From the Molecular Neurobiology Unit, Gerontology Research Center,
NIA, National Institutes of Health, Baltimore, Maryland 21224
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INTRODUCTION |
The NMDA1 subtype of
glutamate receptor plays important roles in
voltage-dependent Ca2+ influx, synaptic
plasticity, and excitotoxic neuronal death in the mammalian central
nervous system (1-3). Functional NMDA receptors contain a key NMDAR1
subunit in combination with one or more members of the NMDAR2(A-D)
subunits (4-7). The expression pattern of the NMDAR1 gene
is widespread in the brain and restricted to neuronal cells (8-10). A
great deal of data suggest that interactions of cis elements in a gene
with tissue-specific trans-acting factors play crucial roles in
determining tissue-specific expression of the gene (11-14). We
previously isolated a 3-kilobase pair promoter of the rat
NMDAR1 gene and observed that a proximal region with 356 bp
is sufficient to confer to the promoter a cell-type specificity in
neuronal-like PC12 cells compared with C6 glioma and HeLa cervical cancer cells (15-17). This proximal promoter region includes a 5
-untranslated region that contains a neuron-restrictive silencer element (NRSE)/RE1-like element. Several neuronal genes contain this
element, and a neuron-restrictive silencer factor/REST, expressed in
nonneuronal tissues, may recognize this element and restrict gene
expression in nonneuronal cells (11, 18).
Recently, several factors were identified that preferentially bind
transcriptional elements on single-stranded DNA and function as
suppressors or activators (19-31). For example, heterogeneous nuclear
ribonucleoprotein K specifically binds to a cytosine-rich single-stranded DNA of the human c-MYC promoter and
transactivates a heterogeneous promoter containing this binding
sequence only when this promoter is present in a circular, supercoiled
conformation and not in a linearized construct (19). The supercoiled
structure is thought to provide a single-stranded region of DNA due to
negative supercoiling of the circular plasmid. Another example is a
cellular nucleic acid binding protein (22). This cellular nucleic acid binding protein is a ~19-20-kDa zinc finger protein that recognizes a purine-rich single strand sequence GTGCGGTG in the sterol regulatory element (21) or a sequence TGGGGAGGG in the CT element of the human
c-MYC promoter and acts as an activator of gene expression (22). Recently, Taira and Baraban (20) identified a protein complex
that recognizes a G-rich strand of an NGFI/Egr binding element (20).
Proteins in this complex are enriched in the rat brain. Their results
suggested that the NGFI/Egr element may be a convergence point for
double and single-stranded binding transcription factors.
The proximal 356-bp promoter of the NMDAR1 gene contains a
GC-rich sequence encompassing GSG and Sp1 elements. The sense strand of
this region also is purine-rich (15). Our previous studies showed that
a disruption of these sequences eliminating GSG and Sp1 binding
decreased the basal and growth factor-regulated promoter activities
(17). In this study, we examined the role of an NRSE and the
interactions of nuclear proteins with a purine-rich single (sense)
strand of GSG/Sp1 region in the control of cell-type-specific expression of the NMDAR1 promoter.
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EXPERIMENTAL PROCEDURES |
Plasmid Construction--
Plasmids of the NMDAR1
promoter/luciferase reporter gene were constructed as described
previously (16, 17). Deletion of the NRSE in the NMDAR1 promoter
construct, pNRL356, was prepared using deletion PCR (32). Primers were
designed and used as follows. For the upper fragment in the
first round of PCR, primer GB46 (17) was paired with a downstream
primer GB126, 5
-GAGCGCGGCGGATG|GCGTGTTTGGCGCT. The lower fragment
was generated with GB40 (17) with an upstream primer, GB125,
5
-AGCGCCAAACACGC|CATCCGCCGCGCTC. A "|" indicates a
deletion of a 17-bp NRSE-containing sequence in both GB126 and GB125.
The second round of PCR was performed with agarose gel-purified products from the first round of PCR under the conditions as described before (17). The final PCR products were cloned into pGEM-T and
subcloned into a luciferase expression vector as reported previously
(17).
Cell Culture and Reporter Gene Assay--
PC12, C6, and HeLa
cells were maintained as described before (16). Methods for
transfection of the NMDAR1 promoter constructs were described
previously (16). A plasmid containing the cytomegalovirus promoter-driven
-galactosidase gene was used to correct for
transfection efficiency. In some experiments, G-25 Sephadex-purified
oligonucleotides were added to transfection mixtures together with
promoter/reporter plasmids.
