Polypyrimidine Tract-Binding Protein-Associated Splicing Factor Is a Negative Regulator of Transcriptional Activity of the Porcine P450scc Insulin-Like Growth Factor Response Element
Randall J. Urban,
Yvonne Bodenburg,
Alexander Kurosky,
Thomas G. Wood and
Slavisa Gasic
Division of Endocrinology (R.J.U., Y.B., S.G.) Department of
Internal Medicine University of Texas Medical Branch Galveston,
Texas 77555-1060
Department of Human Biological Chemistry and
Genetics (A.K., T.G.W.) University of Texas Medical Branch
Galveston, Texas 77555-0645
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ABSTRACT
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The porcine P-450 cholesterol side-chain cleavage
enzyme gene (P450scc) contains a 30-bp region [insulin-like
growth factor response element (IGFRE)] that mediates
insulin-like growth factor I (IGF-I)-stimulated gene expression and
binds Sp1. In this study, we showed that polypyrimidine tract-binding
protein (PTB)-associated splicing factor (PSF), an RNA-binding
component of spliceosomes, binds to the IGFRE. Southwestern analysis
with an IGFRE oligonucleotide showed that a protein (from
Sp1-immunodepleted HeLa extract) fractionated on SDS-PAGE at 100 kDa.
Microsequence analysis of 100-kDa band HeLa proteins detected PSF. DNA
affinity chromatography, using an IGFRE mutant oligonucleotide that
does not bind Sp1, isolated a protein that immunoreacted with PSF
antibody. Deoxyribonuclease I (DNase I) footprint analysis showed
recombinant PSF binds 5' of the Sp1-binding GC box of the IGFRE, and
mutant oligonucleotides further delineated this region to a palindrome,
CTGAGTC. Functional analysis of these mutants by transfection
experiments in a cell line overexpressing the IGF-I receptor (NWTb3)
found that an inability to bind PSF significantly increased the
IGFRE transcriptional activity, while retaining responsiveness to
IGF-I. Moreover, transfection of expression vectors for Sp1 and PSF in
porcine granulosa cells found that Sp1 expression stimulated IGFRE
transcriptional activity while PSF inhibited activity even with
coexpression of Sp1. In conclusion, we identified PSF as an
independent, inhibitory regulator of the transcriptional activity of
the porcine P450scc IGFRE.
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INTRODUCTION
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Insulin-like growth factor-I (IGF-I) is a growth factor that is
important in regulation of normal ovarian function (1, 2). IGF-I, as a
local paracrine factor produced by granulosa cells, stimulates
steroidogenesis (3) and increases immunoprecipitable P-450 cholesterol
side-chain cleavage (P450scc) protein (4) and mRNA concentrations (5).
P450scc is the rate-limiting enzyme in the steroidogenic pathway and is
responsible for cleavage of the
C20C22 bond that frees
the C22C27 side chain of
cholesterol (6).
The mechanisms by which IGF-I stimulates gene transcription are not
understood. Studies have found IGF-I-responsive elements (IGFREs) in
the chicken
1-crystallin gene (7, 8) and rat elastin gene (9, 10, 11).
These regions are GC rich and bind the ubiquitous transcription factor,
Sp1 (8, 11). Rat elastin gene expression is increased by IGF-I
treatment in cultures of neonatal rat aortic smooth muscle cells (9).
This up-regulation of elastin gene expression is mediated by the loss
of binding of complexes to a GC-rich domain that functions as a
negative element for gene transcription (9). Additional evidence
indicates that IGF-I treatment prevents the binding of Sp3 to a
retinoblastoma control element that serves as a repressor for elastin
gene transcription (12).
We previously identified a GC-rich, 30-bp IGFRE in the porcine P450scc
gene (13) that binds Sp1 (14, 15). In this study, we used microsequence
analysis and sequence-specific DNA affinity chromatography on HeLa cell
nuclear extract protein to identify a protein, polypyrimidine
tract-binding protein (PTB)-associated splicing factor (PSF), that
binds to the porcine P450scc IGFRE. Moreover, using expression vectors
in porcine granulosa cell transient transfection experiments, we show
that Sp1 stimulates, and PSF inhibits, transcriptional activity of the
porcine P450scc IGFRE.
