Transcription of the Leydig Insulin-Like Gene Is Mediated by Steroidogenic Factor-1
Stephan Zimmermann,
Anja Schwärzler,
Sabine Buth,
Wolfgang Engel and
Ibrahim M. Adham
Institute of Human Genetics University of Göttingen
37073 Göttingen, Germany
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ABSTRACT
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The Leydig insulin-like gene (Ley I-L), a member
of the insulin-related gene family, is specifically expressed in pre-
and postnatal Leydig cells of the testis and in postnatal theca cells
of the ovary. To determine the functional region of the mouse Ley I-L
promoter and factors controlling the Ley I-L gene expression, we used
2.1 kb of the 5'-flanking region of the mouse Ley I-L gene to generate
chimeric constructs with the chloramphenicol acetyltransferase gene
(CAT). Transient transfections of MA10 Leydig cells,
LTK- fibroblasts, and F9 embryonic cells by a
series of 5'-deleted mouse Ley I-L promoter-CAT constructs revealed
that the sequence between nucleotides -157 to +4 directs the
transcription of the reporter gene in MA10 but not in
LTK- and F9 cells, indicating that the
determinants of Leydig cell-specific expression reside within this
region. Deoxyribonuclease I (DNase I) footprint analysis revealed that
the sequences designated SF-1/1, SF-1/2, and SF-1/3 within three DNase
I-protected regions are homologous to the consensus binding site of the
steroidogenic factor-1 (SF-1). Competition and antibody studies showed
that the three SF-1-binding sites in the Ley I-L promoter have similar
binding affinities for SF-1. Furthermore, transient transfections of
MA10 cells with mutant reporter constructs, in which SF-1/1 or both
SF-1/2 and SF-1/3 were deleted, demonstrated that all three SF-1-
binding sites are required for SF-1-mediated stimulation of Ley I-L
transcription. Cotransfection of an SF-1-containing expression vector
together with a Ley I-L promoter-CAT construct into HeLa cells, which
lack the endogenous SF-1 protein, resulted in CAT gene transcription,
which indicated that SF-1 can transactivate the Ley I-L promoter. These
data demonstrate an essential role of SF-1 in transcriptional
activation of the Ley I-L promoter.
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INTRODUCTION
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Recently, we have characterized a new member of the insulin-like
hormone superfamily, termed Ley I-L, which is specifically expressed in
Leydig cells of the fetal and adult testis (1) and in the theca cells
of the postnatal ovary (2). The Ley I-L cDNA and gene were isolated
from different mammalian species and characterized (1, 2, 3, 4, 5). Sequencing
revealed that the primary structure of the prepro-Ley I-L factor and
exon-intron organization of the gene are more similar to those of
preproinsulin and preprorelaxin than to those of preproinsulin-like
growth factor I and II (1, 4).
The Ley I-L gene is expressed at a high level in the adult testis and
at a much lower level in the adult ovary. Analyses of Ley I-L
transcripts in testis and ovary throughout the pre- and postnatal life
of the mouse revealed a sexual dimorphic pattern of Ley I-L expression
during development. No Ley I-L transcripts are detected in female
embryos of any stage, whereas in male embryos transcripts are first
detected at day 13.5. After birth, the level of Ley I-L transcription
in testis remains constant during the first 3 weeks, increases at the
time at which the first wave of round spermatids undergoes
spermiogenesis, and reaches the highest level in adult testis. These
results suggest that the Ley I-L peptide plays a role in germ cell
maturation. In the female, expression of the Ley I-L gene is first
detected in the ovary at day 6 after birth. This, taken together with
the distinct expression pattern of Ley I-L during the estrous cycle and
pregnancy, implies a functional role of Ley I-L during follicular
development (5).
As a first step in identification of the regulatory elements that
control Ley I-L gene transcription and its cell-specific expression, we
have cloned and sequenced the human, porcine, and mouse Ley I-L gene as
well as their 5'-flanking sequences (4, 5). Alignment of the
5'-flanking region of these genes did not reveal any significant
sequence homologies. However, the TATA-box and two sequence stretches
are conserved and located at equivalent positions in all three genes.
