NF-1C, Sp1, and Sp3 Are Essential for Transcription of the Human Gene for P450c17 (Steroid 17{alpha}-hydroxylase/17,20 lyase) in Human Adrenal NCI-H295A Cells

Chin Jia Lin1, John W. M. Martens1 and Walter L. Miller

Department of Pediatrics and The Metabolic Research Unit, University of California San Francisco, San Francisco, California 94143-0978

Address all correspondence and request for reprints to: Professor Walter L. Miller, Department of Pediatrics, Building MR IV, Room 209, University of California San Francisco, San Francisco, California 94143-0978.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cytochrome P450c17 catalyzes steroid 17{alpha}-hydroxylase and 17,20 lyase activities, which are required for the biosynthesis of cortisol and sex steroids. Human P450c17 is expressed in a cAMP-responsive, cell-specific, developmentally programmed fashion, but little is known about its transcriptional regulation. Expression of deletion mutants of up to 2,500 bp of human 5'-flanking DNA in human adrenal NCI-H295A cells indicated that most regulatory activity was confined to the first 227 bp. Deoxyribonuclease I footprinting of the proximal promoter identified the TATA box, an steroidogenic factor-1 site, and three previously uncharacterized sites at -107/85, at -178/-152, and at -220/-185. EMSAs and methylation interference assays suggested that the -107/-85 site and the -178/-152 site bind members of the NF-1 (nuclear factor-1) family of transcription factors. An NF-1 consensus sequence generated similar DNA/protein complexes, and antibodies against NF-1C2/CTF2 supershifted the complexes formed by the -107/-85 site, the -178/-152 site, and the NF-1 consensus site. Western blots of nuclear extracts from NCI-H295A cells probed with this NF-1 antiserum identified two NF-1 isoforms between 50 and 55 kDa. The presence of NF-1C2 (CTF2) and CTF5 in NCI-H295A cells was demonstrated by RT-PCR and sequencing. Mutation of both the -107/-85 and the -178/-152 NF-1 sites reduced basal transcription by half. Supershift assays showed that the ubiquitous proteins Sp1 and Sp3 both bind to the -227/-184 region, and that mutation of their binding sites reduced transcription by 75%. Mutation of the Sp1/Sp3 site plus the two NF-1 sites eliminated almost all detectable transcription. Thus, Sp1 and Sp3 binding to the -227/-184 site and NF-1C proteins binding to the -107/-85 and the -178/-152 sites are crucial for adrenal transcription of the human gene for P450c17.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
CYTOCHROME P450c17 is the single protein that catalyzes both 17{alpha}-hydroxylase and 17,20 lyase activities in the biosynthesis of steroid hormones (1, 2, 3, 4, 5, 6, 7, 8, 9, 10). P450c17 is the qualitative regulator of steroidogenesis. In its absence, the human adrenal zona glomerulosa produces 17-deoxy C21 steroids, such as aldosterone, which is the principal human mineralocorticoid; similarly, in the absence of P450c17, the human placenta produces progesterone, which is needed to maintain pregnancy. When the 17{alpha}-hydroxylase activity, but not the 17,20 lyase activity, of P450c17 is present, the human adrenal zona fasciculata produces cortisol, the principal human glucocorticoid. When both the 17{alpha}-hydroxylase and 17,20 lyase activities are present, as in the adrenal zona reticularis, testicular Leydig cells, or ovarian theca cells, 17 hydroxy C19 precursors of sex steroids are produced. Thus the expression of P450c17 is of fundamental interest to mammalian physiology and reproduction. When P450c17 is expressed in a particular cell type, the differential regulation of its 17{alpha}-hydroxylase and 17,20 lyase activities is determined at a posttranslational level by phosphorylation of the P450c17 protein (11) and by the availability of electron-donating redox partners (5, 9, 10, 12, 13, 14, 15, 16, 17).

However, a more basic level of regulation concerns the tissue-specific regulation of expression of the gene for P450c17. Both human and rodent P450c17 genes are expressed in a developmentally programmed (18, 19, 20) and hormonally responsive (21, 22, 23) fashion. However, the tissue specificity of P450c17 is complex and differs among mammals. The absence of P450c17 mRNA in ovarian granulosa cells and its presence in theca cells prove the two-cell theory of mammalian ovarian sex steroid production (24); similarly, de novo human placental steroidogenesis is confined to progesterone, with estrogen synthesis requiring precursor steroids produced by the fetal adrenal, as the human placenta produces no P450c17 (24); finally, the rodent uses corticosterone, rather than cortisol, as its principal glucocorticoid, as rodent adrenals produce no P450c17 (24). In the human adrenal, the zona glomerulosa produces no P450c17 (25), but the zona fasciculata and reticularis do, accounting for the production of 17-deoxy C21 steroids in the glomerulosa. Thus the tissue-specific expression of P450c17 is unique among the steroidogenic enzymes (26) and is a major factor in determining the class of steroids produced.

Human P450c17 is encoded by a single gene (27), formally termed CYP17, that is located on chromosome 10q24.3 (28, 29, 30) and is expressed in both the adrenals and the gonads (7, 31). Substantial effort has been directed toward elucidating the factors required for tissue-specific expression of P450c17. To date, studies in rodents have been most successful. In the rat P450c17 promoter, a proximal element conferring both basal and cAMP-induced activity, is located at position -85/-55 and interacts with steroidogenic factor-1 (SF-1) (23, 32). A second cis-acting element containing three putative recognition sequences for zinc-finger nuclear receptors was identified at bases -447/-399 of the rat P450c17 promoter (33). A complex machinery of multiple orphan nuclear receptors including SF-1, COUP-TF (chicken ovalbumin upstream promoter transcription factor), NGF-IB (nerve growth factor inducible protein B), and two previously unknown factors, termed steroidogenic factor inducer of transcription-1 and -2, have been shown to bind to this sequence (33). Recently, steroidogenic factor inducer of transcription-1 has been identified as SET nuclear phosphoprotein, revealing a novel role for this protooncogene product (34). Related work in cattle has identified two sequences important for both basal and hormonally induced activities at positions -243/-225 and -80/-40 of the bovine promoter (35). In the bovine promoter, SF-1 and COUP-TF bind to the -80/-40 element in a mutually exclusive fashion (36). A cooperative interaction between members of the Pbx1 (37) and Meis1 families of proteins (38) regulates the activity of the -243/-225 element.

