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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cytochrome P450 17{alpha}-hydroxylase/17–20 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.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Human cytochrome P450 17{alpha}-hydroxylase/17–20 lyase (P450C17) is a critical branchpoint enzyme in steroid biosynthesis, converting progesterone to 17{alpha}-hydroxyprogesterone in the glucocorticoid pathway and pregnenolone to dehydroepiandrostenedione (DHEA) in the sex steroid pathways. In the fetal testis, 17{alpha}-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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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. 1AGo). Human P450C17 expression commences centrally in a few cells between 41 and 44 dpc (CS17 and 18), 8–11 days after the adrenal primordium first forms (Fig. 1BGo, 44 dpc). By 52 dpc/CS21, the human embryonic adrenal cortex contains an inner cluster of large eosinophilic cells—the fetal zone—that 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. 1Go, 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. 1BGo, 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.

 
SF-1 and DAX1 are expressed within the developing adrenal gland from its inception at 33 dpc/CS15 (Fig. 1Go, 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. 1Go, C–E, 52 dpc). Lower levels of SF-1 and DAX1 expression persist throughout the fetal adrenal cortex at 18 weeks gestation (Fig. 1Go, 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. 2Go). Progressive deletion of the hCYP17 5'-flanking region incrementally decreased SF-1 stimulation, suggesting that multiple promoter elements mediate SF-1 induction (Fig. 2Go).



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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%).

 
Inspection of the 5'-flanking sequence of hCYP17 revealed three potential SF-1 binding sites within the p-381Luc construct (–150/–142,–211/–204, and–290/–276 bp). Mutation of any one of these sites reduced SF-1-stimulation by 25–45%, concurrent mutation of any two sites caused a 70–80% decrease, and combined mutation of all three sites abolished >90% of the SF-1 induction (Fig. 3AGo). 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. 3BGo), 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.

 
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. 1Go), 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. 4AGo); 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. 4BGo), 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. 4Go and 5Go), pGL3 basic vector (Fig. 5Go, 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 (A–C), 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. {Delta} represents mutated element within p-381Luc. Results are standardized to SF-1 stimulation of wild-type p-381Luc (100%) in panels A–C 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.

 
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. 5AGo), 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 at–150/–142 bp (Fig. 3AGo). 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. 5Go, B and C). As shown in Fig. 5BGo, 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{Delta}–150/–142) or severely decreased (p-381Luc{Delta}-211/-204 and p-381Luc{Delta}-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. 5CGo). 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 p–182Luc+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 p–182Luc+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{Delta}-290/-276 and p–381Luc{Delta}-211/-204 (Fig. 5BGo). 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 sites—irrespective of orientation or position—rather 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. 6AGo), 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. 6BGo).



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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.

 
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, 57–62% of wild-type repression; I439S, 44–52% of wild-type repression; Fig. 7Go). 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. 7Go). 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.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The development and function of the fetal adrenal cortex in humans and higher primates differ from other species because a distinct compartment—the fetal zone—is 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 surprising—given 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 1Go 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 1Go). 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

 
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-1’s 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 number—rather than the precise core or flanking sequences—of 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. 6Go).

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 2Go). 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).


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Table 2. Summary of DAX1 Repression of Basal or SF-1-Transactivated Gene Transcription

 
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. 8Go) (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. 3BGo), 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 ).

 

    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 384–398. 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 1Go 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 manufacturer’s 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 manufacturer’s 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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