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RESULTS
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Identification of a Second Protein Binding to the Porcine P450scc
IGFRE
It is known that IGF-I has tissue-specific effects. Therefore, we
suspected a greater degree of complexity of regulation of the porcine
P450scc IGFE than just the binding of Sp1. Using SDS-PAGE of HeLa cell
nuclear extract protein and microsequence analysis, we identified a
second protein binding to the porcine P450scc IGFRE. First, an Sp1
antibody was used to immunodeplete HeLa nuclear extracts of Sp1.
Western blots were prepared with recombinant Sp1, HeLa nuclear extract,
and Sp1-depleted HeLa extract (Fig. 1
).
Next, the blots were probed using conventional Southwestern methodology
with labeled IGFRE oligonucleotide and the mM6 oligonucleotide, which
does not bind Sp1 (14), as shown in the lower panels of Fig. 1
. Both intact and Sp1-depleted extracts contained a protein that bound
to wild-type IGFRE and mM6. This protein had the same SDS-PAGE
electrophoretic mobility as Sp1 (100 kDa) but was not precipitated by
Sp1 antibody.

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Figure 1. Western and Southwestern Analysis of HeLa Nuclear
Extract Protein
The top image (Western) is a Western blot
(Sp1 antibody) of Sp1 recombinant protein (Sp1), 25 µg of HeLa cell
nuclear extract protein (HeLa), and 25 µg of HeLa cell nuclear
extract after immunoprecipitation with Sp1 antibody (Precip). The
lower images are Southwestern blots hybridized with the
porcine P450scc IGFRE (mWT) oligonucleotide and a mutant IGFRE
oligonucleotide (mM6) that does not bind Sp1.
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Having demonstrated that Sp1 and a second protein comigrated during
SDS-PAGE, and knowing that Sp1 contained a blocked N terminus (16), we
microsequenced the 100-kDa band from the HeLa cell extract. Six
100-µg samples of crude HeLa cell extract were used for
analysis (Materials and Methods). N-terminal sequence
analysis of the 100-kDa band gave only one major sequence, shown in
Table 1
under the column
"N-Terminus". Since sequence analysis of proteins, especially
larger proteins, typically demonstrates some background levels of
Pth-amino acids, the occurrence of other proteins in the 100-kDa band
cannot be ruled out; however, their level of occurrence relative to the
major sequence was very low, and no other identifiable sequence was
evident. Furthermore, confirmation of the major sequence was also
established by internal sequence analysis after hydrolysis of the
100-kDa band with trypsin, followed by capillary HPLC of the
hydrolysate and sequencing of selected resulting peaks shown in Fig. 2
and Table 1
. The protein and peptide
sequences established by sequence analysis were searched in the protein
database using the Intelligenetics, Inc. (Mountain View,
CA) FASTDB program (Oxford Molecular Group) and revealed an
exact match with PTB-associated splicing factor (PSF) reported by
Patton et al. (17).

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Figure 2. Capillary HPLC of Peptide Fragments from HeLa Cell
Nuclear Extract after Trypsin Digestion
Peptides were fractionated on a Perkin-Elmer Corp./PE Applied Biosystems Division MicroBlotter
System (Materials and Methods). A, Profile of blank PVDF
control without blotted protein. Asterisks denote dye
marker peaks that correlate column effluent retention time with the
deposition of the effluent on PVDF. B, Profile of tryptic peptides in
100-kDa HeLa cell band. Results of the microsequence analysis of peaks
7 and 4 are given in Table 1 .
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DNA Affinity Chromatography
We partially purified HeLa cell nuclear extract protein for use in
affinity chromatography to determine whether PSF bound to the porcine
P450scc IGFRE. HeLa cells (60 g total) were grown in suspension, and
nuclear protein extract was prepared and partially purified
(Materials and Methods). Partially purified HeLa cell
nuclear extract proteins are shown in Fig. 3
after separation by SDS-PAGE.

