From the Institut d'Oncologie Cellulaire et
Moléculaire Humaine, 129, Route de Stalingrad, F-93000 Bobigny,
France, the ** Laboratoire de Biologie des Tumeurs Humaines, CNRS UMR
1598, Institut Gustave Roussy, F-94805 Villejuif Cedex, France, and the
Laboratoires Fournier, Centre de Recherche, Boîte Postale
90, F-21121 Daix, France
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() |
---|
Human copper-zinc superoxide dismutase
(Cu,Zn-SOD) participates in the control of reactive oxygen intermediate
intracellular concentration. In this study, we show that phorbol
12-myristate 13-acetate (PMA) increases Cu,Zn-SOD mRNA expression
within 30 min. The sequence between nucleotides Superoxide dismutases
(SODs)1 are found in all
aerobic organisms and are thought to participate in the detoxification
of reactive oxygen intermediates by catalyzing superoxide anion
(O Although Cu,Zn-SOD activity has been found modified in some specific
situations including hematopoietic cell differentiation, aging, and
some tumor cells (17-19), the SOD1 gene is often considered as a "housekeeping gene," and its activity is often used as an internal control when variations of manganese-associated SOD activity are measured. We report here the analysis of the proximal region of the
SOD1 gene promoter. Phorbol 12-myristate 13-acetate (PMA) was used as a model agonist to identify trans-
and cis-acting factors for SOD1 gene
expression. We found that SOD1 gene expression is rapidly
enhanced following PMA treatment. A region between nucleotides Cell Culture--
The HeLa cell line was obtained from the
American Tissue Culture Collection. Cells were grown in Dulbecco's
modified Eagle's medium (Life Technologies, Inc.) supplemented with
10% heat-inactivated fetal calf serum, 2 mM glutamine, and
antibiotics at 37 °C and 5% CO2 in a humidified
incubator. Cells were periodically checked for absence of mycoplasma contamination.
Chemicals--
PMA (Sigma) was resuspended in dimethyl sulfoxide
to a stock concentration of 1.5 mM, aliquoted, and stored
at Oligonucleotides--
Oligonucleotides were obtained from
Oligo-Express (Paris, France) and used without any further
purification, except for site-directed mutagenesis, for which
purification on polyacrylamide gel electrophoresis was carried out.
Northern Blotting--
Total cellular RNA were purified using
the conventional guanidium thiocyanate-CsCl method (20). Thirty
micrograms of total RNA were loaded on each gel lane. Electrophoresis,
transfer onto nitrocellulose membrane (Hybond C, Amersham Pharmacia
Biotech), and hybridization were carried out according to standard
protocols (21). The SOD1 probe was obtained by polymerase
chain reaction on total HeLa cDNA using primers
5'-ACCTAAGCTTATGGCGACGAAGGCCGTGTG-3' and
5'-ACTCAAGCTTCCTCAGACTACATCCAAGGG-3' and corresponds to a 490-base pair fragment containing the full-length SOD1
coding sequence. HindIII sites (underlined) were introduced
to facilitate subcloning. An internal control probe was derived from a
cDNA clone encoding human 18 S rRNA and was a gift from Dr. A. Harel-Bellan (Institut Fédératif sur le Cancer, Villejuif,
France). Both probes were labeled by the "random-priming" technique
using a Multiprime kit (Amersham Pharmacia Biotech) following the
manufacturer's instructions. Membranes hybridized with the
SOD1 probe were autoradiographed for 24 h. When the 18 S probe was used, membranes were autoradiographed for 10 min.
Western Blotting--
Protein extraction, polyacrylamide gel
electrophoresis, and blotting were carried out according to standard
procedures (22). A polyvinylidene difluoride membrane (Polyscreen, NEN
Life Science Products) was incubated with anti-human Cu,Zn-SOD primary
antibody from sheep (Calbiochem). Rabbit anti-sheep secondary antibody was from Jackson ImmunoResearch Laboratories, Inc.
125I-Labeled protein A (Amersham Pharmacia Biotech) was
used to detect the rabbit secondary antibody (protein A does not bind
sheep antibodies). 125I was used as the detection method
because of the poor linearity of signals generated with the
peroxidase/light-based systems. The membrane was autoradiographed for
12 h.
