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INTRODUCTION |
The melanoma growth stimulatory activity/growth-regulated protein
(CXCL1)1 chemokine plays an
important role in wound healing, inflammation and tumorigenesis (1).
The CXCL1 gene is not constitutively expressed in normal
retinal pigment epithelial cells (RPE) but can be induced by cytokines
such as interleukin-1 (IL-1) and tumor necrosis factor
and
bacterial products such as lipopolysaccharide. In contrast, Hs294T
malignant melanoma cells exhibit high, constitutive levels of
CXCL1 mRNA. IL-1 treatment of Hs294T cells does not significantly increase the transcription of the gene, although it does
appear to stabilize CXCL1 mRNA (2).
Transcription of the CXCL1 gene is regulated through a
306-bp minimal promoter comprising four cis-acting elements, which include the TATA box (
25 to
30 nt), a NF-
B binding site (
67 to
77 nt), an AT-rich HMGI(Y) binding element nested within the NF-
B
site, an immediate upstream region (IUR;
78 to
93 nt), and a
GC-rich SP1 binding site (
117 to
128 nt) (3). In RPE cells, IL-1
increases nuclear levels of NF-
B p65 (RelA) and NF-
B p50 subunits
(4). This is due to the IL-1-induced activation of the I-
B kinases
(IKK1/IKK2) and subsequent phosphorylation, ubiquitination and
degradation of the IKK substrate, I-
B. In Hs294T cells,
constitutively high nuclear levels of the p50 and p65/RelA proteins can
be correlated with constitutive activity of IKK1/IKK2 and enhanced
degradation of the I-
B protein. (5, 6). The NF-
B element
therefore represents a crucial, inducible component of the putative
CXCL1 enhanceosome.
The IUR is an ~20-bp sequence, which is located immediately upstream
of the NF-
B site in the CXCL1 promoter. Previously, we
demonstrated that this element is essential for basal as well as
cytokine expression of the CXCL1 gene. In particular, point mutations within a putative TCGAT motif abolished basal and
IL-1-induced transcription in reporter gene assays with RPE and Hs294T
cells. Furthermore, in electrophoretic mobility shift assays (EMSA), these mutations blocked the ability of this element to compete with a
constitutive, IUR-specific complex in RPE and Hs294T nuclear extracts
(7). UV cross-linking and Southwestern blot analyses revealed that at
least one protein having a relative molecular size of 115 kDa bound the
IUR element in a sequence-specific (8). In this study, the 115-kDa
IUR-specific protein has been purified by binding site oligonucleotide
affinity chromatography. Peptide sequence analysis identifies this
protein as poly(ADP-ribose) polymerase (PARP).
PARP is a 114-115-kDa nuclear DNA-binding protein, which catalyzes the
transfer of long, branched ADP-ribose chains to either itself or
different classes of target proteins involved in chromatin decondensation, DNA replication, DNA repair, and gene expression (9,
10) ADP-ribosylation by PARP affects such cellular processes as
apoptosis, necrosis, cellular differentiation, and malignant transformation (11). PARP appears to have dual functions in the
regulation of transcription. PARP-mediated ADP-ribosylation of the
transcription factors TATA-binding protein, Yin-Yang (YY1), Sp1 (12),
NF-
B (13), and p53 (14) and alters the sequence-specific DNA binding
of these protein and is thought to cause a reversible silencing of
transcription. On the other hand, PARP has been shown to enhance
activator-dependent transcription in vitro (15). More recently, PARP has been shown to activate muscle-specific gene
expression by interacting with sequences in the MCAT element of the
cardiac troponin T promoter and by ADP-ribosylating the MCAT-specific
transcription enhancer factor 1 (16). In addition, PARP increases the
on-rate binding of nuclear factors to the PAX-6 gene enhancer (17).
Furthermore, PARP has been shown to activate transcription of genes
involved in cell proliferation in cooperation with the transcription
factor, B-MYB (18) and potentiates the transcriptional activation by
the human T-cell leukemia virus type 1 Tax protein (19).
In this study, the IUR element of the CXCL1 promoter
has been further characterized. Evidence presented here indicates that the IUR is a positive cis-acting element and its activity is dependent on its contiguity with the adjacent NF-
B element. A 115-kDa IUR specific activity has been purified by binding site oligonucleotide affinity chromatography and identified as PARP. A specific PARP inhibitor, 3-aminobenzamide, inhibits CXCL1 promoter
activity and decreases the endogenous levels of CXCL1
mRNA in a dose-dependent manner. We propose that PARP
is a potential coactivator of CXCL1 gene transcription.
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MATERIALS AND METHODS |
Cell Lines and Treatments--
Hs294T melanoma cells are a
continuous cell line established from a human melanoma metastatic to
the lymph node. These cells were obtained from American Type Culture
Collection (Rockville, MD). RPE cells are normal retinal pigment
epithelial cells that were cultured by Dr. Glenn Jaffe from the North
Carolina Organ Donor and Eye Bank within 24 h of death. RPE and
Hs294T cells were cultured as described previously (7). In one series
of experiments, Hs294T cells were incubated at 37 °C in the absence of serum for 48 h, during which period, cells received the
indicated doses of 3-aminobenzamide (Sigma-Aldrich) at 24-h intervals.