Initial characterization of human promoter/reporter constructions transfected into various cell lines identified the region between -235 and -184 as playing a major role in basal transcription, and the region between -184 and -104 as playing a major role in hormonally induced expression of the human gene when it was analyzed in mouse adrenal Y1 cells (21). However, these regions exerted much weaker activity when transfected into mouse Leydig MA-10 cells (21). This presented an important conundrum. The endogenous mouse P450c17 gene is not expressed in mouse adrenal tissue or in mouse adrenocortical carcinoma Y1 cells, yet human P450c17 promoter/reporter constructions were active and showed appropriate tropic hormone-induced expression in these cells (21). By contrast, the endogenous mouse P450c17 gene is active in mouse Leydig cells, yet when transfected into mouse Leydig MA-10 cells the human P450c17 promoter/reporter constructions were expressed at about 10% the levels seen Y-1 cells (21). Thus it appeared that mouse cell lines could not provide reliable information about the transcriptional regulation of human P450c17. This concern was substantiated by showing that there were substantial differences in the pattern of expression of human P450c17 promoter constructs when transfected into mouse Y1 or into human adrenal NCI-H295A cells (39). The characterization of expression of the human P450c17 promoter in human adrenal NCI-H295A cells located previously unidentified cis-acting elements in the human P450c17 promoter. We now characterize these elements in substantially greater detail and identify potential trans-acting proteins that appear to activate these elements.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Sequence and Activity of the Proximal Promoter
Our human P450c17 proximal promoter, which was used to create our -63 bp promoter/reporter construct (39), contains four consecutive adenine residues at positions -52 to -49 (21, 27). However, others have reported only three A’s in this region (40). To determine whether this was a polymorphism that might influence promoter activity, we used PCR and direct sequencing of this region from seven people of diverse ethnic origins and always detected the three-A sequence. Thus, the quadruple-A sequence resulted from an unusual polymorphism in our previously published human gene sequence. The -63 construct was rebuilt to contain three A’s using site-directed mutagenesis, and the luciferase activities of the -63 constructs containing either three or four A’s were compared by transfection into NCI-H295A cells. Statistical analysis using the Mann-Whitney U test showed no difference between triple and quadruple A -63 vector (P = 0.92, n = 5) (Fig. 1AGo).



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Figure 1. Activity of Human P450c17 Promoter/Reporter Constructs Transiently Transfected into Human NCI-H295A Cells

A, Activities of the P450c17 sequence variants. There is no significant difference between the presence of three or four adenines at -52/-49 in the -63 construct, or in the presence of C or T at position +27 in the -227 construct. B, Basal activities are shown as a function of -100 construct, arbitrarily set as equal to 100%. The -63, -206, and -227 constructs are significantly more active than the -80 through -2,500 (by Knuskall/Wallis ANOVA). C, Activities after stimulation with 1 mM 8-Br-cAMP presented as a percentage of the basal activity value for each construct. The data are shown as mean ± SEM for four to six independent transfection experiments, each performed in triplicate.

 
Another sequence variant is the presence of thymidine (21, 27) or cytosine (41) at nucleotide +27 in the 5'-untranslated DNA. Some have reported a statistical association of the C variant with the polycystic ovary syndrome (41), breast cancer (42, 43), and colorectal adenomas (44), suggesting that the C variant creates a novel Sp1 recognition site that might increase P450c17 gene transcription. However, neither sequence variant binds Sp1, and the putative association of this sequence polymorphism with breast cancer has been questioned (45). To test this hypothesis directly, we built our -227 construct, with either T or C at position +27, and found no difference in transcriptional activity (Fig. 1AGo) (n = 4, P = 0.39 by Mann Whitney U test). Therefore, if the C variant is indeed associated with any of these disorders, it is as a linked polymorphism and not related to P450c17 transcription.

Promoter Activity to 2,500 bp
Twelve promoter/reporter constructs ranging from 63 bp to 2.5 kb in length were expressed in NCI-H295A cells. The short 63-bp fragment of the human P450c17 promoter conferred substantial basal transcriptional activity compared with the promoterless control luciferase vector (Fig. 1BGo). This transcriptional activity decreased to about one-third to one-half of this level with addition of sequences from -80 to -184, suggesting the presence of an inhibitory element between -80 and -63. The basal transcriptional activity increased with the -206 construct and was maximal with the -227 vector (Fig. 1BGo), consistent with our previous results (39). NCI-H295A cells transfected with each vector were also stimulated with 1.0 mM 8-bromo-cAMP (8-Br-cAMP). The response to cAMP induction was also observed within the first 63 bases of the promoter (Fig. 1CGo). The cAMP-induced activity in the -63 fragment was about 250% of basal activity and remained above 200% with all of the constructs, except for the -206 and -227 bp constructs, consistent with the data obtained previously using 0.1 mM cAMP (39).

Database searching (46) identified several potential regulatory sequences in the -227 construct. The TTTAAAA sequence at -24/-17 corresponds to an atypical TATA box found in the human (27), bovine (47), pig (48), mouse (49), and rat (23) genes. There is a potential consensus recognition sequence for the orphan nuclear receptor SF-1 (TCAAGGTGA) at -48/-40 and for GATA-1 (GGCAAGAGATAAC) at -68/-56. The presence of a putative SF-1 in the initial portion of P450c17 promoter has also been reported for rat (23), mouse (49), pig (48), and cow (47). A potential inverted nuclear factor-1 (NF-1) recognition site was identified at -102/-90, and the antisense strand sequences at -168/-159 and -215/-201 correspond to the recognition sequences for Sp1 and the vitamin D receptor.

Footprinting of the Proximal Promoter
DNA-protein interactions in the human P450c17 promoter were examined using deoxyribonuclease I (DNase I) footprinting. A PCR-generated 345-bp probe was labeled at each end and footprinted, detecting five protected regions (Fig. 2Go). The first protected region corresponded to the TTTAAAA sequence at bases -24/-18. A single large protected region encompassed bases -70/-33, containing recognition sites for SF-1 (-58/-50) and GATA-1 -68/-56 (results not shown). Three other protected regions at -107/-85 (FP1), at -178/-152 (FP2), and -220/-185 (FP3) that had not been described previously were also identified (Fig. 2Go).



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Figure 2. DNase I Footprinting of Proximal Promoter Sequences

A 345-bp double-stranded probe labeled either on the sense or antisense strand was generated by PCR, incubated with different amounts of NCI-H295A cell nuclear extract (NE), and then partially digested with DNase I. A, On the sense strand, five protected regions are seen corresponding to the TATA box (-24/-17), a large footprint (-70/-33) encompassing the SF-1 site at -48/-40 and three upstream footprints termed FP1 (-107/-84), FP2 (-178/-152), and FP3 (-220/-185). B, On the antisense strand, only the region encompassing FP1, FP2, and FP3 (bases -75 to -225) is shown.

 
Identification of DNA/Protein Complexes
Based on our functional results from the transfection experiments, we hypothesized that the proteins forming the footprints at -107/-85 (FP1), -178/-152 (FP2), and -185/-220 (FP3) may be involved in the basal regulation of P450c17 transcription in NCI-H295A cells. To characterize the DNA-protein binding in these regions, we used electrophoretic mobility shift assays (EMSAs). When incubated with nuclear extract from NCI-H295A cells, all three probes produced retarded doublet complexes (Fig. 3Go). A 100- to 300-fold excess of unlabeled competitor prevented the formation of retarded complexes by all three probes. By contrast, a 1,000-fold molar excess of an unrelated, double-stranded DNA fragment containing the SF-1 site from nucleotides -162/-129 of the Z region of the human P450c21 promoter (50, 51), and a mutant DNA fragment derived from bases -155/-131 of human P450scc promoter (52, 53) did not compete for complex formation with either FP1 (Fig. 3AGo) or FP2 (Fig. 3BGo). Similarly, 1,000-fold molar excess of FP1 and FP2 failed to displace the formation of FP3 complexes (Fig. 3CGo). Thus, specific DNA-protein interactions occurred at each footprinted region we analyzed.



