From the Oncology and Molecular Endocrinology
Research Center and the ¶ Unit of Health and Environment, CHUL
Research Center, Ste-Foy, Quebec G1V 4G2, Canada
Received for publication, November 15, 2001, and in revised form, February 28, 2001
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ABSTRACT |
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Poly(ADP-ribose) polymerase-1 (PARP-1) catalyzes
the rapid and extensive poly(ADP-ribosyl)ation of nuclear proteins in
response to DNA strand breaks, and its expression, although ubiquitous, is modulated from tissue to tissue and during cellular differentiation. PARP-1 gene promoters from human, rat, and mouse have been cloned, and
they share a structure common to housekeeping genes, as they lack a
functional TATA box and contain multiple GC boxes, which bind the
transcriptional activator Sp1. We have previously shown that, although
Sp1 is important for rat PARP1 (rPARP) promoter activity, its finely
tuned modulation is likely dependent on other transcription factors
that bind the rPARP proximal promoter in vitro. In this
study, we identified one such factor as NF1-L, a rat liver isoform of
the nuclear factor 1 family of transcription factors. The NF1-L site on
the rPARP promoter overlaps one of the Sp1 binding sites previously
identified, and we demonstrated that binding of both factors to this
composite element is mutually exclusive. Furthermore, we provide
evidence that NF1-L has no effect by itself on rPARP promoter activity,
but rather down-regulates the Sp1 activity by interfering with its
ability to bind the rPARP promoter in order to modulate transcription
of the rPARP gene.
Poly(ADP-ribose) polymerase-1
(PARP-1)1 is a nuclear enzyme
which catalyzes the addition of ADP-ribose units from nicotinamide adenine dinucleotide (NAD+) onto itself and other nuclear
proteins such as histones and topoisomerases (reviewed in Refs. 1 and
2). It is made up of three functional domains, namely the
amino-terminal DNA-binding domain, the central automodification domain,
and the carboxyl-terminal catalytic domain (3). Although PARP-1 is
often associated with DNA repair, because of its rapid and extensive
activation following DNA damage (4, 5), it has also been implicated in
other major nuclear functions such as transcription (6, 7), DNA replication (8, 9) and recombination (10). PARP-1 is also important for
cell differentiation (11-13) and is involved in cell death, likely
acting as a molecular switch between apoptosis and necrosis (14).
PARP-1 is expressed in all organs, albeit at varying degrees, with
highest mRNA expression found in brain, thymus, heart, and testis
(15, 16). PARP-1 mRNA level is regulated at the cell cycle level,
reaching its peak at either the G1 (17-20) or the S phases
(21, 22). It has also been shown that a decrease in PARP-1 mRNA
levels is associated with cellular differentiation (23-26) and
senescence (27), whereas an increase is observed upon activation of
lymphocytes (17, 28) or peripheral blood mononuclear cells (18). All
these studies show that, although PARP-1 is ubiquitously expressed, its
modulation, likely through complex transcriptional regulation, is
critical to major cellular functions.
In order to better understand the transcriptional mechanisms regulating
PARP-1 expression, the PARP-1 gene promoter has been identified and
cloned from three mammalian species, human (29, 30), rat (31), and
mouse.2 All three mammalian
proximal promoters share a similar structure proper to housekeeping
genes; they lack a functional consensus TATA box, are GC-rich, and
contain a consensus initiator sequence surrounding the transcription
start site.3 The human
promoter has been shown to contain binding sites for transcription
factors Sp1, AP-2 (30), YY1 (32), and Ets (33), whereas the mouse
promoter was recently shown to be down-regulated by a complex of
adenovirus E1A protein and pRb (34). In the case of the rat PARP-1
(rPARP) proximal promoter, we have shown that it is highly but not
completely dependent on five Sp1 binding sites (35, 36), and that other
nuclear proteins, including one likely belonging to the nuclear factor
1 (NF1) family of transcription factors (37), bind to and potentially
regulate rPARP promoter transcription.
In this study, we further characterize the rPARP promoter and
demonstrate that it binds the rat liver form of NF1 known as NF1-L
(38). Furthermore, we provide evidence that NF1-L does not actively
modulate rPARP transcriptional activity but rather prevents Sp1 from
binding the rPARP promoter in vitro and thus causes a
concentration-dependent down-regulation of rPARP promoter activity.