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Figure 3. SDS-PAGE of Partially Purified HeLa Cell Nuclear
Proteins
SDS-PAGE stained with Coomassie Blue G-250 showing the partial
purification steps of HeLa cell nuclear extract protein. C, Crude
extract (50 µg); S, extract after Sephacryl S-300 high resolution
column chromatography (20 µg); and D, extract after DEAE-Sepharose
chromatography (25 µg). Molecular mass markers (kDa) comprise the
left lane. The arrow
indicates the 100-kDa band that contains both Sp1 and PSF.
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The partially purified HeLa cell nuclear protein extract was applied
onto a mM6 oligonucleotide affinity column (Materials and
Methods). The mM6 oligonucleotide is a mutant of the porcine
P450scc IGFRE that does not bind Sp1 in gel shift assay (14). Fractions
eluted with increasing salt concentrations were pooled and subjected to
Western blot analysis using a polyclonal antibody to PSF. As shown in
Fig. 4
, the PSF antibody recognized a
single protein band of 100 kDa that showed strong affinity for the
column (0.60.8 M KCl).

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Figure 4. PSF Western Blot of Partially Purified HeLa Cell
Extract from mM6 DNA Affinity Column
Autoradiogram of elution fractions from DNA affinity column prepared
from the mM6 mutant oligonucleotide of porcine P450scc IGFRE (does not
bind Sp1). The Western blot was immunoreacted with a PSF polyclonal
antibody.
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DNase I Footprint Analysis of Recombinant Sp1 and PSF
Footprint analysis was done with recombinant Sp1 and PSF
(Materials and Methods) to define binding regions to the
porcine IGFRE. Recombinant Sp1 protected a 21-bp region partially in
and 3' of the IGFRE (Fig. 5
). Sites of
enhanced DNase cleavage were found directly 5' of the footprint. A
similar pattern of binding of Sp1 to the SV40 promoter has been
previously described (18). The PSF footprint was 5' to the GC box (Fig. 5
).

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Figure 5. DNase I Footprinting of the Porcine IGFRE with
Recombinant Sp1 and PSF
Recombinant Sp1 (Promega Corp., 2 µl) and recombinant
PSF (10 µg) bind to distinct regions of the porcine P450scc IGFRE.
PSF binds 5' of the GC box and Sp1 binds to and 3' from the GC box.
P450scc sequence reactions are shown on the left with
the bracket identifying a visually distinct sequence of
the IGFRE for orientation.
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Identification of Nucleotides Binding PSF in the IGFRE
Having established that PSF binds 5' of the GC box of the IGFRE,
we next defined the nucleotides that bind PSF. A protein with 71%
homology to PSF, named nuclear RNA-binding protein, 54 kDa
(p54nrb), binds with DNA to the nucleotides GTGAC
(19). In the IGFRE a palindrome similar to these sequences was
identified 5' of the GC box, CTGAGTC. Therefore, mutant
oligonucleotides of the IGFRE were made to each side of the palindrome
(Table 2
). These mutant oligonucleotides
did not bind PSF by Southwestern analysis (Fig. 6
). The mutants did bind a protein that
is the same size as PSF and most likely is Sp1 (Fig. 6
).

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Figure 6. Southwestern Analysis of Recombinant PSF with
Mutant P450scc IGFRE Oligonucleotides
Recombinant PSF (15 µg) and porcine granulosa cell nuclear extract
protein (25 µg) were subjected to standard Southwestern analysis with
wild-type IGFRE (mWT) and two mutant oligonucleotides (Table 2 ).
Neither mutant binds recombinant PSF.
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Functional Significance of PSF Binding to the IGFRE
The functional significance of PSF binding to the IGFRE was
determined with transient transfection studies in NWTb3 cells using
luciferase constructs (Materials and Methods) that contained
the wild-type IGFRE (mWT) and the IGFRE mutants (Table 2
) that do not
bind PSF. NWTb3 cells overexpress the IGF-I receptor, and the IGFRE is
functional in these cells (15). This cell line was used rather than
porcine granulosa cells because the only heterologous promoter we have
found active in porcine granulosa cells, cytomegalovirus (CMV), is also
responsive to IGF-I. As shown in Fig. 7
, transient transfection experiments
with luciferase constructs containing mutants of the IGFRE ligated to a
SV40 promoter showed that loss of PSF binding significantly increased
the transcriptional activity of the IGFRE while retaining its
responsiveness to IGF-I.

