(Received for publication, August 29, 1996)
From the Department of Biochemistry, Institute for Brain Research, Faculty of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113, Japan
Neuronal cell-specific expression of the rat m4 muscarinic acetylcholine receptor (mAChR) is regulated by a silencer element. A likely mediator of this silencing is the neuron-restrictive silencer element/repressor element 1 (NRSE/RE1), which is present 837 base pairs (bp) upstream from the transcription initiation site of the m4 mAChR gene (Wood, I. C., Roopra, A., Harrington, C., and Buckley, N. J. (1995) J. Biol. Chem. 270, 30933-30940; Mieda, M., Haga, T., and Saffen, D. W. (1996) J. Biol. Chem. 271, 5177-5182). In the present study, we examined whether this putative NRSE/RE1 functions as a silencer. Transient expression assays using m4 mAChR promoter/luciferase expression vectors showed that the m4 NRSE/RE1 is necessary and sufficient to repress m4 promoter activity in non-neuronal L6 cells. m4 promoter activity was only partially repressed, however, in neuronal NG108-15 cells exogenously expressing the neuronal-restrictive silencer factor/RE1-silencing transcription factor (NRSF/REST). By contrast, the promoter activity of the type II sodium channel (NaII) gene was nearly completely repressed in NRSF/REST-expressing NG108-15 cells. Experiments with expression vectors containing chimeric promoters revealed that the NRSE/RE1 elements derived from both the m4 and NaII genes are independently sufficient to silence NaII gene promoter activity, but only partially repress m4 mAChR gene promoter activity in NRSF/REST-expressing NG108-15 cells. Thus, the repression activity of NRSF/REST depends upon the species of promoter to which it is linked. Gel-shift assays showed that the NRSF/REST is the only protein that binds to a 92-bp segment from the m4 mAChR promoter containing NRSE/RE1. This and the fact that m4 promoter activity was completely repressed in L6 cells suggest that the proteins that bind to the m4 constitutive promoter may be different from those in NG108-15 cells. Deletion analysis of the m4 constitutive promoter revealed that a 90-bp segment immediately upstream from the transcription initiation site contains significant promoter activity. Gel-shift assays revealed that several proteins in nuclear extracts prepared from L6 and NG108-15 cells bind to this 90-bp segment and that some of these proteins are L6 or NG108-15 cell-specific. These data support the idea that the repression activity of NRSF/REST depends upon the species of promoter to which it is linked and upon the proteins that bind to those promoters.
Muscarinic acetylcholine receptors (mAChRs)1 are the members of the G-protein-coupled receptor superfamily. Five subtypes of mAChR (m1-m5) have been identified by molecular cloning (1). Each subtype of mAChR shows a unique distribution in peripheral tissues and brain (2, 3), but mechanisms that underlie differential expression of each subtype have not yet been determined.
Recently, we and others isolated the promoter region of the rat m4 mAChR gene and demonstrated that the neuronal cell-specific expression of this gene is regulated by a silencer element (4, 5). The segment of the promoter region required for this silencing contains a neuron-restrictive silencer element/repressor element 1 (NRSE/RE1). The existence of this element is consistent with the exclusive expression of the m4 gene in neurons, although its expression has also been detected in smooth muscle of rabbit lung (but not human or pig lung) (6-8).
The NRSE/RE1 was initially identified as a silencer element that
regulates neuron-specific expression of the rat SCG10 (9) and rat
sodium channel type II (NaII) genes (10). In addition to these two
genes, the NRSE/RE1 is known to function in neuron-specific expression
of the human synapsin I gene (11, 12), rat Na,K-ATPase 3 subunit
gene (13), and chick neuron-glia cell adhesion molecule gene (14).
Recently, a zinc finger protein termed neuron-restrictive silencer
factor/RE1-silencing transcription factor (NRSF/REST) was cloned and
shown to repress the activities of the constitutive promoters of the
rat SCG10 and NaII genes by binding to the NRSE/RE1 sequence in
non-neuronal cell lines (15, 16).
In the present study, we have shown that the NRSE/RE1 sequence regulates the neuronal cell-specific expression of this gene and that NRSF/REST significantly represses its expression. We also discuss the mechanism underlying NRSF/REST function in terms of the constitutive promoter region to which it is linked.