Sequence of Oligonucleotides--
The wild-type IUR probe,
2xIUR, used in electrophoretic mobility shift assays and
Southwestern blot analyses had the upper strand sequence
5'-ccatcgatctggaactccggttcgatctggaactccggatgc-3' and contained two copies of the IUR sequence, which are underscored. Sequences between the two IUR repeats and those flanking the
probe were included to optimize binding. A mutant IUR oligonucleotide, 2xmIUR, which contained mutations in the TCGAT motif of the IUR element
had the upper strand sequence,
5'-ccaAGTaCctggaactccggtAGTaCctggaactccggatgc-3'. Uppercase characters indicate nucleotide replacements in the
TCGAT motif, and the underscored sequences define the two copies of IUR element.
The oligonucleotide used to multimerize the IUR sequence for DNA
affinity chromatography had the upper strand sequence 5'- gggatcgatctggaactccgggatcgatctggaactcc-3' and
the lower strand sequence 5'-cccggagttccagatcgatcccggagttccagatcgat-3'.
The underscored characters represent the two copies of the IUR
sequence. The two strands when annealed form cohesive ends and were
ligated with T4 polynucleotide kinase (Promega, Madison, WI) to
generate the multimerized IUR DNA comprising up to 24 tandem repeats of
the 2x IUR sequence. A similar strategy was employed to generate the multimerized mutant IUR (mIUR) DNA using the upper strand sequence 5'-gggaAGTaCctggaactccgggaAGTaCctggaactcc-3'.
The uppercase characters represent nucleotide replacements in the
TCGAT motif, and the underscored characters outline the IUR element repeats.
Reporter and Expression Vectors--
The CXCL1 minimal promoter
region (
306 to +45 nt) was inserted in the pGL2 Basic vector
(Promega) either in the correct orientation (pWT.Luc) or in the
opposite orientation (pREV.Luc) relative to the transcription start
site. To separate the IUR element from the NF-
B site, pWT.Luc was
modified to include an NcoI site between the two elements
using the GeneEditor in vitro site-directed mutagenesis sytem (Promega, Madison, WI). The mutagenic oligonucleotide employed for this purpose had the sequence:
5'-tcgggatcgatctggaactccATgggaatttccctggcc-3'. (The
characters in uppercase represent the 2-nucleotide insertion that was
necessary to create the NcoI site, which is underscored.) The modified construct was termed pIN:2 to represent a two nucleotide insertion between the the IUR and NF-
B sites. Similarly, mutant promoters containing a 6-bp (pIN:6) and 12-bp insertion (pIN:12) were
generated using the mutagenic oligonucleotides
5'-gggatcgatctggaactccGGATCCgggaatttccctggcc-3' and
5'-tcgggatcgatctggaactccGGATCCTCTAGAgggaatttccctggcc-3'. (Characters in
uppercase represent the BamHI site and the
BamHI/XbaI sites introduced to create the 6- and
12-bp separations.) To generate the promoter with a 25-bp insertion
(pIN: 25), a 25-bp oligonucleotide having the sequence
5'-catggcagtgagcgcaacgcaattac-3' and flanked by NcoI sites,
was inserted in the NcoI site of pIN:2. A clone pIN:50, in
which the 25-bp insert had dimerized, was also selected. To generate
the pGL2.mIUR construct, the TCGAT motif in the wild type promoter was
mutated to AGTAC by using a mutagenic oligonucleotide 5'-ttccttccggactcgggaAGTaCctggaactccgggaatt-3'. All
constructs were confirmed by restriction analysis and automated plasmid sequencing.
Reagents--
Anti-PARP antibodies were from Santa Cruz
Biotechnology (Santa Cruz, CA). The PARP enzyme was purchased from R&D
Systems (Minneapolis, MN).
Electrophoretic Mobility Shift Assays--
Nuclear extracts were
prepared as described previously (7). All extracts contained a 1×
concentration of Complete protease inhibitor mixture (Roche Molecular
Biochemicals). The 2xIUR or the 2xmIUR probes were radiolabeled by
extension of an annealed primer, 5'-gcatccggagttcca-3' with the Klenow
fragment of Escherichia coli DNA polymerase, dNTPs, and
[
-32P]dCTP. A typical binding reaction involved a
15-min pre-incubation with 10 µg of nuclear extract, 2 µg of
nonspecific competitor poly(dI-dC), 200 ng of single-stranded
oligonucleotide, 20 mM Hepes-NaOH (pH 7.6), 100 mM NaCl, 1 mM dithiothreitol, 2% glycerol, followed by a 20-min incubation with 50,000 cpm (40 fmol) of
radiolabeled probe. In oligonucleotide competitions, 1000-fold molar
excess of cold, double-stranded oligonucleotide was added to the
pre-incubation mix. Complexes were resolved by electrophoresis for
2 h at 170 V on a 6% native, polyacrylamide gel, which was later
dried and processed for autoradiography. EMSA with
affinity-purified fractions contained 50 ng of poly(dI-dC) and 100 ng
of cold, double-stranded 2mR oligonucleotide to ensure specificity of
binding in the fractions. In cases where purified PARP was used, the
reaction mixture contained 50 ng of poly(dI-dC) and about 5-10 fmol of
pure enzyme (catalog no. 4667-50-01) obtained from R&D Systems.
Southwestern and Western Blot Analysis--
Nuclear extracts (25 µg) were heated at 90 °C for 3 min in 50 mM Tris-Cl
(pH 6.8), 100 mM dithiothreitol, 2% SDS, and 10% glycerol, resolved on 4% stacking/8% resolving SDS-polyacrylamide gel
and electrophoretically transferred to nitrocellulose membranes (Bio-Rad). The membranes were rocked for 30 min in phosphate-buffered saline, blocked for 4 h at room temperature with buffer A (20 mM Hepes-NaOH, 50 mM NaCl, 12.5 mg/ml skim milk
powder, 2.5 mg/ml bovine serum albumin, 100 µg/ml native salmon sperm
DNA), and incubated overnight at room temperature in up to 2 ml of
Buffer A + 107 cpm (8-10 pmol) of radiolabeled 2xIUR or
2xmIUR probes. Membranes were subjected to three washes of 15 min each
at room temperature in a buffer containing 20 mM
Hepes-NaOH, 50 mM NaCl, 1 g/liter skim milk powder, 0.025%
Nonidet P-40, prior to drying and autoradiography.