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Figure 7. Functional Activity of Mutant Porcine P450scc
Oligonucleotides in a Mouse Fibroblast Cell Line
The mutant oligonucleotides shown in Fig. 6 to lack PSF binding were
ligated into SV40 luciferase constructs (Materials and
Methods) and transiently transfected in a mouse fibroblast cell
line that overexpresses the IGF-I receptor (NWTb3). Without treatment
(Control), both mutants showed significantly greater luciferase
activity than the wild-type IGFRE. All oligonucleotides were
responsive to treatment (IGF-I, 20 nM). Arbitrary
units are luminescence of the lysate after treatment divided by
absorbance (alkaline phosphatase). ANOVA on ranks with
Student-Newman-Keuls comparison test found mM18 and mM25 to be
significantly increased over wild type in the control conditions, and
all oligonucleotides were significantly stimulated by IGF-I. The
results are the mean ± SE from six
replicates.
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To further assess the function of PSF and Sp1 binding to the porcine
P450scc IGFRE, we performed transient transfection experiments in
primary cultures of porcine granulosa cells with expression vectors for
PSF and Sp1 that used the CMV promoter. As shown in Fig. 8
, expression of Sp1 significantly
increased the luciferase activity of the cotransfected porcine
P450scc IGFRE. PSF expression inhibited the activity of the IGFRE
and also inhibited the IGFRE when Sp1 was concomitantly expressed (Fig. 8
).

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Figure 8. Expression of PSF and Sp1 in Porcine Granulosa
Cells
Porcine granulosa cells were transfected with a pcDNA3 expression
vector (Control, 2 µg), an expression vector for PSF (1 µg), an
expression vector for Sp1 (1 µg), or both (1 µg each). A luciferase
construct of the porcine P450scc IGFRE (-2320 P450scc/luc) was
cotransfected with the expression vectors. Arbitrary units are
luminescence of the lysate after treatment divided by the protein
concentration (13 ). The asterisk indicates statistical
significance. The data represent the mean ± SE
from nine replicates.
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DISCUSSION
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Our previous study used deletion constructs of the porcine P-450
scc 5'-region to identify a 30-bp GC-rich domain (IGFRE) that mediates
IGF-I-stimulated gene expression (13). In this study, we determined
that a protein, PTB-associated splicing factor (PSF), binds to the
IGFRE and acts as a negative regulator of transcriptional activity. We
identified PSF by microsequence analysis and DNA affinity
chromatography using HeLa cell nuclear extract protein. We then
produced recombinant PSF that was used with DNase I footprint analysis
and Southwestern analysis to identify a palindrome, CTGAGTC, that binds
PSF 5' of the GC box in the IGFRE. Transient transfection experiments
showed that Sp1 (stimulation) and PSF (inhibition) have independent,
opposite actions on transcriptional activity of the IGFRE.
PSF was isolated and cloned in 1993 by Patton et al. (17).
It is a 76-kDa protein that migrates anomalously on SDS gels because it
is highly basic. The protein associates with PTB to form spliceosomes
for splicing of pre-mRNA. When compared with the average protein, PSF
has an unusual amino acid composition with high levels of glycine
(15%), proline (16%), glutamine (6.9%), and arginine (8.5%) and low
amounts of hydrophobic residues (20). In particular, the N-terminal
region is rich in proline and glutamine residues. Similar
proline/glutamine-rich regions comprise the transactivation domains of
Sp1 (21, 22). The sequence is also unusual in having many di- and
multiple-repeat residues. PSF is the product of only one gene,
but alternative splicing results in two isoforms that vary in length
from their carboxyl terminus but retain the proline/glutamine-rich
regions and two RNA-binding domains (17).
