From the Departments of Neurochemistry and
** Pharmacology and the § Instituto de Neurociencias,
Universidad Miguel Hernández,
03550 San Juan, Alicante, Spain
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ABSTRACT |
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The Nicotinic acetylcholine receptors
(nAChRs)1 are members of the
gene superfamily of neurotransmitter-gated ion channels (1, 2). These
multimeric receptors are heteromers or, in some cases, homomers of
subunits ( To understand how the regional and developmental expression of nAChR
subunits is controlled, we have started to analyze the transcriptional
mechanisms that regulate expression of the nAChR subunits expressed in
chromaffin cells of the bovine adrenal gland. This cell type represents
a relevant and accessible model in which to study a particular neuronal
nAChR subtype with a well defined function. Previously, we cloned the
bovine Isolation and Analysis of the 5'-Flanking Sequence of the RNase Protection--
Poly(A)+ RNA was directly
selected from a lysate of bovine adrenal medulla by oligo(dT)-Dynabeads
(Dynal, Oslo, Norway) and used in the RNase protection experiments.
Probes were generated with SP6 and T7 polymerases (Boehringer Mannheim,
Barcelona, Spain), [ Plasmid Constructions--
All
The basic strategy for site-directed mutagenesis of the different
elements in region Cell Culture and Reporter Assays--
Chromaffin cells were
isolated from bovine adrenal glands as described (24) and cultured in
90% Dulbecco's modified Eagle's medium (Sigma, Madrid), 10% fetal
calf serum, 10 µM cytosine arabinoside, and 10 µM 5-fluoro-2'-deoxyuridine (Sigma) to prevent fibroblast proliferation. SHSY-5Y human neuroblastoma cells were grown in 90%
Eagle's minimal essential medium with Glutamax-1 (Gibco-BRL, Barcelona) and 10% fetal calf serum. COS cells were grown in 90% Dulbecco's modified Eagle's medium and 10% fetal calf serum.
Plasmids were banded in two gradients of CsCl. Both cell types were
transfected by the calcium phosphate procedure (25). Chromaffin cells
on 48-well plates (5 × 105 cells/well) were incubated
with 0.75 µg of pGL2 vector or an equivalent amount (in molar terms)
of the different constructs derived from this vector and with 0.75 µg
of Electrophoretic Mobility Shift Assay--
Crude nuclear extracts
were prepared from chromaffin and SHSY-5Y cells as described by
Schreiber et al. (26). The DNA fragment corresponding to
region Western Blotting--
Ten µg of nuclear proteins/lane was
separated by 10% SDS-polyacrylamide gel electrophoresis. Western
blotting was carried out as described by Towbin et al. (27).
After the transfer, nitrocellulose membranes were blocked overnight at
4 °C with 5% dry milk in phosphate-buffered saline and incubated in
the same way with the anti-Sp1 antibody (1:500) in phosphate-buffered
saline and 5% dry milk. After incubation with the secondary antibody at room temperature for 2 h, the bands were visualized by a
chromogenic reaction (Sigma Fast, nitro blue tetrazolium, Sigma).
DNase I Footprinting--
The sense strand corresponding to
region Structure of the 5'-Flanking Region of the
The 5'-end of Functional Analysis of the
We next compared the
The
Initially, four GC boxes present between -111 and Characterization of the Regulatory Elements Present at -111 to
Western blot analysis of SHSY-5Y and chromaffin nuclear proteins
indicated that Sp1 protein was indeed expressed in these cells (Fig.
7C). Two proteins bands (~95-100 kDa) were detected with
anti-Sp1 antibodies in chromaffin (lane 15) and SHSY-5Y
(lane 16) nuclear extracts, showing the same size as that
previously described for Sp1 polypeptides (29). The amount of Sp1
protein detected in both extracts was approximately the same, and both bands had the same intensity. By contrast, recombinant Sp1 (lane 17) showed predominantly one species.
Interactions of Sp1 with the GC Boxes in the
To confirm that an additional Sp1 site exists in the proximal region of
the
The functional significance of the fifth GC box was examined in
chromaffin cells by transfecting constructs, made in the context of
p111 Neuronal nAChRs mediate chemical synaptic transmission, probably
regulating transmitter release at many synapses (reviewed in Ref. 5). A
relatively large number of genes that encode nAChR subunits have been
identified (1), having distinct, although overlapping patterns of
expression in the central and peripheral nervous systems. This
diversity constitutes, in large part, the molecular basis on which the
variety of nAChR properties and neural responses to acetylcholine is
established. The nAChR The core promoter region of the The most remarkable feature in the deleted region, between -111 and
-39, was the presence of at least four closely located Sp1 sites
(labeled 1-4 in Fig. 6A). Therefore, these
elements appeared to be suitable candidates for controlling promoter
activity. Consequently, when these elements were simultaneously mutated in the context of p111 In addition to silencing mechanisms, the Finally, we would like to emphasize the role that Sp1 could play in a
potential common regulatory mechanism of the 5 subunit is a component of the neuronal
nicotinic acetylcholine receptors, which are probably involved in the
activation step of the catecholamine secretion process in bovine
adrenomedullary chromaffin cells. The promoter of the gene coding for
this subunit was isolated, and its proximal region was characterized,
revealing several GC boxes located close to the site of transcription
initiation (from
111 to
40). Deletion analysis and transient
transfections showed that a 266-base pair region (
111 to +155) gave
rise to ~77 and 100% of the maximal transcriptional activity
observed in chromaffin and SHSY-5Y neuroblastoma cells, respectively.