L6 (a rat skeletal muscle myoblast cell line) and NG108-15 cells (a hybrid cell line derived from mouse neuroblastoma N18 and rat glioma C6) were cultured as described previously (4).
ConstructionsA series of constructs, in which the NRSE/RE1
sequence was directly linked to the constitutive promoter region of the
rat m4 mAChR gene (the proximal 435-bp sequence of the 5-flanking region; Ref. 4), was obtained as follows. Synthetic
BamHI-BglII fragments containing the wild type or
mutant form of the NRSE/RE1 were cloned in the BamHI site of
the plasmid pUC119 and then re-isolated by digestion with
PstI and KpnI. The luciferase reporter construct pGL2-ScP1600, which contains about 1.6 kb of the 5
-flanking region of
the m4 mAChR gene (previously described as construct C; Ref. 4), was
digested with PstI and KpnI. The largest fragment
that contains the constitutive promoter region of the rat m4 mAChR gene
and the luciferase gene was isolated. This fragment was ligated to the
PstI-KpnI fragment containing the NRSE/RE1
sequence. The sequences of synthetic oligonucleotides are given below.
(Sequences of sense strands are shown. Lowercase residues represent
linker sequences introduced to facilitate cloning.) NaII:
5
-gatctATTGGGTTTCAGAACCACGGACAGCACCAGAG-3
(residues
1023 to
992
from the rat NaII gene promoter region (17)); m4:
5
-gatcCATGTGGAGCTGTCCGAGGTGCTGAATCTGCCTA-3
(residues
865 to
832
from the rat m4 mAChR gene promoter region); m4m: 5
-gatcCATGTGGAGCTGTAAGAGGTGCTGAATCTGCCTA-3
(a mutant form
of m4, where two cytosine residues in m4 oligonucleotide were
substituted with adenine residues). Construct pGL2-P1074dSau92 (Fig.
1A) is a plasmid in which a sequence (residues
895 to
803) was deleted from pGL2-P1074 (Fig. 1A, described as
construct B in Ref. 4). This plasmid was generated as follows.
pGL2-1074 was digested with PstI and partially with
SmaI, then SmaI-PstI fragment, which contains residues
1074 to
435 and a portion of the pGL2-Basic vector (Promega), was isolated. This fragment was digested with Sau3AI and cloned in the SmaI and PstI
sites of pT7 vector (Novagen) to obtain a construct in which the
Sau3AI fragment (residues
895 to
803) was deleted from
the SmaI-PstI fragment. This construct was
digested with PstI and KpnI, and the shorter
fragment was isolated and cloned in the largest of the fragments
derived by digestion of pGL2-ScP1600 with PstI and
KpnI. In constructs pCAT-P435 and pCAT-P1074, promoter
regions contained in pGL2-P435 (described as construct A in Ref. 4) and
pGL2-P1074, respectively, were cloned upstream of the chloramphenicol
acetyltransferase (CAT) gene. These constructs were generated as
follows. pGL2-P1600 was digested with XhoI and partially
with PstI, and the resultant fragments containing
appropriate m4 promoter regions were cloned in the PstI and
XhoI sites of the pBLCAT2 (purchased from ATCC). These
constructs were digested with XhoI followed by blunt-ending, and then partially digested with HindIII. Resultant
fragments containing m4 promoter regions were ligated to the
CAT-containing fragment obtained by digestion of pSDK7 (10) (a gift
from Dr. G. Mandel) with PstI and HindIII, in
which PstI site was blunt-ended. Construct pCAT-Sau92P435,
in which the Sau3AI fragment (residues
895 to
803,
containing the NRSE/RE1 sequence) was directly linked to the
constitutive promoter region of the m4 mAChR gene, was obtained as
follows. The Sau3AI fragment was cloned in the
BamHI site of pBluescript SK(+) (pBS-Sau92). pBS-Sau92 was
digested with XbaI, blunt-ended, then digested with
PstI to re-isolate the Sau3AI fragment. This
fragment was ligated to pCAT-P1074 digested with PstI and
SphI, in which SphI site was blunt-ended.