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Figure 3. Electrophoretic Mobility Band-Shift Experiments

Double-stranded, 32P end-labeled probes corresponding to FP1, FP2, or FP3 were incubated with NCI-H295A cell nuclear extract without or with excess unlabeled competitor. A, Labeled FP1 probe incubated without and with a 30-, 100-, and 300-fold molar excess of unlabeled FP1 or a 1,000-fold molar excess of Z-162/-129 (Oligo 1) or SCC -155/-131 (Oligo 2). B, Labeled FP2 incubated without and with 30-, 100-, and 300-fold molar excess of unlabeled FP2 or 1,000-fold excess of Oligos 1 and 2, as in panel A. C, Labeled FP3 incubated without and with 30-, 100-, and 300-fold excess of unlabeled FP3 or 1,000-fold excess of FP1 or FP2.

 
Identification of Bases Interacting with Proteins
To identify the purine bases responsible for the interaction of each of our three footprinted regions with nuclear proteins, we used methylation interference assays. Double-stranded EMSA probes were labeled on either the sense or antisense strand, partially methylated, incubated with NCI-H295A nuclear proteins, separated on EMSA gels, purified, and cleaved with piperidine. The location of purines that are methylated in the free probes, but not in the retarded ones, are shown in Fig. 4Go. In FP1, a pair of G’s at -99/-98 and a single G at -93 were cleaved on the sense strand in the free, but not in the bound, probe, and two G’s at -90/-89, a G at -97, and two pairs of A’s at -79/-78 and -75/-74 were cleaved on the antisense strand (Fig. 4AGo). In FP2, cleavage detected protected G’s at positions -174, -173, and -166 of the sense strand, and two G’s at -165/-164 and two A’s at -158/-157 were protected on the antisense strand (Fig. 4BGo). In FP3, A’s at -217, -216, -212, and -199 were protected on the sense strand, and a GAG trinucleotide sequence at -197/-195, a pair of A’s at -205/-204, a G at -208, and an A at -190 were protected on the antisense strand (Fig. 4CGo). Since the probes in our assay were methylated before the incubation with nuclear proteins, the protected bases in the bound fraction represent those that are either essential for the DNA-protein binding or important for stabilization of the complex.



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Figure 4. Methylation Interference Assay

Probes FP1, FP2, and FP3 were end-labeled on the sense or antisense strand, methylated, and incubated with NCI-H295A cell nuclear extract. After electrophoresis as for EMSA, the "Free" and "Bound" fractions of each probe were isolated, cleaved with piperidine, analyzed by electrophoresis through denaturing polyacrylamide gel, and subjected to autoradiography. In each panel the cleavage patterns of free and bound fractions are compared for both the sense and antisense strands. The purines that appear to participate in DNA-protein interactions appear as protected band in the bound fraction and are marked with an asterisk.

 
To determine whether the specific nucleotides identified in the methylation interference assay were important for protein interactions with the FP1, FP2, and FP3 sequences, we prepared mutants of the FP1, FP2, and FP3 oligonucleotides in which the protected base pairs in all three sequences and the GAG trinucleotide of FP3 were changed (Table 1Go). The abilities of these mutant sequences to form retarded complexes or to inhibit the formation of wild-type complexes were then tested using EMSA (Fig. 5Go). FP1 probes mutated at positions -99/-98 (FP1-m1) and -90/-89 (FP1-m2) did not inhibit the binding of wild-type FP1 to nuclear proteins (Fig. 5AGo), and when these mutants were incubated directly with NCI-H295A nuclear proteins, they formed less intense complexes (not shown), confirming the importance of the mutated bases. FP2 probes mutated at positions -174/-173 (FP2-m1) and -165/-164 (FP2-m3) similarly decreased the ability of FP2 to compete for NCI-H295A nuclear proteins (Fig. 5BGo). Mutation of the GAG sequence at -197/-195 in the FP3 probe (FP3-m2) completely abolished the formation of FP3 complexes (Fig. 5CGo). Mutation of other protected purines in FP1, FP2, or FP3 (oligonucleotides FP1-m3, FP1-m4, FP2-m2, FP3-m1, and FP3-m3; Table 1Go) did not significantly affect the ability of these probes to bind to nuclear protein. Thus, G pairs present on both the sense and antisense strands of FP1 and FP2 play important roles in forming complexes with nuclear proteins from NCI-H295A cells. Similarly, the GAG trinucleotide sequence in FP3 is essential for its interaction with NCI-H295A nuclear proteins.


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Table 1. Activities of the Plasmids Shown in Fig. 7Go

 


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Figure 5. Electrophoretic Mobility Band-Shifts with Mutant Probes

The purines in the FP1, 2, and 3 oligonucleotides identified from the methylation interference assay were mutated as shown in Table 1Go, and EMSAs were done with NCI-H295A nuclear proteins. A, Wild-type FP1 probe forms a pair of bands in the absence of competitor; these bands are competed by increasing concentrations of unlabeled FP1 and by the FP1-m3 and FP1-m4 mutant probes but not by the FP1-m1 and FP1-m2 mutants. B, Wild-type FP2 probe forms a pair of bands in the absence of competitor; these bands are competed by increasing concentrations of unlabeled FP2 and by the FP2-m2 mutant but not by the FP2-m1 and FP2-m3 mutants. Similarly, the FP2-m2 mutant forms a pair of complexes indistinguishable from wild-type, but the FP2-m1 and FP2-m3 mutants do not. C, Wild-type FP3 probe and the FP3-m1 and FP3-m3 mutants all form a pair of bands, but the FP3-m2 mutant, carrying the mutation in bases -197/-195, does not.

 
Footprints 1 and 2 Are Formed by NF-1C
The antisense strand of FP1 and the sense strand of FP2 have similar motifs of protected bases consisting of a pair of G’s and a single G separated by six nucleotides. Because this pattern resembles the consensus recognition sequence for the transcription factor NF-1 ((C/T)TGGC(N)6CC(N)3) (54), we determined whether FP1 and FP2 are related to an NF-1 recognition sequence. Both FP1 and FP2 formed similar doublet complexes (Figs. 3Go and 6Go) and are able to inhibit each other’s interaction with NCI-H295A nuclear proteins at similar concentrations (Fig. 6AGo). Complexes formed by an oligonucleotide (NF-1-Ad; Table 1Go) of the NF-1 recognition site from adenovirus (55) resemble the complexes formed by FP1 and FP2 (Fig. 6AGo) and could also be inhibited by excess unlabeled FP1 and FP2. Oligonucleotides (NF-1-Ad-m1 and NF-1-Ad-m2) harboring mutations in the consensus binding site for NF-1 could compete neither for complexes formed by the wild-type NF-1-Ad oligonucleotide nor for those formed by FP1 and FP2, but an oligonucleotide mutated outside the NF-1 consensus sequence (NF-1-Ad-m3) retained the ability to compete (Fig. 6BGo and results not shown). Thus, the EMSA with the mutant FP1 and FP2 probes and the competition with the adenoviral NF-1 probe suggest that the FP1 and FP2 footprints result from interactions with an NF-1-like transcription factor. Furthermore, the mutant NF-1 competition studies indicate that NF-1 consensus sequences are required for binding to NCI-H295A nuclear proteins, as suggested by the methylation interference data.