Site-directed mutagenesis of five different GC motifs indicated that
all of them contribute to the activity of the
5 gene, but in a
different way, depending on the type of transfected cell. Thus, in
SHSY-5Y cells, alteration of the most promoter-proximal of the GC boxes decreased
5 promoter activity by ~50%, whereas single mutations of the other GC boxes had no effect. In chromaffin cells, by contrast, modification of any of the GC boxes produced a similar decrease in
promoter activity (50-69%). In both cell types, however, activity was
almost abolished when four GC boxes were suppressed simultaneously. Electrophoretic mobility shift assays using nuclear extracts from either chromaffin or SHSY-5Y cells showed the specific binding of Sp1
protein to fragment
111 to
27. Binding of Sp1 to the GC boxes was
also demonstrated by DNase I footprint analysis. This study suggests
that the general transcription factor Sp1 plays a dominant role in
5
subunit expression, as has also been demonstrated previously for
3
and
4 subunits. Since these three subunits have their genes tightly
clustered and are expressed in chromaffin cells, probably as components
of the same receptor subtype, we propose that Sp1 constitutes the key
factor of a regulatory mechanism common to the three subunits.
INTRODUCTION
Top
Abstract
Introduction
References
2-
9 and
2-
4) that exhibit well defined and
restricted expression patterns in vivo (1). The diversity of
neuronal nAChRs arises, at least in part, from the different combinations of subunits able to form functional nAChRs (3). Thus, it
is clear that their differential expression affects the electrophysiological and pharmacological properties of the resultant receptors (4). Moreover, potential changes in subunit expression in
response to modulation of synaptic function might have important consequences on the signals transduced by nAChRs (5).
3 (6),
5 and
4 (7), and
7 (8) subunits of neuronal
nAChRs, which are expressed in chromaffin cells as components of the
two nAChR subtypes typically present at the peripheral nervous system
(9). We have also shown that nAChRs formed by
7 subunits are
differentially expressed in adrenergic cells (10), probably as the
result of transcriptional regulation, whereas
3,
5, and
4
subunits have a less restricted distribution in adrenergic and
noradrenergic cells (7). Interestingly, the
3,
5, and
4
subunit genes have been found tightly clustered in the avian (11) and
mammalian (12) genomes, with the
3 and
5 genes contiguous and
having opposed transcription polarity. A number of studies have
concentrated on the transcriptional regulation of the
3 and
4
subunits. Deneris and co-workers (13, 14) have shown that the POU
domain factor SCIP/Tst-1 is able to activate the
3 subunit promoter,
probably as a consequence of protein-protein interactions at the level
of the basal transcriptional machinery. Furthermore, an enhancer
located in the 3'-untranslated exon of the
4 subunit (15, 16), at
the
4/
3 intergenic region, activates transcription from the
3
and
4 subunit promoters in a cell type-specific manner, possibly via
a novel ETS domain factor, Pet-1, whose expression is almost restricted
to the adrenal medulla (17). Considerable effort has also been
dedicated to the transcriptional regulation of the
4 subunit. Thus,
Gardner and co-workers (18) have shown that Pur
interacts with a
19-bp element in the
4 promoter. In addition, Sp1 (19) and Sp3 (20)
transactivate the
4 promoter in a synergistic way, an effect
possibly mediated by heterogeneous nuclear ribonucleoprotein K, which
affects the transactivation of
4 promoter activity by Sp1 and Sp3
differentially (21). As no information has been available until now for
the promoter of the
5 subunit, we have chosen to focus on it, with
the aim of finding a possible link in the regulation of the three
subunits. This study reports that at least five positive regulatory
elements exist in the
5 promoter proximal region. These elements,
all of them GC boxes, were shown to interact with Sp1. Since the
3 promoter is also the target of Sp1 (22), we suggest the possible involvement of this transcription factor in a regulatory mechanism common to the
3,
4, and
5 subunits.