Construct pCAT-Nam4, in which the NRSE/RE1 containing
HindIII-BglII fragment derived from the NaII gene
(10) was fused to the constitutive promoter region of the m4 mAChR
gene, was generated as follows. The PstI fragment derived
from m4 mAChR gene (residues
435 to +19) was cloned in the
PstI site of pBluescript and re-isolated by digestion with
BamHI and partially with PstI. This fragment was
ligated to the pSDK7 digested with BglII and
PstI. pCAT-Sau92Na, in which the Sau3AI fragment
derived from m4 mAChR gene (residues
895 to
803, containing the
NRSE/RE1 sequence) was directly linked to the constitutive promoter
region of the NaII gene, was constructed as follows. The
Sau3AI fragment was cloned in the BglII site of the pGL2-Basic vector and then re-isolated by digestion with
HindIII and partially with BglII. This fragment
was ligated to pSDK7 digested with HindIII and
BglII. pEF-REST, in which expression of NRSF/REST is
regulated by the human elongation factor 1
promoter, was obtained as
follows. REST-Express (16) (a gift from Dr. G. Mandel) was digested
with HindIII and XhoI, and the fragment encoding
NRSF/REST was isolated and cloned between the HindIII and
SalI sites of pUC119. The fragment encoding NRSF/REST was
isolated again by digestion with KpnI and HindIII
and cloned in pBluescript SK(+). Finally the fragment encoding
NRSF/REST was isolated by digestion with XbaI and was cloned
in the XbaI site of mammalian expression vector pEF-BOS
(18). Constructs pGL2-NP376, pGL2-NP206, pGL2-NP143, and pGL2-NP1 (Fig.
4) were generated by cloning appropriate
NlaIV-XhoI fragments derived from pGL2-P1074 in
the SmaI and XhoI sites of the pGL2-Basic vector.
pGL2-NP296 (Fig. 4) was obtained by deleting the 1.3-kb
KpnI-NarI fragment from pGL2-ScP1600. pGL2-SmP90
(Fig. 4) was obtained by deleting the 1.5-kb SmaI fragment
from pGL2-ScP1600.
Gel-shift Assays
Nuclear extracts from L6 and NG108-15
cells were prepared essentially as described previously (19) except for
the inclusion of additional proteinase inhibitors: 20 µM
leupeptin, 0.5 mM phenylmethylsulfonyl fluoride, 0.5 mM benzamidine, 1 µg/ml pepstatin, and 18 µg/ml aprotinin. The probe that contains the NRSE/RE1 sequence was prepared by digesting pBS-Sau92 (described under "Constructions")
with HindIII and XbaI and isolating a smaller
fragment. The probe for analyzing the constitutive promoter region was
prepared by isolating a 109-bp SmaI-XhoI fragment
from pGL2-SmP90. DNA fragments were end-labeled with
[-32P]dCTP (Amersham) using the large (Klenow)
fragment of DNA polymerase (New England Biolabs). DNA-protein binding
reactions with silencer probe were carried out in a 20-µl final
volume of reaction buffer containing 12 mM Hepes (pH 7.9),
160 mM KCl, 12.5 mM MgCl2, 0.12 mM EDTA, 12% glycerol, 125 µg/ml poly(dI-dC), 0.3 mM dithiothreitol, 0.3 mM phenylmethylsulfonyl
fluoride, 0.6 µg/ml pepstatin, 0.02 mM leupeptin. For
binding experiments using the promoter probe, the concentration of KCl
and MgCl2 were 100 mM and 0 mM,
respectively, and 125 µg/ml salmon sperm DNA was substituted for
poly(dI-dC). The nuclear extract (approximately 4 µg of protein for
silencer probe, and 10 µg of protein for promoter probe) was added to
the reaction buffer and preincubated for 10 min on ice. After addition of labeled DNA probe (1 × 104 cpm/reaction), the
nuclear extracts was incubated for another 30-60 min at room
temperature. Synthetic oligonucleotides described under
"Constructions" were used as competitor DNA. Each
competitor DNA was added prior to the addition of the probe. The
sequence of oligonucleotide containing Sp1 consensus binding site is
5
-cgATTCGATCGGGGCGGGGCGAGC-3
(lowercase residues represent linker
sequence for labeling). Electrophoresis was performed in 4%
polyacrylamide gels in 0.5 × TBE for 2-2.5 h at 150 V.
