DNA, but also bound to -432/-399 DNA (Fig. 4B
). This binding is indicated by a supershift of the protein-DNA
complex by antibody to COUP-TF. These data suggest that COUP-TF binding
to the -447/-399 DNA occurs at sites 1 and 2. The data are consistent
with a lack of transcriptional activation (i.e. repression)
seen with the -432/-399TK32LUC construct (Fig. 3
).

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Figure 4. COUP-TF Antibody Supershift Assay
Autoradiographs of incubations of labeled oligonucleotide containing a
sequence from -447/-399 bp of the rP450c17 gene (A) or -432/-399 bp
of the rP450c17 gene (B) with MA-10 cell nuclear extract, displayed on
a 5% nondenaturing acrylamide gel. A, Three complexes, I, II, and III,
are identified (lanes 2) and are competed with 500-fold molar excess of
unlabeled oligonucleotide (lane 3). In lane 4, the probe was incubated
with MA-10 cell extract and COUP-TF antibody, resulting in a decrease
in the intensity of complexes II and III and the formation of an
additional, super-shifted complex that we call IIIa. B, One complex is
formed with the -432/-399 DNA. Incubation with a COUP-TF antibody
results in the formation of an additional, supershifted complex.
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Identification of Additional Protein-DNA Complexes within the
-447/-399 Region of the Rat P450c17 Gene
Transfection data using smaller DNA fragments from within the
-447/-399 region suggested that each AGGTCA-like motif may function
independently when isolated from the other motifs, but that these
motifs may function cooperatively, and differently, when arranged in a
group of three motifs. Therefore, we determined whether additional
nuclear proteins bound to sites 1, 2, and 3 when these regions were
isolated from each other. We used -447/-419 (containing sites 2 and
3) or -418/-399 oligonucleotide (containing site 1) as probes in gel
shift experiments (Fig. 5A
). We detected two protein-DNA
complexes, called complexes IV and V, when -447/-419 DNA was used as
probe (lane 2), and we detected one protein-DNA complex, called complex
VI, when -418/-399 DNA was used as probe (lane 9). Complexes IV and V
could be competed using 500-fold excess of unlabeled -447/-419
oligonucleotide (lane 3) but could not be competed using an unlabeled
-418/-399 oligonucleotide (lane 4). Complexes IV and V could also be
competed with -432/-399 DNA (lane 5), even though this DNA lacks site
3. Complex VI could be competed with 500-fold excess unlabeled
-418/-399 oligonucleotide (lane 10), but it could also be competed
using unlabeled -432/-399 (lane 12) and -447/-419 (lane 11)
oligonucleotides. These data indicate that the proteins in complexes IV
and V were different from the protein in complex VI. The data also
suggest that the protein in complex VI had a permissive DNA sequence
requirement, as its binding could be competed with an unrelated, but
similar, DNA sequence found in the -447/-419 oligonucleotide.