Isolation of a SOD1 5'-Flanking Region Clone--
A pGLS Expression Vectors--
pSCTKr24 (an expression vector for
Egr-1) was a gift from Dr. P. Charnay (Ecole Normale Supérieure,
Paris, France). Expression vectors for WT1( Preparation of Nuclear Extracts--
HeLa nuclear extracts were
prepared using a simplified version of the method of Dignam et
al. (26). Briefly, 1-2 × 107 exponentially
growing cells were collected by scraping; rinsed once in
phosphate-buffered saline; and resuspended on ice in 3 pellet volumes
of 20 mM Hepes (pH 7.0), 0.15 mM EDTA, 0.015 mM EGTA, 10 mM KCl, and protease inhibitors
containing 1% Nonidet P-40. Membrane disruption and nucleus integrity
were checked by visual inspection under a microscope. Nuclei were
collected by centrifugation and resuspended in 5 pellet volumes of 10 mM Hepes (pH 8.0), 25% glycerol, 0.1 M NaCl,
0.1 mM EDTA, and proteases inhibitors. After
centrifugation, nuclei were resuspended in 2 pellet volumes of
hypertonic 10 mM Hepes (pH 8.0), 25% glycerol, 0.4 M NaCl, 0.1 mM EDTA, and proteases inhibitors
and incubated for 30 min on a rotating wheel. Extracts were centrifuged
at high speed to remove nuclear debris; supernatants were aliquoted,
quickly frozen in liquid nitrogen, and stored at EMSA--
Complementary single-stranded oligonucleotides were
annealed by incubation at a concentration of 2.5 µg/µl in STE
buffer (100 mM NaCl, 10 mM Tris (pH 8.0), and 1 mM EDTA) at 80 °C for 2 min. The mixture was then slowly
cooled down to 4 °C at a rate of 1 °C/min in a thermal incubator
(Perkin-Elmer). Annealed oligonucleotides were diluted to 25 ng/µl in
STE buffer and stored at Transient Transfections and Luciferase Assays--
HeLa cells
were divided 48 h prior to transfection to generate 40-60%
confluence in 150-mm plates at the time of transfection. Culture medium
was replaced 6 h prior to transfection, and cells were transfected
by electroporation. Fifteen micrograms of test plasmid DNA (pGLS PMA Treatment Increases the SOD1 mRNA Level--
To determine
whether SOD1 gene expression can be induced by PMA
treatment, Northern blot analysis was carried out. As shown in Fig.
1A, a major mRNA species
of 0.7 kilobases was detected with the SOD1 cDNA probe and was
constitutively expressed in HeLa cells. PMA treatment of HeLa cells
induced a 2-fold increase in the SOD1 mRNA level, which
could be detected as soon as 30 min following PMA addition and remained
constant up to 24 h (Fig. 1B). As shown in Fig.
1C, this mRNA induction was followed by a 1.7-fold
increase in the Cu,Zn-SOD protein level after 4 h of PMA
treatment. Interestingly, in contrast with the observations obtained
for the steady-state mRNA levels, the Cu,Zn-SOD protein level
decreased to initial values after 8 h of PMA treatment, suggesting
the existence of post-transcriptional and/or post-translational regulation mechanisms.
A PMA-responsive Element Is Located between Nucleotides Sp1, Egr-1, and a Possible Sp1-like Protein Bind to the Mapping of Sp1-, Egr-1-, and Sp1-like Protein-binding
Sites--
Sequence analysis of the The Sp1/Egr-1 Site Is Essential for Basal and Egr-1-induced SOD1
Proximal Promoter Expression--
To test the functionality of the
Egr-1 site, the pGLS The Sp1/Egr-1 Site Is a Target for WT1--
Since numerous Egr-1
sites have been described to bind other Egr-related transcription
factors such as the Wilms' tumor protein (27), we tested the ability
of two splicing variants of WT1 to modulate transcription controlled by
the SOD1 proximal promoter. HeLa cells were transiently
transfected with pGLS We report the analysis of the human Cu,Zn-SOD gene proximal
promoter. Deletion analysis of the SOD1 gene promoter
suggests the existence of several regulatory sequences spread along the 5'-flanking region of the SOD1 gene. Indeed, previous
reports had allowed identification of several cis-acting
sequences. Two AP-2 sites located between positions Comparison of the Sp1/Egr-1 site with consensus Sp1-, Egr-1-, or
WT1-binding sites (Fig. 7) revealed that
this site does not fit consensus sequences described so far (32). In
addition, in most other sequences recognized by both Sp1 and Egr-1, the binding sites of the two proteins overlap on six nucleotides, but are
not identical (27). In our study, Sp1 and Egr-1 sites seem to be
identical or at least do not seem to differ by more than two
nucleotides since we could not find a mutation that affects binding of
only one of the two factors. This non-canonical Sp1/Egr-1/WT1 binding
sequence might represent the prototype of a novel binding site involved
in transcriptional regulation by members of the zinc finger
transcription factor family. Another sequence is recognized by a
possible Sp1-like protein. Surprisingly, the Sp1-like site fits
perfectly the Sp1 consensus sequence, but does not seem to be involved
in SOD1 transcription regulation in HeLa cells since a
mutation at this site does not lead to significant modification of
transcriptional activity.