The nucleotide sequence of one of the conserved sequences is identical
to the consensus binding site for steroidogenic factor-1 (SF-1) (6),
also designated as Ad4BP. SF-1, an orphan nuclear receptor, has been
found to regulate several genes in testis, ovary, adrenal cortex, and
pituitary gland (7, 8, 9, 10, 11, 12, 13, 14, 15) by binding to specific cis-acting
elements in the regulatory region of these genes. The conservation of
the consensus sequence of the SF-1-binding site in the 5'-flanking
region of mammalian Ley I-L genes and the observation that SF-1
expression precedes the Ley I-L expression in testis and ovary suggests
that SF-1 regulates the expression of the Ley I-L gene (5).
In the present study, we demonstrate that SF-1 binds to the
SF-1-binding sites in the Ley I-L promoter and transactivates the mouse
Ley I-L promoter.
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RESULTS
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The Proximal 157-bp Region of the Ley I-L Promoter Is Required for
Leydig Cell-Specific Transcription
To determine the region necessary for transcriptional regulation
of the Ley I-L gene, MA10, LTK-, and F9 cells were
transiently transfected with chloramphenicol acetyl transferase (CAT)
constructs containing different lengths of the 5'-flanking region of
the mouse Ley I-L gene obtained by progressive deletion (Fig. 1
). CAT activities were determined in cell extracts
(Fig. 1
). For control experiments, these cell lines were also
transfected with pSV40-CAT and pCAT basic.

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Figure 1. Expression of CAT Activities in MA10,
LTK-, and F9 Cells
Left, Partial map of murine Ley I-L promoter illustrating
the restriction sites that were used for construction of the
5'-deletion constructs and schematic diagram of various Ley I-L-CAT
fusion constructs. The Ley I-L start of transcription is indicated by
an arrow. Restriction enzymes are abbreviated as follows: B,
BamHI; P, PstI; S, SstI; E,
EcoRI; X, XhoI. The CAT activity in MA10,
LTK-, and F9 transiently transfected cells with each
construct is illustrated at the right. The relative CAT
activity was correlated to ß-galactosidase activity and expressed as
a percentage of pSV40-CAT. The relative CAT activity represents at
least three independent transfections, each done in duplicate.
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The progressive removal of 5'-sequences of the Ley I-L gene from
nucleotide position -2109 to -690 resulted in a slight increase of
the promoter activity in MA10 cells. Further deletion of the sequence
from -690 to -157 did not significantly affect the expression of the
CAT reporter gene. However, a drastic decrease of CAT activity was
observed when the sequence from -157 to -53 was deleted. These
results suggest that the region between -157 to -53 of the Ley I-L
promoter contains regulatory elements sufficient to direct the
transcription of the reporter gene in the Leydig cells (MA10).
The cell type-specific activity of the Ley I-L promoter was
demonstrated by measuring CAT activity in extracts of F9 and
LTK- cells, which do not express Ley I-L (our unpublished
data). No significant CAT activity was found in these cell lines,
either transfected with p2109-CAT or with other constructs. These
results demonstrate that the 5'-flanking region of the Ley I-L gene can
direct the cell type-specific expression of the Ley I-L gene.
The Transcription Factor SF-1 Binds to the Ley I-L Promoter
As shown above, the region between bp -157 and -53
was determined to be sufficient for the regulation of the Ley I-L
promoter in MA10 cells. DNase I footprinting analysis was carried out
to identify the binding sites involved in the Ley I-L regulation. A DNA
fragment spanning the region -157/+4 was labeled at each strand and
allowed to bind to MA10 nuclear proteins. In the experiments depicted
in Fig. 2
, four protected DNA segments
located between -43 to -37 (F1), -62 to -53 (F2), -110 to -102
(F3), and -142 to -130 (F4) were detected. The motif CCAAGGCC in
the protected site F2 was found to be conserved in sequence and located
at an equivalent position in the mouse, porcine, and human Ley I-L gene
and was identical to the consensus sequence
of the SF-1-binding site (5). A further search of the nucleotide
sequence in F1, F3, and F4 revealed that the sequences TCAAGGTC on the
coding strand of F3 and TCACGGTC on the noncoding strand of F2 (Fig. 3
) share homology with the consensus
sequence of the SF-1-binding site. We named to the SF-1-binding site
homologous sequences in the protected F2, F3, and F4 segment as an
SF-1/1, SF-1/2, and SF-1/3 element, respectively.

