Expression Profiles of SF-1, DAX1, and CYP17 in the Human Fetal Adrenal Gland: Potential Interactions in Gene Regulation
Neil A. Hanley,
William E. Rainey,
David I. Wilson,
Stephen G. Ball and
Keith L. Parker
Departments of Internal Medicine & Pharmacology (N.A.H.,
K.L.P.) Division of Reproductive Endocrinology (W.E.R.)
Department of Obstetrics & Gynecology University of Texas
Southwestern Medical Center Dallas, Texas 75390-8857
School of Biochemistry & Genetics (N.A.H., D.I.W.) Department
of Medicine (D.I.W., S.G.B.) University of Newcastle Newcastle
upon Tyne, United Kingdom
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ABSTRACT
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Cytochrome P450 17
-hydroxylase/1720 lyase
(P450C17) is a critical branchpoint enzyme for
steroid hormone biosynthesis. During human gestation,
P450C17 is required for the production of
dehydroepiandrostenedione sulfate by the fetal adrenal cortex and
for testicular production of androgens that mediate male sexual
differentiation. In this study, we investigate the regulation of the
human CYP17 gene by two orphan nuclear receptors,
steroidogenic factor 1 (SF-1) and DAX1. In human embryos, SF-1 and DAX1
are expressed throughout the developing adrenal cortex from its
inception at 33 days post conception (dpc). In contrast,
P450C17 expression, which commences between 41
and 44 dpc, is limited to the fetal zone. The 5'-flanking region of the
human CYP17 gene contains three functional SF-1 elements
that collectively mediate a
25-fold induction of promoter activity by
SF-1. In constructs containing all three functional SF-1 elements, DAX1
inhibited this activation by
55%. In the presence of only one or two
SF-1 elements, DAX1 inhibition was lost even though SF-1
transactivation persisted. These data suggest that efficient repression
of SF-1-mediated activation of the human CYP17 gene by DAX1
requires multiple SF-1 elements. Opposing effects of SF-1 and DAX1 may
fine tune the differential responses of various SF-1 target genes in
different endocrine tissues.
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INTRODUCTION
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Human cytochrome P450 17
-hydroxylase/1720
lyase (P450C17) is a critical branchpoint enzyme
in steroid biosynthesis, converting progesterone to
17
-hydroxyprogesterone in the glucocorticoid pathway and
pregnenolone to dehydroepiandrostenedione (DHEA) in the
sex steroid pathways. In the fetal testis, 17
-hydroxylase activity
is essential for the production of testosterone, which stimulates male
sexual differentiation in utero. The combined actions of
P450C17 and sulfotransferase in the human fetal
adrenal cortex of both sexes produce enormous quantities of
DHEA sulfate (DHEAS,
50 mg/day); DHEAS serves as
substrate for estrogen biosynthesis by the placenta and thereby helps
to maintain pregnancy (1). Although steroidogenesis by the human fetal
adrenal is regulated at least partly by pituitary ACTH (2), little is
known about the mechanisms that initiate P450C17
expression in the human fetal adrenal cortex or that regulate its
transcription from the human CYP17 gene
(hCYP17).
The orphan nuclear receptor steroidogenic factor 1 (SF-1,
officially designated NR5A1) is critical for development and function
of the steroidogenic organs. Many cell transfection studies have
highlighted important roles for SF-1 in the transcriptional activation
of the genes that encode steroidogenic enzymes (3), while targeted
disruption of the mouse gene encoding SF-1 defined essential roles in
adrenal and gonadal development (4, 5, 6). SF-1 likely plays similar roles
in humans, as evidenced by adrenal insufficiency and male-to-female sex
reversal in a patient with a heterozygous mutation in the human gene
that encodes SF-1 (7).
Another orphan nuclear receptor, DAX1 [dosage-sensitive sex
reversal, adrenal hypoplasia congenita (AHC), critical region on the X
chromosome gene 1], colocalizes with SF-1 during mouse development (8)
and inhibits SF-1-mediated transactivation of target genes in both
steroidogenic and nonsteroidogenic tissues. In addition to the
cytochrome P450 steroid hydroxylases, promoters shown previously to be
inhibited by DAX1 include: steroidogenic acute regulatory protein
(StAR) (9, 10), Type II 3ß-hydroxysteroid dehydrogenase
(Type II 3ßHSD) (10), and Müllerian
inhibiting substance (MIS) (11)].