For Western analysis, membranes were probed with 800 µg/10 ml
anti-PARP rabbit polyclonal antibody (catalog no. sc-7150) from Santa
Cruz Antibodies (Santa Cruz, CA) using procedures recommended by the
antibody manufacturer, and the signal was visualized by enhanced
chemiluminescence (ECL) assay (Amersham Pharmacia Biotech) according to
the manufacturer's recommendations.
Northern Analysis--
Total RNA from Hs294T cells treated over
a 48-h period with various doses of 3-aminobenzamide was isolated using
the Ultraspec RNA isolation system (Biotecx Laboratories, Houston, TX)
according to the manufacturer's protocol. Purified RNA (20 µg/lane)
was used in Northern analysis, which was performed exactly as described previously (2). After probing for levels of CXCL1 mRNA, blots were
stripped by boiling for 10 min in water and re-probed with radiolabeled, cyclophilin cDNA. Bands corresponding to the CXCL1 and cyclophilin were analyzed by PhosphorImager using ImageQuant software (Molecular Dynamics). Values for CXCL1 mRNA were
normalized against cyclophilin mRNA. The experiment was repeated
three times in triplicate. Mean normalized values obtained from samples
receiving the highest dose of 3-aminobenzamide (3-AB) were compared
with values from samples that received Me2SO alone and were
found to be statistically different as determined by Student's
t test (paired).
Transient Transfection and Reporter Activity Assay--
Hs294T
were plated in 60-mm dishes at a density of 2 × 105.
The following day, cells at nearly 70% confluence were transfected using the LipofectAMINE/Plus method (Life Technologies, Inc.) with 1 µg of the appropriate reporter construct containing either the wild
type CXCL1 promoter (WT) or promoters containing insertions between the
IUR and NF-
B elements or nucleotide replacements within the TCGAT
motif of the IUR element (mIUR.Luc). In addition, all samples received
1 µg of pRSV-
-gal. Cells were harvested 48 h after
transfection, and luciferase activity was measured using the luciferase
assay system (Promega) and Monolight 2010 Luminometer (Analytical
Luminescence Laboratory, San Diego, CA).
In transfection experiments where 3-aminobenzamide was used, cells were
first transfected with 1 µg of pRSV-
-gal reporter as well as
reporter constructs driven by either the wild type (CXCL1.Luc) or the
mutant CXCL1 (mIUR.Luc) promoters. Six hours after transfection, the
medium was replaced by medium containing either Me2SO or
3-aminobenzamide within a dose range of 1-5 M. A second
change of Me2SO- or 3-AB-containing medium was given at
24 h after transfection. Cells were harvested 48 h after
transfection and processed as described above.
All values were normalized to
-galactosidase expression to correct
for transfection efficiency. Each experiment was repeated three
separate times in triplicate. In promoter mutagenesis experiments, mean
normalized values from samples showing the highest inhibition of
promoter activity were compared with those obtained from samples transfected with the wild type promoter and were found to be
significantly different according to Student's paired t
test. Similarly, mean normalized values of the samples treated with the
highest dose of 3-AB were compared with values obtained of the sample
containing the wild type promoter treated with Me2SO alone
and were found to be significantly different according to Student's
paired t test (p < 0.01). Statistical
measurements were performed using Microsoft Excel software.
Purification of the 115-kDa IUR-specific Protein--
Binding
site oligonucleotide affinity chromatography was performed using a
modified protocol of Kadonaga et al. (20). Briefly, multimers of the IUR were cross-linked to cyanogen bromide-activated agarose to generate the IUR-agarose column. Similarly, a control column
containing mIUR multimers (mIUR-agarose) was made. Cells equivalent to
60 liters of HeLa suspension culture were obtained from National Cell
Culture Center (Minneapolis, MN). Approximately 2.5 g of nuclear
extract was prepared from these cells. First, nonspecific DNA-binding
proteins were competed with poly(dI-dC) at a protein:DNA ratio of 30:1,
incubated at room temperature for 6 h, and eliminated by high
speed centrifugation. Unbound proteins in the supernatant were
subjected to binding site oligonucleotide chromatography, which
involved two passages through a 3-ml settled bed volume of the mutant
IUR-agarose column and an overnight incubation with a 3-ml bed volume
of IUR-agarose resin, followed by two passes through the same column.
After 5 washes of 3 ml each in a buffer containing 50 mM
NaCl, fractions were eluted from the IUR-agarose column by a step
gradient of buffers containing 0.2-1.0 M NaCl. In EMSA and
Southwestern assays, the 400 mM NaCl fraction consistently tested positive for an activity with a relative molecular size of 115 kDa that specifically bound the 2xIUR probe. SDS-PAGE and silver
staining revealed selective enrichment of the 115-kDa protein.
To further purify the 115-kDa protein, the 400 mM NaCl
fraction was pooled, dialyzed against water, lyophilized, and
reconstituted in the 1× SDS-PAGE loading dye. The sample was then
subjected to continuous-elution SDS-PAGE on a 7% polyacrylamide gel
using a model 491 Prep Cell (Bio-Rad).