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Figure 5. Analyses of Protein-DNA Interactions with
-447/-419 and with -418/-399 rP450c17 DNA
A, Gel shift assay of MA-10 cell nuclear extract to rP450c17
oligonucleotide probes -447/-419 (lanes 17) and -418/-399 (lanes
814) Incubation of the -447/-419 probe with MA-10 cell nuclear
extracts yields two protein-DNA complexes, IV and V, indicated on the
left side of the autoradiogram. Competition for these
complexes was examined using 500-fold unlabeled wild type
oligonucleotides -447/-419 (lane 3), -418/-399 (lane 4),
-432/-399 (lane 5), or by mutant -447/-419 oligonucleotides mutant
2 (lane 6) and mutant 3 (lane 7). Incubation of the -418/-399 probe
with MA-10 cell nuclear extracts yields one protein-DNA complex, VI
(lane 9), indicated on the right side of the
autoradiogram. Competition for this complex was by unlabeled wild type
oligonucleotides -418/-399 (lane 10), -447/-419 (lane 11),
-432/-399 (lane 12), -447/-399 (lane 13) or by mutant -418/-399
oligonucleotide mutant 1 (lane 14). B, Direct binding of MA-10 cell
nuclear proteins to mutant oligonucleotides. Wild type oligonucleotide
-447/-419 was used as probe in lanes 14; -447/-419 mutant 2
oligonucleotide was used as probe in lanes 57; -447/-419 mutant 3
oligonucleotide was used as probe in lanes 810. Two protein-DNA
complexes are formed with wild type -447/-419 oligonucleotide
(complexes IV and V; lanes 12) and is competed by 500-fold molar
excess unlabeled oligonucleotide (lane 3); complex V is formed with
mutant 2 oligonucleotide probe (lane 6) and competed with 500-fold
excess unlabeled mutant 2 probe (lane 7); complex IV is formed with
mutant 3 oligonucleotide probe (lane 9) and competed with 500-fold
excess unlabeled mutant 3 probe (lane 10). Complex IV formation is
competed by -84/-55 rP450c17 oligonucleotide probe (lane 4). C, Rat
recombinant SF-1 binding to -447/-399 oligonucleotide. Autoradiogram
of a gel shift assay using -447/-399 oligonucleotide as probe (lane
1) with rat recombinant SF-1 prepared from bacteria (lane 2). SF-1-DNA
complex is competed by 500-fold molar excess -447/-399
oligonucleotide (lane 3) and by -447/-419 mutant 3 oligonucleotide
(lane 5) but not by -447/-419 mutant 2 oligonucleotide (lane 4). D,
Protein-DNA interactions within the -447/-399 region of the rat
P450c17 gene. In vivo, this region of the rat P450c17
gene is bound by at least three factors, COUP-TF, SF-1, and an
unidentified nuclear protein. All these proteins appear to bind to
AGGTCA-like sequences. COUP-TF binds at site 1, and either at site 2
and/or 3, and therefore needs an intact DNA sequence. There are also
additional nuclear factors in MA-10 and Y-1 cells that will bind to
this region. They are not apparent on gel shift assays, either due to
the relative abundance of the nuclear proteins, or the binding
affinities of these proteins, relative to the affinity of COUP-TF. By
separating the AGGTCA-like elements in vitro, thereby
displacing COUP-TF binding, we identified two additional nuclear
proteins that bind to this DNA. Since these proteins increase
transcription (either basal transcription or both basal and
cAMP-induced transcription), we call them steroidogenic factor inducer
of transcription, StF-IT-1 and -2.
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We used various mutant oligonucleotides as competitors of the wild type
probes to determine which bases were required to form protein-DNA
complexes. Mutant 2 competed with the -447/-419 probe for formation
of complex V (lane 6), but also competed slightly for complex IV.
Mutant 3 competed mainly for formation of complex IV (lane 7), but also
slightly competed for complex V. These data indicate that the protein
in complex IV binds to site 2 (Table 1
) and the protein in complex V
binds to site 3. Mutant 1 did not compete with the -418/-399 probe
for formation of complex VI (lane 14), indicating that this complex
probably also requires an ERE half-site.
To identify specific bases required for formation of complex IV, V, and
VI, we used mutant oligonucleotides (Table 1
) as probes in gel shift
experiments (Fig. 5B
). Mut 2 -447/-419 oligonucleotide generated a
single protein-DNA interaction, corresponding to complex V (lane 6).
Mut 3 -447/-419 oligonucleotide formed a single protein-DNA
interaction corresponding to complex IV (lane 9). Mut 1 -418/-399 did
not generate complex VI (not shown).
Our data indicated that SF-1 bound to the intact -447/-399 DNA (Fig. 2A
). We determined whether SF-1 also bound to -447/-419 or to
-418/-399 and whether it was the protein in either complex IV, V, or
VI. We used oligonucleotide -84/-55, to which SF-1 binds in another
region of the rat P450c17 gene (2, 18) (see Fig. 2A
), as a competitor
of the -447/-419 DNA (Fig. 5B
) or of the -418/-399 DNA (not shown).