In this study, we investigate the expression profiles of SF-1,
DAX1, and P450C17 during the development of the
human adrenal gland. We further study the regulation of
hCYP17 gene expression by SF-1 and DAX1. Our results provide
new insight into the onset of P450C17 expression
in the human embryonic adrenal cortex and suggest that
hCYP17 is especially sensitive to DAX1-mediated repression
via actions at multiple SF-1 response elements.
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RESULTS
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Expression Patterns of P450C17,
SF-1, and DAX1 in the Human Embryonic Adrenal Cortex
The adrenal primordium in human embryos arises from
intermediate mesoderm as a distinct blastema at 33 days post conception
(dpc), which is equivalent to Carnegie Stage 15 (CS15) (12, 13, 14). Lying
posteromedial to the urogenital ridge, it rapidly increases in size
during the embryonic period (up to 56 dpc/CS23; Fig. 1A
). Human P450C17
expression commences centrally in a few cells between 41 and 44 dpc
(CS17 and 18), 811 days after the adrenal primordium first forms
(Fig. 1B
, 44 dpc). By 52 dpc/CS21, the human embryonic adrenal cortex
contains an inner cluster of large eosinophilic cellsthe fetal
zonethat stain strongly for cytoplasmic P450C17
expression. This region is surrounded by the small, densely packed
basophilic cells of the definitive zone, which do not express
P450C17 (asterisk in Fig. 1
, A and B,
52 dpc). By 18 weeks gestation, P450C17
expression is less clearly defined, with strongly expressing cells
within a transitional zone (15) flanked by more weakly staining cells
in the inner fetal zone and scattered expression in the outer
definitive zone (arrow, Fig. 1B
, 18 weeks gestation).

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Figure 1. Expression of P450C17, SF-1, and DAX1
in the Human Embryonic and Fetal Adrenal Cortex
Transverse sections show hematoxylin and eosin (H & E) staining (A and
C), expression of P450C17 by indirect immunohistochemistry
counter-stained with toluidine blue (B), or darkfield photomicrographs
of in situ hybridization with the SF-1 and DAX1
antisense probes (D and E). gr, Gonadal ridge; a, adrenal; m,
mesonephros; t, testis; DZ, definitive zone; TZ, transitional zone; FZ,
fetal zone. At 52 dpc, the definitive zone is marked by an
asterisk in panels A and C. Scale
bars = 80 µm.
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SF-1 and DAX1 are expressed within the developing adrenal gland from
its inception at 33 dpc/CS15 (Fig. 1
, D and E); their levels in both
sexes exceed those in the urogenital ridge (12, 16). The detection of
SF-1 transcripts is greater than for DAX1 at all embryonic stages. In
contrast to the absence of P450C17 expression in
the definitive zone, both transcripts are distributed throughout the
entire adrenal cortex at 52 dpc/CS21 (Fig. 1
, CE, 52 dpc). Lower
levels of SF-1 and DAX1 expression persist throughout the fetal adrenal
cortex at 18 weeks gestation (Fig. 1
, D and E).
SF-1 Activates Transcription of the Human CYP17 Gene
through Multiple cis-Elements
Although previous studies have implicated SF-1 in the expression
of hCYP17, the role of DAX1 has not been explored. Using
transient transfection studies in NCI-H295R human adrenocortical cells,
which preferentially secrete DHEAS (W. E. Rainey, unpublished
observation), we examined the regulation of the hCYP17 gene
by SF-1. Cotransfection of SF-1 with promoter constructs containing
either 2,845 bp (p-2845Luc) or 381 bp (p-381Luc) of hCYP17
5'-flanking region stimulated luciferase activity by
25-fold over
basal expression (Fig. 2
). Progressive
deletion of the hCYP17 5'-flanking region incrementally
decreased SF-1 stimulation, suggesting that multiple promoter elements
mediate SF-1 induction (Fig. 2
).

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Figure 2. 5'-Deletion Analysis of the Human
CYP17 Promoter
Promoter constructs containing the indicated lengths of 5'-flanking
sequence (1 µg) were transfected with (+) or without (-) human SF-1
(1 µg) and cell extracts assayed for luciferase activity. EV, Empty
vector. Shown are the fold-increases over basal for SF-1-stimulated
constructs and means ± SEs from at least three
independent experiments standardized relative to the SF-1-stimulated
activity of p-381Luc (100%).