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Figure 2. DNase I Footprinting Analysis of Ley I-L Promoter
Using Nuclear Extracts of MA10 Cells
DNase I footprinting experiments were performed with the coding
(left) and noncoding (right) strand of
the DNA fragment extending from -157 to +4. The probes were incubated
with nuclear protein (+) or without protein (-). The protected regions
on each strand are indicated by boxes. The line G+A
represents the Maxam and Gilbert sequence ladder obtained with the
respective probes.
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Figure 3. Mapping of DNase I-Protected Segments in the
Sequence of Ley I-L Promoter
The sequence of the murine Ley I-L is shown. The DNase I footprints
identified in Fig. 2 are boxed and designated F1F4.
Bold sequences correspond to the identified SF-1-binding
sites. The sequences of the oligonucleotides used in EMSA experiments
are underlined. The Ley I-L start of transcription is
indicated by an arrow and +1.
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To examine whether the same nuclear protein binds to the sequence
elements SF-1/1, SF-1/2, and SF-1/3 of the Ley I-L promoter, three
oligonucleotides containing the sequences of SF-1/1, SF-1/2, and SF-1/3
sites were designed (Fig. 3
) and used in electrophoretic mobility shift
assay (EMSA) (Fig. 4
). When the SF-1/1
oligonucleotide was incubated with the nuclear extracts of MA10 cells,
one DNA/protein complex was observed (Fig. 4A
, lane 1). The 100-fold
competition with the unlabeled SF-1/2 and SF-1/3 oligonucleotides
abolished the formation of the complex (Fig. 4A
, lanes 2, 3), whereas
competition with a nonspecific oligonucleotide (N1) spanning the
sequence -131 to -112 of the Ley I-L promoter (Fig. 3
) and the SF-1/1
M oligonucleotide, which has a 3-bp substitution in the
SF-1/1 site did not affect the formation of this complex (Fig. 4A
, lanes 4 and 5). Protein complexes to the SF-1/2 and SF-1/3
oligonucleotide had the same mobility as that formed with the SF-1/1
oligonucleotide (Fig. 4B
, lanes 1 and 4). The protein binding to the
SF-1/2 and SF-1/3 oligonucleotides could be effectively abolished with
a 100-fold excess of the SF-1/1 as well as the SF-1/2 or SF-1/3
oligonucleotides (Fig. 4B
, lanes 23 and lanes 56). These results
indicate that the same nuclear protein in the MA10 cells interacts with
the sequence elements SF-1/1, SF-1/2, and SF-1/3.

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Figure 4. The Affinity Binding of SF-1 to the Three
SF-1-Binding Sites in the Ley I-L Promoter and Tissue Specificity of
SF1/1-Binding Protein Analyzed by EMSA
A, The double-stranded SF-1/1 oligonucleotide (Fig. 3 ) was end-labeled
and incubated with the nuclear extract of MA10 cells in the absence
(lane 1) or in the presence of 100 molar excess of unlabeled SF-1/2
(lane 2), SF-1/3 (lane 3), N1 (lane 4), and SF-1/1 M (lane
5) oligonucleotides. This specific DNA-protein complex (SF-1) is
indicated by an arrow. B, Double-stranded SF-1/2,
SF-1/3, and N1 oligonucleotides were end-labeled and incubated with
nuclear extracts of MA10 cells in the absence (lanes 1, 4, and 7) or
presence of 100 molar excess of unlabeled competitors (lanes 2, 3, 5,
and 6). C, The nuclear extract of MA10 cells was incubated without
(lane 1) or with polyclonal SF-1 antibody (lane 2) or normal rabbit
serum (lane 3) for 30 min and then incubated with the labeled SF-1/1
oligonucleotide. D, Nuclear extracts from MA10 cells (lane 1), testis
(lane 2), spleen (lane 3), and kidney (lane 4) were incubated with the
SF-1/1 oligonucleotide to test the presence of SF-1/1-binding
activity.