71 and
29 is
essential for both basal and PMA-induced gene expression. This region
includes an Sp1-binding site that is also recognized by a possible
Sp1-like protein and by Egr-1 in a PMA-inducible manner. Egr-1 and two splicing variants of the Egr-related protein WT1 were able to transactivate the SOD1 promoter in co-transfection
experiments. Sp1 and the possible Sp1-like proteins bind to two
overlapping, but distinct sequences. However, Egr-1 and Sp1 seem to
interact with two sites that are either identical or very close to each other. None of these sites fit the consensus sequences previously reported for these proteins. Analysis of various mutants of the SOD1 proximal promoter revealed that the region that binds
Sp1 and Egr-1 is required for both basal and Egr-1-driven expression. Interplay between different members of the Sp1 family, Egr-1, and
different splicing variants of WT1 in the SOD1 proximal
promoter may provide clues about the physiological function of
Cu,Zn-SOD.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
REFERENCES
2) dismutation, yielding hydrogen peroxide
(H2O2) and molecular oxygen (O2)
(1). Three distinct SODs have been described in mammalian cells: a mitochondrial manganese-associated SOD, whose function is likely to
detoxify superoxide anion generated by partial O2 reduction during oxidative phosphorylation; an extracellular SOD, which is found
in extracellular fluids and bound to the extracellular matrix; and a
cytosolic Cu,Zn-SOD, whose endogenous role remains poorly understood
(2, 3). The fundamental importance of SOD activity is illustrated in
various organisms by the phenotype of mutants that do not express a
form of SOD. In bacteria and yeast, SOD deficiency leads to severe
growth deficits under aerobic conditions (4, 5). The life span of
Drosophila that lack SOD is shortened. Conversely,
Drosophila that overexpress SOD and catalase can live up to
150% longer (6, 7). Manganese-associated SOD-deficient mice die
shortly after birth (8, 9). Human Cu,Zn-SOD is encoded by a gene
(SOD1) located in the 21q22 region (10). This region is
involved in the most common genetic disease, known as trisomy 21 or
Down's syndrome. However, although a 1.5-fold increase in
SOD1 gene expression may be associated with the disease, several Down's syndrome patients carrying a partial trisomy 21 have a
normal Cu,Zn-SOD activity (11). It has been reported that some cases of
familial amyotrophic lateral sclerosis, a serious neurodegenerative
disease, are associated with dominant mutations in the SOD1
gene (12, 13). However, a gain of function is likely to cause familial
amyotrophic lateral sclerosis, rather than a defect of Cu,Zn-SOD
activity (14). Accordingly, in mice, Cu,Zn-SOD homozygous null mutants
are viable and do not exhibit the neuropathological symptoms
characteristic of familial amyotrophic lateral sclerosis patients.
However, these Cu,Zn-SOD
/
mice exhibit an enhanced
neuronal cell death following axotomy or ischemia, suggesting a
requirement for Cu,Zn-SOD under stress (15, 16).
71 and
29 was essential for both basal and PMA-induced expression. This
region could be further refined to a single Sp1/Egr-1/WT1-binding site
located between nucleotides
59 and
48. These findings suggest that
competition between Sp1, Egr-1, and variants of WT1 (and perhaps other
Sp1- or Egr-related proteins) for the same binding site may play a role
in the regulation of SOD1 gene expression in response to a
variety of biological signals.
EXPERIMENTAL PROCEDURES
80 °C. For Northern blotting, electrophoretic mobility shift
assay (EMSA), and transfection experiments for which PMA has been used,
a control including dimethyl sulfoxide alone was added to ensure the
absence of solvent-generated artifact. No effect of dimethyl sulfoxide was found in any of the assays.
EMBL4
human genomic library (a gift from Dr. Malek Djabali, CNRS,
Marseille-Luminy, France) was screened according to standard procedures
(21). The probe used to screen this library was generated by polymerase
chain reaction using oligonucleotides
5'-ACGCGGATCCGCCATTTTCGCGTACTGCAAC-3' and
5'-TGCAGGATCCTCGCAACACAAGCCTCCC-3'. These primers amplify a
458-base pair fragment corresponding to positions
271 to +187 of the
SOD1 sequence described by Levanon et al. (23).