Although the DNA-binding domain in PSF has not been identified,
computer-assisted analysis of the proteins amino acid sequence shows
the highest probability of consensus DNA-binding domains such as SPXX
(23) and nuclear factor I (24) are in the N-terminal region. A protein,
named nuclear RNA-binding protein, 54 kDa
(p54nrb), that is 71% identical to PSF has been
identified (25). The DNA-binding domain of p54nrb
has been localized to the N terminus while the activation domain
resides in the C terminus (19). This protein has been shown to contain
two RNA-binding domains similar to PSF and binds to and stimulates
transcription through a DNA response element for murine intracisternal
A particles (19).
In our initial identification of the porcine P450scc IGFRE, we
hypothesized that additional transcription factors must bind to the
IGFRE because of the cell-specific effects of IGF-I (13). In this study
we report the identification of PSF as an additional protein regulating
transcriptional activity of the IGFRE. However, PSF is an essential
component of the spliceosome and (like Sp1) is an ubiquitously
expressed cellular protein. Therefore, while we have uncovered a
fascinating piece to the puzzle of cell-specific transcriptional
effects of IGF-I, we do not yet understand the mechanism. The discovery
of a protein (PSF) that is intimately involved in mRNA production, but
can also feedback and control transcription of mRNA, presents
intriguing possibilities for control of gene expression that could have
much broader implications than merely IGF-I stimulation of P450scc gene
expression.
The interactions of PSF and Sp1 in the porcine P450scc IGFRE are
complex and cannot be determined in these experiments. Sp1 and PSF do
not bind to overlapping sites within the IGFRE, but PSF inhibits
Sp1-driven transcriptional activity of the IGFRE. While this is
apparently incongruent data from a two-dimensional orientation, it
could be explained by consideration of function of the IGFRE in three
dimensions. Sp1 is a frequent proximal transcriptional enhancer of
genes, and studies have shown that multiple cofactors and coactivators
can assemble in complex spatial arrangements to activate RNA polymerase
II for gene transcription (26). Our results indicate that PSF in some
manner negatively influences such a complex for the porcine P450scc
IGFRE.
Increased expression of P450scc mRNA occurs in a severe form of
polycystic ovarian syndrome (PCOS), hyperthecosis (27). A hallmark of
PCOS is a markedly elevated serum insulin concentration that could
stimulate IGF-I receptors in the ovary (28). Several studies have
proposed a genetic predisposition toward the development of PCOS
(28, 29, 30, 31). Moreover, genetic linkage studies indicate the P450scc gene
as a possible candidate gene in families with PCOS (32, 33). Therefore,
a mutation in the palindrome of the IGFRE that binds PSF or a mutation
to the DNA-binding region of PSF could impair PSF binding and
predispose a woman with such a mutation to enhanced expression
of P450scc in the presence of increased insulin concentrations.
Additional studies are necessary to study the interactions of Sp1 and
PSF with the P450scc IGFRE and to explain how such interactions could
result in abnormalities of ovarian function.
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MATERIALS AND METHODS
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Materials
Recombinant Sp1 protein and restriction enzymes were obtained
from Promega Corp. (Madison, WI). Nitrocellulose filters
were obtained from Micron Separations, Inc. (Westboro, MA).
Polyvinylidene difluoride (PVDF) membranes were purchased from
Bio-Rad Laboratories, Inc. (Hercules, CA). For kinase
labeling,
[
-32P]-deoxyadenine-5'-triphosphate (dATP)
was obtained from Amersham Pharmacia Biotech
(Arlington Heights, IL). The mouse fibroblast cell line, NWTb3,
was previously described (15) and was obtained from Dr. Charles Roberts
(Department of Pediatrics, University of Oregon, Eugene, OR). The
antibody to Sp1 was obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The PSF antibody used in the affinity
chromatography experiments was a gift from Dr. James Patton (Vanderbilt
University, Nashville, TN). All reagents for sequence analysis were
purchased from Perkin-Elmer Corp./PE Applied Biosystems Division (Norwalk, CT). Trypsin, modified sequencing
grade, was a product of Promega Corp. (Madison, WI). All
HPLC reagents used were of HPLC grade.