Transient transfections and luciferase assays were performed as described previously (4). For co-transfection assays, NG108-15 cells were seeded in Corning 12-well microtiter plates (1.4 × 105 cells/well) and were cultured for 1 day before transfection. Transfections were carried out with 0.25 µg of pEF-REST, 0.25 µg of reporter plasmids, and 1.4 µl of LipofectAMINETM reagent (Life Technologies, Inc.) in each well. Equal amounts of pEF-BOS was substituted for pEF-REST as a control. Cells were harvested 50 h after transfection, and CAT assays were performed essentially as described previously (20).
Previously we found that neuronal cell-specific
expression of the rat m4 mAChR gene is regulated by a silencer element
and that the promoter region contains a putative NRSE/RE1 in an
inverted orientation. In this study, we used transient transfection
assays to determine whether the NRSE/RE1 of the m4 mAChR gene is a
functional silencer. For this purpose, we constructed luciferase
reporter plasmids in which the NRSE/RE1 is directly linked to the
constitutive promoter region of the m4 mAChR gene (the proximal 435-bp
sequence of the 5-flanking region; Ref. 4) (Fig.
1A). Fusion of the NRSE/RE1 derived from the
m4 mAChR gene repressed luciferase induction by the m4 mAChR
constitutive promoter approximately 10-fold in L6 myoblast cells, which
do not express the endogenous m4 mAChR gene. This repression was
independent of the orientation of the NRSE/RE1 (m4-f and m4-r,
respectively, contain the NRSE/RE1 in the same and reversed orientation
compared to that in the genome). Similar repression was shown with the
NRSE/RE1 derived from the NaII gene (Na-f and Na-r). By contrast,
luciferase activities were not changed by the presence of NRSE/RE1
sequences in NG108-15 cells, which express the endogenous m4 mAChR
gene. We also examined the effect of the mutant form of the NRSE/RE1
(constructs m4m-f and m4m-r). In the mutant form, two cytosine residues
of the m4 NRSE/RE1 were substituted with adenine residues (Fig.
1B). Homologous mutations in the NRSE/RE1 of the rat SCG10
and human synapsin I genes are known to abolish the binding of a
repressor protein and its silencer activity (9, 11). As expected, this
mutant did not have silencer activity. Deletion of a Sau3AI
fragment (residues
895 to
803) from pGL2-P1074, pGL2-P1074dSau92,
resulted in recovery of luciferase activities in L6 cells (Fig.
1A). The extent of derepression was more than 10-fold
compared to pGL2-P1074, and activities were comparable to ones observed
in NG108-15 cells. These data indicate that the NRSE/RE1 sequence of
the m4 mAChR gene functions as a silencer in a way similar to NRSE/RE1
sequences found in other neuron-specific genes and that the NRSE/RE1 is necessary and sufficient to repress the expression of the m4 mAChR gene
in non-neuronal cells.
To determine if there
is any nuclear protein that binds to this silencer element, we carried
out gel-shift assays using nuclear extracts from L6 cells and NG108-15
cells. We detected a nuclear protein that bound to a 92-bp
Sau3AI fragment containing the NRSE/RE1 (residues 895 to
803) only in L6 nuclear extracts (Fig. 2A). This binding was inhibited by addition of an excess of the NRSE/RE1 sequences derived from the m4 mAChR gene, but was not inhibited by the
mutant form of the m4 mAChR NRSE/RE1 (the same mutant as described
above) or by a Sp1 binding sequence (as a negative control), showing
that binding is specific to the NRSE/RE1. Addition of an excess of the
NRSE/RE1 derived from the NaII gene also inhibited this binding,
suggesting that the same protein, probably NRSF/REST, binds to the
NRSE/RE1 derived from both m4 mAChR and NaII genes. The extent of the
mobility shift of this band was about the same as that obtained with
the HindIII-BglII fragment derived from the rat
NaII gene promoter (residues
1051 to
937; Ref. 10) containing the
NRSE/RE1 sequence (data not shown). When the Sp1 consensus binding
sequence was used as a probe, we found binding proteins in both L6 and
NG108-15 extracts, suggesting that the cell type-specific binding
activity to the NRSE/RE1 is not due to a failure in preparing the
nuclear extracts (Fig. 2B). These data and results of
transient expression assays described above indicate that a repressor
protein binds to the NRSE/RE1 and represses the expression of the m4
mAChR gene in non-neuronal cells.