After four successive screening rounds, one positive clone was obtained and purified. Identification of this clone was further assessed by
restriction mapping analysis and comparison with the predicted restriction map derived from the sequence described by Kim et al. (24).
x Constructs--
Polymerase chain reaction fragments
corresponding to various lengths of the SOD1 5'-flanking
region were generated using Pfu DNA polymerase (Stratagene)
to ensure high fidelity amplifications. The proximal primer (reverse
primer) spans positions
3 to +17 and was the same in all reactions:
5'-CCGAAAGCTTGAGACTACGACGCAAACCAG-3'. A HindIII
site (underlined) was added to facilitate subsequent cloning. Forward
primers were designed according to the size of the desired fragment:
1499, 5'-CCGACTCGAGCCCTTGGCAAGTTTACAATG-3',
952,
5'-CCGACTCGAGGTGGTCCCAGGTACTTGGGA-3',
750,
5'-CCGACTCGAGTATTTCCCTTGAAAGGTAAG-3',
552,
5'-CCGACTCGAGACCGAATTCTGCCAACCAAA-3',
355,
5'-CCGACTCGAGTGGCCAAACTCAGTCATAAC-3',
157,
5'-CCGACTCGAGACGCGCCCCTTGCCCCGCCC-3',
71,
5'-CCGACTCGAGATTGGTTTGGGGCCAGAGTG-3', and
29,
5'-CCGACTCGAGTATAAAGTAGTCGCGGAGAC-3'. The XhoI
site (underlined) was introduced at the 5'-extremity of all forward
primers to allow directional cloning into the reporter pGL2-Basic
vector (Promega). Amplifications were carried out using 100 ng of
bacteriophage DNA as a template and 15 cycles in a thermal cycler
(Perkin-Elmer). The different polymerase chain reaction fragments were
introduced into the promoterless pGL2-Basic Photinus pyralis
luciferase reporter vector (Promega) between XhoI and
HindIII sites. One recombinant clone for each construct was
chosen, and plasmid DNA was extracted and purified by alkaline lysis
and ion-exchange chromatography (QIAGEN, Inc.) following the
manufacturer's instructions. The nucleotide sequences of
construct inserts were determined by dideoxy chain termination
fluorescent automated sequencing (Euro Sequences Genes Services,
Montigny-Le-Bretonneux, France). These sequences were compared with the
corresponding regions of the sequence previously described by Kim
et al. (24) to check for polymerase-related misincorporation of nucleotides. Site-directed
mutagenesis was performed on pGLS
71-derived single-stranded DNA
according to the protocol described by Kunkel (25).
KTS) and WT1(+KTS) were
kindly provided by Dr. F. Cabon (Institut Fédératif sur le Cancer).
80 °C until use.
Protein concentration was measured by Bradford assay microtitration
using the Coomassie Plus kit (Pierce) according to the manufacturer's instructions. Typical protein concentration obtained under these conditions was in the 3-6 µg/µl range.
20 °C until further use. 5'-End labeling
of double-stranded oligonucleotides was performed by polynucleotide
kinase reaction (New England Biolabs Inc.) using 25 ng of
oligonucleotide and 30 µCi (1.1 MBq) of [
-32P]ATP
(3000 Ci/mmol; Amersham Pharmacia Biotech). Labeled probes were
purified by spin column exclusion chromatography (G-50, Amersham Pharmacia Biotech). For a typical EMSA experiment, 5 µg of nuclear extract were diluted in buffer containing (final concentrations) 40 mM Hepes (pH 8.0), 50 mM KCl, 0.05% Nonidet
P-40, 1% dithiothreitol, 10 µg/ml poly(dI·dC), and 100 µg/ml
sheared salmon sperm DNA in a total volume of 20 µl. Antibody (1 µg) or unlabeled competitor DNA (25 ng) was added (when required),
and the mixture was incubated for 20 min at room temperature. Labeled
probe (0.25 ng) was added to the mixture, and the reaction was
submitted to an additional 20-min incubation at room temperature.
Samples were run on a 5% nondenaturing polyacrylamide gel in 0.5×
Tris borate/EDTA. Following electrophoresis, the gel was dried and
autoradiographed. Anti-Sp1 (sc-059X) and anti-Egr-1 (sc-189X)
antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).
29 to
pGLS
1499) were combined with 1 µg of pRLTK (Renilla
reniformis luciferase under the control of a minimal herpes
simplex virus thymidine kinase promoter; Promega) and introduced into
107 cells by electroporation (200 V, 900 microfarads; Easy
CellJect, Eurogentec). Transfected cells were then plated in three
wells of a six-well plate. After 16 h, the culture medium was
replaced with fresh medium, and cells were collected and assayed for
luciferase activities 42-44 h after transfection. Luciferase
activities (P. pyralis and R. reniformis) were
quantitated with the Dual-luciferase kit (Promega) in a luminometer
(Berthold) following the manufacturers' instructions. The mean of
Photinus luciferase activities measured in the three wells
of the triplicates was plotted after adjusting for Renilla
luciferase activities. Two to five independent transfections were
performed for each experiment with similar results. For each assay, a
representative experiment is displayed in the figures.