The -84/-55 oligonucleotide did not compete with -418/-399 DNA for
formation of complex VI (not shown) but did compete with -447/-419
for formation of complex IV, but not complex V (lane 4). Thus, complex
IV is due to an interaction of SF-1 with site 2. These data were
confirmed by displacement of complex IV with an antibody against mouse
SF-1 (not shown) and by using recombinant rat SF-1 and -447/-399 as
probe (Fig. 5C
). SF-1 bound to this DNA, and binding was not competed
by Mut 2 -447/-419, but was competed with Mut 3 -447/-419. Thus,
SF-1 binds to site 2 but not to site 3.
We also determined whether any of the proteins in complexes IV, V, or
VI was COUP-TF by adding antibody to COUP-TF in the binding reaction.
Although COUP-TF bound to the intact -447/-399 oligonucleotide, it
did not bind to either the -447/-419 or the -418/-399
oligonucleotides because formation of complexes IV, V, and VI was not
supershifted by antibody to COUP-TF (not shown). Likewise, the data in
Fig. 4
demonstrated that COUP-TF binds to -447/-399 DNA and to
-432/-399 DNA. These findings suggest that COUP-TF binds
simultaneously to two sequences: one is within the -447/-419 region,
most likely site 2, and the second is in the -418/-399 region, and
binding requires these two regions to be colinear. These results
further indicate that COUP-TP does not bind to sites 2 and 3 in the
-447/-419 region alone. COUP-TF binding to sites 1 and 2 likely
represses the activating function of the protein that binds to site 1
alone. Thus, removing COUP-TF binding, by separating sites 1 and 2,
results in increased transcription (Fig. 3
).
Thus it appears that there are two different sets of proteins that bind
to the -447/-399 region of the rat P450c17 gene. One set of proteins
(SF-1, COUP-TF, and an additional unidentified protein) binds to this
region when it is intact, and an additional two proteins bind when the
region is cut into two pieces. As these proteins affect basal and/or
cAMP transcription, we call these two novel proteins Steroidogenic
Factor Inducer of Transcription-1 and -2, or StF-IT-1 (which forms
complex VI) and StF-IT-2 (which forms complex V). The data further
indicate that in vivo, binding of COUP-TF to the intact DNA
and formation of a ternary protein-DNA complex consisting of complexes
I, II, and III would preclude binding of StF-IT-1 or StF-IT-2 (Fig. 5E
). Protein binding and transcriptional activation data are summarized
in Table 2
.
Cell-Specific Expression of StF-IT-1 and StF-IT-2
To determine the tissue distribution of expression of StF-IT-1 and
StF-IT-2, we performed gel shift experiments using nuclear extracts
from mouse adrenal Y-1, mouse adrenal AN4Rpp7 (23), human placental
JEG-3, rat glial C6, monkey COS-1, and human HeLa cells, and from mouse
testis, adrenal, and liver, using either the -447/-419 or the
-418/-399 oligonucleotides as probe (Fig. 6
). One
protein-DNA complex is formed with extracts from MA-10, Y-1, AN4Rpp7,
and rat glioma C6 cells, and from mouse testis and adrenals when the
-418/-399 DNA is used as probe. HeLa cell extracts also formed a
single complex of slightly lesser mobility than the complex from the
other cells. Thus, StF-IT-1 is expressed in Leydig, adrenal, and glial
cells and may also be expressed in nonsteroidogenic HeLa cells. The
slight difference in mobility with HeLa cell extract may represent
human/mouse differences in the molecular weight of StF-IT-1.

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Figure 6. Gel Shift Assay of Nuclear Extracts from Various
Cell Lines
A, Tissue specificity of StF-IT-1. A -418/-399 oligonucleotide probe
was incubated with nuclear extracts from mouse MA-10, Y-1, AN4Rpp7
(23), rat C6 glioma, and human HeLa cells, and from mouse adrenal and
testis. Lanes 25 and 78 show one protein-DNA complex, and lane 6
shows a protein-DNA complex of different mobility. B, Tissue
specificity of StF-IT-2. A -447/-419 oligonucleotide probe was
incubated with nuclear extracts from mouse MA-10, Y-1, rat C6, monkey
COS-7, human HeLa, and JEG-3 cells and from rat liver, mouse adrenal,
and mouse testis. Two protein-DNA complexes IV and V are found with
extracts from MA-10 and Y-1 cells (lanes 2 and 4) and from mouse
adrenal and testis (lanes 9 and 10), and one protein-DNA complex
migrating faster than complex V is found with extracts from COS-7 and
JEG-3 cells (lanes 5 and 6). No complexes are formed with extracts from
HeLa or C6 cells or from extracts of rat liver (lanes 3, 7, and 8).