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Inspection of the 5'-flanking sequence of hCYP17 revealed
three potential SF-1 binding sites within the p-381Luc construct
(150/142,211/204, and290/276 bp). Mutation of any one of
these sites reduced SF-1-stimulation by 2545%, concurrent mutation
of any two sites caused a 7080% decrease, and combined mutation of
all three sites abolished >90% of the SF-1 induction (Fig. 3A
). Electrophoretic mobility shift
assays (EMSAs) showed that oligonucleotide probes corresponding to each
of these putative SF-1 elements formed two specific complexes with
NCI-H295R nuclear extracts. Complex 1 comigrated with the complex
formed by in vitro translated SF-1 and was either abolished
(211/204 bp) or supershifted (150/142 and 290/276 bp) by
preincubation with anti-SF-1 antiserum (Fig. 3B
), suggesting strongly
that it results from the binding of SF-1.

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Figure 3. Characterization of Three Functional SF-1 Sites in
the Human CYP17 Promoter
A, The human CYP17 promoter contains three functional
SF-1 sites. Potential SF-1 binding elements within wild-type p-381Luc
were mutated (+, wild-type element; m, mutated element), 1 µg of each
plasmid was cotransfected with the human SF-1 expression vector (1
µg), and cell extracts were assayed for luciferase activity.
Luciferase activities are standardized relative to wild-type p-381Luc,
which contains all three wild-type elements (100% = 25-fold
stimulation over basal). Shown are the means ± SEM of
two independent experiments. B, SF-1 binds to the hCYP17
promoter elements. [32P]-labeled probes (20 fmol)
corresponding to the 150/142, 211/204, and 290/276 bp sites
were incubated with NCI-H295R nuclear extract (NE, 2 µg) and resolved
by electrophoresis on a nondenaturing 4% polyacrylamide gel. Lane 1,
free probe; lane 2, probe + in vitro translated SF-1
(0.4 µl); lane 3, probe + NE; lane 4, probe + NE + 100-fold excess
unlabeled probe; lane 5, probe + NE + anti-SF-1 antiserum.
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DAX1 Inhibits SF-1-Mediated Transactivation of hCYP17
in a Dose-Dependent Manner
SF-1, DAX1, and P450C17 have overlapping
expression profiles in the human embryonic adrenal cortex (Fig. 1
), and
DAX1 reportedly interferes with SF-1-dependent transcriptional
activation (9, 10, 11, 17, 18, 19). We therefore studied the effect of DAX1 on
SF-1-mediated stimulation of the hCYP17 promoter. DAX1
inhibited SF-1 activation of the p-381Luc construct in a
concentration-dependent manner (Fig. 4A
);
when cotransfected in equal amounts, DAX1 inhibited the activity
achieved by SF-1 alone by
55%. This DAX1 inhibition was partly
overcome by cotransfection with increasing amounts of SF-1 (Fig. 4B
),
suggesting a competition between the two proteins. Similar results were
obtained with the hCYP17 promoter constructs in multiple
cell types [e.g. mouse Y1 adrenocortical cells, monkey
kidney COS-1 cells, a human ovarian thecal tumor cell line (20), and
primary cultures of adult human adrenocortical cells] using either
cytomegalovirus- or Rous sarcoma virus-based expression vectors (data
not shown). In contrast to the DAX1 inhibition of SF-1-stimulated
luciferase activity, transfection with DAX1 alone weakly stimulated
(
3-fold) the expression of a number of plasmids, including
hCYP17 promoter constructs (Figs. 4
and 5
), pGL3 basic
vector (Fig. 5
, A, C, and D), or a
cytomegalovirus-based ß galactosidase vector (data not shown). This
nonspecific activation, which has been observed previously (17), may
reflect squelching of corepressors by DAX1.

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Figure 4. Titration of DAX1 Inhibition of SF-1 Action
A, DAX1 inhibits SF-1-stimulated hCYP17 reporter
activity in a dose-dependent manner. The p-381Luc construct was
cotransfected with a constant amount of SF-1 (+, 1 µg) and increasing
amounts of DAX1 (0.25, 0.5, 1.0 µg). DAX1 alone (+, 1 µg) did not
inhibit basal activity of p-381Luc. B, Increasing SF-1 partially
overrides DAX1 inhibition. p-381Luc was cotransfected with a constant
amount of DAX1 (+, 0.5 µg) and increasing amounts of SF-1 (0.25, 0.5,
0.75, 1.0 µg). Results are standardized to stimulation of p-381Luc
with SF-1 alone (100%, 25-fold stimulation over basal) in panels A
(+, 1.0 µg) and B (+, 0.5 µg). Shown are the means ±
SEM of at least two independent experiments.