RESULTS
View larger version (26K):
[in a new window]
Fig. 1.
Expression of the SOD1 gene in
HeLa cells following PMA treatment. A, Northern blot. Thirty
micrograms of total RNA from HeLa cells treated for various times with
150 nM PMA were hybridized to a full-length human SOD1
cDNA probe. The positions of the SOD1 transcript is
indicated. The same membrane was reprobed with a human 18 S rRNA probe
as an internal loading control. B, autoradiography scanning
of the Northern blot shown in A. Relative amounts (corrected
for 18 S rRNA expression) of SOD1 transcript in PMA-treated
cells are compared with untreated control cells. C, Western
blot. Twenty micrograms of total proteins from HeLa cells treated for
various times with 150 nM PMA were probed with an anti-SOD1
antibody (upper panel). The signal was quantitated by
autoradiography scanning (lower panel). kb,
kilobase.
71 and
29--
To identify the most proximal element responsible for basal
expression and PMA induction in the SOD1 5'-flanking region,
we performed a luciferase assay using a series of constructs
transfected in HeLa cells. These constructs carry different
5'-deletions of the SOD1 promoter linked to the P. pyralis luciferase gene. As shown in Fig.
2, constructs pGLS
1449 to pGLS
71
displayed basal promoter activity as well as PMA inducibility. In
contrast, luciferase expression from transfected construct pGLS
29 (in
which the regulatory sequences immediately upstream of the TATAA box
have been deleted) was similar to that observed with the promoterless
pGL2-Basic control vector and was barely detectable. It is noteworthy
that both basal and PMA-induced promoter activities gradually decreased following the progressive removal of 5'-regulatory flanking sequences, indicating the existence of several cis-acting positive
regulatory sequences spread along 1.5 kilobases of the SOD1
promoter proximal region. This observation led us to define the
71/+1
region as the minimal sequence required to provide SOD1
promoter activity. Kinetics experiments showed that PMA induction could
be detected as soon as 30 min following treatment for constructs
pGLS
71 and pGLS
1499 (data not shown).
View larger version (26K):
[in a new window]
Fig. 2.
Basal and PMA-induced expression of various
5'-deletions of the SOD1 promoter linked to the
luciferase gene in HeLa cells. Cells were transfected with
different pGLS x constructs or with the promoterless
pGL2-Basic control vector together with a Renilla luciferase
expression vector for normalization of transfection efficiencies. Cells
were treated with 150 nM PMA 16 h prior to harvesting
(black bars) or were left untreated (white
bars).
60/
38
Region--
The
71/
29 region seems to be critical for both basal
and PMA-induced expression of the SOD1 promoter. To further
define the sequences involved in this regulation and to identify
trans-acting factors bound to this region, we performed a
series of band shift experiments using overlapping oligonucleotides
covering this region. We used three different probes spanning
nucleotides
71 to
51,
60 to
38, and
50 to
29, respectively.
Incubation of the
71/
51 and
50/
29 probes with HeLa nuclear
extracts (PMA-treated or not) did not reveal any specific retarded
complex (data not shown). In contrast, incubation of the
oligonucleotide corresponding to the
60/
38 sequence with nuclear
extracts from untreated HeLa cells revealed two major shifted complexes
(complexes I and III) (Fig.
3B). Incubation of the same
probe with extracts from PMA-treated cells allowed the detection of a
third complex (complex II) (Fig. 3B). Minor complexes were
also inconsistently observed (complexes IV and V). Transcription
factors containing zinc finger DNA-binding domains have been found to
be involved in the regulation of several GC-rich proximal promoters
(27). Because the
60/
38 probe contains 77% G or C nucleotides, we
investigated the possible involvement of Sp1 and Egr family members in
the complexes identified above. Thus, we carried out competition
experiments using consensus binding sequences for Sp1 and Egr family
members. As shown in Fig. 3, complexes I and III became barely
detectable following incubation with an excess of Sp1 unlabeled
competitor oligonucleotide, whereas complex II was not affected. On the
other hand, the PMA-induced complex II was efficiently displaced by an
Egr consensus oligonucleotide, whereas complexes I and III were not.
Control experiments including consensus NF-
B- and AP-1-binding sites
did not reveal any change in electrophoretic profiles (data not shown).