Plasmid Constructs
The full-length PSF cDNA clone was obtained from Dr. James
Patton (Vanderbilt University) in a pET-15b expression vector (17). The
cDNA was excised and cloned into a cytomegalovirus mammalian expression
vector, pcDNA3 (Invitrogen, San Diego, CA), maintaining
the open reading frame. The construct was verified by sequencing. The
Sp1 expression vector, pCMV-Sp1, was obtained from Dr. Robert Tjian
(University of California, Berkeley, CA). Cotransfected in porcine
granulosa cell Sp1 and PSF expression experiments was the -2320
P450scc/luc construct that has been previously described (13). Briefly,
this construct contains the entire sequenced 5'- region of P450scc
including the IGFRE cloned into a promoterless luciferase vector
(34).
The mWT luciferase reporter gene construct used in transfection
experiments in NWTb3 cells has been previously described (14). The mM18
and mM25 oligonucleotide mutants of the IGFRE were cloned in pSVPLUC, a
modified pGEM3 plasmid containing the luciferase gene and the
enhancerless SV40 early region promoter (34). These constructs were
made identically to mWT and verified by sequencing.
Transient Transfection Experiments
Transfection of NWTb3 cells was done as previously described
(15). Briefly, cells were cultured in DMEM + 10% FBS and 500 µg/ml
Geneticin (Life Technologies, Inc., Gaithersburg, MD).
Transient transfection was done by lipofection (Tfx-50 Reagent,
Promega Corp.). Cells were harvested and measured for
luminescence 48 h after cotransfection because this time was
previously determined to show increases in P450scc mRNA after IGF-I
treatment (13).
For porcine granulosa cell transfection experiments, granulosa cells
were isolated from 1- to 5-mm follicles of immature swine and cultured
at 1.5 x 106 cells per 35-mm well on a
six-well plate with 2 ml of Eagles MEM with 3% FBS per well. After
overnight culture, cells were transfected with 2 µg of -2320
P450scc/luc construct and 1 µg PSF and 1 µg Sp1 expression vectors
per well. LipofectAMINE PLUS Reagent (Life Technologies, Inc.) was used to transfect cells. DNA-PLUS-LipofectAMINE
Reagent complexes were left on cells for 4 h before treatment
media were added directly to wells containing complexes and then
incubated at 37 C for 48 h. Cells were rinsed with PBS
before harvesting. The Luciferase Assay System (Promega Corp.) was used to harvest cellular lysate and to perform
luciferase assays. Light production was measured with a Turner
TD-20e luminometer (Turner Designs, Sunnyvale, CA). A protein assay
reagent from Bio-Rad Laboratories, Inc. was used to
measure protein concentrations of the lysates to normalize the
experiments as previously described (13).
Sequence Analysis of HeLa Cell Nuclear Extract Protein
Samples of crude HeLa cell nuclear extract protein (100 µg)
were fractionated by discontinuous SDS-PAGE under reducing conditions.
Gel electrophoretically resolved proteins were electroblotted onto PVDF
membrane using transfer buffer containing 0.05% SDS for 3.5 h.
Proteins electroblotted onto PVDF were visualized by staining with
Coomassie Blue G-250. Protein bands at 100 kDa were excised for both
N-terminal and internal amino acid sequence analyses.