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The DNA/protein complex was further analyzed using an antibody
inhibition assay with polyclonal antibodies directed against the
purified bovine SF-1 (16). As shown in Fig. 4C
, the SF-1-antiserum
(lane 2) but not rabbit preimmune serum (lane 3) disrupted the
formation of the SF-1/1 oligonucleotide-nuclear factor complex. EMSA
was also performed with nuclear extracts of 15-day-old mouse testis,
spleen, kidney, and MA10 cells. Binding of a nuclear protein to the
SF-1/1 oligonucleotide was observed with nuclear extracts of MA10 cells
and testis but not with extracts of spleen and kidney (Fig. 4D
), which
do not express the SF-1 gene. These results confirm that the SF-1
protein is the component of the complex formed using the SF-1/1,
SF-1/2, and SF-1/3 oligonucleotides.
SF-1 Transactivates the Ley I-L Promoter
The results of EMSA have demonstrated that SF-1 binds to the three
SF-1 sites of the murine Ley I-L promoter. To examine whether the SF-1
can activate the transcription of the Ley I-L promoter by binding to
the three sites and to establish the contribution of each of the three
SF-1 elements to the promoter activity, a series of mutant reporter
constructs were generated, in which one, two, or all three of the SF-1
elements were deleted (Fig. 5
). The
constructs were introduced into MA10 cells, and the relative CAT
activity was determined as a percentage of the activity of the
wild-type p157-CAT construct. Transfection of the
p157
SF-1/1-CAT with a deletion of 6 bp in the SF-1/1 site
resulted in a reduction in promoter activity to 19% relative to
the p157-CAT. A similar reduction in promoter activity was observed
when the construct p100-CAT with progressive deletion of the sequence
-157 to -101, was introduced into MA10 cells. A drastic decrease in
CAT activity was observed with p100
SF-1/1-CAT, in which all three
SF-1 sites were absent. The possibility that the reduction in CAT
activity in the cell lysate transfected with p100-CAT was due to the
absence of a further binding site in a deleted sequence (-157/-100)
can be excluded, because the results of DNase I footprinting (Fig. 2
)
and EMSA (Fig. 4B
, lane 7) with the oligonucleotide N1 spanning the
nucleotide sequence between SF-1/2 and SF-1/3 sites failed to
demonstrate a further binding site.

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Figure 5. Effect of SF-1-Binding Sites Located in the Ley I-L
Promoter on the Expression of the CAT Reporter Gene
Schematic diagram on the left represents the Ley I-L-CAT
fusion constructs used in this experiment. The sequence of SF-1-binding
sites in the Ley I-L promoter is indicated with shaded
boxes, and the deleted SF-1-binding site with a
triangle. The relative CAT activity in MA10 cells
transfected with each construct is illustrated on the
right. The relative CAT activity was normalized relative
to ß-galactosidase activity. All CAT activities are expressed as a
percentage of the CAT activity obtained with p157-CAT. Each
bar represents average relative CAT activity from at
least three independent transfections, each assayed in duplicate.
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To examine more directly whether the SF-1 could transactivate the Ley
I-L promoter, cotransfection experiments were performed in HeLa cells,
which are known to lack the endogenous SF-1 protein (12). The
cotransfection of the p157-CAT together with the expression vector
pRc/RSV did not result in CAT activity. However, cotransfection of the
reporter construct p157-CAT with the expression vector pRSV/Ad4BP (17),
which encodes SF-1, resulted in a significant transactivation of the
Ley I-L promoter in HeLa cells (Fig. 6
).