Plasmid Constructs--
The rPARP promoter fragment pCR3
(Fig. 1), spanning region
The expression plasmid pPacNF1-L was constructed by amplifying the
NF1-L insert from vector NF1-21/pBS (kindly provided by Dr Paolo
Monaci, Istituto di Ricerche di Biologia Molecolare, Rome, Italy),
using the synthetic oligonucleotides 5'-NF1
(5'-GAATTCCTCGAGGCAGTTATGTATTC-3') and 3'-NF1
(5'-GAATTCCTCGAGGGTGGTCTGTCTGG-3'). A unique
XhoI site (underlined in the primer sequences) was included
for cloning purposes. The PCR reaction was performed in a final volume
of 50 µl, and the mixture contained 200 ng DNA, 250 µM
each dNTP, 50 mM KCl, 1.5 mM MgCl2,
10 mM Tris-HCl, pH 9.0, 2.5 µM each synthetic primer, and 2.5 units of Pfu DNA polymerase (Stratagene, La
Jolla, CA). Using a GeneAmp 2400 thermal cycler, the samples were
subjected to an initial denaturation at 94 °C for 30 s and were
then processed through 32 cycles of denaturation at 94 °C for
30 s, annealing at 47 °C for 30 s, and elongation at
72 °C for 120 s, followed by a final elongation of 420 s
at 72 °C. The resulting PCR product was run through a 1% agarose
gel in 1× TAE, 0.1 µg/ml EtBr, and was isolated using the QIAEX II
gel extraction kit. The purified DNA fragment was then digested with
XhoI (Amersham Pharmacia Biotech) and ligated into the
XhoI-linearized vector pPac, which was obtained by removing
the XhoI-inserted Sp1 cDNA from vector pPacSp1 (kindly provided by Dr. Guntram Suske, Institut für Tumorforschung,
Philipps Universität, Marburg, Germany). The recombinant plasmid
was then sequenced by chain-termination dideoxy sequencing (39) to
confirm the reading frame.
Protein Extracts and Electrophoretic Mobility Shift Assay
(EMSA)--
Crude nuclear extracts from rat pituitary GH4C1 cells
(kindly provided by Dr David D. Moore, Massachusetts General Hospital, Boston, MA) were prepared essentially as described previously (36, 40)
and kept frozen in small aliquots at
For EMSA, the 140-bp HindIII-XbaI pCR3 or
pCR3/F2-F3-F4m fragments or double-stranded synthetic oligonucleotides
bearing either the L3 sequence from the rPARP promoter
(5'-CACAGCAGTCGCGCTGGAACCCAACCCCGCCATG-3'; Ref. 37) or the target
sequence for human HeLa CTF/NF-I (5'-GATCTTATTTTGGATGAAGCCAATATGAG-3'; Ref. 42) were 5'-32P-end-labeled as described
previously (43) and used as probes. Approximately 2 × 104 cpm (except in Fig. 2, where 4 × 104
cpm were used) of labeled DNA probe was incubated for ~8 min at room
temperature with GH4C1 nuclear extract or NF1-L-enriched CM-Sepharose fraction in the presence of 1 µg or 200 ng,
respectively, of poly(dI-dC)·poly(dI-dC) (Amersham Pharmacia Biotech)
and 50 mM KCl in buffer D (10 mM HEPES, 10%
v/v glycerol, 0.1 mM EDTA, 0.25 mM
phenylmethylsulfonyl fluoride). The DNA-protein complexes were
separated by electrophoresis on native polyacrylamide gels (4% for the
pCR3/F2-F3-F4m probe, 6% for the pCR3 probe, 8% for the
oligonucleotide probes) run against Tris/glycine buffer at 4 °C, as
described previously (43).
Competition experiments were performed using EMSA conditions
similar to those described above, except that the protein extracts were
incubated with the pCR3-labeled probe in the presence of molar excesses
(as specified in the caption to Figs. 2 and 5A) of unlabeled
double-stranded oligonucleotides bearing the target sequence of
CTF/NF-I (see above), the high affinity binding site for Sp1
(5'-GATCATATCTGCGGGGCGGGGCAGACACAG-3') (44), the PARP-1 promoter
initiator sequence
(5'-GATCGCGCCGCCAGGCATCAGCAATCTATCCTG-3'),3 L3, or L3m (see
above). In the case of the pCR3/F2-F3-F4m fragment, the GH4C1 protein
extract was incubated with the labeled probe in the presence of molar
excesses (as specified in the caption to Fig. 6) of the
HindIII-XbaI pCR3 fragment (which was
isolated from a 1.5% agarose gel in 1× TAE, 0.1 µg/ml EtBr and
purified using the QIAEX II gel extraction kit), or the unlabeled
oligonucleotides CTF/NF-I or Sp1 (see above).
For supershift experiments, similar EMSA conditions as described above
were used, except that following formation of the DNA-protein complexes, 1.5 µl of pre-immune rabbit serum, anti-NF1-L rabbit serum
(kindly provided by Prof. P. C. van der Vliet, Laboratory of
Physiological Chemistry, Utrecht University, Utrecht, The Netherlands) or anti-Sp1 antibody (Santa Cruz Biotechnology, Santa Cruz, CA) was
added and incubation allowed to proceed for another 8 min.
DNA Footprinting Procedures--
Deoxyribonuclease I (DNase I)
footprinting analysis of the rPARP promoter fragment pCR5 (36), which
encompasses the pCR3 region, was performed using 5 µl of the
NF1-L-containing CM-Sepharose fraction. Briefly, after pCR5
was 5'-end-labeled on the top strand, 3 × 104 cpm
labeled probe was incubated for 10 min at room temperature with the
protein extract and treated with DNase I (Worthington, Freehold, NJ) as
described previously (45). Digestion products were resolved by
electrophoresis on a 8% polyacrylamide sequencing gel.
