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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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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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).
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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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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after separation by SDS-PAGE.
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, the PSF antibody recognized a
single protein band of 100 kDa that showed strong affinity for the
column (0.6–0.8 M KCl).
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). 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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). 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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) 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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, 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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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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 protein’s 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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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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-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 Eagle’s 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 1–2 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 (20–200 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 100–150 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.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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1-crystallin gene by insulin-like growth
factor I and insulin requires DNA cis elements in chicken. Proc Natl
Acad Sci USA 87:3353–33571-crystallin promoter are developmentally regulated. Biochem Biophys
Res Commun 183:659–665[CrossRef][Medline]
inhibits transcriptional activity of the porcine P-450 11A
insulin-like growth factor response element. J Biol Chem 271:31699–31703-hydroxylase enzymes in ovarian
hyperthecosis. Fertil Steril 71:328–333[CrossRef][Medline]
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