Interaction of DNA with Nuclear Proteins--
Crude
nuclear extracts were prepared from cultured PC12, C6, and HeLa cells
using a modified Dignam method (16). In some experiments, PC12 cells
were treated with 100 ng/ml NGF for different times before harvesting
for nuclear extraction.
DNA probes or competitors used in electrophoretic mobility shift assays
(EMSAs) are as follows. Single strand DNA probes encompassing the GSG/Sp1 element are GB59 (sense),
5
-GCGTCAGGAAGCGGGGGCGGTGGGAGGGGTAGAACGCGTAGGT, and GB60 (antisense),
5
-ACCTACGCGTTCTACCCCTCCCACCGCCCCCGCTTCCTGACGC; double-stranded
oligonucleotide probe for the NMDAR1 promoter was formed by annealing
GB59 and GB60. Sequences of all competitors are listed in Fig. 5,
A and C. Single-stranded sense and antisense Sp1
consensus oligonucleotides (22-mer) were synthesized following the
sequence of Briggs et al. (33). EMSA experiments were done as described before (16). Briefly, crude nuclear proteins at 4.5-9
µg/reaction were preincubated with poly(dI:dC) and in some experiments with the addition of competitors at 50-fold excess of
probe. Synthesized double or single-stranded oligonucleotides were
labeled at the 5
end with [
-32P]ATP and
T4 kinase. Radiolabeled probes were purified by G-25 Sephadex chromatography, and single-stranded probes were freshly denatured by heating for each experiment. 10-50 fmol of probe were
used in each reaction. In supershift EMSAs, a polyclonal Sp1 antibody
purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA) was added
to reaction mixtures 20 min after the addition of the probe.
DNA-protein complexes were separated on 5% native polyacrylamide gels
(PAGE), and dried gels were exposed to x-ray film or quantitatively
analyzed on a PhosphorImager SI system as described before (17).
UV cross-linking experiments were carried out using a method described
by Chodosh (34). A modified sense strand probe, GB59-2, was
synthesized by replacing the deoxyguanine at position 23 in GB59 with a
8-bromo-deoxyguanine and purified by a reverse-phase chromatographic
column at Paragon Biotech, Inc. (Baltimore, MD). Radioactive
32P labeling of GB59-2 and its reaction with nuclear
extracts were performed as described for EMSAs. Reaction mixtures were
then irradiated with UV light at 254 nm for 15 min on ice. Irradiated samples were mixed with one volume of 2 × sample buffer and
denatured at 85 °C for 3 min. DNA/protein cross-linked products were
fractionated on SDS-PAGE (Novex, San Diego, CA) and visualized by
exposing dried gels to x-ray film. The apparent molecular weight of
each complex was estimated based on protein molecular weight markers, SeeBlue pre-stained standard (Novex), run simultaneously on the same
gel.
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RESULTS |
Neuron-restrictive Silencer Factor/REST Participates in the Control
of Cell-specific Expression of the NMDAR1 Promoter--
Using a
luciferase reporter gene assay in a transient transfection system, we
observed that activity of the 356-bp NMDAR1 promoter showed a 309-fold
induction over a promoter-less plasmid in PC12 cells but only 2-fold
and 8.5-fold induction in HeLa and C6 glioma cells respectively (Table
I). This reporter activity is consistent
with the endogenous NMDAR1 mRNA levels that are easily measurable
by RNase protection assay in PC12 cells but not detectable in C6 and
HeLa cells (16). A similar activity for the 3-kb promoter was seen in
each cell line (16). These results strongly suggest that elements
important for the cell-type-specific expression reside in this proximal
promoter region. As reported previously (12, 18), a NRSE/RE1-like
sequence was found in the NMDAR1 5
-untranslated region encoded by exon
1 and is part of the 356-bp promoter construct (16). To test its
functional role in silencing NMDAR1 promoter activity in nonneuronal
cells, we deleted the 17-bp core sequence of the NRSE in the 356-bp
promoter and examined the activity of the mutated promoter in both HeLa and C6 cells. Fig. 1 shows that deletion
of the NRSE had no effect on activity in PC12 cells but increased
activity in C6 cells 4.6-fold and in HeLa cells 2.7-fold when compared
with the wild-type promoter activity in each cell line. This result
strongly suggested that NRSE participates in the cell-type-specific
expression of the NMDAR1 gene. However, after the NRSE/RE1
deletion, the fold increase in activity of the NMDAR1 promoter over
promoter-less plasmid was only 40-fold in C6 cells and 5.4-fold in HeLa
cells. This is in contrast to a 309-fold induction over promoter-less
plasmid in PC12 cells. This lack of a similar fold induction in C6 and HeLa cells suggests the possibility that additional factors,
particularly activators, may participate in the control of the NMDAR1
promoter in PC12 cells. This assumption prompted us to search for other nuclear factors involved in this control.