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Figure 5. DAX1 Inhibition of SF-1-Stimulated
hCYP17 Reporter Activity Requires Multiple SF-1 Sites
Progressively shorter wild-type promoter constructs, singly or multiply
mutated SF-1 elements within p-381Luc (AC), and p-182Luc with extra
-150/-142 binding sites (D) (all 1 µg) were cotransfected with SF-1
(1 µg) and DAX1 (1 µg) and cell lysates were assayed for luciferase
activity. represents mutated element within p-381Luc. Results are
standardized to SF-1 stimulation of wild-type p-381Luc (100%) in
panels AC and wild-type p-182Luc (100%) in panel D. Shown are the
fold-increases over basal for SF-1-stimulated promoter constructs and
means ± SEM of at least two independent experiments.
EV, Empty vector.
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DAX1 Inhibition of SF-1-Stimulated hCYP17 Requires
Multiple SF-1 Binding Sites
In cotransfection experiments with hCYP17 promoter
constructs shorter than 381 bp, DAX1 did not inhibit SF-1-stimulated
reporter activity. For example, DAX1 only minimally inhibited
expression of the p-228Luc construct (Fig. 5A
), despite the fact that
this reporter still responds to SF-1. Similarly, DAX1 did not inhibit
SF-1-mediated activation of p-182Luc, even though this construct
retains the functional SF-1 site at150/142 bp (Fig. 3A
). Taken
together, these data suggest either that the shorter constructs lack a
specific promoter region important for DAX1 inhibition or that DAX1
requires multiple SF-1 sites to inhibit SF-1-stimulated transcription
of the hCYP17 gene.
To differentiate between these models, we carried out
cotransfection experiments using combinations of the mutated SF-1
elements within the p-381Luc construct (Fig. 5
, B and C). As shown in
Fig. 5B
, constructs containing mutations of any of the three SF-1
binding sites were still significantly induced by SF-1 (range 12- to
17-fold), but DAX1 inhibition was either absent
(p-381Luc
150/142) or severely
decreased (p-381Luc
-211/-204 and
p-381Luc
-290/-276). Although SF-1 still
stimulated promoter activity by 5- to 8-fold, mutation of any two SF-1
sites within p-381Luc completely abolished DAX1 inhibition (Fig. 5C
).
Coupled with the observation that SF-1 can override DAX-1 inhibition of
reporter gene expression, these findings suggest that DAX1 requires the
presence of multiple SF-1 sites to effectively inhibit SF-1
transactivation of the hCYP17 promoter.
To determine whether the apparent need for multiple sites reflects
a requirement for an absolute number of sites or for specific
sequences of the SF-1 binding elements, we placed multiple copies of
the 150/142 binding site at the 5'-end of the wild-type p-182Luc
construct. The p-182Luc+2(150/142) and
p182Luc+1(150/142) constructs contain,
respectively, a total of three or two copies of the 150/142 SF-1
binding site. SF-1 induced luciferase activity of the
p182Luc+2(150/142) construct by 44-fold and
DAX1 inhibited promoter activity to a degree similar to that observed
with the wild-type p-381Luc construct (which also contains three SF-1
sites in a different combination of orientation and spacing). This
repression was not altered when the spacing of the most 5'-element was
modified by 20 or 40 bp (data not shown). Although SF-1 cotransfection
induced promoter activity of
p-182Luc+1(150/142) by 20-fold, DAX1
inhibition was reduced to the levels seen with
p-381Luc
-290/-276 and
p381Luc
-211/-204 (Fig. 5B
). In the wild-type p-182Luc construct, which contains a single copy
of the 150/142 element, SF-1 still induced luciferase activity by
10-fold, but was not inhibited by DAX1. These data suggest that
DAX1 inhibition of hCYP17 requires a minimal number of
functional SF-1 sitesirrespective of orientation or positionrather
than a particular sequence of the binding elements and their flanking
nucleotides.