Electro-elution was performed at a flow-rate of 0.1 ml/min. Aliquots of
electro-eluted fractions were screened by electrophoresis on 4%
stacking/10% resolving SDS-PAGE gels, and polypeptides were detected
by silver-staining. Fractions that contained the 115-kDa protein were
pooled and lyophilized. Reconstituted sample was re-electrophoresed
through a 4% stacking/8% resolving SDS-PAGE gel, and the band
containing the 115-kDa protein was excised and sent for sequencing. Two
different, nearly homogeneous, preparations of the 115-kDa protein were
sequenced separately in two different laboratories: by microcapillary
reverse phase chromatography electrospray ionization tandem mass
spectrometry using an ion trap mass spectrometer (LCQ; Finnigan MAT,
San Jose, CA) in the laboratory of Dr. R. Aebersold at the University
of Washington (Seattle, WA) and by mass spectrometry/mass
spectrometry/matrix-assisted laser desorption ionization/time of flight
spectroscopy at the sequencing facility at the W. M. Keck
Foundation (Yale University, New Haven, CT).
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RESULTS |
The Activity of the CXCL1 Promoter Depends on the Position and
Orientation of the IUR Element--
The IUR element in the
CXCL1 promoter plays an important role in the basal and
cytokine induced expression of the CXCL1 gene. The IUR
contains a TCGAT motif designated here as
T1C2G3A4T5.
Previously, we showed that a T5
C point mutation
inhibits basal and IL-1-induced CXCL1 promoter activity. To
further characterize the IUR element, the effect of multiple
replacements in the TCGAT motif on CXCL1 promoter activity
was examined. In addition we investigated whether dislocating the IUR
relative to the NF-
B site has any effect on transcriptional activity
of the CXCL1 promoter. We created reporter gene constructs that either retained the 306-bp CXCL1 promoter in the wild type configuration (WT) or had a 2-, 6-, 12-, or 25-bp insertion between the
IUR and NF-
B elements (IN:2, IN:6, IN:12, and IN:25, respectively). Two additional promoter mutants were also constructed, one which reversed the entire 350-bp CXCL1 promoter relative to the
luciferase reporter (Rev) and a second, which had a
T1C2G3A4T5
to
A1G2T3A4C5
conversion in the IUR element (mIUR). These constructs were then transfected into Hs294T malignant melanoma cells and luciferase activity measured 48 h later. Results in Fig.
1A indicate that transcription
from the CXCL1 promoter is inhibited by nearly 90% when the
entire promoter is oriented in a direction opposite to the
transcription start site (Rev). Furthermore, a 75% inhibition in
promoter activity is observed when the
T1C2G3A4T5
motif in the IUR element is replaced by
A1G2T3A4C5
(mIUR). A similar inhibition of promoter activity is seen when the IUR
and NF-
B elements are separated by insertions (Fig. 1B). Although promoter activity appears to be unaffected when the IUR and
NF-
B elements are separated by a 2-bp insert. However, inserts that
dislocate the IUR element from the NF-
B site by 6, 12, and 25 bp
strongly inhibit promoter activity in a distance dependent manner. This
inhibition, however, is independent of helical phase of the two
elements, because introduction of a half (IN:6) or two and a half
helical turn (IN:25) has the same effect as a full helical turn
(IN:12), ruling out the possibility that the insert-mediated inhibition
of CXCL1 promoter activity is due to a change in the helical
phase between the two elements. Together, the data suggest that the IUR
is a positive cis-acting element and that the transcriptional activity
of the CXCL1 promoter is strictly dependent on the distance of the IUR element relative to the NF-
B site.

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Fig. 1.
The IUR element is a positive cis element,
which requires the TCGAT motif as well as contiguity with the adjacent
NF- B element. A, 5 × 105 Hs294T cells were transfected with RSV- -gal vector
and one of the following luciferase reporter constructs: 1, a reporter
construct lacking the CXCL1 promoter (pGL2); 2, a reporter
construct driven by the wild-type CXCL1 promoter in the correct
orientation relative to the transcription start site (WT);
3, a reporter construct with the CXCL1 promoter in the opposite
direction (REV); 4, a mutant CXCL1 promoter in which the
TCGAT motif was altered to AGTAC (mIUR). B,
5 × 105 Hs294T cells were transfected with
RSV- -gal vector as well as luciferase reporter constructs driven by
the wild type CXCL1 promoter or mutant promoter which had an insertion
of 2 bp (IN:2), 6 bp (IN:6), 12 bp
(IN:12), or 25 bp (IN:25) between the IUR and the
NF- B elements. 48 h after transfection, cells were harvested
and luciferase activity was measured and normalized to
-galactosidase activity. Shown here is a representative of three
independent experiments performed each time in triplicate. Mean
normalized values obtained from the sample showing the highest
inhibition in promoter activity were compared with those from samples
transfected with the wild type promoter and were found to be
significantly different according to Student's t test
(paired). The asterisk (*) indicates p < 0.01.