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Table I
Activity of the NMDAR1 promoter in different cells
Cells were cultured and transfected with indicated NMDAR1 promoter
luciferase constructs as described under "Experimental Procedures."
The relative luciferase activity was normalized to cotransfected
-galactosidase activity. Promoter activity was then calculated based
on the -fold increase in the relative luciferase activity over a
promoter-less vector, pGL2Basic. Numbers are mean values ± standard
error.
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Fig. 1.
Effect of deletion of the NRSE/RE1 element on
NMDAR1 promoter activity. A construct carrying the deleted
NRSE/RE1 element in the NMDAR1 promoter, pNRL356 NRSE, was
transfected into the indicated cells. Wild-type construct, pNRL356, was
used as a control in the same experiment. The relative luciferase
activity was obtained as described in Table I, and fold increase in
mutant over wild-type construct was calculated for each cell line.
wt, wild type.
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Novel Purine-rich Single-stranded DNA-binding Proteins May
Participate in the Regulation of the NMDAR1 Promoter in PC12
Cells--
As shown in Table I, deletion of a 117-bp 5
sequence
(
356/
240) from the 356-bp promoter abolished activity of the
reporter gene. An overlapping GSG/tandem Sp1 element in this deleted
sequence was previously shown to be important for promoter activity
(17). Also, it is well known that the ubiquitously expressed
transcription factor Sp1 and inducible NGFI/Egr proteins interact with
this element. Therefore, we tested the binding activity of this
sequence in an EMSA. Results in Fig.
2A show that a 43-bp
double-stranded DNA encompassing the GSG/Sp1 region formed complexes
with nuclear extracts similar to those reported previously using a
112-bp fragment as probe (16). However, we also observed
single-stranded DNA binding activity with both sense and antisense
oligonucleotides of the same region as the 43-bp dsDNA. One complex was
formed on the antisense strand and showed more binding in PC12 nuclear extracts than in HeLa extracts. The nuclear extracts of HeLa cells formed only one band with the sense strand. We named this band single-stranded binding protein complex 1, SBPC1. Interestingly, in
addition to SBPC1, PC12 cell extracts formed a second, slower migrating
band. We named this band as SBPC2. We quantified the binding activities
of SBPC1 and SBPC2 by phosphorimaging. Interestingly, a high
SBPC2/SBPC1 ratio as shown in Fig. 2B correlated with a strong promoter activity in PC12 cells, whereas a low ratio was observed in HeLa cells corresponding to a low promoter activity. This
suggests that both SBPC1 and SBPC2 may participate in the regulation of
NMDAR1 promoter activity. To confirm this possibility, we performed two
types of experiments: cotransfecting single-stranded DNA as competitor
with the NMDAR1 luciferase reporter gene and testing the effect of NGF
treatment of PC12 cells on the formation of these complexes.

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Fig. 2.
EMSA of double- and single-stranded NMDAR1
promoter oligonucleotides with nuclear proteins. A,
interaction of the GSG/tandem Sp1 element of the NMDAR1 promoter with
nuclear proteins. EMSA was performed as described under "Experimental
Procedures." Radiolabeled probes for the NMDAR1 gene
including double- and single-stranded oligonucleotides were incubated
with 4.5 µg of nuclear extracts and fractionated on 5% nondenaturing
PAGE. Autoradiograms were obtained by exposing the dried gel to x-ray
film for 8 h at 80 °C. B, interaction of
single-strand GSG/Sp1 element of the NMDAR1 promoter with different
nuclear extracts. Sense strand probe, GB59, was labeled with
[ -32P]ATP and incubated with 4.5 µg of nuclear
extracts from PC12, HeLa, or C6 cells. The dried gel was exposed to a
PhosphorImager plate for quantitation before exposure to an x-ray film.
Quantitated radioactivity in each complex was used to calculate the
ratio of SBPC 2 to 1. The ratios are listed for each cell line on the bottom of autoradiogram shown in the figure.
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22-mer oligonucleotides were cotransfected with pNRL356 into PC12
cells. Those single-stranded oligonucleotides known to compete with
formation of SBPC (see below results) dramatically reduced reporter
activity in a dose-dependent manner. In contrast, an oligonucleotide known not to compete for SBPC complex formation did not have an inhibitory effect (Fig.