For dimethylsulfate (DMS) methylation interference, the 140-bp
HindIII-XbaI pCR3 fragment was 5'-end-labeled on
the top strand and partially methylated with DMS essentially as
described (46). Once methylated, 6 × 104 cpm labeled
probe was incubated with 10 µl of the NF1-L-containing CM-Sepharose fraction in buffer D and DNA-protein complexes were separated by native polyacrylamide gel electrophoresis as described above. The NF1-L-DNA complex was then visualized by
autoradiography and isolated by electroelution (47). The isolated
labeled DNA was finally treated with piperidine (46) and further
analyzed on a 8% sequencing gel.
Cell Culture, Transient Transfection, and CAT Assay--
Rat
GH4C1 cells were grown in Ham's F-10 medium (Sigma-Aldrich, Oakville,
Ontario, Canada) supplemented with 10% fetal bovine serum (Life
Technologies, Inc., Burlington, Ontario, Canada) and 20 µg/ml
gentamycin, under 5% CO2 at 37 °C.
Drosophila Schneider line 2 cells (SL2, ATCC CRL-1963) were
grown in Schneider insect medium (Sigma-Aldrich) supplemented with 10%
fetal bovine serum and 20 µg/ml gentamycin at 27 °C without
CO2.
The GH4C1 cells were transiently transfected with either pCR3 or its
mutant derivative pCR3-L3m by the calcium phosphate precipitation method as described (48), using 15 µg of test plasmid and 5 µg of
the human growth hormone (hGH) gene-encoding plasmid pXGH5 (49). In the
case where the expression vectors NF1-L/pSI (kindly provided by Dr
Winnie Eskild, Institute of Medical Biochemistry, University of Oslo,
Oslo, Norway) or NF1-X/pcDNA3 (kindly provided by Dr. Bin Gao,
Medical College of Virginia, Richmond, VA) were added, GH4C1 cells were
transfected with GeneSHUTTLE-20 cationic liposome (Quantum
Biotechnologies, Montréal, Quebec, Canada), using 1 µg of test
plasmid, 0.5 µg of pXGH5, and the expression vectors as described in
the caption to Fig. 5B. The SL2 cells were transfected by
the calcium phosphate precipitation method, using 13 µg of test
plasmid, 5 µg of the Rat NF1-L Binds the rPARP Promoter in Vitro at a Site Overlapping
an Sp1 Binding Site--
We have previously demonstrated that,
although Sp1 specifically binds to (Fig. 2, A and
B) and strongly transactivates the GC-rich rPARP promoter,
it is not sufficient to account for all of its transcriptional activity
(36). Furthermore, a nuclear protein likely belonging to the NF1 family
of transcription factors was shown to bind to the rPARP minimal
promoter region, which extends from
Using DNase I and DMS methylation interference footprinting, we
precisely mapped the binding site of the NF1-like protein to the L3
region of the rPARP proximal promoter (Fig.
3, A and B), which
we had previously identified (37) as a potential NF1 target site due to
its similarity both to the consensus sequence recognized by members of
the NF1 family (42) and to the proximal silencer-1 element from the rat
growth hormone gene (52). Interestingly, the footprinted L3 region
completely overlaps the F2 region (see Figs. 1 and 3), which was shown
to bind Sp1 (36).
In order to unequivocally identify the rat liver L3-binding protein as
a genuine member of the NF1 family, we performed supershift experiments
using an antiserum specific to the rat liver form of NF1 known as NF1-L
(38). As shown in Fig. 4A, the
rat liver DNA-binding protein formed a complex (NF1-L) of
similar electrophoretic mobility using either the NF1 consensus binding
site or the L3 sequence as the labeled probe, and this complex was
efficiently supershifted by the anti-NF1-L antiserum (C1).
As shown in Fig. 4B, the same rat liver protein bound the
pCR3 DNA fragment to form a specific complex (NF1-L), which
was supershifted by the anti-NF1-L antiserum (C1), but not
by the pre-immune rabbit serum (PS) or by an anti-Sp1
antibody (Sp1Ab). We therefore conclude that the rat liver
protein that binds to the rPARP proximal promoter region is NF1-L.