The Effect of Copy Number of SF-1 Elements on DAX1 Repression of
Other Steroidogenic Promoters
To investigate whether DAX1 also represses other promoters most
efficiently through multiple SF-1 sites, we studied the human
CYP11B1 (hCYP11B1) and mouse Cyp21
(mCyp21) promoters. Previous analyses of the 5'-flanking
region of the mCyp21 gene identified three SF-1 binding
elements, the most proximal of which was located 65 bp upstream of the
transcription initiation site (21). By itself, this single SF-1 site
(mCyp21p-65Luc) responds poorly to SF-1 (Fig. 6A
), and the 1.4-fold induction was not
inhibited by DAX1. Strikingly, when five copies of the 65 element are
placed upstream of the PRL core promoter (mCyp21p-65x5Luc),
SF-1 induced luciferase activity by 38-fold; in this setting, DAX1
inhibited SF-1-induced transactivation to a degree comparable to that
seen with the hCYP17 promoter constructs containing three
SF-1 elements. Transactivation of the hCYP11B1 gene by SF-1
occurs almost exclusively through a single SF-1 site at 243 bp (N.
Hanley and W. Rainey, unpublished observation). Although the
hCYP11B1p-1102Luc construct, which contains this site, was
induced 12-fold upon cotransfection with SF-1, DAX1 failed to inhibit
this transactivation (Fig. 6B
).

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Figure 6. DAX1 Inhibition Requires Multiple SF-1 Sites in
Other Steroidogenic Promoters
The mCyp21 p-65Luc and p-65x5Luc vectors (A) and
hCYP11B1 p-1102Luc vector (B) were cotransfected with
SF-1 (1 µg) and DAX1 (1 µg), and cell lysates were assayed for
luciferase activity. Results are standardized to SF-1 stimulation of
the p-65x5Luc vector (A; 100%) and hCYP11B1 p-1102Luc
vector (B; 100%). Shown in parentheses are the -fold
increases over basal for SF-1-stimulated promoter constructs and
means ± SEM of at least three independent
experiments.
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Impaired Repression of SF-1-Stimulated hCYP17 Reporter
Activity by DAX1 Mutations That Cause AHC
We next investigated whether three DAX1 mutations that impair
adrenal development in AHC could still repress SF-1 transactivation of
the hCYP17 promoter. These naturally occurring single amino
acid substitutions are predicted to disrupt the hydrophobic core of the
putative DAX1 ligand binding domain in helix 3 (R267P), helix 9
(V385G), or helix 11 (I439S). In the presence of three SF-1 elements
(p-381Luc or p-182Luc+2(150/142)), each
mutation repressed considerably less efficiently than did wild-type
DAX1 (R267P, 5762% of wild-type repression; I439S, 4452% of
wild-type repression; Fig. 7
). Although
the V385G mutant differentially repressed the two
SF-1-stimulated constructs (34% of wild-type DAX1 activity with
p-381Luc compared with 51% with
p-182Luc+2(150/142)), this difference was
statistically insignificant (Fig. 7
). Like wild-type DAX1, none of the
AHC mutations repressed reporter activity with the p-182Luc construct
containing a single SF-1 element (data not shown).

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Figure 7. DAX1 Mutations Causing AHC Impair DAX1 Repression
of hCYP17 Constructs Containing Three SF-1 Elements
The hCYP17 p-381Luc and
p-182Luc+2(150/142) vectors were cotransfected with
SF-1 (1 µg) and in the presence (+) or absence () of either
wild-type (wt) or mutated DAX1 (1 µg), and cell lysates were assayed
for luciferase activity. Results are standardized to SF-1 stimulation
of the two reporter constructs (100%). Shown in
parentheses are the fold increases over basal for
SF-1-stimulated promoter constructs and means ± SEM
of at least three independent experiments.
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DISCUSSION
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The development and function of the fetal adrenal cortex in humans
and higher primates differ from other species because a distinct
compartmentthe fetal zoneis disproportionately enlarged and
produces enormous quantities of DHEAS. Insight into human adrenal
development has relied largely on limited access to second trimester
material and inference from clinical syndromes such as congenital
adrenal hyperplasia. In humans, the fetal adrenal cortex is first
distinct from the adrenal-gonadal pool of SF-1-positive cells at 33 dpc
(12). The restricted expression pattern of
P450C17 within the cortex at 52 dpc argues that a
distinct fetal zone already has formed. In contrast, SF-1 and DAX1 are
expressed throughout the cortex, including the outer definitive zone
that lacks P450C17 expression. The cytochrome
P450 cholesterol side-chain cleavage enzyme
(P450SCC) also is expressed centrally in the
embryonic adrenal cortex from 42 dpc onward, overlapping the expression
of P450C17 (N. Hanley, unpublished observations).
