NF-1C, Sp1, and Sp3 Are Essential for Transcription of the Human Gene for P450c17 (Steroid 17
-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.
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ABSTRACT
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Cytochrome P450c17 catalyzes steroid 17
-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.
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INTRODUCTION
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CYTOCHROME P450c17 is the single protein
that catalyzes both 17
-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
-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
-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
-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.
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RESULTS
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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 As 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 As using site-directed mutagenesis, and the luciferase
activities of the -63 constructs containing either three or four As
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. 1A
).

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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.
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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. 1A
) (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. 1B
). 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. 1B
), 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. 1C
). 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. 2
). 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. 2
).

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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.
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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. 3
). 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. 3A
) or FP2 (Fig. 3B
). Similarly, 1,000-fold molar excess of FP1 and FP2
failed to displace the formation of FP3 complexes (Fig. 3C
). 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.
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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. 4
. In FP1, a pair of Gs 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 Gs at -90/-89, a G
at -97, and two pairs of As at -79/-78 and -75/-74 were cleaved
on the antisense strand (Fig. 4A
). In FP2, cleavage detected protected
Gs at positions -174, -173, and -166 of the sense strand, and two
Gs at -165/-164 and two As at -158/-157 were protected on the
antisense strand (Fig. 4B
). In FP3, As at -217, -216, -212, and
-199 were protected on the sense strand, and a GAG trinucleotide
sequence at -197/-195, a pair of As at -205/-204, a G at -208,
and an A at -190 were protected on the antisense strand (Fig. 4C
).
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.
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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 1
). 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. 5
). 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. 5A
), 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. 5B
).
Mutation of the GAG sequence at -197/-195 in the FP3 probe (FP3-m2)
completely abolished the formation of FP3 complexes (Fig. 5C
). Mutation
of other protected purines in FP1, FP2, or FP3 (oligonucleotides
FP1-m3, FP1-m4, FP2-m2, FP3-m1, and FP3-m3; Table 1
) 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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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 1 , 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.
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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 Gs 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. 3
and 6
) and are able to
inhibit each others interaction with NCI-H295A nuclear proteins at
similar concentrations (Fig. 6A
). Complexes formed by an
oligonucleotide (NF-1-Ad; Table 1
) of the NF-1 recognition site from
adenovirus (55) resemble the complexes formed by FP1 and
FP2 (Fig. 6A
) 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. 6B
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 1 ) 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 others 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.
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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. 7A
), 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. 7B
). 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
5055 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 2 ). 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.
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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 5055 kDa in NCI-H295A nuclei, but
no detectable NF-1-like proteins in JEG-3 nuclei (Fig. 7C
). 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. 7D
). 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. 1
). 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 1
and expressed in NCI-H295A cells using a promoterless
vector as a control (Fig. 8
).
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 1
). 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 1
). 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 1 ) 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 1 .
|
|
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. 1
, 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 1
). 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. 8
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. 9
). 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. 5C
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. 10
).
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. 10A
). 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. 9 . 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. 9
to transfect
each of these cell types (Fig. 10B
). 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. 9
): 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. 10A
and
the functional data in Fig. 10B
. 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. 11A
). Similarly, unlabeled FP3.1 could
compete for the complexes formed by FP3, whereas FP3.2 could not (Fig. 11B
). 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. 11C
). 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 1 ) 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. 11D
). 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. 11E
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. 11D
). 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. 11F
).
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. 10A
) as well as with
the ability of the FP3 element to activate transcription in a variety
of cell lines (Fig. 10B
).
 |
DISCUSSION
|
---|
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. 12
). 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 5055
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
711 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. 7B
), 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 6080% 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 7880 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. 11B
). 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. 11E
), suggesting this may be a general phenomenon.
 |
MATERIALS AND METHODS
|
---|
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 1
). 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
luc. The
fidelity of all constructs was verified by restriction enzyme digestion
and sequencing.
Site-Directed Mutagenesis
Using the appropriate mutant oligonucleotide (Table 1
),
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% Hams 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
Waymouths 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
[
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 2
).
Before annealing, the sense oligonucleotides were 5' end-labeled with
[
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 300500 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 1530 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.
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 dT1218 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 1
), 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. 
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.
 |
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