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Figure 6. Activation of the Ley I-L Promoter in HeLa Cells in
the Presence of SF-1
Shown in upper panel are CAT activities obtained from
HeLa cells transfected with 10 µg p157-CAT construct together with
2.5 µg mammalian expression vector pRc/RSV (Invitrogen, San Diego,
CA) or SF-1 expression vector. The control assays were performed by
transfections with pCAT basic (promoterless) or pSV40-CAT (SV40
promoter). Histograms in the lower panel are derived
from quantitation of CAT assays. The relative CAT activity was
expressed as percentage of that with pSV40-CAT construct. Results are
given as means and SDs from three independent
transfections, each assayed in duplicate.
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DISCUSSION
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In this report, we demonstrate that SF-1 binds to and activates
the murine Ley I-L promoter. Cotransfection of a plasmid producing SF-1
with a Ley I-L-CAT reporter construct containing the sequence between
-157 to +4 of the Ley I-L gene into HeLa cells clearly demonstrates
that SF-1 is required for activation of the Ley I-L promoter.
SF-1 was first recognized to regulate genes coding for steroidogenic
enzymes in the adrenal gland and testicular Leydig cells (7, 8).
Recently, the arrest of gonadal development in SF-1-deficient mice (18)
has led to the suggestion that SF-1 also activates other genes that are
required for the differentiation of the reproductive system. It has
been established that SF-1 regulates the specific expression of
Müllerian inhibitor substance (MIS) gene in Sertoli cells (12)
and the genes coding for the
-subunit of LH and FSH (13) and for the
ß-subunit of LH in pituitary gonadotropes (14). Because the Ley I-L
gene is also regulated by SF-1 and Ley I-L deficient mice show a
failure of outgrowth and differentiation of the gubernaculum, which is
essential for testicular descent (our unpublished results), it is
evident that the Ley I-L gene is involved in the SF-1-regulated gene
cascade required for sexual differentiation.
Our analysis of the regulatory region of the Ley I-L gene revealed that
the sequence from -157 to +4 is sufficient for Leydig cell-specific
transcription. DNase I footprint experiments revealed three protected
segments between -157/-53 that contain DNA sequences homologous to
the consensus SF-1-binding site. Deletions of these sequences led to a
drastic decrease of the CAT activity in the MA10 cells. Antibody
inhibition and competition assay indicated that each of the three
SF-1-binding sites in the Ley I-L promoter possess equal binding
affinities to SF-1. Moreover, transient transfection experiments in
MA10 cells showed that mutant reporter constructs, in which the
sequence for the SF-1/1-binding site or for both SF-1/2- and
SF-1/3-binding sites were deleted, could be activated by SF-1 to a
similar extent, approximately 1719% of the wild-type construct. In
conclusion, the three SF-1-binding sites in the Ley I-L promoter have
similar binding affinities for SF-1 and contribute to transactivation
of the Ley I-L promoter by SF-1. Contribution of more than one SF-1
binding site in the transcriptional activation of other SF-1-regulated
genes has been demonstrated for the gene coding for the ß-subunit of
LH. The mutation of only one of the SF-1-binding sites in the
5'-flanking region of this gene did not fully diminish promoter
activity (14).
Furthermore, we demonstrated that SF-1 transactivates the Ley I-L
promoter in cotransfection experiments in HeLa cells. Since SF-1 is
able to activate the promoter of Ley I-L in HeLa cells, it is likely
that transactivation occurs mainly via a direct binding of SF-1 to the
three SF-1 binding sites, and that a cofactor or a ligand is not
required for the transactivational properties of the SF-1 on the Ley
I-L promoter. These data are consistent with previous reports that
showed that SF-1 apparently activates rat LHß and salmon gonadotropin
IIß subunit genes in a ligand-independent manner (14, 19). In
contrast, the regulation of the MIS gene by SF-1 requires a
SF1-specific ligand or a cofactor (12). SF-1 by itself was unable to
activate the MIS promoter in HeLa cells. Removal of the SF-1
ligand-binding domain enabled the truncated SF-1 to stimulate the MIS
promoter in HeLa cells and led to the hypothesis that a cofactor
specific for Sertoli cells was required for the SF-1 activation of the
MIS gene. SF-1 is present in gonads, adrenal cortex, and brain, but the
expression of certain SF-1-regulated genes is restricted to one of
these organs. Therefore, other factors or SF-1 ligands must further
distinguish gonadal from adrenal and brain-specific expression of
SF-1-regulated genes. A synergistic interaction of SF-1 with other
factors or a SF-1 cofactor specific for these organs may contribute to
cell-specific and organ-specific expression of SF-1 responsive
genes.