Altogether, these results suggest that complexes I and III contain two
Sp1-related proteins, whereas complex II contains a member of the Egr
family. Indeed, Fig. 3 shows that complex I was completely supershifted following incubation with an anti-Sp1 monoclonal antibody, whereas complex II and III were not. Conversely, complex II disappeared when
the binding reaction was performed in the presence of an anti-Egr-1
antibody. The absence of supershift for this complex may be explained
if the epitope recognized by the anti-Egr-1 antibody is part of (or
located near) the Egr-1 DNA-binding site. Antibody binding would
compete DNA binding, resulting in the disappearance of complex II
rather than in a supershifted complex. Alternatively, antibody binding
might result in a conformational change in Egr-1 structure, which would
become unable to bind DNA. Altogether, these results allow the
identification of Sp1 and Egr-1 as components of complexes I and II,
respectively. Complex III is likely to contain an Sp1-related protein
since this complex is efficiently competed by an excess of unlabeled
Sp1 oligonucleotide competitor, although it is not supershifted by an
anti-Sp1 monoclonal antibody. Thus, complex III may contain an Sp1
proteolytic fragment lacking the epitope recognized by the anti-Sp1
antibody or contains another possible Sp1-like protein. The minor
complexes IV and V are likely to contain degradation products of
Sp1-related proteins since they are efficiently competed by a consensus
Sp1-binding site.
View larger version (57K):
[in a new window]
Fig. 3.
Gel shift analysis of nuclear factors bound
to the 60/
38 region of the SOD1 promoter.
A, shown is the sequence of the
71/
30 region of the
SOD1 promoter. The
60/
38 region used as a probe for gel
shift assays is underlined. The TATAA box is indicated in
italics. Sequences of Sp1 and Egr-1 oligonucleotides used as
competitors. Binding sites are underlined. B,
EMSA was performed using nuclear extracts from HeLa cells (PMA-treated
or not) and a 32P-labeled double-stranded
60/
38
oligonucleotide as a probe. Competition and supershift assays were
carried out using Sp1 binding sequence, Egr binding sequence, anti-Sp1
or anti-Egr-1 antibodies.
60/
38 region did not allow the
identification of any obvious Egr-1-binding site. To identify Sp1- and
Egr-1-binding sites within the
60/
38 region, we used mutant oligonucleotides derived from the
60/
38 sequence in band shift assays. Nucleotides were substituted by groups of three, allowing complete coverage of the
60/
38 sequence using seven mutant
oligonucleotides (Fig. 4A).
The EMSA results displayed in Fig. 4B show that mutations between positions
59 and
48 disrupted all the complexes seen with
the wild-type oligonucleotide. Oligonucleotide m(
47/
45), carrying
mutations on nucleotides
47,
46, and
45, bound Sp1 and Egr-1, but
poorly bound the possible Sp1-like protein. Mutations in the 3'-region
of the
60/
38 sequence had no effect on the recognition of the
corresponding oligonucleotides. The identities of the different factors
bound to mutant oligonucleotides have been assessed by competition and
supershift assays with antibodies (data not shown). Thus, in this
region, binding sites for Egr-1 and Sp1 are indistinguishable and cover
positions
59 to
48, whereas a possible Sp1-like factor binds to an
overlapping sequence located between nucleotides
59 and
45. These
results were confirmed using the
60/
38 oligonucleotide as a probe
and various mutant oligonucleotides as competitors. As expected,
oligonucleotides carrying mutations between positions
59 and
48
were unable to compete for Sp1 and Egr-1 binding (Fig. 4C).
Conversely, competitors mutated at positions
59 to
45 did not
compete for the binding of the possible Sp1-like factor, confirming
assessment of binding site positions. Surprisingly, oligonucleotide
m(
44/
42) did not completely compete the possible Sp1-like complex,
suggesting that another unidentified protein may be present within this
complex.
View larger version (45K):
[in a new window]
Fig. 4.
Mapping of the Sp1-, Sp1-like protein-, and
Egr-1-binding sites in the 60/
38 region. A, shown
are the sequences of the mutant oligonucleotides used either as probes
(B) or competitors (C) in EMSAs. B,
EMSAs were performed using different 32P-labeled mutant
oligonucleotides corresponding to the
60/
38 region with nuclear
extracts from HeLa cells. The positions of Sp1, Egr-1, and Sp1-like
protein shifted bands are indicated. C, EMSA was performed
using the wild-type (wt)
60/
30 oligonucleotide as a
32P-labeled probe and different mutant oligonucleotides as
competitors.