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Figure 6. Similarity of FP1 and FP2 to NF-1

A, Labeled oligonucleotides for FP1, FP2, and a probe of the consensus adenovirus NF-1 recognition site (NF-1-Ad; Table 1Go) were incubated with NCI-H295A cell nuclear extract and unlabeled oligonucleotides. The probes are indicated below the figure; the competitors are indicated above. FP1 and FP2 were able to disrupt each other’s interaction with nuclear proteins, although FP2 appeared to bind more strongly. NF-1-Ad formed a pair of complexes indistinguishable from those formed by FP1 or FP2, and the formation of complexes with NF-1-Ad could be inhibited by either FP1 or FP2. B, Wild-type NF-1-Ad probe forms a pair of bands in the absence of competitor. These bands are competed by unlabeled wild-type NF-1-Ad oligonucleotide and by the NF-1-Ad-m3 oligonucleotide that is mutated outside the consensus region, but not by two mutant oligonucleotides (NF-1-Ad-m1 and NF-1-Ad-m2) that are mutated in the consensus binding sequence.

 
Supershift assays with a rabbit polyclonal antiserum raised against human NF-1C2/CTF-2, which recognizes only NF-1C proteins (56, 57) indicated that an NF-1C protein contributes to the complexes formed by FP1 and FP2. The complexes formed by FP1, FP2, and by the NF-1-Ad consensus oligonucleotide were supershifted by the antibody (Fig. 7AGo), but the complexes formed by FP3 were not. The supershifted complexes were very large and did not enter an 8% polyacrylamide gel, but did enter a 4% gel, showing that the antiserum formed discrete complexes and not merely an aggregate (Fig. 7BGo). Thus, supershift assays confirm that an NF-1-like protein that is recognized by antiserum to NF-1C2/CTF2 interacts with the DNA in FP1 and FP2 but not in FP3.



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Figure 7. NF-1C-Like Proteins Bind to FP1 and FP2

A, Antibody supershifts of EMSA complexes formed by FP1, FP2, FP3, and NF-1-Ad analyzed on 8% polyacrylamide gel. FP1, FP2, and NF-1-Ad form doublet complexes with the antiserum to NF-1C2/CTF2. In the presence of this antiserum, the complexes are either supershifted (note the radioactive material remaining in the well) or competed; the antiserum has no effect on the complexes formed by FP3. B, Analysis of the FP1, FP2, and NF-1-Ad complexes with and without antiserum to NF-1C2/CTF2 by an extended run on 4% polyacrylamide gel. In each case the antiserum forms a supershifted complex (indicated with an asterisk) that enters the gel. The FP1 complex without the antibody consistently ran slightly faster than the FP2 and NF-1-Ad complexes, despite a longer length of the FP1 probe. C, Western blot. Nuclear proteins (40 µg) from NCI-H295A and JEG-3 cells were separated on SDS 10% polyacrylamide gel transferred to a nylon membrane and probed with the antiserum raised to NF-1C2/ CTF2. Two bands between approximately 50–55 kDa were detected in nuclear extracts from NCI-H295A cells; no proteins were detected in nuclear extracts from JEG-3 cells. D, Presence of NF-1C in NCI-H295A cells. RT-PCR was performed (+RT) with total RNA from NCI-H295A cells using primers specific for all known NF-1C/CTF mRNA species (Table 2Go). For comparison, the cDNAs of NF-1C/CTF1, -2, and -3 were also amplified separately. The controls shown are a cDNA sample amplified in the absence of reverse transcriptase (-RT); and a water control (W) for the PCR reaction. The samples were displayed on a 0.7% agarose gel. Two cDNA products were identified in NCI-H295A cells. Complete sequencing identified the larger band as NF-1C2/CTF2 and the smaller band as NF-1C5/CTF5.

 
Because there are multiple isoforms of NF-1C, we sought to determine which NF-1 proteins are present in the NCI-H295A nuclear extracts. Western blots of nuclear extracts from NCI-H295A cells and human cytotrophoblast JEG-3 cells probed with the antiserum to NF-1C2/CTF2 showed two bands of approximately 50–55 kDa in NCI-H295A nuclei, but no detectable NF-1-like proteins in JEG-3 nuclei (Fig. 7CGo). Since JEG-3 cells have little NF-1 protein and lack NF-1 transcriptional activity (58), the absence of bands in the JEG-3 sample indicates that the antiserum is highly specific. To identify the isoforms of NF-1C present in NCI-H295A cells, we performed RT-PCR using primers designed to amplify the full length of all known alternatively spliced NF-1/CTF mRNA species (Fig. 7DGo). NCI-H295A cells contained only two NF-1C mRNA species; cloning and sequencing of these two bands identified the upper one as NF-1C2/CTF2 and the lower one as NF-1C5/CTF5.

Function of the DNA/Protein Complexes
To test the functional importance of each of the three footprinted promoter elements, we recreated the mutations that prevented binding in the EMSAs in the -227 promoter/reporter construct, which gave the highest basal transcriptional activity (Fig. 1Go). Mutants carrying each mutation singly, or the multiple mutations FP1+FP2 or FP1+FP2+FP3, were prepared using the Mut-FP1, Mut-FP2, and Mut-FP3 oligonucleotides listed in Table 1Go and expressed in NCI-H295A cells using a promoterless vector as a control (Fig. 8Go). The -227 wild-type construct was 31 times more active than the promoterless control. Mutation of FP1 alone exerted no effect; mutation of FP2 alone decreased promoter activity by approximately 23%, and mutation of both the FP1 and FP2 sites reduced promoter by about 53% (Table 1Go). Thus, FP1 exerts little, if any, activity on basal P450c17 gene expression in human adrenal cells, whereas FP2 seems to be more important. The FP3 site is much more important to the basal activity of the -227 construct than the FP1 and FP2 sites. Ablation of the FP3 site alone reduced promoter activity by about 71%, and mutation of FP3 in conjunction with mutation of the FP1 and FP2 sites reduced promoter activity to the level of the promoterless control (Table 1Go). Thus, FP3 is of fundamental importance to the basal transcription of human P450c17 gene expression in NCI-H295A cells and the cooperation between FP1 and 2 and FP3 is required for full activity.