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Purification and Identification of 115-kDa IUR-binding
Protein--
We have previously demonstrated that the IUR element
binds a 115-kDa protein in Southwestern blot assays (8). To identify the 115-kDa protein, which binds the IUR, we purified the IUR-specific factor by binding site oligonucleotide affinity chromatography. HeLa
nuclear extract proved to be a convenient and abundant source for both
proteins. We obtained frozen pellets of HeLa cells from 63-liter
suspension cultures from the National Tissue Culture Center. The yield
of nuclear extract from a 63-liter suspension culture was about
1.5-2.0 g. The IUR-specific activities were reproducibly purified to
near homogeneity in three separate experiments. Making use of the
following protocol, nuclear extract from HeLa cells was first
challenged with nonspecific competitor DNA and chromatographed
through the mutant IUR-agarose column, which contained multimers of the
double-stranded mIUR oligonucleotide. The unbound fraction was then
chromatographed through an IUR-agarose column, which contained
multimers of double-stranded IUR oligonucleotide. Bound fractions from
both columns were washed and eluted by a step gradient of NaCl ranging
from 50 mM to 1000 mM. Proteins specific to the
IUR element emerged in the unbound (flow-through) fraction of the
mutant IUR column and eluates from the bound fraction of this column
consistently tested negative for IUR specific activity in EMSA (data
not shown). Eluates from the IUR-agarose column were assayed for IUR
specific activity by EMSA and Southwestern blot analysis. Fig.
2 is a representative of three separate
experiments. The results from an EMSA (Fig. 2A) indicate
that an IUR specific activity reproducibly eluted in the 400 mM fraction of the IUR-agarose column. This correlated well
with results from Southwestern blot analysis (Fig. 2B),
where a 115-kDa protein in the 400 mM fraction bound the
2xIUR element in a sequence-specific manner. Proteins from bound and
unbound fractions from the IUR-agarose column were resolved by SDS-PAGE
(Fig. 2C) and detected by silver staining. SDS-PAGE analysis
consistently showed that the 400 mM fraction of the
IUR-agarose column essentially contains five bands with relative
molecular sizes of 180, 115, 85, 42, and 29 kDa. Of these, the 115-kDa
band was considerably enriched in this fraction.


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Fig. 2.
Binding site oligonucleotide affinity
purification of the 115-kDa IUR-specific protein. In
electrophoretic mobility shift assays (A), crude HeLa
nuclear extract (2 µg) (lane 1) or 200 ng of
bound fractions eluting at 0.2 M NaCl (lanes
2-5), 0.4 M NaCl (lanes
7-10), 0.6 M NaCl (lanes
11-14), or 1 M NaCl (lanes
16-18) from the IUR-agarose column, were tested for binding
to the 2R probe in EMSA. All samples contained a 250-fold molar excess
of cold oligonucleotide corresponding to the 2mR probe. B,
Southwestern blot analysis: Polypeptides in crude HeLa nuclear extract
(25 µg) (lanes 1 and 3) or the 0.4 M NaCl fraction (100 ng) eluted from the IUR-agarose column
were separated on 8% SDS-PAGE gels, transblotted on nitrocellulose
membranes, and probed with radiolabeled oligonucleotides corresponding
to either the 2R (lanes 1 and 2) or
the 2mR (lanes 3 and 4) probes. The
relative molecular size is indicated on the left.
C, silver-stained SDS-PAGE profile of the 0.4 M
NaCl fraction. Analysis: polypeptides in crude HeLa nuclear extract
(500 ng) or the 0.4 M NaCl fraction (100 ng) eluted from
the IUR-agarose column was separated on 8% SDS-PAGE gels and the
protein was visualized by silver-staining. D, SDS-PAGE
electro-elution. The 0.4 M IUR-agarose eluate was then
fractionated by electro-elution through a 7% SDS-PAGE using the model
491 Prep Cell (Bio-Rad). Fractions were collected, every eighth
fraction was electrophoresed on an 8% SDS-PAGE gel, and proteins were
stained by silver staining.
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To further purify the 115-kDa protein, the 400 mM
IUR-agarose fractions were pooled, dialyzed and lyophilized to dryness. The reconstituted 400 mM fraction was then electrophoresed
through a preparative SDS-PAGE Miniprep cell and 265 fractions of 1.0 ml each were collected. Electro-eluted fractions were analyzed by
SDS-PAGE/silver-staining. Fig. 2D shows results of such an experiment. The 115-kDa protein electro-elutes reproducibly in fractions 135-160 of the Miniprep cell.
Two separate preparations of the 115-kDa protein, purified to near
homogeneity, were independently sequenced at two different institutions: by mass spectrometry/mass spectrometry/matrix-assisted laser desorption ionization/time of flight spectroscopy mass
spectroscopy at W. M. Keck Foundation (Yale University, New Haven,
CT) and by microcapillary reverse phase chromatography electrospray
ionization tandem mass spectrometry using an ion trap mass spectrometer
(LCQ, Finnigan MAT, San Jose, CA) at the University of Washington
(Seattle, WA) by S. Gygi and R. Aebersold). Each preparation had about
10-20 pmol of the 115-kDa protein. Both sources identified the 115-kDa protein as the human PARP (EC no. 2.4.2.30).
The DNA Affinity-purified 115-kDa Protein Is PARP--
We tested
the 400 mM fraction from the IUR-agarose column by Western
blot analysis (Fig. 3A) to
determine whether or not the 115-kDa is PARP. Anti-PARP antibodies
successfully detected a 115-kDa protein in the fraction, thus
confirming that the affinity purified 115-kDa protein is PARP.

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Fig. 3.
PARP can bind the IUR element. A,
Western blot analysis. Crude HeLa nuclear extract (25 µg),
unbound/flow-through fraction from the IUR-agarose column (25 µg) and
100 ng of 0.2 M (lanes 3 and
4) or 0.4 M (lanes 5 and
6) IUR-agarose fractions were separated on 8% SDS-PAGE gels
and probed with anti-PARP antibody. Arrow ( ) indicates
the relative mobility of the PARP protein. B, EMSA.
Partially purified, commercially available PARP was tested for binding
to the 2xIUR (lanes 1-3) or the 2xmIUR
(lanes 4-6) probes. Nonspecific complexes were
competed with poly(dI-dC) in the range of 0.2- 1.0 µg. The
arrow ( ) indicates the specific complex formed by PARP
with the 2xIUR probe, and NS represents nonspecific
complexes.