The pattern of Ley I-L expression during pre- and postnatal development
of the testis and postnatal development of the ovary is correlated with
the presence of SF-1 in these developmental stages. Marked differences
were observed between the expression of Ley I-L and SF-1 in the testis
during postnatal life. The amount of Ley I-L transcript increases after
the third postnatal week and reaches its highest level in adult testis
(5), whereas the level of SF-1 transcript drops slightly at postnatal
day 15 and remains at a low level thereafter (12, 16). These
differences between relative expression of Ley I-L and SF-1 in
postnatal testis could be explained by the presence of other
transcription factors or hormonal influences that act in synergism with
SF-1 to drastically enhance the expression of Ley I-L in adult testis.
Such synergistic interactions have been observed between SF-1 and
estrogen receptor in the regulation of the salmon gonadotrophin IIß
subunit gene in the pituitary gland (19). In the mouse, serum levels of
LH and FSH increase sharply between day 20 and 35 (20, 21), precisely
when Ley I-L expression increases. Therefore, transgenic experiments
with reporter constructs containing the Ley I-L promoter may directly
address whether the SF-1 binding sites are a key regulator of Ley I-L
promoter activity in vivo as has been shown for the LHß
(22) and MIS (23) genes and whether the increase of the Ley I-L
expression in adult testis is the result of interactions between SF-1
and hormone-stimulated responses.
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MATERIALS AND METHODS
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Plasmid Construction
The 3.5-kb BamHI genomic fragment (5) containing the
5'-flanking region, exons 1 and 2 of the Ley I-L gene, was subcloned
into pBluescript IIKS+ (pBam-1). A promoterless CAT basic
vector (Promega, Madison, WI) and the genomic subclone (pBam-1) were
used for preparation of the chimeric expression constructs (Fig. 1
).
Using the subclone pBam1 as a template, a fragment -690 bp/+4 and
-157/+4 with an artificial HindIII site at the 5'-end and
an XbaI site at the 3'-end was amplified by PCR and
subcloned into the plasmid pCAT basic to produce p690-CAT and p157-CAT.
The nucleotide sequence of these truncated clones was confirmed by
sequencing. The p100-CAT was generated by digestion of p157-CAT with
HindIII/XhoI and religated. The
HindIII/XhoI fragment -2109/-101 was isolated
from the pBam-1 clone and ligated with the
HindIII/XhoI-digested p157-CAT construct to
create p2109-CAT. The p1361-CAT vector was constructed by digestion of
the p2109-CAT construct with PstI and the -1361/+4-CAT
fusion fragment was purified from the agarose gel and subcloned by
blunt-end ligation.
The vector p157
SF-1/1-CAT in which the conserved sequence CCAAGG
(-61/-56) was deleted was generated by a two-step PCR-directed
mutagenesis by the following procedures. First, a fragment -157/-41
was prepared using a 5'-HindIII site-GCACCTGGGAGAGGACTTC-3'
as 5'-primer and 5'-CGCGCCGCCCATGGGCG GGAACACAGCCAA-3' as 3'-primer,
and a fragment -63/+4 was prepared using
5'-GCTGTGTTCCCGCCCATGGGCGGCGCGAGG-3' as 5'-primer and 5'-TGGTGGC
AGGAGGCAGTGGGC-XbaI site-3' as 3'-primer. The p157-CAT
construct was used as the template to amplify the two fragments.