Thus, both enzymes necessary to convert cholesterol to
DHEA are present in the presumptive fetal zone of the
human embryonic adrenal by 52 dpc, earlier than previously described
and before documented production of ACTH by the anterior pituitary (2).
In contrast, the lack of P450C17 expression
within the definitive zone at this early stage implies that this zone
can produce neither DHEA nor cortisol.
To date, studies of CYP17 gene regulation have not
identified mechanisms that are conserved across species such as human
(22, 23, 24), bovine (25, 26), rodent (27, 28, 29), and pig (28, 30). Previous
analyses of hCYP17 gene expression in NCI-H295R cells
identified a region in the proximal 63 bp of the promoter that
contributes to basal transcription, with an additional minor element
located between 206 and 184 bp (24). Relative to other species, the
hCYP17 promoter responded only minimally to cAMP. Although
our finding that SF-1 regulates promoter activity is not
surprisinggiven its extensive role as a transcriptional activator of
steroidogenic enzymes (3)our results define for the first time three
distinct promoter elements by which SF-1 regulates the
hCYP17 gene. The 150/142 and 211/204 elements have
been shown previously to bind SF-1 (31), implicating the sequences in
Table 1
as central to SF-1 action on
these elements. In contrast, the 290/276 site has not been
characterized previously either by functional or DNA-binding studies.
All three sites differ somewhat in sequence from the corresponding
elements in CYP17 genes from other species (Table 1
). In
particular, the nuclear hormone receptor half-sites at 62/40 bp of
the bovine CYP17 promoter (25) are not conserved completely
in the human sequence. Taken together, these data define three SF-1
responsive elements within the proximal 381 bp of the
hCYP17 promoter that support a functional role for SF-1 in
the regulation of P450C17 expression within the
human fetal adrenal cortex.
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Table 1. Upper Strand Sequence Alignment of the SF-1
Elements of the Human, Porcine, Bovine, Rat, and Murine
CYP17
Genes
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Two different mechanisms have been proposed to explain DAX1-mediated
inhibition of SF-1 action. Several groups have demonstrated in
vitro protein-protein interaction between DAX1 and SF-1 (17, 18),
potentially recruiting corepressors such as NCoR (nuclear receptor
corepressor) to the transcription factor complex (18). Others
have proposed that DAX1 binds directly to DNA at stem-loop structures,
thereby inhibiting gene expression (9). Unlike the promoters of the
mouse Star and Dax1 genes, the proximal promoter
of the hCYP17 gene contains no potential stable hairpin loop
structures. Moreover, DAX1 inhibition is dose-dependent, can be
partially overridden by increasing amounts of SF-1, and requires intact
SF-1 binding elements. These features argue that a direct interaction
between DAX1 and SF-1 bound to its response element inhibits SF-1s
stimulation of hCYP17 gene expression. Our data further
demonstrate that all three SF-1 binding sites are required for DAX1 to
maximally inhibit SF-1-stimulated hCYP17 gene transcription.
Experiments described above further suggest that the critical factor is
the numberrather than the precise core or flanking sequencesof SF-1
elements and that precise position and orientation are relatively
unimportant. Notably, SF-1 still increased reporter activity of
p-182Luc, despite the complete lack of inhibition by DAX1. SF-1 target
genes often contain multiple functional SF-1 binding sites within their
5'-flanking regions. If the need for multiple SF-1 sites for effective
inhibition by DAX1 is true in a broader setting, this result carries
implications for the concerted regulation of the steroidogenic pathway
and the transcriptional regulation of other SF-1 target genes. In
support of this, DAX1 repression can be augmented when the
mCyp21 65 bp element is concatenated and DAX1 does not
repress the wild-type human CYP11B1 promoter that is
transactivated almost exclusively through a single functional SF-1 site
(Fig. 6
).