71 construct was transfected in HeLa cells
together with an expression vector for Egr-1. The results shown in Fig.
5A indicate that the SOD1 proximal promoter-driven luciferase expression
increased up to 4-fold when increasing amounts of Egr-1 expression
vector were added. We then analyzed the response to Egr-1 of various mutant promoters. Mutant construct pGLS
71 m(
41/
39) is derived from oligonucleotide m(
41/
39), which binds Sp1, Egr-1, and the possible Sp1-like protein. Mutant construct pGLS
71 m(
47/
45) is
derived from oligonucleotide m(
47/
45), which efficiently binds Sp1
and Egr-1, but poorly binds the possible Sp1-like factor. Mutant
construct pGLS
71 m(
50/
48) is derived from oligonucleotide m(
50/
48), which does not bind any of the three proteins. The results displayed in Fig. 5B show that all constructs but
one, pGLS
71 m(
50/
48), displayed promoter activity and Egr-1
inducibility. Luciferase activity driven by an SOD1 proximal
promoter mutated at positions
50 to
48 was almost abolished, both
for constitutive and Egr-1-induced expression, confirming the crucial
role of the Sp1/Egr-1 site. Interestingly, luciferase expression (both
basal and Egr-1-induced) driven by construct pGLS
71 m(
47/
45) was comparable to that of the wild-type pGLS
71 control, suggesting that
the possible Sp1-like factor(s) does not play a major role in
SOD1 transcription in HeLa cells. We used the same mutant
constructs to map the PMA-induced element within the SOD1
proximal promoter. Fig. 5C shows that, in a manner similar
to the data described above, mutation of nucleotides
50 to
48
strongly reduced PMA-induced luciferase expression, whereas other
mutants tested remained inducible by PMA. These data suggest that the
PMA-induced element and the Sp1/Egr-1 site are identical. Thus,
PMA-induced activation of an Egr family member would result in
SOD1 proximal promoter induction via the Sp1/Egr-1-binding
site.
View larger version (17K):
[in a new window]
Fig. 5.
Basal and Egr-1- and PMA-induced expression
of wild-type and mutant derivatives of the SOD1 proximal
promoter. A, HeLa cells were co-transfected with 15 µg of wild-type pGLS 71 construct and increasing amounts of an
expression vector for Egr-1 under the control of a cytomegalovirus
promoter. B, 15 µg of wild-type pGLS
71 construct or
different mutants of pGLS
71 were transfected alone (white
bars) or were co-transfected with 6 µg of Egr-1 expression
plasmid (gray bars). C, HeLa cells were
transfected with 15 µg of wild-type pGLS
71 construct or different
mutants of pGLS
71 and treated with 150 nM PMA 16 h
prior to harvesting (gray bars) or left untreated
(white bars).
71 together with increasing amounts of
expression vectors for either the WT1 splicing variant containing the
tripeptide KTS or the WT1 variant lacking KTS. The results depicted in
Fig. 6 show that, in contrast to what is
observed in many systems, both splicing variants of WT1 transactivated
the SOD1 proximal promoter. Excessive amounts of WT1
expression vectors were inhibitory, a "squelching" phenomenon often
observed in other systems and thought to be an artifact linked to
episomal expression in transient transfection assays (28). In
accordance with the results obtained for Egr-1, mutations in the
50/
48 region completely abolished basal and WT1-induced expression,
whereas other mutations (m(
47/
45) and m(
41/
39)) had no
significant effect on WT1-induced expression (data not shown). These
results suggest that the two WT1 splicing variants bind to the same
sequence as Egr-1 in the SOD1 proximal promoter. However,
since WT1 often induces transcriptional repression rather than
activation through sites that respond to Egr-1 in a positive manner
(27, 29), we cannot exclude the possibility that the observed enhancer
effect of WT1 overexpression may be indirect.
View larger version (23K):
[in a new window]
Fig. 6.
WT1-induced expression of the SOD1
proximal promoter. HeLa cells were co-transfected with 15 µg of pGLS 71 construct and increasing amounts of an expression
vector for either WT1(+KTS) or WT1(
KTS) under the control of a
cytomegalovirus promoter.