Subsequently, these two fragments were mixed and used for another round
of PCR in the presence of the 5'-primer and the 3'-primer which have
been used for amplification of the -157/-41 and -63/+4 fragments,
respectively. The final PCR product was digested with
HindIII/XbaI, subcloned into a promoterless CAT
vector, and the internal deletion was confirmed by sequencing. The
p100
SF-1/1-CAT was generated by digestion of p157
SF-1/1-CAT with
HindIII/XhoI followed by blunt-end ligation.
Cell Culture
The mouse fibroblast LTK-, mouse embryonic
carcinoma F9 cells, and human HeLa cells were cultured in DMEM
(Life Technologies, Inc., Gaithersburg, MD) supplemented
with 10% heat-inactivated FCS (Boehringer, Mannheim,
Germany) under 5% CO2. All media included penicillin
(100 U/ml) and streptomycin (100 µg/ml). The mouse tumor Leydig cell
line MA10 was maintained as described (24).
Transfection and CAT Assay
Mouse MA10, LTK-, and F9 cells were cotransfected
with 10 µg of the tested plasmid and 2 µg of pCMV-ß-galactosidase
control plasmid (CLONTECH, Palo Alto, CA) by the calcium phosphate
coprecipitation method (25). After exposure to the DNA precipitate for
1216 h, cells were washed and fresh medium was then added. The cells
were harvested 48 h later, and CAT activities were measured in the
cell lysates (26). One tenth of cell lysate was used to determine
ß-galactosidase activity. For each CAT reaction, cell extract was
combined with 4 µl of [14C] chloramphenicol (Amersham,
Arlington Heights, IL), 10 µl of 20 mg/ml acetyl-coenzyme A (Sigma,
St. Louis, MO), and 250 mM Tris, pH 7.8, to a final volume
of 150 µl. The mixtures were incubated at 37 C for 2 h, and the
reactions were stopped by extraction with 1 ml ethyl acetate.
Acetylated and nonacetylated chloramphenicol were separated by TLC.
Nuclear Extract
Nuclear protein extracts from MA10, LTK-, and F9
cells were prepared according to Dignam et al. (27), whereas
nuclear extracts from tissues were prepared by a modified method of
Gorski et al. (28). Briefly, 0.51 g of tissue was minced
and homogenized in 10 ml homogenization buffer using a Teflon glass
homogenizer. The homogenate was layered over 3.2 ml of homogenization
buffer (10 mM HEPES, pH 7.3, 25 mM KCl, 0.15
mM spermine, 0.5 mM spermidine, 1
mM EDTA, 2 M sucrose, 2 µg/ml leupeptin, 2
µg/ml pepstatin A, 10% glycerol) and centrifuged in a SW40 rotor at
25,000 rpm for 30 min at -4 C. The pelleted nuclei were resuspended in
2 ml of nuclear lysis buffer (20 mM HEPES, pH 7.9, 420
mM NaCl, 1.5 mM MgCl2, 0.2
mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride,
0.5 mM dithiothreitol, 2 µg/ml leupeptin, 2 µg/ml
pepstatin A, 25% glycerol) and lysed using an all-glass Dounce
homogenizer. The lysate was mixed for 30 min at 4 C and then
centrifuged in a microcentrifuge at 12,000 rpm for 20 min at 4 C. The
supernatant was dialyzed several hours against dialysis buffer (20
mM HEPES, pH 7.9, 100 mM KCl, 0.2
mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride,
0.5 mM dithiothreitol, 2 µg/ml leupeptin, 2 µg/ml
pepstatin A, 20% glycerol), frozen in liquid nitrogen, and stored at
-70 C. The concentration of the nuclear proteins was measured by a
protein assay kit (Bio-Rad, Richmond, CA).