EXPERIMENTAL PROCEDURES
5
Subunit--
A cDNA probe containing 152 bp of the 5'-end of exon
2 was used to screen a bovine genomic library in EMBL-3 SP6/T7
(, Heidelberg, Germany) as described
previously (8). Several overlapping bacteriophage clones were purified
and characterized.
-32P]CTP (Amersham Pharmacia
Biotech, Madrid, Spain), and the corresponding linearized templates (in
the pSPT18 vector, Boehringer Mannheim). A 431-bp
BglII-PvuII fragment of the
5 gene that
included 163 bp 5' to exon 1 and 249 bp downstream in the same exon was
subcloned into the BamHI and HincII sites of
pSPT18. After linearization of the plasmid with EcoRI, a
probe of 479 nucleotides was synthesized with SP6 polymerase. As
control, a cRNA sense fragment of 368 nucleotides was synthesized by
linearizing the same template with HincII and using T7
polymerase. This cRNA protected a fragment of 358 nucleotides upon
RNase treatment. Parallel experiments were carried out with a smaller
probe that overlapped the first one. For this purpose, a 341-bp
fragment of the
5 gene that included 319 bp of the 5'-end of the
first probe (downstream of the HincII site mentioned above)
was also subcloned into pSPT18. After linearization of the plasmid with
EcoRI, a probe of 403 nucleotides was synthesized with SP6
polymerase. The same control sense cRNA used above then produced a
protected fragment of 328 nucleotides when used instead of adrenal
medulla RNA (see Fig. 2 for further explanations). RNase protection
experiments were performed using an RNase protection kit (Boehringer
Mannheim) as indicated by the manufacturer. Protected fragments were
separated on a 7 M urea and 6% acrylamide gel along with
several other labeled RNAs of known size, which were also synthesized
and used for calibration.
5 promoter-luciferase gene
fusions were made in the pGL2-Basic vector (Promega, Madison, WI),
introducing in its polylinker, upstream of the luciferase gene, the
suitable
5 promoter fragments. These fragments were generated with
restriction enzymes and directly cloned into pGL2-Basic or subcloned
first in pBluescript and then transferred to pGL2-Basic. The vector
pGL2-Control, which expresses the luciferase gene under the regulation
of the SV40 promoter and enhancer sequences, was used to check
luciferase activity. Deletion analysis of the most promoter-proximal
region was performed by generating polymerase chain reaction fragments
with suitable sense oligonucleotides and an antisense primer
(5'-CTTTATGTTTTTGGCGTCTTCC-3') that anneals to the pGL2-Basic vector
downstream of the site of transcription initiation.
111 to
40 of the
5 promoter (see Fig. 6)
consisted of the following steps. (a) We performed
polymerase chain reaction (25 cycles at 94 °C for 10 s,
62 °C for 30 s, and 68 °C for 45 s) amplification of
p111
5LUC (or its single or double mutants when double or quadruple
mutants were desired, respectively) with appropriate mutagenic primers
in the sense orientation, which generated restriction sites useful for
further mutant constructions and to confirm mutagenesis. We used the
same oligonucleotide mentioned above as antisense primer. The
introduced mutations are indicated in lowercase letters in Fig.
6A (sites 1-4) and Fig. 10A (site 5). The mutant
sequences did not create any known binding site for transcription
factors as predicted by the MatInspector data base search (23).
(b) Polymerase chain reaction products were cloned into
pBluescript, sequenced, and transferred to the appropriate construct
into the pGL2-Basic vector.
-galactosidase expression vector pCH110 (Amersham Pharmacia
Biotech, Barcelona) as a control of transfection efficiency. SHSY-5Y
cells (105 cells/well) or COS cells (5 × 104 cells/well) on 24-well plates were incubated with 1.5 µg of the different
7 constructs and 1.5 µg of pCH110 per well.
Cells were harvested after 48 h and lysed with reporter lysis
buffer (Promega).
-Galactosidase and luciferase were then determined
in the lysates with the corresponding assay systems (Promega).
Luciferase activity was normalized to values obtained with the
p163
5LUC (see Fig. 3) or p111
5LUC (see Fig. 6) plasmid in the
same cell type. When comparing
5,
7, and
4 promoters (see Fig.
4), representative constructs for each of the subunits, giving the
maximal promoter activity, were chosen. They are indicated in the
corresponding figure legends.