Exogenous Expression of NRSF/REST Partially Represses Promoter Activity of the m4 mAChR Gene
A strong candidate for this
repressor protein is NRSF/REST, which was recently cloned and shown to
repress the activities of the constitutive promoters of the rat SCG10
and NaII genes in non-neuronal cell lines by binding to the NRSE/RE1
sequence (15, 16). NRSF/REST is also known to be expressed in L6 cells (16). To examine whether NRSF/REST can repress m4 mAChR promoter activity, we co-transfected a NRSF/REST expression plasmid with CAT
reporter plasmids into NG108-15 cells (Fig. 3).
Exogenous expression of NRSF/REST repressed promoter activity of
NRSE/RE1-containing reporter plasmid pCAT-Sau92P435 (residues 895 to
803 and
435 to +19) approximately 2-fold (Fig. 3A).
Activity of pCAT-P435 (residues
435 to +19), which does not contain
the NRSE/RE1, was not repressed by exogenous expression of NRSF/REST,
suggesting that NRSF/REST functions via the NRSE/RE1. We carried out
the same co-transfection assay with reporter constructs containing the
promoter of the NaII gene as a positive control. The promoter activity
of the rat NaII gene was repressed approximately 10-fold by exogenously
expressing NRSF/REST protein in NRSE/RE1-dependent manner (Fig.
3A, pSDK7 and pSDK7/dHB). Since promoter activity of the m4
mAChR gene is approximately 15-fold higher than that of the NaII gene
(pCAT-P435 versus pSDK7/dHB), we cannot directly compare the
effect of exogenous NRSF/REST expression on the m4 mAChR promoter to
that on the NaII promoter. But when pCAT-Sau92P435 or pSDK7 were
introduced into L6 cells, their activities were repressed almost
completely (Fig. 3B), although the difference of activities
between pCAT-P435 and pSDK7/dHB was more than that in NG108-15 cells
(approximately 20-fold). These data suggest that exogenous expression
of NRSF/REST represses promoter activity of the m4 mAChR gene to a much
lesser extent than that of NaII gene and is not sufficient for the
repression of the m4 mAChR gene expression. There are two possible
explanations for this difference between NaII and m4 mAChR genes. One
is that NRSF/REST binds to the NRSE/RE1 of the m4 mAChR gene with lower
affinity than that of NaII gene. The other is that NRSF/REST can bind
to the NRSE/RE1 effectively but other factor(s) are also required for
full repression of the m4 mAChR gene. To discriminate between these two
possibilities, we constructed chimera reporter plasmids of m4 mAChR and
NaII gene promoter regions and co-transfected with the NRSF/REST
expression plasmid (Fig. 3C). Exogenous expression of
NRSF/REST repressed promoter activity of pCAT-Sau92Na by approximately 90% but that of pCAT-NaP435 only by 50%, showing that the NRSE/RE1 derived from the m4 mAChR gene represses the constitutive promoter of
the NaII gene to an extent similar to that of the NRSE/RE1 derived from
the NaII gene. These results suggest that the character of the
constitutive promoter is important for NRSF/REST function, supporting
the hypothesis that some factor(s) other than NRSF/REST are required
for silencing.
The 90-bp Segment Proximal to the Transcription Start Site of the m4 mAChR Gene Produces Significant Promoter Activity
If the above
explanation is correct, how is it that the m4 mAChR gene is completely
repressed in L6 cells? We have shown that the NRSE/RE1-containing 92-bp
Sau3AI fragment is sufficient to repress the promoter
activity of the m4 mAChR gene in L6 cells (Figs. 1 and 3B)
and that NRSF/REST is apparently the only protein that binds to that
fragment (Fig. 2). NRSF/REST is also the only protein that binds to the
92-bp Sau3AI fragment in NG108-15 cells that express
NRSF/REST exogenously. These results suggested that the difference in
the ability of NRSF/REST to silence in L6 and NG108-15 cells may depend
upon differences in the proteins that bind to the constitutive m4
promoter in these cell lines. We therefore decided to analyze the
constitutive promoter region of the m4 mAChR gene to test this
possibility. For this purpose we constructed luciferase reporter
plasmids containing various lengths of the 435-bp constitutive promoter
region and carried out transient transfection assays (Fig.