EMSA
From the sequence around the three footprinted regions F2, F3,
and F4, double-stranded DNA fragments were prepared by annealing of
complementary oligonucleotides. The sequences of the sense strands of
the double-stranded oligonucleotides were: SF-1/1:
5'-agcttTCCCGCCAAGGCCC-ATGG (-66/-49); SF-1/1 M:
5'-agcttTCCCGACAATTCC CATGG; SF-1/2:
5'-agcttACACAGCCCCTGACCGTGAC-TCG (-121/-99); SF-1/3: 5'-agctt
GACTTCAAGGTCCCAAGCTGGAC (-144/-122); N1: 5'-agcttCAAGCTGGACACACAG
CCCC (-131/-112). The SF-1/1 M has three nucleotide
substitutions in the consensus SF-1 binding site. The localization of
the oligonucleotides within the promoter sequence are given in
parentheses. Five nucleotides were added to the 5'-end of the synthetic
oligonucleotides to use for labeling reaction with Klenow fragment and
[
-32P]dCTP (Amersham). In each gel shift assay, 10
µg nuclear extract were preincubated in a 20 µl reaction mixture
containing 20 mM HEPES, pH 7.9, 0.5 mM EDTA, 25
mM KCl, 25 mM NaCl, 0.5 mM
dithiothreitol, 7% glycerol, and 24 µg
poly(deoxyinosinic-deoxycytidylic)acid. After 10 min, approximately
1 x 104 cpm of 32P-labeled probe was
added, and the incubation was continued for 20 min on ice, then loaded
onto 6% nondenaturing polyacrylamide gel. The gels were fixed in a
solution of 10% acetic acid and 30% methanol, dried, and exposed to
Hyperfilm-MP (Amersham). In competition assay, 100-fold excess of
unlabeled double-stranded oligonucleotide was incubated for 15 min
together with the nuclear extracts before addition of the
32P-labeled probe. For antibody cross-reaction, 1 µl of
anti-SF-1 antibody (16) or rabbit preserum was incubated with the
nuclear proteins for 30 min before addition of the
32P-labeled probe.
DNase I Footprinting
The DNA fragments used for DNase I footprint analysis of the Ley
I-L promoter were prepared from the p157-CAT construct by digestion of
the plasmid with either XbaI or HindIII to label
the coding and noncoding strand, respectively. Ends were filled in with
[
-32P]dCTP using the Klenow fragment of
Escherichia coli DNA polymerase I, and the fragments were
subsequently released by XbaI or HindIII
digestion as appropriate. In the binding reaction, 30100 µg MA10
nuclear extract and 5 x 104 cpm of
32P-labeled probe were used under the same conditions as in
EMSA in a total volume of 100 µl. The reactions were chilled on ice
for 20 min. After the concentration of CaCl2 had been
adjusted to 2.5 mM, DNase I (3 U) was added, and the
digestion was carried out at 20 C for 1 min. The reaction was
terminated by adding 90 µl of prewarmed (37 C) stop solution (20
mM Tris, pH 7.5, 20 mM EDTA, 0.4% SDS, 80
µg/ml proteinase K, 80 µg/ml yeast tRNA), and the probe was
extracted with phenol-chloroform-isoamylalcohol. The reaction products
were precipitated and analyzed on a 6% sequencing gel along with a
Maxam-Gilbert G+A sequencing reaction as a size marker.
 |
ACKNOWLEDGMENTS
|
---|
We thank Dr. Ken-ichirou Morohashi, University of Kyushu
(Fukuoka, Japan), for providing the SF-1 antibody and plasmid
RSV/Ad4BP, and Dr. Ascoli, University of Iowa (Iowa City, IA), for
providing the MA10 cells. We also thank Mrs. Angelika Winkler and Mr.
Peter Richter for secretarial and photographic assistance.
 |
FOOTNOTES
|
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Address requests for reprints to: Dr. Ibrahim M. Adham, Institut für Humangenetik der Universität, Gosslerstrasse 12d, 37073 Göttingen, Germany.
This work was supported by a grant from the Deutsche
Forschungsgemeinschaft through SFB 271 (to I.M.A.).
Received for publication December 1, 1997.
Revision received January 27, 1998.
Accepted for publication January 30, 1998.
 |
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