Members of the NF1 Family Indirectly Down-regulate the rPARP
Promoter Activity--
To characterize in more detail the effect of
NF1 on the rPARP promoter, the L3 element was mutated at positions
critical for its recognition by NF1 (Fig. 3 and Ref. 37) in order to
yield the mutant derivative pCR3-L3m. This mutated L3 sequence was
previously shown to abolish NF1 binding (37) and thus would give
valuable information in assessing the functional role of NF1 on rPARP
promoter transcriptional activity. When the mutated pCR3-L3m construct was transiently transfected into rat pituitary GH4C1 cells, its activity was similar to that directed by wild-type pCR3 (Fig. 5A), suggesting that NF1 had
no obvious effect on rPARP transcription. This is consistent with
previous observations using the mutant derivative pCR3/F2-F3-F4m, in
which all three Sp1 binding sites (F2, F3, and F4; see Fig. 1) were
mutated (36). This mutated construct had practically no transcriptional
activity when transiently transfected into GH4C1 cells (36) despite the
fact that GH4C1 cells displayed efficient NF1 binding activity on the
pCR3/F2-F3-F4m DNA fragment (Fig. 5B). Indeed, binding of an
NF1 protein from GH4C1 cells to labeled pCR3/F2-F3-F4m was efficiently
competed by excesses of unlabeled NF1 oligonucleotide and by unlabeled pCR3 wild-type fragment (Fig. 5B). These results suggest
that an NF1 protein from GH4C1 cells can bind to but has no direct effect on the rPARP promoter. This GH4C1 protein displays different electrophoretic mobility than liver-enriched NF1-L (compare Figs. 4B and 5B); this may suggest that those are two
distinct proteins from the NF1 multigene family (53) or that, as
suggested by Paonessa et al. (38), liver NF1-L is a cleavage
product of a larger polypeptide which may be the GH4C1 NF1 protein.
However, we believe the latter to be unlikely, as the anti-NF1-L
antiserum does not supershift the NF1 protein from GH4C1 nuclear
extract (data not shown).
Cotransfection of the wild-type pCR3 along with plasmids encoding high
expression levels of members of the NF1 family, such as NF1-L (38) and
NF1-X (54), led to a 2-fold decrease of transcriptional activity
directed by pCR3 in GH4C1 cells (Fig. 6A, black
columns, and data not shown). In contrast, overexpression of
NF1-L practically had no influence on the NF1-site mutated derivative
pCR3-L3m fragment (Fig. 6A, white
columns). To gain more insight into the mode of action of
NF1 on the rPARP promoter, we took advantage of the
Drosophila SL2 cells, which lack classical NF1 and Sp1
activities (55, 56). NF1-L alone had no effect on the transcriptional
activity of pCR3, in contrast to Sp1, which increased pCR3 activity
over 3-fold (Fig. 6B). However, coexpression of both Sp1 and
NF1-L led to a concentration-dependent decrease of the
transcriptional activation mediated by Sp1 on the pCR3 promoter
fragment (Fig. 6B). In fact, when equal amounts of Sp1 and
NF1-L expression vector DNA were cotransfected, NF1-L completely abolished Sp1-mediated activation of pCR3 transcription, which returned
to basal level (Fig. 6B).
We then used the pCR3 fragment and its Sp1 mutant derivative
pCR3/F2-F3-F4m labeled probes in EMSA with the
NF1-L-enriched rat liver protein extract and the GH4C1
nuclear protein extract, which supports strong Sp1 binding to the pCR3
probe (see Fig. 2A and Ref. 36). When the pCR3 probe was
incubated with a constant amount of NF1-L and increasing amounts of
GH4C1 extract, NF1-L binding to pCR3 was abolished by the increasing
amounts of Sp1 (Fig. 7A,
left panel). On the other hand, NF1-L binding to
the Sp1 mutant probe was expectedly unaffected by increasing amounts of
GH4C1 nuclear extract (Fig. 7A, right
panel). The reverse is also true, i.e. increasing
amounts of NF1-L abolished Sp1 binding to pCR3 (Fig. 7B,
left panel), suggesting that binding of NF1-L and
Sp1 to the rPARP promoter is mutually exclusive. This and the SL2
transfection experiments show that NF1-L acts indirectly on rPARP
promoter transcriptional activity by preventing Sp1 from binding to its
target and therefore leads to decreased transcriptional activation of
the rPARP promoter.
Poly(ADP-ribosyl)ation is a major post-translational modification
potentially involved in chromatin architecture (1) and likely to affect
important nuclear functions such as DNA repair, recombination,
replication, and transcription. Although PARP-1, the main
poly(ADP-ribosyl)ating enzyme (2), is expressed in a variety of cells
and organs, its transcriptional regulation during cell cycle
progression, cell proliferation, and differentiation is likely to
impact on the timing and extent of PARP-1 activity and function.
Indeed, it has been shown that PARP-1 mRNA levels increase just
before maximal enzymatic activity during thymocyte proliferation (22),
astrocyte proliferation and differentiation (25), and lymphocyte
activation (28). Furthermore, elevated levels of PARP-1 mRNA
apparently account for the enhanced poly(ADP-ribosyl)ation observed in
Ewing's sarcoma cells (24).
In this study of the rPARP proximal promoter, we investigated the
regulatory mechanisms involved in its expression and have highlighted
the roles of both Sp1 and NF1 in fine-tuning rPARP gene transcription.
Whereas Sp1 clearly up-regulated rPARP transcription (Ref. 36 and Fig.