DISCUSSION
134 and
127 and
positions
118 and
111 are involved in the promoter response to
Panax ginseng extracts (30). An Sp1 site (positions
104 to
89) and a CCAAT/enhancer-binding protein (C/EBP) site (positions
64
to
55) have also been described (31). Our data demonstrate that,
although several regulatory sequences are present within 1.5 kilobases
of 5'-upstream sequences, the SOD1 promoter region located
between nucleotides
71 and +1 is sufficient to provide both basal and
PMA-induced promoter activities. Within this region, we identified a
site located between nucleotides
59 and
48 that is able to bind Sp1
constitutively and Egr-1 following PMA treatment. This sequence can
also mediate transactivation by two splicing variants of the Wilms'
tumor protein WT1. Interestingly, the C/EBP-binding site (positions
64 to
55) described by Seo et al. (31) partially
overlaps the Sp1/Egr-1 site (positions -59 to
48) described in this
study, suggesting the existence of a complex interplay between
different trans-acting factors for the transcriptional
regulation of the SOD1 gene.
View larger version (16K):
[in a new window]
Fig. 7.
A, putative positions of Sp1-, Sp1-like
protein-, and Egr-1-binding sites on the SOD1 proximal
promoter according to this study and to consensus sequences previously
described (32). B, consensus binding sequences for Sp1 and
Egr-1 according to the Transfac data base (32). Differences from the
proposed sites on the SOD1 proximal promoter are indicated
by boxes.
The apparent lack of phenotype in SOD1 null mice (15) indicates that Cu,Zn-SOD is probably not necessary for normal development and life, but rather is involved in the response to stress. Indeed, the only phenotype described for these mice is a partial defect in motor neuron regeneration following section. Interestingly, Egr-1 has been described as a key factor in response to mechanical stress in endothelial cells (33). Thus, Egr-1 regulation of the SOD1 gene provides a link between this key factor and Cu,Zn-SOD in response to mechanical stress in endothelial cells. Accordingly, a recent report has described an induction of Cu,Zn-SOD activity in human aortic endothelial cells in response to shear stress (34). This increase in activity was, at least in part, mediated by increased transcription of the SOD1 gene and was not observed in aortic smooth muscle cells. Although it was not demonstrated that this increase was mediated by Egr-1, we propose that Egr-1 regulation of SOD1 participates, via the antioxidant properties of Cu,Zn-SOD, in an atheroprotective pathway.
The function of the tumor suppressor protein WT1 in SOD1
gene regulation remains unknown. A connection between WT1 and apoptotic processes has been described in several reports. WT1 stabilizes p53 and
inhibits its ability to induce apoptosis in response to various stimuli
(35). Down-regulation of endogenous WT1 by antisense oligonucleotides
induces apoptosis in myeloid leukemia cell lines (36). On the other
hand, a pro-apoptotic role for WT1 has been reported: overexpression of
WT1 induces apoptosis in an osteosarcoma cell line (37), and
p53-independent programmed cell death was induced in U2OS and Saos-2
cells (38), HepG2 and Hep3B cells (39), and the myeloblastic leukemia
M1 cell line (40) following WT1 induction. In most cases, the
pro-apoptotic effect of WT1 seems to be mediated by the splicing
variant that lacks the KTS tripeptide (38, 39). In addition, WT1 has
been described as able to down-regulate transcription of the
anti-apoptotic bcl-2 gene. Although reactive oxygen
intermediates are not likely to be involved in all apoptotic processes,
sympathetic neurons and neuronally differentiated PC12 cells undergo a
reactive oxygen intermediate-dependent apoptosis when
deprived of nerve growth factor. This phenomenon is prevented by
injection of Cu,Zn-SOD (41). Taken together, these results suggest a
function for the WT1 pathway of SOD1 regulation in the
prevention of stress-induced apoptosis in neuronal cells.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. P. Charnay for the gift of the Egr-1 expression construct and Dr. F. Cabon for the gift of WT1 expression vectors. We are grateful to V. Sallé-Vogade and A. Brussels for technical help and to Drs. A. Harel-Bellan, D. Trouche, and M. Lipinski for invaluable stimulating discussions and critical reading of the manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported by grants from the Institut d'Oncologie Cellulaire et Moléculaire Humaine (Bobigny, France) and the Association pour la Recherche contre le Cancer.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.
§ Present address: Lab. de Biologie Cellulaire du Noyau, CNRS UMR 7592, Inst. Jacques Monod, Université Paris VII, Tour 43, 2, Place Jussieu, F-75251 Paris, France.
¶ Present address: Lab. d'Immuno-pharmacologie, CNRS UPR 0415, Inst. Cochin de Génétique Moléculaire, 22, Rue Méchain, F-75014 Paris, France.
To whom correspondence should be addressed. Tel.:
33-1-42-11-49-20; Fax: 33-1-42-11-54-94; E-mail: cjaulin{at}igr.fr.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: SODs, superoxide dismutases; Cu, Zn-SOD, copper-zinc superoxide dismutase; PMA, phorbol 12-myristate 13-acetate; EMSA, electrophoretic mobility shift assay..
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() |
---|