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Figure 8. Functional Assays of the Roles of FP1, FP2, and FP3

Mutations that disrupt the DNA-protein interactions of each sequence (Mut-FP1, Mut-FP2, and Mut-FP3 in Table 1Go) were introduced into a vector containing the first 227 bases of the human P450c17 promoter creating vectors that carry mutations of each individual site, a combined mutation of FP1 and FP2, or all three sites. The FP3 mutation was also introduced into a vector carrying the first 2,500 bases of the P450c17 promoter. All vectors were transiently expressed in NCI-H295A cells, and the results are shown as a percentage of the activity of the 227-bp wild-type vector, set at 100%. The data are the means ± SEM of four to nine individual experiments, each done in triplicate. The statistical analysis of these results is shown in Table 1Go.

 
To assess the potential importance of elements upstream from the 227-bp basal promoter, we mutagenized the same three bases mutated previously in FP3 in the -2,500 construct. Consistent with the data in Fig. 1Go, the 2,500-bp promoter fragment showed 51% of the activity of the 227-bp fragment. Mutation of FP3 in the 2,500-bp promoter reduced activity to 22% of the level of the wild-type 227-bp basal promoter, which is similar to the reduction to 29% of the wild-type seen with the 227-bp promoter (Table 1Go). Thus, elements up to 2,500 bp upstream cannot compensate for the loss of FP3, confirming that the DNA/protein interaction at FP3 is crucial for full adrenal expression of P450c17.

The data in Fig. 8Go indicate that FP3 is the most important cis-acting element driving basal transcription of the P450c17 promoter constructs in NCI-H295A cells. To determine whether FP3 alone is sufficient to drive transcription, we fused the wild-type FP3 oligonucleotide and the inactive FP3-m2 oligonucleotide in one or more copies in both orientations upstream from the minimal promoter of the herpes simplex thymidine kinase (TK) gene (TK32/luc) and transfected these constructs into NCI-H295A cells (Fig. 9Go). In the forward orientation, one, two, or three copies of FP3 caused a 10-, 35-, or 53-fold induction of luciferase activity, indicating dose dependency, but four copies did not increase luciferase activity further, indicating the effect was saturated with three copies. In the reverse orientation, one or two copies of FP3 caused a 10- or 32-fold induction, showing that the dose-dependent induction was independent of the orientation. By contrast, the FP3-m2 mutant that was unable to yield a band shift in Fig. 5CGo had a minimal effect on TK32/luc activity. This indicates that the FP3 element alone can act as an enhancer element while the mutant FP3 element cannot.



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Figure 9. FP3 Is Sufficient to Promote Transcription

NCI-H295A cells were transfected with a minimal promoter vector (TK32/luc) without (TK) and with one (1F), two (2F), or three (3F) copies of the FP3 oligonucleotide in the forward and one (1R) or two (2R) copies in the reverse orientation. NCI-H295A cells were also transfected with a TK32/luc containing one, two, or three copies of the inactive FP3-m2 oligonucleotide in the forward and one or two copies in the reverse orientation. FP3, but not FP3-m2, acts as an enhancer element that is active in NCI-H295 cells in an orientation-independent manner. Data are the means ± SEM of three experiments, each performed in triplicate.

 
To determine whether FP3 contributes to the tissue specificity of P450c17, we examined its ability to bind nuclear proteins from various cell types and to enhance transcription from the heterologous minimal TK32/luc promoter in these cells (Fig. 10Go). In EMSA analysis, the wild-type FP3 oligonucleotide formed two DNA/protein complexes with nuclear proteins from all cell types examined (NCI-H295A, MA-10, Y-1, JEG-3, COS-1, HeLa, HepG2); by contrast, the mutant FP3-m2 oligonucleotide did not form a complex with nuclear proteins from any of these cells (Fig. 10AGo). With Y-1 nuclear extract, the more rapidly migrating complex appeared to be smeared or to consist of three fainter components; this difference was consistent but its significance is not clear. In all cases, both bands could be competed by a 100-fold excess of unlabeled probe (not shown). Thus, the protein(s) binding to FP3 are widely distributed among human steroidogenic cells (NCI-H295A, JEG-3), mouse steroidogenic cells (MA-10, Y-1), and human and monkey nonsteroidogenic cells (COS-1, HeLa, HepG2).



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Figure 10. FP3 Acts in a Tissue-Independent Fashion

A, EMSA of a 32P end-labeled probes corresponding to FP3 (WT) and FP3-m2 (m2) were incubated with nuclear extracts from NCI-H295A, MA-10, Y-1, JEG-3, COS-1, HeLa, and HepG2 cells. The nuclear proteins from all cell types formed similar complexes, although the relative abundance of the different complexes varied. The FP3-m2 mutant oligonucleotide did not form complexes with any of the extracts. B, FP3 promotes transcription in both steroidogenic and nonsteroidogenic cells. MA-10, Y-1, JEG-3, COS-1, HeLa, and HepG2 cells were transfected with the empty TK32/luc vector (TK) or with TK32/luc containing one, two, or three copies of FP3 (1F, 2F, 3F), and one or two copies of FP3-m2 (M1F, M2F) in the forward orientation; data displayed as in Fig. 9Go. FP3, but not the mutant, promoted transcription in a dose-dependent fashion irrespective of the cell type.

 
To determine whether the FP3 binding protein(s) in the different cells could transactivate via an FP3 element, we used the same wild-type and mutant FP3/TK32/luc promoter constructs used in Fig. 9Go to transfect each of these cell types (Fig. 10BGo). Although there are minor quantitative differences, the activities in each of these six cell types are remarkably similar to the activities seen in NCI-H295A cells (Fig. 9Go): one, two, and three copies of FP3 increase TK32/luc activity in a dose-dependent fashion, while the FP3-m2 mutant has no significant effect. We could determine no quantitative or qualitative correlation among the minor differences seen in the EMSA data in Fig. 10AGo and the functional data in Fig. 10BGo. Thus, the protein(s) binding to FP3 are widely expressed and active in various cellular contexts, and hence do not appear to contribute to the tissue-specific distribution of P450c17.

Footprint 3 Is Formed by Sp1 and Sp3
To identify the proteins binding to FP3, we first located its protein binding site(s) more precisely. Oligonucleotide FP3.1, comprising the proximal sequences of FP3 (-204/-182), formed complexes that looked like the FP3 complexes while oligonucleotide FP3.2, containing the distal region (-219/-198), could not (Fig. 11AGo). Similarly, unlabeled FP3.1 could compete for the complexes formed by FP3, whereas FP3.2 could not (Fig. 11BGo). Recent data (59) indicate the presence of an SF-1 site at -205 to -211; the lower band formed by oligonucleotide FP3.2 could be competed by an SF-1 oligonucleotide (data not shown). EMSA with a series of scanning mutants (FP3-m2 to m10) showed that the sequence AGCTCCTCCTCCG is required for binding, and that underlined bases (-197/-190) were most important (Fig. 11CGo). This sequence closely resembles the consensus binding sites for the zinc finger proteins Sp1 (60) and WT-1 (61), and Egr-1/krox-1A (62).