|
|
When the -447/-419 oligonucleotide was used as probe, two protein-DNA
complexes were formed only with extracts from Leydig and adrenal cells
or from testis and adrenals (Fig. 6B
). Extracts from COS-1 and JEG-3
cells gave one major protein-DNA complex that migrated faster than
complex V, and extracts from C6, mouse liver, or HeLa cells showed a
diffuse protein-DNA complex or no complex at all. Thus, StF-IT-2
(complex V) is expressed in steroidogenic cells and may be expressed in
COS-1 cells, but not in HeLa cells or rat liver. Again, differences in
the mobilities of the protein-DNA complex in COS-1 or JEG-3 cells
vs. the rodent cell lines may reflect species differences in
the molecular weight of StF-IT-2.
NGF-IB Increases Transcription from -447/-419 Rat P450c17 DNA
The DNA sequence between -447/-419 contains the sequences
5'-CAAAGGTTA-3' (site 2) and 5'-ATAAGGTCA-3' (site 3) on the noncoding
strand of DNA, which are variant sites for the nuclear receptor NGF-IB,
whose consensus binding site is 5'-AAAAGGTCA-3' (24, 25). NGF-IB, a
member of the immediate early response gene family, is involved in the
transcriptional regulation of the related steroidogenic enzyme,
P450c21, in adrenal Y-1 cells (26). Therefore, we determined whether
NGF-IB was involved in the regulation of the rat P450c17 gene in mouse
Leydig MA-10 cells. Our bacterially expressed rat NGF-IB binds to
-447/-419 DNA (Fig. 7A
, lane 2). This binding is not
competed by Mut 2 -447/-419 (lane 4) but is competed by Mut 3
-447/-419 (lane 5), thus indicating that NGF-IB binds to site 2.
NGF-IB binding is also competed by WT -447/-399, or by WT -432/-399
oligonucleotides, consistent with binding at site 2.

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Figure 7. Luciferase Assays of Reporter Gene Constructs
Cotransfection of various wild type and mutant rP450c17-TKLUC reporter
gene constructs, or the TK minimal promoter alone (TKLUC), together
with a plasmid expressing NGF-IB, into cultured mouse Leydig MA-10
cells (A and C) and cultured mouse adrenocortical Y-1 cells (B).
Luciferase activity from reporter constructs alone is shown in
open bars, and from cells cotransfected with an NGF-IB
expression vector is shown in hatched bars.
Transfections were performed in triplicate for each construct. This
figure is one representative of three separate transfection experiments
with each cell type. Error bars represent ±SD.
|
|
To determine whether NGF-IB participates in P450c17 gene regulation, we
cotransfected a vector expressing NGF-IB cDNA (26) and either the WT
-447/-419 TK32LUC, mutants 2 or 3 -447/-419 TK32LUC into MA-10 and
Y-1 cells (Fig. 7B
and C), or -447/-399
1- and
447/-399
2-TK32LUC into MA-10 cells (Fig. 7D
). MA-10 and Y-1 cells
yielded qualitatively similar results, but stimulation of transcription
by NGF-IB in MA-10 cells was much greater than in Y-1 cells. In MA-10
cells, NGF-IB caused a 10-fold increase in the activity of
-447/-399-TK32LUC, a 7-fold increase with -447/-419-TK32LUC, and a
negligible increase with -418/-399-TK32LUC. When site 1 is mutated in
the -447/-399 DNA (Fig. 7D
), NGF-IB elicits a stimulation similar to
its effect on wild type -447/-399 DNA. However, when site 2 is
mutated, NGF-IB has no effect on transcription. These data are
consistent with NGF-IB binding to site 2 to increase transcription
directly or they could indicate that NGF-IB binding prevents repression
by COUP-TF. Since both the -447/-399 and -447/-419-TK32LUC plasmids
contain these ERE half-sites but are stimulated differently by NGF-IB
in Y-1 cells, it is also possible that NGF-IB may interact with a
protein (e.g. StF-IT-1) that binds to sequences between
-418/-399.