6B), the role of NF1 appeared more ambiguous; although
mutation of the NF1 binding site in the rPARP fragment pCR3 had no
effect on its transcriptional activity in rat pituitary GH4C1 cells
(Fig. 5A), overexpression of NF1-L or NF1-X in the same
cells down-regulated pCR3 activity (Fig. 6A and data not shown). In Drosophila SL2 cells, which lack endogenous Sp1
and NF1 (55, 56), NF1-L had no direct effect on the transcriptional activity mediated by the pCR3 fragment, but inhibited the Sp1-mediated up-regulation of this rPARP promoter fragment (Fig. 6B).
NF1-L likely acted in a concentration-dependent fashion by
preventing and/or displacing Sp1 from binding to the rPARP proximal
promoter (Fig. 7). NF1-L has been previously shown to negatively
regulate rat growth hormone (41) and peripherin (57) gene transcription.
Disruption of the Sp1 gene has revealed that, although important for
embryonic development, it is dispensable for cell growth and
differentiation (58), suggesting that it may not be relevant to
differentiation- and cell cycle-related PARP-1 transcription. On the
other hand, Sp1 is one of at least 16 members of the Sp/XKLF family of
transcription factors able to bind GC or GT boxes (59), and it is
therefore plausible that other members of this family take over Sp1
function in Sp1-deficient cells. Nevertheless, a number of genes have
been shown to rely on Sp1 for either cell cycle-regulated expression,
including thymidine kinase (60) and dihydrofolate reductase (61), or
differentiation-associated expression, such as acetylcholine receptor
The NF1 family is composed of four members encoded by distinct genes,
NF1-A, -B, -C, and -X (53), producing distinct protein products, which
can form homo- or heterodimers (64). In addition, all four NF1
mRNAs can be differentially spliced to yield a multitude of
proteins with subtle differences in their transactivation capacities (58, 65). Disruption of the NF1-A gene leads to severe neurological defects, with the majority of mice dying perinatally, yet other organs
seem to develop normally (66). As in the case of the Sp1 knockout
study, other members of the NF1 family may compensate for the absence
of NF1-A. Nevertheless, proteins of the NF1 family have been implicated
in adipocyte differentiation (67) and in liver regeneration (68). In
adipocytes, the adipocyte p2 gene expression, which is dependent on
NF1, is induced upon differentiation (67), as is expression of PARP-1
(13), even though Sp1 expression is down-regulated in the early stages
of adipocyte differentiation (69). On the other hand, in liver
regeneration, NF1 concentration decreases (68) whereas PARP-1
expression increases (20), which is consistent with Sp1 activity being
more prominent (70). That NF1 proteins exhibit stimulatory activity in
one case (i.e. adipocyte p2 gene expression) and inhibitory
activity in the other (i.e. the rPARP promoter) has been
previously observed and shown to be dependent on the promoter or the
cell type (71).
Some correlation is observed between the level of PARP-1 mRNA in
mouse organs (15) and that of Sp1 (72), although it is not complete.
Indeed, mouse thymus and testis express nearly as much PARP-1 (15)
despite that Sp1 levels are 8 times higher in thymus (72), whereas high
expression of Sp1 mRNA in the lung (72) does not result in
proportional PARP-1 expression (15), thereby confirming that Sp1 is an
important but not the only transcription factor involved in PARP-1 gene
expression. Interestingly, NF1 proteins are also highly expressed in
the lung (65) and, in light of the present study, may contribute to
down-regulation of PARP-1 expression in the lung.
Composite elements are transcriptionally active DNA sequences, which
bind two or more transcriptional regulators, and they have been found
in many vertebrate gene promoters (73). They provide an efficient way
of fine-tuning gene expression in different cellular situations, by
exploiting either the synergistic or antagonistic effects of the
transcription factors they bind to respectively overactivate or
down-regulate transcription. In the present study, we provide evidence
that the rPARP gene promoter is regulated by the antagonistic effect of
NF1 over Sp1 transactivation on a composite element. Both of these
transcription factors have been found in composite elements on other
gene promoters. Antagonistic binding of Sp1 has been reported with
erythroid cell-specific factor NF-E1 on the human We have shown that Sp1-activated transcription of the rPARP gene
promoter is susceptible to subtle regulation via the mutually exclusive
binding of NF1-L. As Sp1 and NF1-L are both individual members of
extended families of transcription factors, it is expected that
transcriptional regulation through a composite Sp1/NF1 element, such as
those reported here on the rPARP promoter, on the rat liver-type
arginase promoter (77), the murine collagen
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
103 to +13 relative to the mRNA start