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Figure 11. FP3 Binds Sp1 and Sp3

EMSAs were done with the indicated labeled probes and competitors (Table 1Go) and NCI-H295A nuclear extract. A, The two upper bands formed by the FP3 probe are also formed by the FP3.1 probe, whereas the faster-migrating SF-1 band is formed by FP3.2. B, Labeled FP3 was competed with a 300-fold excess of FP3.1 or FP3.2. C, Labeled FP3 was competed with a 300-fold excess of wild-type FP3 and a series of FP3 scanning mutants. Mutants m2, -8, and -5 fail to compete the upper band, while mutants m7 and m9 compete poorly, defining the site AGCTCCTCCTCCG. D, Antibody supershifts. Left panel, Antiserum to Sp1, but not to SET, WT-1, or NF-1 supershifts the upper band formed by the FP3 probe. Right panel, Antiserum to Sp1 supershifts the upper band, and antiserum to Sp3 shifts the lower band as well as part of the slower migrating complex, and a mixture of both sera shifts both bands, formed either with the FP3 probe (four left lanes) or a consensus Sp1 probe (four right lanes). E, Unlabeled Sp1 oligonucleotide, but not WT-1 oligonucleotide, competes for both upper bands formed by the FP3 probe. F, An indistinguishable pair of upper bands is formed by bovine and mouse-sequence FP3.1 probes.

 
Antibodies to WT-1, NF-1, and SET [a protein recently implicated in rodent P450c17 transcription (34)] did not affect the mobility of the complexes, but antiserum to Sp1 clearly decreased the abundance of the slower (but not the faster) migrating complex (Fig. 11DGo). To determine the identity of the faster migrating complex, we tested an antiserum to a related protein, Sp3. Antiserum to Sp3 removed the faster migrating complex completely and also decreased the abundance of the slower migrating complex. Adding Sp1 and Sp3 antisera together left a small amount of the slower migrating complex. Furthermore, an oligonucleotide containing the Sp1 consensus sequence could compete for the complexes formed by FP3, but oligonucleotides containing the consensus sequences for WT-1, Egr-1, and SET (34) could not (Fig. 11EGo and data not shown). The Sp1 consensus oligonucleotide also formed complexes that were indistinguishable from those formed by the FP3 oligonucleotide, and gave the same pattern as FP3 when treated with the antisera to Sp1 and Sp3 (Fig. 11DGo). Finally, the Sp1/Sp3 binding site is conserved in the murine and bovine genes for P450c17, and both the mouse and the bovine FP3.1 oligonucleotides formed identical-appearing complexes (Fig. 11FGo). Thus, Sp1 and Sp3 are the principal proteins binding to FP3. The expression of Sp1 and Sp3 is ubiquitous (63), which is consistent with the presence of similar-appearing band shifts with nuclear extracts from multiple cell lines (Fig. 10AGo) as well as with the ability of the FP3 element to activate transcription in a variety of cell lines (Fig. 10BGo).


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Several studies have examined the transcription of the P450c17 genes of the rat (23, 32, 33, 34) and cow (35, 36, 37, 38), but few studies have examined the human gene. Our previous work (21) showed that human P450c17 promoter/reporter constructs work well in mouse adrenal Y1 cells, even though the rodent adrenal does not express P450c17 (24) and that these promoter/reporter constructs worked poorly in mouse Leydig MA-10 cells, even though the rodent testis expresses abundant P450c17. Thus, examination of the human P450c17 promoter in rodent cells yields data of uncertain significance. The development of the NCI-H295 cell line (64) offers an attractive alternative, as these cells express their endogenous P450c17 gene under appropriate physiologic regulation (22). The NCI-H295A cells simply provide a faster growing, more readily transfectable form of NCI-H295 cells that adhere to culture plates (39).

Examination of functional elements in the rodent, bovine, and human P450c17 promoters, as revealed by transfection of cells from the corresponding species, shows substantial differences among these three mammalian P450c17 promoters (Fig. 12Go). Thus, observations in one species cannot reliably be applied to the P450c17 gene of another species. This pronounced species-specific variation in the transcriptional regulatory strategies of P450c17 may account for the different patterns of tissue-specific regulation seen among different mammals.



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Figure 12. Comparison of the Reported Proximal Promoter Elements of the P450c17 Genes from Three Species

The footprinted regions we found in the human promoter are shown as open boxes. The numbers indicate the reported positions of each binding site. The promoters from all three species contain an SF-1 site in close proximity to the transcription initiation site, but the other sites are substantially different. The data for the rat (23 32 33 ) and bovine gene (35 36 37 38 ), and the SF-1 data for the human gene (59 ) are taken from the work of other laboratories.

 
Our footprint, band shift competition, and supershift data indicate that proteins related to the NF-1 family of transcription factors bind to FP1 and FP2. NF-1 proteins, which can mediate both initiation of transcription and DNA replication, are encoded by four distinct genes (65). Each NF-1 gene (NF-1A, NF-1B, NF-1C, and NF-1X) can undergo alternative splicing, generating various isoforms of NF-1 proteins (66). NF-1 proteins have a conserved N-terminal region that mediates both DNA binding and dimerization (67). Depending on the promoter context, some NF-1 members are stronger inducers of transcription than others (68, 69). NF-1 isoforms form stable dimers in all possible combinations (70), but NF-1 homodimers are transcriptionally more active than heterodimers (68). Some isoforms of NF-1 can also repress transcription (71).

Antiserum to NF-1C2/CTF2 detected two proteins of approximately 50–55 kDa in NCI-H295A nuclear extracts. This antiserum is specific for NF-1C/CTF (57), indicating that splice variants of NF-1C/CTF bind to FP1 and FP2. Human NF-1C/CTF is encoded by a single gene of 11 exons, and the full-length NF-1C/CTF protein contains 499 amino acids. The carboxy-terminal 100 amino acids encoded by exons 7–11 contain the proline-rich region and the CTD-related motif that are important for transactivation (72, 73). NF-1C2/CTF2 is a splice variant that lacks the region encoded by exon 9, causing a frame shift that terminates translation in exon 10 (73, 74). NF-1C2/CTF2 has been thought to exert little transcriptional activity apparently because it lacks part of the transactivation domain (74). NF-1C3/CTF3 is similar to NF-1C2/CTF2 except it also lacks exon 3 and presumably has little activity (73). NF-1C5/CTF5 also lacks a substantial part of the transactivation domain (exons 9 and 10) but it retains exon 11 in the correct reading frame and is very active in promoting transcription (69). NF-1C7/CTF7 lacks exons 7, 8, and 9 but retains exons 10 and 11 and has an activity that is intermediate between NF-1C1 and NF-1C5 (74).

Using PCR primers in exons 1 and 11 that should amplify all forms of NF-1C, we found that NCI-H295A cells contain NF-1C2/CTF2 and NF-1C5/CFT5; thus it appears that NF-1C2/CTF2 and NF-1C5/CFT5 are the principal contributors to the activity of FP1 and FP2. Because there are slight differences in size of the complexes formed by FP1 and FP2 (Fig. 7BGo), it is possible that FP1 and FP2 exert slightly different preferences for NF-1C2 and NF-1C5. It is not clear whether the difference in orientation of the NF-1 sites in FP1 and FP2 is significant.