3). PC12 cells treated with 100 ng/ml NGF
for 30, 60, and 120 min showed a significant ~65% reduction in SBPC1
and an ~80% increase in SBPC2 complex formation (Fig.
4). These results suggest that
single-stranded binding proteins may contribute to both basal and
trophic factor-induced NMDAR1 promoter activities.

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Fig. 3.
Effect of cotransfected sense strand
oligonucleotides on the NMDAR1 promoter activity in PC12 cells.
Different amounts of 22-mer oligonucleotides were cotransfected with
the NMDAR1 promoter luciferase construct, pNRL356, into PC12 cells as
described under "Experimental Procedures." The sequences of each
oligonucleotide competitor are listed in Fig. 5A. The
luciferase activity was measured and used to calculate promoter
activity as a percentage of control (pNRL356 activity transfected
without oligonucleotide). The luciferase of the control was 17,131 ± 176.7 fg (mean ± S.E.).
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Fig. 4.
Differential effects of NGF treatment on
SBPC1 and 2 binding in PC12 cells. PC12 cells were treated with
100 ng/ml NGF for 30, 60, and 120 min, and nuclear extracts were
prepared for EMSAs. Nuclear extracts from nontreated PC12 cells served as a control. Two different amounts of nuclear extracts, 4.5 and 9 µg/reaction, were used. Binding activities in SBPC1 and 2 were quantitated as in Fig. 2B and expressed as a percentage of
control.
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SBPC1 Binding Involves a dG4 Core, Whereas SBPC2
Binding Requires a Core Plus Flanking Sequence--
In an experiment
to define the binding elements of SBPC1 and SBPC2, EMSAs were performed
with multiple competing 22-mer oligonucleotides overlapping the sense
strand probe and flanking sequences shown in Fig.
5A. A typical binding pattern
is shown in Fig. 5B. Using phosphorimaging, we quantitated
SBPC1 and SBPC2 formation in the presence of each competitor and
compared the remaining complex to a control without competitor.
Oligonucleotides containing dG4 sequence effectively
reduced SBPC1 binding to <15% control (competitors 3-7), whereas
competitor 4 containing a core sequence of TG3A with a
15-nucleotide-flanking region reduced SBPC2 binding to only 23%
control. Competitor oligonucleotides at the 3
end of the probe
(competitors 7-10), resulting in the loss of 5
sequence, reduced the
competition compared with competitors 4-6. This competition study
suggests that SBPC1 and SBPC2 protein complexes may recognize slightly
different elements located within this region. This concept was further
supported by experiments with other oligonucleotide competitors (Fig.
5, C and D). Competitor 12 containing
dG4 but lacking TG3A sequence competed SBPC1
but not SBPC2. Competitor 14 containing TG3A in addition to
all of the sequence of competitor 12 was able to compete for both SBPC1
and SBPC2. However, removing the sequence 5
to TG3A
(competitors 14-17) resulted in a loss of SBPC2 competition.
Competitor 17 lacking dG4 failed to compete any complexes although it
contains a TG3A core sequence.

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Fig. 5.
Sequence specificity of SBPC1 and SBPC2
formation. A, sequences of sense strand probe and
competitors. A 43-mer probe (GB59) and 22-mer oligonucleotide
competitors are listed above and below the NMDAR1
promoter sequence (numbering according to Ref. 15). TG3A
and dG4 elements are underlined. B,
competition of SBPC1 and SBPC2 formation on the sense strand of the
NMDAR1 promoter. PC12 cell nuclear extracts were preincubated with the indicated competitors at a 50-fold molar excess over probe before the
addition of radiolabeled sense probe, GB59. C, sequence GB59 and shorter competitors. The sense probe (GB59) and various competitors are listed. D, SBPC1 and SBPC2 are competed by different
oligonucleotides. EMSA experiments were done as described in
B with competitors listed in C.
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Sp1 Binding Is Not Involved in the Formation of SBPC1 and
SBPC2--
Considering that the Sp1 element (TGGGAGGGG) in the NMDAR1
promoter overlaps the apparent SBPC1 and SBPC2 binding sequence, we
performed experiments to clarify whether these complexes contained Sp
family members. In an EMSA, neither SBPC1 nor SBPC2 was changed by a
Sp1 antibody, which we previously showed was able to supershift Sp1
binding on the double-stranded probe (Fig.