When site 2 is mutated (mutant 2 -447/-419-TK32LUC), NGF-IB elicits a
3-fold stimulation in luciferase activity in MA-10 cells, but not in
Y-1 cells, even though the gel shift data (Fig. 7A
) indicated that
NGF-IB did not bind to this DNA. When site 3 is mutated (mutant 3
-447/-419 TK32LUC), NGF-IB elicits a 3-fold stimulation in luciferase
activity in MA-10 cells, compared with a 7- to 10-fold stimulation with
the wild type. This stimulation of mutant-TK-LUC constructs was not
seen in Y-1 cells, as neither mutant 2- nor 3-TK32LUC could be
stimulated by cotransfection with NGF-IB. These data suggest that
NGF-IB binds to site 2, that by itself, NGF-IB does not activate
transcription, and that the action of NGF-IB may require interaction
with another protein that binds to DNA at site 3 (i.e.
StF-IT-2). The slight stimulation of Mut3-TK-LUC in MA-10 cells
indicates that NGF-IB binding to DNA in those cells may result in some
transcriptional activation by itself; however, it is puzzling that
Mut2-447/-419-TK32LUC, a plasmid to which NGF-IB does not bind, is
activated similarly.
Our gel shift data (Fig. 5
) indicated that SF-1 bound to the same site
(site 2) as NGF-IB in MA-10 and in Y-1 cells. Like NGF-IB, the
functional data (Fig. 3
, mutant 3-447/-419-TK32LUC construct) also
indicated that although SF-1 binds to mutant 3, this binding does not
activate transcription. However, when proteins bind both to sites 2 and
3 (i.e. SF-1 and StF-IT-2; Fig. 5
, A and B), transcription
is activated (Fig. 3
; -447/-419 TKLUC construct). Thus, it appears
that SF-1, like NGF-IB, must interact with StF-IT-2 and, together, this
protein-protein-DNA complex induces transcription.
 |
DISCUSSION
|
---|
Multiple Nuclear Proteins Regulate Steroidogenic P450 Genes
The transcriptional regulation of the genes encoding the
steroidogenic enzymes is complex, involving tissue-specific,
gene-specific, and species-specific factors. One such factor,
SF-1/Ad4BP, is a member of the orphan nuclear receptor family and binds
as a monomer to sequences having the core consensus sequence AGGTCA. An
SF-1-binding site has been found within 100 bp of the cap site of all
the steroidogenic P450 enzyme genes and regulates these genes in
various steroidogenic tissues (1, 2, 18, 20, 27, 28, 29, 30, 31, 32, 33, 34, 35). However, SF-1 is
not absolutely required for steroidogenic gene regulation. P450scc gene
transcription is regulated in C6 glioma cells (32) and in JEG-3 cells
(36, 37), and rat P450c17 is regulated in rat Rcho-1 (38) and in human
JEG-3 placental cells (preliminary data) even though C6, Rcho-1, and
JEG-3 cells do not express SF-1.
Multiple Members of the Orphan Nuclear Receptor Gene Family
Regulate P450c17 Gene Transcription
A second factor that is involved in steroidogenic P450 gene
regulation is NGF-IB, which is also a member of the orphan nuclear
receptor family. NGF-IB binds as a monomer to the same AGGTCA core
sequence as SF-1, but its DNA sequence requirements 5' to this core
region are distinct from those required by SF-1. NGF-IB regulates the
transcription of the mouse P450c21 gene in adrenal Y-1 cells (26, 39)
and of the rat P450c17 gene in Leydig MA-10 cells (Fig. 7
). Ablation of
the NGF-IB gene in transgenic animals has no effect on adrenal or
gonadal function (40). Thus, NGF-IB expression is not uniquely crucial
to rodent adrenal or gonadal development or steroidogenesis; it is
possible that other proteins can compensate for the lost NGF-IB
function in these knockout mice. This hypothesis is consistent with our
demonstration that multiple factors, in addition to NGF-IB, regulate
expression of P450c17 in both adrenocortical and Leydig cells.