Our data provide the first evidence that NF-1 proteins are expressed in adrenal cells and contribute to the expression of P450c17. Whether or not the absence of their expression in JEG-3 cells is sufficient to explain the absence of P450c17 expression in these cells is unclear. Differential expression of NF-1 isoforms has been implicated in the tissue-specific expression of aldolase A in a subset of muscle fibers (55). It is conceivable that FP1 and FP2 might play a regulatory role in determining the tissue-specific expression of human P450c17. Interestingly, neither NF-1 site is conserved in rodent P450c17 promoters, which are inactive in the rodent adrenal.

The DNA/protein interaction of FP3 appears to be crucial for human P450c17 promoter activity, as disruption of FP3 eliminates 60–80% of basal transcription in both the 227-bp basal promoter and in the 2,500-bp construct. Despite its importance for P450c17 in human adrenal cells, it is clear that FP3 is not a tissue-specific regulator of P450c17 expression, as FP3 binding and transactivation activity are present in all cell lines tested, including nonsteroidogenic cells and steroidogenic cells that do not express P450c17.

Scanning mutagenesis and antibody supershift/competition experiments show that FP3 binds the ubiquitously expressed zinc-finger transcription factors, Sp1 and Sp3. The identification of these factors is consistent with the ability of this DNA segment to foster transcription in a wide variety of nonsteroidogenic cell lines. Three human Sp3 proteins are generated from one Sp3 mRNA through alternative transcription initiation (75). Consistent with previous reports, the two Sp3 species of approximately 78–80 kDa comprise the faster migrating FP3 complex, and the 110-kDa form of Sp3 contributes to the slower migrating complex (75). The single form of Sp1 is also 110 kDa (76) and comigrates with the slower migrating Sp3/FP3 complex. The differences in the intensity of these two complexes formed by nuclear extracts from different cell lines probably reflect different levels of Sp1 and Sp3 in these cells.

After this work was completed, a paper focusing on the role of SF-1 in human P450c17 expression reported the presence of an SF-1 site at -211/-204 (59). However, our FP3.2 probe (-219/-199), which encompasses this region, did not compete for the principal bands formed by the FP3 oligonucleotide but did compete for a fainter, more rapidly migrating band (Fig. 11BGo). Other preliminary data in our laboratory are consistent with this faster-migrating band representing SF-1; thus, the data from both groups are consistent. However, the binding of SF-1 to the FP3 region seems to be of lesser importance because the mutated FP3 element, which contains a disrupted Sp1/Sp3 binding site but an intact SF-1 site, has little activity in both nonsteroidogenic and steroidogenic cells. A synergistic interaction between SF-1 and Sp1 has been reported for the bovine P450scc promoter (77); hence, the ubiquitous transcription factor Sp1 might increase tissue-specific expression. Sp1 binding is conserved in the human, rodent, and bovine P450c17 promoters (Fig. 11EGo), suggesting this may be a general phenomenon.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Plasmids
The P450c17 promoter plasmids in the vector pMG3 have been described previously (39); all extend from +63 to the base number indicated in the construction name; base numbers are counted from the transcriptional start site (27). The -2.5 kb P450c17 deletion/luciferase plasmid was prepared by direct subcloning into pMG3 luciferase vector. The shorter constructs were prepared by PCR using the -2.5 kb c17pMG3 as the template. Plasmids pBS-CTF1, CTF2, and CTF3 contain the complete cDNA of the respective NF-1C/CTF in the SmaI site of pBluescript (73).

For site-directed mutagenesis studies of cis-regulatory elements, a fragment of the human P450c17 promoter extending from bases -1 to -227 was generated by PCR using the -2.5 kb c17pMG3 plasmid (39) as template, and primers -1 and -227 (Table 1Go). The PCR fragment was then cloned into pCRII vector (pCRII-TOPO, Invitrogen, Carlsbad, CA) by TA cloning, and the correct orientation and sequence were verified by direct sequencing. This -227c17pCRII plasmid was then used as template for the site-directed mutagenesis experiments. The mutated fragments were cleaved from pCRII with BamHI and XhoI and substituted into the -227c17pMG3 plasmid by replacement cloning. For mutation of FP3 in the -2,500 c17pMG3 vector, the mutant segment was inserted by replacement cloning of an 844-bp PvuII/XhoI fragment.

For FP3 enhancer activity studies, single and multiple copies of the wild-type and the FP3-m2 mutant of the FP3 oligonucleotide were inserted in both orientations into the BamHI site of TK32/luc. This vector contains the basal promoter element (nucleotides -32 to +55) of the herpes simplex virus thymidine kinase promoter fused to the luciferase gene of the empty luciferase vector {Delta}luc. The fidelity of all constructs was verified by restriction enzyme digestion and sequencing.

Site-Directed Mutagenesis
Using the appropriate mutant oligonucleotide (Table 1Go), mutations were introduced into -227c17pCRII by oligonucleotide-mediated mutagenesis using Pfu DNA polymerase and selective digestion of the original template with DpnI (78). Double mutants were created by the simultaneous use of two nonoverlapping mutant oligonucleotides as described (79).

Transfections and Luciferase Assay
An adherent subpopulation of NCI-H295 cells, termed NCI-H295A (39), was grown in RPMI 1640 medium supplemented with 2% FBS, antibiotics (100 U/ml penicillin, 100 µg/ml streptomycin, 0.25 µg/ml amphotericin), 5 µg/ml insulin, 5 µg/ml transferrin, and 5 ng/ml selenite, as described previously (39). Mouse Y1 adrenal carcinoma cells (a generous gift from Dr. Bernard Schimmer, University of Toronto, Toronto, Ontario, Canada) were grown in 50% DMEM-H21:50% Ham’s F12 with 15% heat-inactivated horse serum (HS), 2.5% FBS, and antibiotics. Monkey kidney COS-1 cells, human HepG2 hepatocarcinoma cells, and human HeLa cervical carcinoma cells were grown in DMEM-H21 medium supplemented with 10% FBS and antibiotics. Human JEG-3 choriocarcinoma cells were grown in DMEM-H21 medium supplemented with 5% HS, 5% FBS, and 0.2 mM gentamycin. Mouse Leydig MA-10 cells were grown in Waymouth’s medium supplemented with 15% HS, 2.5% HEPES buffer, and 0.2 mM gentamicin. All cell lines were maintained at 37 C and 5% CO2.

Twenty-four hours before transfection, cells were divided into 2-cm, six-well plates (Falcon 3046, Becton Dickinson, Lincoln Park, NJ) at approximately 50% confluence. For the NCI-H295A cells, the RPMI 1640 medium was replaced by DMEM-H16 supplemented with 10% FBS and antibiotics. Cells were then transiently transfected overnight (6 h for JEG-3 cells) using calcium phosphate precipitates of promoter/reporter constructs (2.5 µg/well). The medium was changed after the transfection period and the cells were grown for an additional 24 h in the presence or absence of 1 mM 8-Br-cAMP. The cells were then lysed and luciferase activity in the cell extract was assayed using the Dual-luciferase Reporter Assay System (Promega, Madison, WI). Cotransfection of 125 ng/well of a Renilla luciferase reporter vector (pRL-CMV, Promega) was used as a control for transfection efficiency. The measured firefly luciferase activity was normalized by Renilla luciferase and the result was expressed as relative luciferase activity.