4). Serial deletions of pGL2-P435 from the 5 side gave
similar profiles of luciferase activities in L6 and NG108-15 cells. In
both cell lines, a reporter plasmid containing a 90-bp fragment just
upstream to the transcription initiation site (pGL2-SmP90) produced
significant luciferase activities (more than 20-fold increase compared
to pGL2-NP1). These data suggest that important regulatory elements
required for the m4 mAChR gene expression are contained within this
proximal 90-bp fragment, although there may be other positive and
negative regulatory elements that also modify gene expression.
We next used gel-shift assays to examine whether any
nuclear proteins bind to this 90-bp fragment. We detected several
proteins that bind to the 109-bp SmaI-PstI
fragment (residues 90 to +19) in both L6 and NG108-15 nuclear
extracts (Fig. 5). Some of these were NG108-15-specific
(bands a and b) or L6-specific (bands
d-f), suggesting that different proteins bind to this
SmaI-PstI fragment in NG108-15 and L6 cells. No
band disappeared upon addition of an excess of Sp1 binding sequence,
indicating that no bands are shifted-up bands due to Sp1 binding, even
though this SmaI-PstI fragment contains Sp1
consensus binding sequences (residues
87 to
82 and
83 to
78).
Bands c and f were weakly competed by an excess
of unlabeled SmaI-PstI fragment but not by a Sp1
binding sequence, suggesting that these bands resulted from specific
binding to the SmaI-PstI fragment.
In this study, we have shown that the NRSE/RE1 regulates neuronal
cell-specific expression of the rat m4 mAChR gene (Fig. 1). Nuclear
extracts from the non-neuronal cell line L6 contain a protein that
binds to the NRSE/RE1 derived from the m4 mAChR gene and is not present
in nuclear extracts of m4 mAChR-expressing NG108-15 cells (Fig. 2). The
m4 mAChR gene is the first example of a NRSE/RE1-regulated gene among
genes whose products are involved in neurotransmission, such as
neurotransmitter-synthesizing enzymes, neuropeptide, and receptors. The
choline acetyltransferase gene is a candidate for another such gene,
since silencers have been implicated in its cholinergic neuron-specific
expression, and a sequence highly homologous to the NRSE/RE1 is present
in the 5-flanking region of the rat gene (21-24). Although the
NRSE/RE1 regulates the neuron-specific expression of m4 mAChR gene, an additional mechanism is necessary to explain the restricted expression of this gene to specific subsets of neurons. Such a mechanism remains
to be elucidated.
We examined whether NRSF/REST mediates repression of the m4 mAChR gene via the NRSE/RE1 using co-transfection assays in NG108-15 cells with CAT reporter plasmids. Initially we tried these co-transfection assays with reporter plasmids containing a luciferase reporter gene such as pGL2-P435 and pGL2-P1074, but exogenous expression of NRSF/REST unexpectedly resulted in NRSE/RE1-independent repression of luciferase activities. In addition, exogenous expression of NRSF/REST repressed luciferase activities even when the reporter plasmids containing NaII constitutive promoter or SV40 early promoter were used (data not shown). Exogenous expression of NRSF/REST may result in its interaction with luciferase gene or luciferase and somehow down-regulate luciferase activity. For this reason, we used the CAT gene as a reporter gene in co-transfection assays. In previous studies, co-transfection assays with NRSF/REST expressing plasmids were also carried out using CAT gene as a reporter (15, 16). Promoter independent down-regulation of the luciferase gene by T3 and T3 receptors has also been reported previously (25). The mechanism of this down-regulation is unknown.