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Recombinant PARP Binds the CXCL1 IUR Element in a Sequence-specific
Manner--
Results in Fig. 2 demonstrate that the 115-kDa protein
(PARP) emerged in the unbound fraction of the mutant IUR-agarose column and eluted in the 400 mM fraction bound to IUR-agarose
column. This strongly suggested that the PARP protein could
discriminate between the mutant and wild-type IUR sequences. To verify
this, we used purified, recombinant PARP in EMSA and compared PARP
binding to 2xIUR and 2xmIUR probes (Fig. 3B). The use of
poly(dI-dC) in the reaction mixture was found to be necessary since the
recombinant protein was not a homogeneous preparation. Specificity to
IUR was observed at relatively high concentrations of the nonspecific competitor DNA. Three shifted complexes could be detected with the
2xIUR probe. Two complexes, which were specific to 2xIUR, did not bind
the 2xmIUR probe. This result indicates that PARP can form a
sequence-specific complex with the IUR element of CXCL1. We would like
to point out that the complex generated by the purified recombinant
protein appears to differ in mobility and intensity to the one obtained
from crude nuclear extracts. Moreover, there are two IUR-specific
complexes generated with the recombinant protein as opposed to a single
complex observed with crude nuclear extracts. The reasons for these
discrepancies are unclear at this time. However, it is likely that the
complex observed in crude nuclear extract could be a composite of more
than one protein in addition to PARP.
3-AB Decreases Levels of CXCL1 mRNA in Hs294T Cells--
PARP
catalyzes the transfer of multiple ADP-ribose units to target nuclear
proteins. This ADP-ribose transferase activity is believed to inhibit
DNA binding as well as trans-activation functions of the target
proteins. 3-AB, a specific PARP inhibitor, blocks ADP-ribose
transferase functions of PARP. We asked the question whether inhibition
of PARP catalytic activity by 3-AB might have any effect on CXCL1
expression in Hs294T cells.
Hs294T cells were either left untreated or treated with 3-AB in a dose
range of 1.0 mM to 5.0 mM. Forty-eight hours
later, cells were harvested and RNA from these samples was isolated. CXCL1 mRNA levels were measured by Northern analysis (Fig.
4A). Values, normalized
against levels of an internal control cyclophilin B, are graphically
represented in Fig. 4B. Results indicate that 3-AB treatment
inhibited CXCL1 mRNA levels by nearly 60% over untreated or Me2SO-treated controls. The data suggest that
PARP-mediated ADP-ribosylation is essential for CXCL1 gene
expression. The ADP-ribose transferase activity of PARP has been shown
to be involved in negatively regulating gene expression by abrogating
the DNA binding of such factors as NF-
B, YY1, C/EBP
, and Sp1. In
addition, ADP-ribosylation of these factors does not appear to require
DNA binding of PARP. It is therefore possible that the effect of 3-AB
on CXCL1 mRNA levels may be indirect and could involve
other trans-activators of the CXCL1 promoter such as NF-
B
and Sp1.

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Fig. 4.
3-AB inhibits MGSA/GRO expression.
A, Northern analysis. Hs294T cells were treated for 48 h with indicated concentrations of 3-AB. RNA from samples was resolved
on a 1.4% formaldehyde-agarose gel, transblotted to nitrocellulose
membrane, and probed with an MGSA/GRO cDNA probe (A,
top panel). Blots were stripped and re-probed
with a cDNA probe for cyclophilin B transcripts (A,
bottom panel). Blots were densitometrically
scanned using a PhosphorImager (Molecular Dynamics). Values for
MGSA/GRO mRNA were normalized against those for cyclophilin
mRNA. B, a graphical representative of six independent
experiments, each performed in triplicate is shown. The results in all
six experiments were qualitatively identical. Error
bars represent S.D. values. The mean normalized value
obtained from samples receiving the highest dose of 3-AB was compared
with those from samples receiving Me2SO and were found to
be significantly different according to Student's paired t
test. The asterisk indicates p < 0.01.
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To test the possibility that the effect of 3-AB on CXCL1 mRNA
levels is at the level of the CXCL1 promoter and involved the IUR
element, luciferase reporter constructs driven by either the wild type
CXCL1 promoter or a promoter containing replacements in the
IUR element of this promoter were transiently transfected in Hs294T
cells and cells were treated with 3-AB in a dose range of 1-5
mM. Cells were harvested 48 h after transfection, and
luciferase activity was measured. Fig. 5
shows a representative of four independent experiments performed in
duplicate. The data indicate that 3-AB inhibited CXCL1
promoter activity in a dose-dependent manner. Maximum
inhibition (50%) was observed at 5 mM 3-AB. There was no
effect on the mutant IUR promoter (mIUR.Luc), although the mIUR
promoter contained intact binding sites for NF-
B, HMGI(Y), and Sp1.
The results in Fig. 2 show that the 115-kDa protein identified to be
PARP bound the wild type IUR but not the mutant mIUR sequence and that
recombinant PARP specifically bound the IUR but not the mIUR sequence
(Fig. 4). Results shown in Fig. 5 further demonstrate that the
PARP-specific inhibitor, 3-AB, specifically inhibits the wild type
CXCL1, but not the mIUR promoter, indicating that PARP mediated
ADP-ribosylation is involved in the trans-activation of the CXCL1
promoter. This rules out the possibility that 3-AB-mediated inhibition
of CXCL1 mRNA levels is due to abrogation of other trans-acting
factors of the CXCL1 promoter.