6). A 70-fold excess of double-stranded
Sp1 consensus oligonucleotide failed to compete for the SBPC1 binding.
Only the sense strand of Sp1 consensus competed for SBPC1 binding. This
sense consensus contains a multiple dG sequence, also present in
oligonucleotides that competed for SBPC1 binding (Fig. 5). Furthermore,
both SBPC1 and SBPC2 showed a faster migration than complexes detected
with either a 24-bp Sp1 consensus double-stranded oligonucleotide (data
not shown) or a 43-bp double-stranded probe of the NMDAR1 promoter
(Fig. 2A). Therefore, these data suggest that the Sp1
protein is not involved in the single strand binding complexes seen on
the NR1 promoter.

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Fig. 6.
Sp1 protein is not involved in the formation
of SBPC1 and SBPC2 complexes. EMSAs were performed as detailed
under "Experimental Procedures" using PC12 nuclear extracts.
Increasing amounts of polyclonal antibody against Sp1 protein were
added to reaction mixtures 20 min after the addition of probe.
Single-stranded sense or antisense consensus oligos of Sp1 elements
were used as competitors.
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SBPC1 Contains a 31.5-kDa Protein, and SBPC2 Consists of Several
Larger Proteins--
To obtain evidence of direct nuclear protein
interactions with the sense strand of the GSG/Sp1 region in the NMDAR1
promoter, we modified the oligonucleotide probe GB58 shown in Fig.
5A by replacing residue 23, guanidine, with a
bromo-guanidine. After 5
-radiolabeling of this modified probe and
incubating it with nuclear extracts, reaction mixtures were irradiated
with UV light to cross-link the DNA-protein complexes. As shown in
the right-most lane of Fig.
7A , the DNA probe itself
migrated as approximately 6.5 kDa in a SDS-PAGE gel. HeLa cells showed
a major band with an apparent molecular mass of 38 kDa, corresponding
to a 31.5-kDa protein after subtracting the contribution of the probe
(6.5 kDa) based on one probe per protein molecule stoichiometry. This
band is relatively weak in PC12 cells and C6 cells. The same band
disappeared after adding oligonucleotide that removed only SBPC1 in
both PC12 or HeLa nuclear extracts (data not shown). These results
along with those in Fig. 2B suggest that this 31.5-kDa
protein may be the major component of the SBPC1 complex. The SBPC2
complex contains several proteins with larger molecular masses
(P, Fig. 7B).

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Fig. 7.
UV cross-linking of nuclear proteins with
sense strand of the GSG/tandem Sp1 element in the NMDAR1 promoter.
Cross-linking of nuclear proteins with radiolabeled GB59-2
oligonucleotide encompassing the GSG/tandem Sp1 element was performed
as described under "Experimental Procedures." Cross-linked products
in 10 µg of protein were fractionated on 10-20% Tricine-SDS/PAGE
for panel A and on 8% Tris-glycine SDS/PAGE for panel
B. Autoradiograms were obtained after exposing dried gels to x-ray
film. A control of probe alone was included to locate its migrating
position. C, H, and P represent
nuclear extracts from C6 glioma, HeLa, and PC12 cells,
respectively.
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DISCUSSION |
Neurons express many genes in common with other cell types and in
addition express their own unique set of genes important in determining
neuron-specific functional characteristics (11, 14, 18, 35). One of
these genes is the NMDAR1 gene, which is an important
subunit of all NMDA receptors (4-9). It is expressed widely in the
brain but exclusively in neurons. A factor important in determining the
distribution pattern of proteins is the transcriptional regulation of
gene expression. Our previous work and the results reported here
suggest that the NMDAR1 gene proximal promoter is able to
confer neuronal-specific expression. This region of the promoter
contains GSG and overlapping tandem Sp1 elements that we previously
showed were important for both basal and neurotrophic factor-induced
promoter activities. Strong activity was observed in neuronal-like PC12
cells but not in C6 glioma and HeLa cells. In addition, the
NMDAR1 gene contains a consensus NRSE in the 5
-untranslated
region. Deletion of this element relieved the suppression of promoter
activity in C6 and HeLa cells without having any effect on PC12 cell
activity, suggesting that this element plays an important role in
neuron-specific expression of the NMDAR1 gene. Nonneuronal
cells express a factor, neuron-restrictive silencer factor/REST, that
binds to NRSEs located in several neuronal-specific genes and represses
their expression (11, 12). Neuronal cells, including PC12 cells, do not
express this repressor, thus allowing cell-specific gene expression.