site, and its Sp1 triple-mutant derivative pCR3/F2-F3-F4m, have been
described previously (36). The site-directed mutant pCR3-L3m was
produced by the polymerase chain reaction (PCR), using pCR3 as template
and the synthetic oligomers L3m
(5'-GCATGCCTGCAGTCGCGCTAAAAAAAAACCCCG-3', mutated nucleotides are shown in boldface) and pCAT-3'
(5'-CTCAGATCCTCTAGAGTCG-3'). Unique PstI and
XbaI sites (underlined in the primer sequences) were
included for cloning purposes. Amplifications were performed in a final
volume of 50 µl, and reaction mixtures contained 100 ng of DNA, 250 µM each dNTP (Amersham Pharmacia Biotech,
Baie-d'Urfé, Quebec, Canada), 50 mM KCl, 1.5 mM MgCl2, 10 mM Tris-HCl, pH 9.0, 2 µM each synthetic primer, and 3.5 units of Taq
DNA polymerase (Amersham Pharmacia Biotech). Using a GeneAmp 2400 thermal cycler (PE Biosystems, Streetsville, Ontario, Canada), the
samples were subjected to an initial denaturation at 94 °C for
180 s and were then processed through 35 cycles of denaturation at
94 °C for 20 s, annealing at 61 °C for 20 s, and
elongation at 72 °C for 40 s, followed by a final elongation of
120 s at 72 °C. The resulting PCR products were run through a
1.5% agarose gel in 1× TAE (40 mM Tris acetate, 1 mM EDTA) containing 0.1 µg/ml ethidium bromide (EtBr) and
were isolated using the QIAEX II gel extraction kit (Qiagen,
Mississauga, Ontario, Canada). The purified DNA fragments were then
digested with PstI (Amersham Pharmacia Biotech) and XbaI (Amersham Pharmacia Biotech) and ligated upstream of
the chloramphenicol acetyltransferase (CAT) reporter gene in the
PstI-XbaI-linearized vector pCATbasic
(Promega, Madison, WI). The DNA insert of each recombinant plasmid was
sequenced by chain-termination dideoxy sequencing (39) to confirm the mutations.
80 °C until use. The
NF1-L-containing rat liver carboxymethyl (CM)-Sepharose fraction has been described previously (41).
-galactosidase (lacZ) gene-encoding plasmid pAc5/V5-His/LacZ (Invitrogen, Carlsbad, CA), and
the pPacSp1 or pPacNF1-L expression vectors as described in the legend
to Fig. 6A. CAT activities were measured as described previously (50) and normalized to either hGH secreted into the culture
medium (for GH4C1), which was assayed using a radioimmunoassay kit
(Medicorp, Montréal, Quebec, Canada), or lacZ produced
in the cells (for SL2), which was measured as described (51). Each single value is expressed as either 100× (% CAT in 4 h)/ng of hGH for GH4C1, or (% CAT in 4 h)/(lacZ/min/ml) for
SL2, and corresponds to the mean of at least two individual
transfection experiments performed in triplicate. Standard deviation
for the CAT assay is provided for each transfected plasmid and for each
cell type. Only values 3 times greater than the background level caused
by the reaction mixture alone (usually corresponding to ~0.15%
chloramphenicol conversion) were considered significant.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
103 to +13 (pCR3, see Fig.
1 and Ref. 37). In fact, EMSA analysis,
using a heparin-Sepharose- and CM-Sepharose-enriched rat liver nuclear
extract, revealed the specific binding of a protein that is clearly
distinct from Sp1 (Fig. 2A).
The interaction of this rat liver DNA-binding protein with the rPARP
promoter likely occurs through a putative NF1 binding site (L3)
previously identified on the pCR3 fragment (36, 37), since formation of
the DNA-protein complex was completely abolished by competition with an
excess of unlabeled oligonucleotide bearing the target sequence for
human CTF/NF-I (42) (Fig. 2A). In contrast, competition using unlabeled oligonucleotides bearing unrelated sequences such as
that for Sp1 or the initiator sequence from the PARP
promoter3 had no effect on pCR3-rat liver protein complex
formation (Fig. 2A). However, when using a crude nuclear
protein extract from rat pituitary GH4C1 cells, only Sp1 forms a
complex with the pCR3 fragment, since its formation is prevented by an
Sp1-specific antiserum but not by a pre-immune or an
NF1-L-specific antiserum (Fig. 2B).
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Fig. 1.
DNA sequence of the rPARP proximal promoter
fragment pCR3. The rPARP promoter sequence that is contained on
the plasmid pCR3 is depicted, along with the position of the three
previously identified Sp1 binding sites (F2, F3,
and F4) (36). In addition, the position of a target sequence
for an NF1-like nuclear protein that binds the rPARP promoter is
provided (L3; boxed nucleotides) (37).
Numbers indicate positions relative to the rPARP mRNA
start site (indicated by the arrow).
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Fig. 2.
EMSA analysis of NF1-L and Sp1 binding to the
rPARP basal promoter. A, the 140-bp
HindIII-XbaI pCR3 fragment from the rPARP
promoter was 5' end-labeled and incubated with crude nuclear extracts
obtained from rat pituitary GH4C1 cells or with a CM-Sepharose enriched
preparation of NF1-L (CM-Sep), either alone or in the
presence of a 500-fold molar excess of unlabeled, double-stranded
oligonucleotides (NF1, Sp1, or Inr).