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Fig. 5.
3-AB inhibits CXCL1 promoter activity.
5 × 105 cells were first transfected with 1 µg of
pRSV- -gal reporter as well as reporter constructs driven by either
the wild type (MGSA.Luc) or the mutant CXCL1 promoter (mIUR.Luc). Six
hours after transfection, the medium was replaced by medium containing
either Me2SO or 3 -AB within a concentration range of 1-5
mM. Cells were harvested 48 h after transfection and
luciferase activity was measured. Values obtained were normalized to
-galactosidase activity. The experiment was performed three times in
triplicate. Error bars represent standard
deviations. The mean normalized values obtained from samples receiving
the highest dose of 3-AB as compared with those from samples receiving
Me2SO were significantly different according to Student's
paired t test The asterisk indicates
p < 0.01.
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Binding of PARP to the IUR Element Does Not Require
ADP-ribosylation--
Since 3-AB treatment had a dramatic effect on
endogenous CXCL1 mRNA levels as well as on the CXCL1
promoter activity in reporter assays, we considered the possibility
that binding of PARP to the IUR element might involve the
ADP-ribosylation activity of the factor. Hs294T cells were treated with
3-AB within a dose range of 1-5 mM. Cells were harvested
after 48 h of 3-AB treatment, and nuclear extracts were prepared.
Fig. 6 shows a representative EMSA
experiment in which nuclear extracts from Me2SO-treated or 3-AB-treated cells were probed with radiolabeled 2xIUR oligonucleotide. The results indicate that there was no difference in the binding of the
IUR-specific protein, PARP, with 3-AB treatment. The data suggest that
ADP-ribosylation may not be involved in binding of PARP to the IUR
element.

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Fig. 6.
3-AB has no effect on binding to the IUR
element. Hs294T cells were treated with either Me2SO
alone or with 1-5 mM 3-AB for 48 h. Cells were
harvested, and nuclear extracts were made. 10 µg of nuclear extract
was incubated with 32P-labeled 2xIUR oligonucleotide probe
in the presence of 1-2 µg of poly(dI-dC). The reaction mixtures
electrophoresed on 6% native polyacrylamide gels, which were then
dried and processed for autoradiography. The arrow indicates
the specific complex associated with the IUR probe. Shown here is a
representative of three independent experiments. Results were
qualitatively similar in all three experiments.
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The data presented in this study provide evidence that the IUR element
is a positive cis-acting element in the CXCL1 promoter whose
function depends on its contiguity with the adjacent NF-kB element. The
IUR binds a 115-kDa factor, which has been purified and identified as
PARP. Although binding of PARP does not require the ADP-ribose
transferase function of PARP, this activity appears to be important for
its ability to transactivate the CXCL1 promoter and induce
CXCL1 mRNA levels. A model for the regulation of
CXCL1 transcription is proposed.
 |
DISCUSSION |
The CXCL1 gene is expressed at a high constitutive
level in several disorders including acute and chronic inflammation and malignant melanoma. Our earlier findings indicate that the
CXCL1 gene is under the control of a 306-bp minimal promoter
containing four cis-acting elements, including a 20-bp region called
the IUR, located immediately upstream to the NF-
B binding sequence (7). In an effort to understand the mechanism by which the CXCL1 gene is regulated, we have further characterized the
IUR element in the CXCL1 promoter and examined its role in
CXCL1 gene regulation.
To examine whether the IUR has a positive or negative role in
CXCL1 regulation, we first made mutations in the
CXCL1 promoter, which either altered the TCGAT motif or
separated it from the adjacent NF-
B element. When tested in
luciferase reporter assays, these mutations effected an almost 50%
inhibition in CXCL1 promoter activity. The data indicate
that the IUR is a positive regulatory sequence in the CXCL1
promoter and that the contiguity of the IUR and NF-
B element is
critical for optimal promoter activity.
In electrophoretic mobility shift assays, we previously identified
constitutive binding to the IUR element and detected a 115-kDa protein
that bound to the IUR element in Southwestern blot analyses. Mutations
that altered the TCGAT motif eliminated binding in both assays,
suggesting that these interactions were specific to the TCGAT motif of
the IUR element (8). Taken together with our current observations from
reporter assays, the evidence points to the possibility that p115 is a
positive transcriptional regulator of the CXCL1 gene.
In this study we have purified the 115-kDa protein binding to the IUR
element using a two-step procedure involving binding site
oligonucleotide affinity chromatography and electrophoretic separation
through a Bio-Rad Mini-prep cell. Pre-incubation of the nuclear extract
through a mutant IUR-Sepharose matrix eliminated nonspecific proteins
and ensured that bound fractions eluting from the wild-type
IUR-Sepharose matrix represented proteins that selectively bound the
wild-type but not the mutant IUR oligonucleotide.
Peptide sequence analysis has established the identity of the 115-kDa
IUR-specific protein as PARP. We show here by Western blot analysis
that the 115-kDa PARP is enriched in the IUR affinity-purified fraction, that peptide sequence of the purified 115-kDa matches that of
PARP, that a commercial preparation of the PARP enzyme can discriminate
between the wild-type and mutant IUR sequences, and that treatment of
cells with 3-AB, a specific PARP inhibitor, significantly reduces CXCL1
mRNA levels in Hs294T cells and inhibits CXCL1 promoter
activity in transient transfection assays. These data indicate that
PARP transactivates the CXCL1 promoter and its ADP-ribose transferase
activity is essential for this process.