Internal sequence analysis of PVDF-blotted proteins was carried out
similarly as described by Fernandez et al. (35). Excised
PVDF membrane bands were cut into 1-mm2 pieces
and prewetted with 100% methanol. The PVDF membrane pieces were
destained with 0.5 ml of 0.1% trifluoroacetic acid (TFA) in 50%
acetonitrile in a 2-ml polypropylene microfuge tube for 1 min followed
by five washes with 0.5 ml of distilled water. Each of the
aqueous washes included 5 min of sonication. An aliquot of 50 µl of
hydrolysis buffer (100 mM Tris-HCl, 1% reduced
Triton X-100, and 10% acetonitrile, pH 8.0) was subsequently added to
the PVDF membrane pieces. Disulfide bonds were reduced by the addition
of 5 µl of 45 mM dithiothreitol and reaction
for 30 min at 55 C. Alkylation was then initiated by addition of 5 µl
of 100 mM iodoacetic acid followed by reaction
for 30 min at 25 C in the dark. After brief centrifugation, the
hydrolysis buffer was removed and the PVDF membrane pieces were washed
once with 50 µl of hydrolysis buffer. Trypsin hydrolysis was then
conducted in 50 µl of hydrolysis buffer to which were added 2 µl of
freshly prepared trypsin (0.1 µg/µl). Trypsin hydrolysis proceeded
for 4 h at 37 C, after which time another 2 µl of trypsin was
added followed by incubation overnight at 37 C. After hydrolysis the
reaction mixture was sonicated for 5 min and centrifuged briefly, and
the supernatant was removed. The PVDF membrane pieces were treated with
3.7 µl of 5% TFA and then sonicated for 5 min after addition of 25
µl of hydrolysis buffer. The sample was centrifuged and the
supernatant was saved. The PVDF sample was similarly sonicated with 25
µl of 0.1% TFA in 50% acetonitrile and centrifuged and finally
sonicated in 25 µl of 0.1% TFA and centrifuged. All supernatants
were combined, the volume was reduced by vacuum centrifugation to about
20 µl, and the sample was stored at -20 C. The combined trypsin
hydrolysate was subsequently fractionated by HPLC on a C18
reversed-phase capillary column (0.5 mm x 150 mm) using a
Perkin-Elmer Corp./PE Applied Biosystems
Division model 173A microblotter system. Peptides were eluted at a flow
rate of 5 µl/min with a gradient eluant of 0.1% TFA (solvent A) and
0.085% TFA in acetonitrile (solvent B). The gradient conditions were
5% B to 45% B over 145 min. Eluted peptides were monitored at 215 nm
and continuously collected on a PVDF membrane strip. An HPLC
hydrolysate control was prepared exactly like the sample hydrolysate
except that a blank region of the PVDF membrane was used. Peak
fractions that were present in the sample hydrolysate but not in the
control hydrolysate were subjected to microsequence analysis.
Selected peptides on PVDF membrane prepared as described above were
subjected to automated N-terminal sequence analysis using a
Perkin-Elmer Corp./PE Applied Biosystems
Procise protein/peptide sequence (model 494-HT) configured with four
blot cartridges. Peptide samples were pretreated with 12 µl of
BioBrene Plus solution (PE Applied Biosystems, Foster City, CA)
[BioBrene (100 µl/ml)-0.1% TFA-methanol (2:1:7)]. Pulsed-liquid
chemistry sequencing methodology was used for all samples.
Partial Purification of Crude HeLa Cell Extract
Crude nuclear extract from HeLa cells grown in suspension was
prepared using a large-scale nuclear protein preparation method
previously described (13). Approximately 700 mg of nuclear extract
were precipitated by 53% saturated ammonium sulfate and centrifuged at
35,000 x g for 15 min. The pellet was resuspended in
TM buffer (50 mM Tris-HCl, pH 7.9, containing
12.5 mM MgCl2, 1
mM EDTA, 1 mM
dithiothreitol, and 20% glycerol) to a final concentration of 30 mg/ml
(16). The soluble protein extract was applied to an H. Prep Sephacryl
S-300 High Resolution column (Pharmacia Biotech)
equilibrated with TM buffer containing 0.1 M KCl.
Protein elution was monitored by absorbance at 280 nm. The fractions
containing Sp1 were determined by Western analysis using Sp1 antibody.
Fractions containing Sp1 from five column runs were combined (46 mg
protein/80 ml) and applied to a 10 ml diethylaminoethyl
(DEAE)-Sepharose CL-6 B column equilibrated with TM buffer containing
0.1 M KCl. The major unretained protein fraction
from the DEAE column was collected in one fraction (
25 mg of
protein). This fraction was precipitated in 53% saturated ammonium
sulfate and resuspended in TM buffer containing 0.1
M KCl at a concentration of approximately 1
mg/ml.
Sequence-Specific DNA Affinity Purification
Preparation of DNA for coupling to Sepharose and coupling of DNA
to Sepharose followed the method of Kadonaga and Tjian (36). The
oligonucleotide coupled to Sepharose was the mM6 oligonucleotide that
in electrophoretic mobility shift assay does not bind Sp1 (14).
Partially purified HeLa cell nuclear extract protein from the DEAE
Sepharose column was used with 15 µg/ml of poly (dI-dC)·poly
(dC-dI) as the competitor DNA. DNA affinity chromatography was
performed as described by Kadonaga and Tjian (36).