Previous reports of DAX1-mediated repression of SF-1 transactivation
generally are consistent with this model. These data can be divided
into three categories according to the nature of the promoter linked to
the reporter gene: steroidogenic (9, 10, 32), nonsteroidogenic (9, 11, 19), and synthetic (17, 18) (Table 2
). In
the steroidogenesis pathway, genes such as human StAR (33)
and CYP11A (31) that contain multiple SF-1-responsive
elements are strongly repressed by DAX1. In contrast, only a single
SF-1 site has been defined in the proximal 5'-flanking region of the
Type II 3ßHSD promoter, and DAX1 did not
significantly repress its promoter activity (10, 32). Similarly, DAX1
repression of promoter activity of nonsteroidogenic SF-1 target genes
is most marked in the setting of multiple SF-1 sites. Finally,
synthetic promoter constructs containing multiple SF-1-responsive
elements are repressed most strongly by DAX (17, 18).
Collectively, these studies suggest that DAX1 represses SF-1-mediated
transactivation poorly in the absence of multiple SF-1 sites. Thus, a
complete understanding of the interplay of transcription factors can
emerge only from analyses of the promoter elements in the context of
the intact promoter. In addition, our data suggest that this inhibition
occurs through SF-1 sites, rather than via independent binding of DNA
by DAX1. These data are consistent with previous studies implicating
DAX1 repression via the recruitment of corepressors (illustrated
schematically in Fig. 8
) (18). Modifying
the number of available SF-1 sites, either by modulating chromatin
structure or by affecting the binding of other factors to SF-1 sites
(e.g. complex 2 in Fig. 3B
), would alter both the magnitude
of SF-1 transactivation and the ability of DAX1 to inhibit
SF-1-stimulated gene transcription. Such a mechanism could fine tune
the differential responses of various SF-1 target genes, facilitating
regulation of the steroid biosynthetic pathway within steroidogenic
tissues.

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Figure 8. Schematic Model of the Human CYP17
Promoter Showing the Mechanism of SF-1 Repression by DAX1
DNA-bound SF-1 transactivates gene expression through the basal
transcriptional apparatus. When this occurs via a single SF-1 binding
site, DAX1 is incapable of repressing SF-1 action. Weak repression by
DAX1 occurs with two functional SF-1 binding sites. DAX1 is a strong
repressor when three functional SF-1 binding sites are present. DAX1
repression is mediated through the recruitment of corepressors to the
DNA-bound SF-1-DAX1 complex (18 ).
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MATERIALS AND METHODS
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Human Embryo Collection, Immunohistochemistry, and in
Situ Hybridization
Human embryos were collected and staged by the Carnegie
classification after ethical permission had been obtained from the
Newcastle Health Authority and with the appropriately signed consents
(13, 14). Fetal material was similarly obtained after second trimester
termination.
Polyclonal anti-human P450C17 antiserum was
raised in rabbits immunized with a peptide containing amino acid
residues 384398. This antiserum produced a single band of the
appropriate size on immunoblotting of human adrenal cell lysate (data
not shown). Indirect immunohistochemistry was carried out on dewaxed
trypsinized sections previously fixed in 4% paraformaldehyde. Slides
were incubated with primary antiserum (1:1,000 dilution) overnight at 4
C. Biotinylated antirabbit secondary antibody (Sigma, St.
Louis, MO) was incubated for 2 h at 4 C, before treatment at room
temperature with streptavidin-horseradish peroxidase, and the color
reaction was developed with diaminobenzidine. Negative controls
included preabsorption of antibody with the antigen and omission of
primary or secondary antibodies.
In situ hybridization was performed as described previously
for SF-1 and DAX1 using
[35S]-labeled probes on 5-µm transverse
sections (12). Sections for morphological study were within 10 µm of
the corresponding section used for in situ hybridization
analysis. For optimal use of material, experiments were carried out on
both right and left sides of sectioned embryos.
Cloning and Plasmid Constructs
Human SF-1 cDNA (34) was subcloned into the expression
vector, pcDNA3.1Zeo+ (Invitrogen, San Diego, CA). Human
DAX1 cDNA is available as an expressed sequence tag (GenBank accession
no. S74720) and was subcloned into pcDNA3.1Zeo+. AHC mutants were
generated by PCR using complimentary upper and lower primers, which
incorporated the single base pair mutations. The 5'-flanking sequence
of the human CYP17 gene is available (GenBank accession no.