The ability of PARP to activate or suppress specific gene expression
appears to be dependent on at least two factors: 1) the ADP-ribose
transferase activity of PARP (12) and 2) DNA binding of PARP (13).
Reversible repression of gene expression by PARP can be correlated with
its catalytic activity and may not involve DNA binding of the enzyme.
PARP-catalyzed ADP-ribosylation of TATA-binding protein, YY1, p53,
NF-kB, Sp1, CREB (12), Oct-1 (21), and retinoid X receptors (22) has
been shown to occur before DNA binding and prevented the formation of
transcriptionally active complexes. In contrast, DNA-bound PARP appears
to be associated with trans-activation. PARP has been identified as the
active component of the transcription factor TFIIC (19) In addition, PARP has been demonstrated to enhance activator-dependent
transcription during which DNA binding, but not the ADP-ribose
transferase activity, of the unmodified PARP seemed to be necessary for
transcriptional activation (15). More recently, PARP has been shown to
trans-activate the cardiac troponin T promoter by binding to a
5'-TGTTG-3' sequence in the MCAT cis element and cooperating with the
muscle-specific transcription enhancer factor 1 (16). In the latter
case, both DNA binding and ADP-ribose transferase functions appear to
be essential for coactivation of the troponin gene promoter.
We examined the possibility that PARP-mediated ADP-ribosylation plays a
role in the regulation of CXCL1 gene expression. We addressed this question by examining the effects of 3-aminobenzamide, a
specific inhibitor of PARP-mediated ADP-ribosylation, on 1) CXCL1 mRNA levels, 2) CXCL1 promoter
activity, and 3) specific binding to the IUR oligonucleotide probe. We
show here that inhibition by 3-AB reduces CXCL1 mRNA
levels and blocks CXCL1 promoter activity up to 50% over
untreated controls in a dose-dependent manner. The level of
inhibition seen with 3-AB appears to be similar to that observed when
the TCGAT motif in the CXCL1 promoter is altered. In addition, the
activity of the mutant IUR promoter appears to be insensitive to 3-AB
treatment, indicating that 3-AB effects target events associated with
the TCGAT motif. Inhibition of PARP-mediated ADP-ribosylation, however,
had no effect on the complex associated with the IUR probe in EMSA
indicating that, although ADP-ribosylation is required for
trans-activation of the CXCL1 promoter, it may not be
necessary for binding to the IUR element in the promoter.
Our data indicate the following: 1) contiguity with NF-
B element
appears to be necessary for trans-activation through the IUR element;
2) the 115-kDa PARP binds to the IUR element in the CXCL1
promoter; 3) PARP is associated with trans-activation of the CXCL1
promoter; 4) the ADP-ribose transferase activity is necessary for
trans-activation; 5) this activity is not necessary for binding of PARP
to the IUR element. Based on these findings, we have proposed a model
for the regulation of the CXCL1 promoter.
We have uncovered a novel role for the PARP in the regulation of the
CXCL1 chemokine gene. The CXCL1 minimal promoter, under the
control of five cis-acting elements including the Sp1, IUR, NF-
B,
HMGI(Y), and TATA box binding sites, is a template for a multiprotein
complex, the CXCL1 enhanceosome. The TCGAT motif of the IUR
element and the 115-kDa PARP, which specifically binds the IUR, appear
to possess distinct positive roles in CXCL1 regulation. PARP
appears to be associated with the trans-activation of the gene. One
mechanism of PARP-mediated trans-activation of the CXCL1 promoter may involve the NF-
B site because dislocation of the IUR
element from this site inhibits trans-activation of the promoter, possibly by disrupting interactions between PARP and Rel factors. We
have no evidence of protein-protein interactions between PARP and the
NF-
B factors at this time. However, a recent report has demonstrated
that NF-
B-dependent transcription activation is severely
compromised in mice that are deficient in PARP (13). PARP-deficient
mice appear to have extensive skin disorder and extreme sensitivity to
x-ray irradiation, a phenotype closely resembling the one observed in
mice deficient for IKK2, the kinase involved in the NF-
B activation
pathway. Based on the findings by Hassa et al. (13), PARP
deficiency may contribute to a failure in NF-
B signaling.
In a separate study, we have identified a second protein binding to the
IUR element that is also specific to the TCGAT motif. Preliminary
evidence indicates that the 170-kDa IUR-specific factor could be a
repressor of CXCL1 transcription. A second mechanism of
PARP-mediated trans-activation could involve displacement or inactivation by ADP-ribosylation of the putative 170-kDa
trans-repressor of the CXCL1 promoter binding to a site at
or near the IUR element.
Tumor models involving PARP are yet to be developed. In this context,
the role of PARP as an anti-apoptotic factor may be important. During
the onset of apoptosis, PARP is cleaved into two fragments, 85 and 29 kDa, by caspase-3, a crucial component of the apoptotic cascade and
this is thought to contribute to cell death (24). However, direct
evidence for PARP as an agent of cell proliferation is forthcoming. In
this regard, the DNA binding and transcriptional regulatory properties
of PARP may be more revealing. PARP has been previously shown to
modulate DNA binding and specificity of the anti-tumor factor, p53
(14). More of such evidence will be crucial toward our understanding of
its role in cell proliferation and tumorigenesis. Our findings that
PARP binds the CXCL1 promoter and is likely involved in its trans-activation, therefore, present an important step in that direction.
Future investigations in this area could examine interactions between
PARP and other constituents of the CXCL1 enhanceosome, compare levels or post-translational modification status of these factors in normal and melanoma cells, and trace the signaling pathways
that modulate their activity. These investigations could elucidate the
role for PARP in disorders such as chronic inflammation and melanoma.