DNA-protein complexes were then resolved by EMSA as detailed under
"Experimental Procedures." The position of both the NF1-L/pCR3
(NF1-L) and Sp1/pCR3 (Sp1) DNA-protein complexes
is shown, along with that of the free probe (U).
P, labeled probe with no proteins added. B, the
140-bp HindIII-XbaI pCR3 labeled probe used in
A was incubated with crude nuclear proteins from GH4C1 cells
either alone or in the presence of antibodies directed against either
Sp1 (Sp1Ab) or NF1-L (NF1Ab). As a negative
control, incubation was also performed in the presence of pre-immune
serum (PS). The position of the Sp1/pCR3 complex
(Sp1) is provided, along with that of the free probe
(U).
View larger version (20K):
[in a new window]
Fig. 3.
Footprinting analysis of NF1-L binding to the
rPARP promoter. A, a 282-bp
HindIII-XbaI obtained from the recombinant
plasmid pCR5 (36) was 5' end-labeled at its HindIII site and
incubated with the CM-Sepharose-enriched preparation of NF1-L and then
subjected to DNase I digestion. A control sample treated with DNase I
but with no protein added (U) and a DNA "G" sequencing
reaction performed on the same probe (G) were also included.
The DNA sequence of the L3 protected region is indicated along with its
positioning relative to the rPARP mRNA start site. B,
the 140-bp HindIII-XbaI pCR3 fragment used in
Fig. 2 was 5'-end-labeled on the top strand, partially methylated with
DMS and incubated with CM-Sepharose-enriched NF1-L. The NF1-L/pCR3
complex (C) was resolved by EMSA and isolated by
electroelution, along with the free probe (U). The labeled
DNA from the complex was then treated with piperidine and further
analyzed on a sequencing gel. The position of the two G residues whose
methylation by DMS interferes with binding of NF1-L is shown, along
with the surrounding rPARP promoter sequence.
View larger version (33K):
[in a new window]
Fig. 4.
Binding of NF1-L to the rPARP promoter as
revealed by supershift analyses in EMSA. A,
CM-Sepharose-enriched NF1-L (CM-Sep) was incubated in the
presence of a 5' end-labeled double-stranded oligonucleotide bearing
either the sequence of the high affinity CTF/NF-I consensus binding
site (NF1) or that of the rPARP L3 element (L3).
The binding reaction was performed in either the absence or the
presence of a polyclonal antibody directed against rat liver NF1-L
(NF1Ab). Formation of DNA-protein complexes was then
monitored by EMSA as in Fig. 2. The position of both the NF1-L and a
supershifted complex (C1) resulting from the binding of the
NF1Ab to the NF1-L/labeled probe complex is indicated, along with that
of a nonspecific signal (NS) that often appears in EMSA.
U, unbound fraction of the labeled probes. B, the
140-bp HindIII-XbaI pCR3 labeled probe used in
Fig. 2 was incubated with the CM-Sepharose-enriched preparation of
NF1-L either alone or in the presence of antibodies directed against
either NF1-L (NF1Ab) or Sp1 (Sp1Ab). As a
negative control, incubation was also performed in the presence of
pre-immune serum (PS). The position of both the NF1-L/pCR3
complex (NF1-L) and its corresponding supershifted
counterpart (C1) is provided, along with that of the free
probe (U).
View larger version (42K):
[in a new window]
Fig. 5.
Transient transfection and EMSA analyses of
pCR3 derivatives that bear mutations in either the NF1 or Sp1 target
sites. A, the L3 element from the rPARP promoter was
mutated in the context of the pCR3 plasmid in order to prevent its
recognition by NF1. Both the wild-type pCR3 and its L3 mutated
derivative (pCR3-L3m) were transiently transfected into rat pituitary
GH4C1 cells in order to assess the effect of NF1 binding to the L3
element on the transcriptional activity of the rPARP pCR3 promoter
fragment. CAT activities were measured and normalized as detailed under
"Experimental Procedures." Standard deviation is provided for both
plasmids. B, a 140-bp HindIII-XbaI
fragment derived from the recombinant plasmid pCR3/F2-F3-F4m that bears
mutations in all three rPARP promoter proximal Sp1 binding sites was 5'
end-labeled and incubated with crude nuclear proteins from GH4C1 cells
in the presence of increasing concentrations (150-, 400-, and 1250-fold
molar excesses) of various unlabeled competitor DNAs (the wild-type
pCR3 fragment or double-stranded oligonucleotides bearing either the
NF1 or Sp1 consensus sequences). Binding of NF1 to the Sp1-mutated
labeled probe was monitored by EMSA as in Fig. 2. The position of the
NF1 complex is indicated, along with that of a weak but specific
complex of unknown identity (*). U, unbound fraction of the
labeled probe. C+, labeled probe incubated with proteins but
without competitor DNA.
View larger version (17K):
[in a new window]
Fig. 6.