A third factor that regulates P450c17 gene transcription is
COUP-TF, a ubiquitous transcription factor that binds as a dimer to two
core AGGTCA sequences. These sequences are usually found as direct
repeats, spaced 012 bp apart, or can be palindromic repeats (21, 22).
COUP-TF binding to the rat P450c17 gene is unusual because the spacing
is 13 bp (if COUP-TF binds to sites 1 and 2),or 28 bp (if COUP-TF binds
to sites 1 and 3). Although COUP-TF binding usually decreases
transcription, there are additional elements in the COUP-TF binding
site of the rat P450c17 gene that bind other factors that activate
transcription. Nevertheless, disruption of the COUP-TF binding sequence
increases transcription, suggesting that COUP-TF acts as a negative
transcriptional modulator.
A fourth transcriptional factor that may regulate steroidogenic P450
genes is the homeobox protein Pbx (41). Pbx1a and Pbx1b bind to the
-243/-225 region of the bovine P450c17 gene and enhance
cAMP-mediated transcription. Although sequences similar to the
Pbx-binding site have not been found in the rat P450c17 gene, it is not
known whether Pbx also plays a role in the transcriptional regulation
of this gene in the rat.
Identification of Novel Nuclear Proteins that Regulate Rat P450c17
Gene Transcription in Both Leydig and Adrenocortical Cells
We have also identified two proteins that bind to ERE half-sites
in different regions of the rat P450c17 gene to activate transcription.
We have named these proteins StF-IT-1 and StF-IT-2. These proteins
appear to be novel as they have not been characterized as binding to
and regulating the transcription of other steroidogenic P450 genes.
Both of these factors are found in mouse testis, Leydig MA-10,
adrenals, adrenocortical Y-1 and AN4Rpp7 cells, indicating that they
are not expressed in one steroidogenic cell type only. StF-IT-1 binds
to DNA and increases basal transcription. StF-IT-2 binding alone has no
effect on P450c17 transcription but, in concert with other proteins
such as SF-1 or NGF-IB, StF-IT-2 also induces transcription. Once bound
to DNA, the interaction of these two proteins increases both basal and
cAMP-induced transcription. Thus, the rat P450c17 gene is regulated by
a number of factors that appear to bind to AGGTCA-like sequences,
suggesting that they are bound by multiple orphan nuclear
receptors.
Mechanism of Action of SF-1 and NGF-IB
The mechanism of SF-1 action is not well understood. As more genes
that are bound by SF-1 are identified, there is increasing evidence
that SF-1 functions by multiple mechanisms. These mechanisms may be
tissue-specific or may depend upon the DNA sequence in the target gene.
SF-1 can 1) bind DNA without altering transcription; 2) bind and
activate basal transcription; and 3) bind and mediate a cAMP response.
In the -84/-55 region of the rat P450c17 gene, SF-1 binding elicits
both increased basal and cAMP-stimulated transcription (2, 18), whereas
binding to-447/-419 (Fig. 5
) has no direct effect on transcription
(Fig. 3
). In the rat P450scc gene, SF-1 binding can elicit both basal
(32) and cAMP-stimulated transcription (27, 32). Similarly, SF-1
induces both basal and cAMP-induced transcription of the human P450arom
gene (30).
We now show that bases -447 to -418 of the rat P450c17 gene
participate in yet another mechanism of SF-1 action; although SF-1
binding alone has no effect on either basal or cAMP-stimulated
transcription, it appears to interact with the novel DNA-binding
protein, StF-IT-2. This SF-1/StF-IT-2 interaction, but neither protein
by itself, increases transcription. Equivalent results were also seen
for NGF-IB, which also required binding of StF-IT-2 to DNA for
transcriptional activation. The nature of these interactions is unknown
but requires that both SF-1 (or NGF-IB) and StF-IT-2 interact with the
DNA. This may be similar to the synergism seen between the estrogen
receptor and SF-1 in activating transcription of the salmon
gonadotropin IIß subunit gene (42), a gene closely related to
mammalian LHß. In both our case and in the case of estrogen
receptor/SF-1 interaction, the two proteins may interact physically to
synergize increased transcription. The role of StF-IT-2 in interacting
with SF-1 or NGF-IB is different from the role of the retinoid X
receptor in interacting with NGF-IB, as in the latter case, the
retinoid X receptor need not interact with the DNA directly (43).