Deletion of the NRSE in the NMDAR1 gene relieved suppressed
activity in C6 and HeLa cells (Fig. 1), but the resulting elevation in
activity of the deleted construct was between 8 and 60 times less than
that observed with wild-type promoter in PC12 cells. This is similar to
results reported by Li et al. (36) with the synapsin I gene
promoter and suggested to us that other factors, present in PC12 cells,
may be responsible for the more efficient expression in this cell
line.
We showed previously that a GC-rich region of the NMDAR1 promoter
encompassing a GSG and overlapping Sp1 sites was protected in DNase
footprinting experiments and was able to form several complexes with
double-stranded oligonucleotides in EMSAs (16). Since this region is
also critical for efficient expression of activity, we further examined
this region for other interacting factors that might be important in
transcriptional regulation. Until recently, the importance of
single-stranded DNA-binding proteins in regulating gene expression has
been under-appreciated. Several groups now have identified proteins
that selectively bind to single-stranded DNA transcriptional elements
(19-31). We found that nuclear extracts from several cell types were
able to form complexes with single-stranded oligonucleotides derived
from the proximal GC-rich region of the NMDAR1 promoter. Complexes were formed on both sense and antisense oligonucleotides (Fig.
2A). These complexes were different from those that formed
when a double-stranded probe from the same region was used (Fig.
2A). PC12 cell extracts contained more of a slower migrating
complex, SBPC2, on the sense strand than C6. In contrast HeLa cell
extracts lacked this complex but contained more of the faster migrating
complex, SBPC1. An approximately 20-nucleotide G-rich region of the
promoter sequence appeared to be critical for complex formation, since
a series of competitors containing at least a G3 sequence
effectively competed for the binding at both sites (Fig. 5). In UV
cross-linking studies, an ~31.5-kDa protein formed the faster
migrating complex and larger, >~64-kDa proteins formed the slower
complex. Although the components of these complexes are unknown, the
SBPC1 complex may be somewhat similar to the GS1 (complex binding
selectively to the G-rich strand of the Egr
response element) protein complex recently described by Taira and
Baraban (20). They reported that this complex, enriched in rat brain
extracts, contains proteins of 36 and 30 kDa and is effectively
competed by G-rich DNA and, less effectively, by RNA oligonucleotides.
The composition of the SBPC2 complex is not known.
Our data suggest that these single-strand complexes may be important in
modifying the activity of the NMDAR1 promoter. When single-stranded
oligonucleotides known to compete with complex formation (competitors 3 and 5, See Fig. 5A) were cotransfected with reporter
plasmid, the reporter activity was dose-dependently reduced
(Fig. 3). Another single-stranded oligonucleotide (competitor 1, see
Fig. 5A) derived from the NMDAR1 gene promoter 5
of the GC-rich region that did not compete for complex formation did not interfere with reporter activity. In separate experiments, SBPC2
complex was increased when PC12 cells were treated with NGF, a
treatment known to increase NMDAR1 promoter activity (17), whereas
SBPC1 was dramatically decreased. Thus two treatments, one inhibiting
reporter activity (competitive oligonucleotide) and one increasing
reporter activity (NGF), also influence the amount of single-stranded
complex formation. We have yet to determine the exact relationship
between the changes in complex 1 and 2 upon NGF treatment and changes
in promoter activity. In any case, this GC-rich region of the NMDAR1
promoter apparently plays an important role in regulating gene
expression.
Several genes lacking a TATA-box motif in the proximal promoter region
contain a GC-rich region similar to the NMDAR1 gene. These
types of gene promoters were thought to be responsible for constitutive
expression. It is now becoming apparent that these promoters also may
be under regulatory control and induced by various treatments (37-40).
It is possible that multiple transcription factors may be competing for
the same DNA elements and that activation of transcription may involve
displacement of one group of factors in favor of another that leads to
more efficient transcription (41, 42). Likely candidates on the NMDAR1
promoter might be Sp1 and Egr transcription factors, which efficiently
bind to GC-rich double-stranded DNA elements. In addition, the current
work suggests that other distinct factors interacting exclusively with
single-stranded oligonucleotides of the same region also may form
complexes and play a role in regulation. In fact, we showed by
competition experiments and supershift EMSAs that Sp1 protein does not
interact with the single-stranded probes. Thus the GC-rich region of
the NMDAR1 gene promoter may be a target for multiple
transcription factors whose interactions with both double-stranded and
single-stranded forms of the promoter region ultimately determine the
transcriptional activity.