DNase I Footprinting
For DNase I footprinting, a 345-bp double-stranded DNA was prepared by PCR using primers +15 and -330 (39). Either the forward or reverse oligonucleotide was 5' end-labeled using [{gamma}32P]ATP (Amersham Pharmacia Biotech) and T4 polynucleotide kinase (New England Biolabs, Inc., Beverly. MA) resulting in double-stranded DNA labeled on the sense or antisense strands, respectively. One picomole of labeled probe was incubated with or without nuclear protein in 50 µl of binding buffer (6 mM HEPES, pH 7.9; 0.09 mM EDTA, pH 8.0; 84 mM NaCl, 0.3 mM MgCl2, 6% glycerol, 1% PEG, mol wt 8,000), and 1 µg poly dI-dC at 27 C for 15 min. After the incubation, CaCl2 was added to a final concentration of 2.5 mM and MgCl2 to a concentration of 10 mM, and the probe was digested with 0.003 U of DNase I for 25 sec (control samples) or 40 sec (experimental samples). The footprinted DNA was extracted with phenol/chloroform, precipitated with ethanol, and analyzed by electrophoresis on a denaturing 6% acrylamide gel containing 7 M urea. A 33P-labeled, PCR sequencing reaction of human P450c17 promoter with either oligonucleotide +15 or -330 was used as the ladder for fragment size.

EMSA
Nuclear extracts were prepared from NCI-H295A, JEG-3, COS-1, HepG2, MA-10, HeLa, and Y1 cells as described (80). Double-stranded DNA probes were prepared by annealing the desired sense and antisense oligonucleotides (Table 2Go). Before annealing, the sense oligonucleotides were 5' end-labeled with [{gamma}32P]ATP and T4 polynucleotide kinase, and then purified using Sephadex G50 spin columns (Amersham Pharmacia Biotech). The protein binding reactions were carried out in 20 µl of buffer [25 mM Tris-HCl, pH 7.5, 50 mM KCl, 1 mM dithiothreitol, 5% glycerol] with 300–500 cpm of labeled probe, 1.5 µg of NCI-H295A nuclear protein, and 1 µg of poly dI-dC. For comparison of the complexes formed by FP3 and FP3-m2, 8 µg of nuclear protein were used from each cell line. The mixtures were incubated at room temperature for 15–30 min in the presence or absence of nonlabeled competitor oligonucleotides and in the presence or absence of rabbit polyclonal antiserum to human NF-1C2/CTF-2 (antiserum 8199; a kind gift from Dr. Naoko Tanese, New York University School of Medicine), goat polyclonal antiserum to human Sp1 (PEP2), and Sp3 (D-20) (Santa Cruz Biotechnology, Santa Cruz, CA), mouse monoclonal antiserum to human WT-1 (F6) (Santa Cruz Biotechnology), and a polyclonal antiserum to SET (81). Free, shifted, and supershifted probes were separated through 4% or 8% nondenaturing polyacrylamide gel in 45 mM Tris-borate (pH 8.0), 1 mM EDTA.


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Table 2. Oligonucleotides Used in PCR Cloning, EMSA, and Site-Directed Mutagenesis

 
Methylation Interference Analysis
The same oligonucleotides used as probe in the EMSA were end-labeled and double-stranded probes were prepared as described. About 106 cpm of each labeled probe was methylated with 1 µl of 99% dimethyl sulfate (Aldrich Chemical Co., Milwaukee, WI) in 100 µl of 50 mM sodium cacodylate (pH 8.0), 0.1 mM EDTA at room temperature for 10 min. The methylation was stopped by adding 35 µl of 2.5 M ß-mercaptoethanol, 3.0 M ammonium sulfate, pH 7.0, and the methylated probe was purified using a Sephadex G-50 spin column. EMSAs were performed using partially methylated probes (105 cpm), 10 µg of nuclear protein, and 2 µg of poly dI-dC. DNA bands were identified by autoradiography, gel pieces containing the retarded (protein-bound) and free DNA were excised, and the probes were eluted under constant shaking at room temperature into gel elution buffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 300 mM NaCl). After purification by phenol-chloroform extraction and ethanol precipitation, both free and bound probes were then cleaved with 100 µl of 1 M piperidine, lyophilized, and analyzed by electrophoresis on a denaturing 8% polyacrylamide gel with 8 M urea.

Western Blotting
Equal amounts (40 µg) of nuclear protein from JEG-3 and NCI-H295A cells were separated on a 10% polyacrylamide/SDS gel and transferred to Immobilon-P polyvinylidene difluoride membranes (Millipore Corp., Bradford, MA). Immunodetection was performed using a 1:5,000 dilution of the NF-1 antiserum. A secondary peroxidase-conjugated antibody was used in combination with the ECL chemiluminescent detection method (Amersham Pharmacia Biotech).

RT-PCR
Single-stranded cDNA was prepared from 5 µg of total RNA using Superscript II (Life Technologies, Inc., Rockville, MD) and oligo dT12–18 as primer. Full-length NF-1/CTF cDNA species were amplified by 30 cycles of PCR (95 C for 60 sec, 63 C for 60 sec, and 72 C for 60 sec) using 40 pmol of primers CTF-F and CTF-R (Table 1Go), and a 20:1 mixture of Taq (Promega Corp.) and Pfu (Stratagene, La Jolla, CA) polymerases in 50 µl containing 20 mM Tris-HCl (pH 8.55), 1.5 mM MgCl2, 150 µg/ml, 16 mM (NH4)2SO4. As a size control, full size NF-1C/CTF1, -2, and -3 were amplified under the same conditions from 100 pg of the cDNA cloned in pBS-CTF1, pBS-CTF 2, and pBS-CTF 3, respectively (73).


    ACKNOWLEDGMENTS
 
We thank Dr. Naoko Tanese (New York University School of Medicine) for the generous gift of the antiserum to NF-1C2/CTF2. Drs. Lin and Martens thank Dr. Jonathan T. Wang for his helpful comments and discussions and Dr. Christa Flück for assistance with the final experiments.


    FOOTNOTES
 
This work was supported by NIH Grants DK-42154, HD-34449, and DK-37922 to W.L.M., and by a fellowship (97/07170-4) from Fudação de Amparo à Pesquisa do Estado de São Paulo (Brazil) to C.J.L.

1 These authors contributed equally to this manuscript. Back

Abbreviations: 8-Br-cAMP, 8-bromo-cAMP; DNase, deoxyribonuclease; HS, horse serum; NF-1, nuclear factor-1; SF-1, steroidogenic factor-1; TK, thymidine kinase.

Received for publication November 29, 2000. Accepted for publication April 26, 2001.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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