Displacement of COUP-TF as a Mechanism for Transcriptional
Activation
Our experiments also show that both SF-1 and NGF-IB may displace
COUP-TF binding from DNA, and that this displacement of the repressive
action of COUP-TF may increase transcription. By this mechanism, both
NGF-IB and SF-1 by themselves would not be activators of transcription
but would activate transcription by removing a protein with repressor
function, thereby allowing other activating proteins (e.g.
StF-IT-1 and StF-IT-2) to bind to DNA.
Transcriptional Regulation by cAMP
The -447/-399 region of the rat P450c17 gene contains a
cAMP-responsive element that is distinct from all consensus CRE and CRS
sequences previously described in other steroidogenic genes (1, 2, 18, 19, 28, 30, 31, 33, 34, 37, 41, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56). This element may be related to
SF-1 binding, as we have previously shown that SF-1 can mediate
cAMP-induced transcriptional activation from another region of the rat
P450c17 gene (18).
SF-1 may play a role in cAMP regulation of other steroidogenic genes.
Others have shown that SF-1 can mediate cAMP-stimulated transcription
of the P450aro and P450scc genes (27, 30, 32). Our previous data
demonstrated that SF-1 could be phosphorylated by protein kinase A
(18), suggesting that this might be a mechanism by which SF-1 mediates
cAMP effects. Since the -447/-399 region of the rat P450c17 gene is
also bound by SF-1 and is also regulated by cAMP, the actions of SF-1
may be similarly modified by protein kinase A at this region. However,
when SF-1 binds to the -447/-419 region of the rat P450c17 gene, it
does not appear to mediate cAMP stimulation. It is thus intriguing that
a single protein has the ability to function by several different
mechanisms. The different elements of the rat P450c17 gene provide an
outstanding template for further studies of these mechanisms of orphan
nuclear receptor action.
 |
MATERIALS AND METHODS
|
---|
Preparation of Nuclear Extracts
Nuclear protein extracts from mouse Y-1 adrenal and MA-10 Leydig
cells and from mouse tissues were prepared as described (2, 57).
Protein concentrations were determined by the BCA protein assay system
(Pierce Chemical Co., Rockford, IL).
Gel Shift Assays
Gel shift assays were performed as described previously (2).
Oligonucleotides corresponding to -447/-399, -418/-399, -447/-419
of the rat P450c17 gene (2), and mutants of these oligonucleotides were
used. These oligonucleotides are shown in Table 1
. Bases that are
underlined in the mutants are different from those in the
wild type sequences. Oligonucleotide probes were end labeled using
[
-32P]ATP and T4 polynucleotide kinase and mixed with
the nuclear proteins in the presence of 100 µg/ml
polydeoxyinosinic-deoxycytidylic acid, 50 µg/ml salmon sperm DNA, 5
mM dithiothreitol, and 1 mg/ml BSA, and incubated at room
temperature for 40 min. One quarter of the total reaction was loaded
onto a 6% nondenaturing polyacrylamide gel, using 0.5 x
Tris-borate-EDTA as a running buffer, to separate the free labeled
probe from probe bound by nuclear protein. The dried gel was then
exposed to x-ray film.
DNase I Footprinting Assay
DNase I footprinting assays were performed as described (2, 58).