Co-transfection experiments in rat GH4C1 and
Drosophila SL2 cells. A, both the
wild-type rPARP promoter-bearing plasmid pCR3 (black
columns) and its mutated derivative pCR3-L3m
(white columns) were transiently transfected into
GH4C1 cells along with the NF1-L expression plasmid (0.5 or 1 µg). As
a negative control, pCR3 was also cotransfected along with the empty
vector pSI (which was used for constructing the NF1-L expression
plasmid). CAT activities were measured and normalized as detailed under
"Experimental Procedures." Values are expressed as percentage of
CAT activity relative to the level directed by either pCR3 and pCR3-L3m
when cotransfected with the control plasmid pSI. Standard deviation is
provided for each value shown. B, the rPARP promoter-bearing
plasmid pCR3 was transfected into Drosophila SL2 cells along
with the empty vector pPac, or with expression plasmids encoding either
NF1-L (0.2, 0.5, or 1 µg) or Sp1 (1 µg), either individually or in
combination. CAT activities were measured and normalized to
-galactosidase as described under "Experimental Procedures."
Standard deviation is provided for each selected condition.
View larger version (71K):
[in a new window]
Fig. 7.
EMSA analysis of the mutually-exclusive
binding of NF1-L and Sp1 to their overlapping target sites on the rPARP
promoter. A, the 140-bp
HindIII-XbaI pCR3 rPARP fragment
(pCR3, left) or its mutated derivative
(pCR3/F2-F3-F4m, right) were 5' end-labeled and
incubated with a constant amount of CM-Sepharose-enriched NF1-L (3 µl) and increasing concentrations of crude nuclear proteins from
GH4C1 cells (1, 2, 4, 8, and 12 µg). The position of the shifted
DNA-protein complexes yielded by both Sp1 and NF1-L is indicated, along
with the position of the free probe (U). B, same
as in panel A except that increasing amounts (1, 2, 4, 8, and 12 µl) of the CM-Sepharose-enriched NF1-L were used in
combination with a constant amount of nuclear proteins from GH4C1 cells
(3 µg).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(62) and keratin K3 (63). Interestingly, these Sp1-related events
are dependent upon modification of Sp1 activity, either by its
association with other regulators such as cell cycle-related
transcription factor E2F (60, 61), by its phosphorylation (62) or by
its competition with other transcription factors such as AP-2 (63). The
analysis of the keratin K3 promoter (63) is particularly relevant to the present study, in that the ratio between Sp1 and AP-2, rather than
actual regulation of Sp1, seems to be critical to the modulation of
keratin K3 expression.
-globin promoter
(74), where it may account for tissue-specific expression, with AP-2 on
the rabbit K3 keratin promoter (63), where it is associated with
differentiation-specific expression, and with NF-
B on the human
fas promoter (75), where it is involved in lymphocyte
activation-related up-regulation. In the case of NF1, its mutually
exclusive binding has been reported with AP-2 on the hGH promoter (76),
but also, and consistent with our results, with Sp1, on the rat
liver-type arginase promoter (77), on the murine collagen
1 (1)
promoter (78), and on the rat
1B adrenergic receptor
gene promoter (79). In the latter two studies, mutually exclusive
binding of Sp1 and NF1 has been suggested to confer an important
mechanism of tissue-specific expression. Interestingly, and in contrast
to the results presented here on the rPARP promoter, Sp1 actually
down-regulates NF1-mediated activation of the collagen
1 (1)
promoter (78).
1 (1) promoter (78), or
the rat
1B adrenergic receptor gene promoter (79), will
lead to complex patterns of activation/down-regulation depending on the
cell type or the cellular situation analyzed, as members of both
families may be differentially expressed in such instances.
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ACKNOWLEDGEMENT |
---|
We thank Steeve Leclerc for technical assistance.
![]() |
FOOTNOTES |
---|
* This work was supported by the Natural Sciences and Engineering Research Council of Canada Grant OGP0138624 (to S. L. G.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Supported by a scholarship from the Medical Research Council of Canada.
Senior scholar from the Fonds de la Recherche en Santé
du Québec. To whom correspondence should be addressed: Oncology
and Molecular Endocrinology Research Center, CHUL Research Center, 2705 Laurier Blvd., Ste-Foy, Québec G1V 4G2, Canada. Tel.:
418-654-2296; Fax: 418-654-2761; E-mail:
sylvain.guerin@crchul.ulaval.ca.
Published, JBC Papers in Press, March 7, 2001, DOI 10.1074/jbc.M010360200
2 T. Ogura, unpublished data.
3 M. A. Laniel and S. L. Guérin, unpublished observations.
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ABBREVIATIONS |
---|
The abbreviations used are: PARP-1, poly(ADP-ribose) polymerase-1; bp, base pair(s); CAT, chloramphenicol acetyltransferase; CM-Sepharose, carboxymethyl-Sepharose; DMS, dimethylsulfate; EMSA, electrophoretic mobility shift assay; EtBr, ethidium bromide; hGH, human growth hormone; NAD+, nicotinamide adenine dinucleotide; NF1, nuclear factor 1; PCR, polymerase chain reaction; rPARP, rat PARP-1; SL2, Drosophila Schneider line 2.
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