L41825 and M63871). All promoter constructs were cloned into pGL3 Basic
(Promega Corp., Madison, WI) and numbered relative to the
transcriptional start site. Wild-type hCYP17 promoter
constructs were generated by PCR using specific forward primers
(2,914 bp, 5'-TGGGACTCTGATTGGCATTAT; 381 bp,
5'-GAAGATCTACTTTAACAGTCCCTG; 228 bp, 5'-GAAGATCTCCTCAGAGGGTGAT)
and/or by the use of unique restriction endonuclease sites. Additional
details are available from the authors upon request. The
mCyp21 promoter constructs have been described previously
(21) and the hCYP11B1 p-1102Luc construct was a gift from
Dr. Perrin White.
Mutations of the SF-1 binding sites within p-381 were created by PCR
with oligonucleotides shown below (wild-type sequence with mutated
nucleotides above and below shown in
bold).
To incorporate additional SF-1 binding sites into
p-182Luc, oligonucleotide linkers were synthesized and annealed to
recreate the wild-type 150/142 element with overhangs compatible
with unique restriction sites in the 5'-multiple cloning sites of pGL3
Basic. One or two linkers were then cloned directly upstream of the
p-182Luc promoter sequence. All constructs were verified by sequencing.
The base pair numbering of our constructs does not strictly align with
the GenBank accession no. M63871, which lacks a SacI
restriction site described in the published reference (23). The
wild-type sequence and numbering of the human CYP17 promoter
used in this study was verified by direct sequencing of PCR products
amplified from human genomic DNA. Sequences in Table 1
were aligned by
Clustal.
Cell Culture, Transfections, and Luciferase Assays
NCI-H295R cells (American Type Culture Collection,
Manassas, VA; CRL-2128) were cultured as described previously in 5%
CO2 at 37 C (35). Twenty-four hours before
transfection, cells were plated on 12-well plates at a density of
150,000 cells per well. Cells were transfected with Fugene 6 using
the manufacturers protocol (Roche Clinical Laboratories, Indianapolis, IN) in a serum-free
DMEM/F12 medium (LifeTechnologies, Gaithersburg, MD).
Total DNA transfected per well (up to 3 µg) was kept constant by
adjusting the amount of empty pcDNA3.1Zeo+ vector. Luciferase assays
were performed 24 h after transfection according to the
manufacturers instructions (Promega Corp., Madison,
WI).
EMSA
Nuclear extract was prepared from NCI-H295R cells as
described previously (36). Human SF-1 was synthesized by
coupled in vitro transcription/translation from the
pcDNA3.1Zeo+ vector by T7 polymerase using the TNT reticulocyte lysate
system (Promega Corp.). Anti-SF-1 antibody was developed
and used as described previously (37). Oligonucleotides corresponding
to the wild-type SF-1 response elements were annealed and labeled with
[32P]dCTP by Klenow polymerase.
Nuclear extract (2 µg) and each radiolabeled probe (40,000 dpm)
were incubated at room temperature for 20 min in 20 µl of reaction
mixture (20 mM HEPES; pH 8.0, 80 mM KCl, 1
mM EDTA, 10% glycerol, 1 mM dithiothreitol,
0.5 mg/ml BSA, and 0.025 mg/ml poly dI-dC as nonspecific competitor).
Where appropriate, nuclear extract and antibody were preincubated on
ice for 20 min before addition of probe and reaction mixture. The
resulting DNA/protein complexes were resolved by electrophoresis on 4%
nondenaturing polyacrylamide gels in high ionic strength Tris-glycine
buffer (38). The gel was dried and visualized after autoradiography at
70 C overnight.
 |
ACKNOWLEDGMENTS
|
---|
The authors acknowledge the use of material provided by the
Newcastle University Developmental Resource, Newcastle upon Tyne,
UK.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Keith L Parker, M.D., Ph.D., Division of Endocrinology & Metabolism, Department of Internal Medicine, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas 75390-8857. E-mail:
kparke{at}mednet.swmed.edu
N.A.H. is a Wellcome Trust Clinical Training Fellow. D.I.W. is
supported by the Knott Trust. This work was funded by NIH Grants
DK-54480 (K.L.P.) and DK-43140 (W.E.R.).
Received for publication February 18, 2000.
Revision received September 5, 2000.
Accepted for publication October 9, 2000.
 |
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