An oligonucleotide corresponding to bases -447/-399 of the rat
P450c17 gene was cloned into the BamHI site of pUC19. The
recombinant plasmid was first digested with EcoRI and
labeled by Klenow fragment of DNA polymerase I and
[
-32P]dATP. The labeled plasmid was then digested with
HindIII and purified on a 6% nondenaturing polyacrylamide
gel. The probe was mixed with 25 µg nuclear proteins from Y-1 and
MA-10 cells in buffer containing 10 mM Tris-Cl, pH 7.9, 5
mM MgCl2, 1 mM CaCl2, 2
mM dithiothreitol, 100 mM KCl, and 2 mg/ml poly
dI/dC. Samples were kept on ice for 30 min, prewarmed to 26 C for 1 min
before DNase I was added (0.02 U/reaction), and then incubated at 26 C
for 90 sec. Reactions were stopped by digestion with proteinase K (20
mg/ml) in 0.1% SDS, extracted once with phenol/chloroform,
precipitated with ethanol, and separated on 8% polyacrylamide
denaturing gels. The protected regions were detected by
autoradiography.
Construction of the Rat P450c17 Oligonucleotide-TK-LUC Expression
Plasmids
Rat P450c17 oligonucleotides were cloned into a luciferase
expression vector with a minimal promoter from TK gene of herpes
simplex virus (TK32LUC) as described (2). The chimeric constructs were
confirmed by DNA sequencing to determine the copy number and sequences.
Plasmids containing a single copy of the wild type and mutant
oligonucleotides cloned in the 5'
3' direction were used for
transfection experiments.
Transfection of Y-1 and MA-10 Cells
Mouse adrenocortical Y-1 (59) and mouse Leydig MA-10 cells (60)
were grown as described (2). Plasmid DNAs were transfected into Y-1 and
MA-10 cells by calcium phosphate precipitation. When vectors expressing
NGF-IB were cotransfected with reporter luciferase constructs, the
molar ratio of DNA for these two plasmids was 1:1. DNA concentrations
were equalized in all samples by addition of the cloning vector pKS.
DNA precipitates were kept on the surface of the cells for 12 h
before being replaced by fresh medium. 8-Bromo-cAMP (1 mM)
was added for an additional 6 h before cells were harvested.
Luciferase assays and data analysis were as described elsewhere (61),
using a Monolight 1500 Luminometer (Analytical Luminescence Laboratory,
San Diego, CA) using D-Luciferin (Sigma, St. Louis, MO) as substrate
for the light reaction.
Preparation of Rat Recombinant SF-1 and Rat Recombinant
NGF-IB
A rat SF-1 cDNA fragment (28), kindly provided by B. A. White
(University of Connecticut, Farmington, CT) was cloned into the
prokaryotic expression vector pET (Novagen, Madison, WI). Comparison of
the rat SF-1 sequence to the full-length mouse SF-1 sequence (62)
suggests that the rat cDNA fragment encodes amino acids 20293 and
lacks part of the ligand-binding domain of SF-1. SF-1 was overexpressed
in bacteria strain BL21 and purified as inclusion bodies as described
(63). Renatured SF-1 protein was used in gel shift assays.
A rat NGF-IB PvuII fragment encoding 477 amino acids was
kindly provided by J. D. Milbrandt (Washington University, St. Louis,
MO) and was similarly cloned into the prokaryotic expression vector
pET. NGF-IB was overexpressed in bacteria and purified as inclusion
bodies. Renatured NGF-IB was used in gel shift assays.
 |
ACKNOWLEDGMENTS
|
---|
We thank Dr. Ming-Jer Tsai (Baylor College of Medicine, Houston,
TX) for the anti-COUP antibody, and Dr. Jeffrey D. Milbrandt
(Washington University, St. Louis, MO) for the NGFI-B expression
vector.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Synthia H. Mellon Ph.D., Department of Obstetrics/Gynecology, University of California, San Francisco, Box 0556, San Francisco, California 94143-0556.
This work was supported by NIH Grants HD-27970 (to S.H.M.) and HD-11979
(to the Reproductive Endocrinology Center, UCSF) and a grant from the
Academic Senate, University of California, San Francisco (to S.H.M.).
P.Z. was supported in part by a grant from the Rockefeller Foundation
(to the Reproductive Endocrinology Center, UCSF).
Received for publication July 17, 1996.
Revision received February 11, 1997.
Accepted for publication March 11, 1997.
 |
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