DNase I Footprint Analysis
The porcine -500 P450scc/luc construct (13) was linearized with
HindIII and radioactively labeled using Klenow enzyme and
three nucleotides. The probe was separated from the plasmid by
digestion with BamHI and isolated on an 8% nondenaturing
polyacrylamide gel. The band was identified by autoradiography, and the
probe was electroeluted from the gel slice using a Bio-Rad Laboratories, Inc. 422 electroeluter. The recovered probe was
phenol-chloroform extracted and ethanol precipitated for further
purification. The Core Footprinting system (Promega Corp.)
was used to perform the DNase I footprinting analysis. Protein samples
containing probe were allowed to incubate on ice for 30 min before
DNase digestion. Samples were heated for 2 min at 95 C and then chilled
on ice before electrophoresis on an 8% polyacrylamide sequencing gel
with a P450scc sequence ladder.
PSF Expression in Eschericia coli
Competent BL21DE3 plysS cells (Novagen, Madison, WI) were
transformed with PSF (pET15b) DNA and used as an inoculum for an
overnight culture. Expression of PSF was performed by propagation of
the respective transformed host cells on super broth to an
A650 of 0.65.
Isopropyl-ß-D-galactopyranoside was added to a final
concentration of 1 mM and incubation was continued for
4 h at 37 C. Cells were collected by centrifugation at 3000
x g for 10 min at 4 C. Cells were suspended (0.2 g/ml) in
20 mM Tris HCl, pH 7.5, 500
mM NaCl, 1 mM phenylmethyl
sulfonyl fluoride (PMSF) (Life Technologies, Inc.), 1
µg/ml leupeptin (Amersham Pharmacia Biotech), 1 µg/ml
pepstatin A (Amersham Pharmacia Biotech), and 1 µg/ml
aprotinin (Amersham Pharmacia Biotech) and frozen once at
-80 C. The thawed cell suspension was sonicated before centrifugation
at 16,000 x g for 20 min at 4 C. The supernatant was
removed and Probond Ni-resin (Invitrogen) was added (1 ml
resin/10 ml supernatant). After a 1-h incubation at 4 C with constant
stirring, the resin was collected by centrifugation (2000 x
g, 5 min 4 C) and suspended in 50 ml of 20
mM Tris HCl pH7.5, 500 mM
NaCl, 1 mM PMSF. The resin was collected using a
1 x 10 cm column (at 4 C) and then washed with 100 ml of 20
mM Tris HCl, pH 7.5, 500 mM
NaCl, 20 mM imidazole (Sigma). A 200
ml linear gradient (20200 mM imidiazole) was
used to elute the resin (16 ml/h) and 4 ml fractions were collected and
analyzed (20 µl) by electrophoresis on polyacrylamide gels (10%,
1/37). PSF eluted as a
broad peak between 100150 mM imidazole. Protein
concentrations were determined using Bradford reagent (Bio-Rad Laboratories, Inc.). Expressed PSF protein was isolated and
given to Bio-Molecular Technology, Inc. (Frederick, MD) for generation
of a polyclonal antibody to PSF.
Statistical Analysis
Statistical differences between transient transfection
experiments in NWTb3 and porcine granulosa cells were determined by
Kruskal-Wallis one-way ANOVA on ranks with Student-Newman-Keuls
multiple comparison test. P values of
0.05 were considered
statistically significant. Data are presented as mean ±
SE.
 |
ACKNOWLEDGMENTS
|
---|
We thank Steve Smith for performing the sequence analysis.
 |
FOOTNOTES
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Address requests for reprints to: Randall J. Urban, M.D., 8.138 MRB, 1060, Division of Endocrinology, University of Texas Medical Branch, Galveston, Texas 77555-1060.
This work was supported by NIH Grants HD-36092 (R.J.U.) and NS-29261
(A.K.) and the University of Texas Medical Branch Educational Cancer
Center Protein Chemistry Laboratory.
Received for publication September 23, 1999.
Revision received March 15, 2000.
Accepted for publication March 22, 2000.
 |
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