Exogenous expression of NRSF/REST in NG108-15 cells represses promoter
activity of the m4 mAChR gene by approximately one-half in a
NRSE/RE1-dependent manner (Fig. 3). Since the extent of
repression was much less than that observed for the NaII gene, we
concluded that repression of the m4 mAChR gene expression by exogenous
expression of NRSF/REST is partial in NG108-15 cells. Co-transfection
assays with m4 mAChR-NaII promoter chimera reporter plasmids showed
that NRSF/REST can bind to the NRSE/RE1 derived from the m4 mAChR gene and represses the NaII gene promoter almost completely, but does not
repress the m4 mAChR gene promoter completely. This result is
consistent with data obtained by gel-shift assays suggesting that the
same protein binds to the NRSE/RE1 derived from the m4 mAChR and NaII
genes (Fig. 2). We therefore propose that the difference in repression
by NRSF/REST between the m4 mAChR and NaII genes is a consequence of
differences in their constitutive promoter regions. Three possible
reasons may be given to explain the fact that the 92-bp
Sau3AI fragment (residues 895 to
803) allows complete
silencing in L6 cells, but not in NG108-15 cells (Fig. 3). First,
another repressor protein, which is expressed in L6 cells but is not
expressed in NG108-15 cells, binds to the 92-bp Sau3AI
fragment at a site other than the NRSE/RE1. Second, a cofactor expressed only in L6 cells is required for NRSF/REST to repress the
promoter of the m4 mAChR gene sufficiently. The first two cases do not
seem likely, since data obtained by gel-shift assays suggest that only
one species of protein binds to this 92-bp Sau3AI fragment
(Fig. 2), although there might be other shifted-up bands undetectable
in these conditions. The third possibility is that activator proteins
that bind to the constitutive promoter region are different between L6
and NG108-15 cells and these are regulated differently by NRSF/REST.
Analysis of the constitutive promoter region of the m4 mAChR gene
showed that the proximal 90-bp region produces significant promoter
activity (Fig. 4). Gel-shift assays showed that several nuclear
proteins bind to this region (Fig. 5). Some of them were specific to
nuclear extracts from L6 or NG108-15 cells. The existence of
cell-specific nuclear proteins that bind to the promoter region is
consistent with the third case described above. Gel-shift assays using
the constitutive promoter region of the NaII gene as a probe showed
that patterns of shifted-up bands were similar among nuclear extracts
from both NaII-expressing and -nonexpressing cell lines (10). This fact may explain the difference in repression by NRSF/REST between the m4
mAChR and NaII genes. Further study will be required to prove this
hypothesis.
The proximal 90-bp region contains an inverted CAAT box (residues 62
to
58), a putative CRE-like sequence (residues
46 to
39, 7 bases
match 8 bases consensus sequence TGACGTCA), and consensus binding site
for Sp1 (residues
87 to
78), although Sp1 does not seem to bind to
this site (Fig. 5). Shifted-up bands may contain factors that bind
these sequences and/or contribute to the transcription initiation
complex. Further analyses will be required for the characterization of
these nuclear proteins.
In summary, we have shown that the NRSE/RE1 regulates neuronal cell-specific expression of the m4 mAChR gene. Exogenous expression of NRSF/REST repressed its expression significantly but not completely. We propose that the ability of NRSF/REST to repress transcription depends on the species of promoter to which it is linked and activator proteins that bind there. We therefore analyzed the constitutive promoter region of the m4 mAChR gene and found that proximal 90-bp region produced significant promoter activity. Several nuclear proteins bind to this region, some of which are cell type-specific. These data support the idea that the repression ability of NRSF/REST depends on the species of promoter and promoter-binding proteins.
We thank Dr. G. Mandel (State University of New York at Stony Brook) for the REST-express and pSDK7 plasmids, Dr. S. Nagata (Osaka Bioscience Institute, Osaka, Japan) for mammalian expression vector pEF-BOS, and the Japanese Cancer Research Resources Bank for L6 cells.
Previously, Wood et al. (5) detected a
transcription initiation site at a location different from one we
determined (at 293 in our numbering). During preparation of this
manuscript, they showed that the NRSE/RE1 represses transcription of
the m4 mAChR gene from their transcription initiation site in
non-neuronal cells (26). They claimed that inclusion of our
transcription initiation site had little effect on promoter activity.
However, our data are inconsistent with theirs. Deletion of their
transcription initiation site from the 5
side reduced, but left
significant promoter activity (Fig. 4). Furthermore, deletion of our
transcription initiation site from the 3
side reduced promoter
activity to approximately one-third (data not shown). These data
indicate that transcription from our transcription initiation site
produces a substantial part of the total transcription of the m4 mAChR gene. Taken together, these data suggest that there are at least two
basal promoters in the m4 mAChR gene. In our study, luciferase expression produced from reporter plasmids that contains both transcription initiation sites were shown to be repressed by the NRSE/RE1 (Fig. 1).