111 to
27 was obtained by digesting pBluescript subclones
with EcoRI-KpnI and end-labeled by Klenow filling
with [
-32P]dATP. The DNA-protein binding reaction
volumes were 20 µl containing 10 mM Tris, pH 7.5, 50 mM NaCl, 1 mM EDTA, 1 mM
dithiothreitol, 10% glycerol, 5 µg of bovine serum albumin, 2 µg
of poly(dA-dT)·(dA-dT) (Amersham Pharmacia Biotech), 2 µg of
nuclear extract protein, and 20,000 cpm of 32P-labeled
probe. Reactions were incubated for 10 min at room temperature; the
labeled probe was added; and the incubation was continued for an
additional 20-min period. For competition studies, the nuclear extract
was incubated with the competing probe prior to the labeled probe
during 20 min. Supershift assays were performed by preincubating
nuclear extracts with 2 µl of antibodies against different
transcription factors (Santa Cruz Biotechnology, Santa Cruz, CA) or
rabbit IgG (Sigma) for 3 h on ice before probe addition.
111 to
27 of the
5 promoter was end-labeled by Klenow
filling with [
-32P]dATP. Assays were performed with
the Sure Track Footprinting kit (Pharmacia) according to the
manufacturer's instructions. Recombinant Sp1 was incubated with the
radiolabeled double-stranded fragment (~25,000 cpm) using the binding
reaction conditions described above in the EMSA experiments (except for
the absence of EDTA and the presence of 2.5 mM
MgCl2). Immediately following the 30-min incubation at room
temperature, 0.5 mM CaCl2 and 1 mM
MgCl2 were added to the reactions. This was followed by the
addition of 1 unit of DNase I. The reactions were incubated at room
temperature for 1 min and stopped with the addition of stop solution
(SDS/EDTA). The DNA was prepared for loading onto a 7 M
urea and 8% polyacrylamide sequencing gel (~15,000 cpm/lane), and
the Maxam-Gilbert A/G chemical sequencing reaction was included as
reference ladder.
RESULTS
5 Subunit
Gene--
To examine the requirements for
5 subunit transcription,
its promoter region was isolated and analyzed. A bovine genomic library
was screened, and several overlapping clones were isolated. Clone
5-21 contained ~16 kilobases of bovine genomic sequence including exons 1 and 2 and ~1.6 kilobases of 5'-flanking region. This region was further subcloned and sequenced (Fig.
1A). Comparison of this
sequence to a data base of binding sequences of known transcription
factors revealed the main features of the promoter/regulatory region of
the
5 gene: the lack of a TATA box and the presence of several GC
boxes, all of them concentrated into ~110 bases located 5' to the
transcription initiation site. Two perfect Sp1 consensus sites
((G/T)GGGCGGGGC) were present within this GC-rich region. Additional
sites with one mismatch, regarding perfect consensus sequences, were
also observed for Egr-1, Ap-1, Oct-1, and Ap-2. It is interesting to
note the presence of a contiguous direct repeat of two 52- and 42-bp
monomers (Fig. 1B), each of them containing one of the
perfect Sp1 sites and two other elements to which this transcription
factor could potentially bind.
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Fig. 1.
The 5'-region of the bovine
5 subunit gene. A, the nucleotide
sequence of a fragment of genomic clone
5-21 carrying exon 1 (with the protein sequence indicated underneath in
italics), the 5'-end of intron 1 (denoted by an
arrowhead), and ~1400 bp of 5'-flanking sequence is
indicated. The translation start codon is underlined, and
the major transcription initiation site (position +1) is denoted by the
arrow. B, shown is the alignment of the 52- and 42-bp direct
repeats in the
5 subunit core promoter region. Potential Sp1-binding
sites are underlined. The center of each of these sites is
separated by ~10-11 bp, or a multiple of this number, indicating
that they are approximately located on the same side of the DNA, as
each turn of B-DNA contains 10.5 bp (see Ref. 37).
5 mRNA was mapped by RNase protection analyses
(Fig. 2). A 479-residue antisense
riboprobe (Fig. 2, Probe 1) yielded two main
protected fragments of ~250 and 249 bases. The major one mapped
transcription initiation to an adenosine present in a group of three
(position +1, black arrow in Fig. 1) and located ~40 bp
downstream of the GC-rich repeats. To improve precision in the
determination of the transcription initiation site, a second overlapping probe was used (Fig. 2, Probe 2). In
this case, two protected fragments were also observed, which were 157 and 156 bp long and mapped transcription initiation to the same place. Therefore, these were tentatively considered the main transcription initiation sites. Other initiation sites may exist, as this is a
typical feature of promoters without TATA boxes, and in fact, minor
protected fragments of smaller size and intensity were also observed.
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Fig. 2.
Determination of the
5 subunit gene transcription initiation
site(s). The 5'-end of the
5 subunit mRNA was mapped by
RNase protection using two
5 probes whose structures are illustrated
in the lower part of the figure. T,
undigested probes, 479 (Probe 1, lane 1) and 403 (Probe 2, lane 5) bases (b); U, probes
digested in the presence of yeast tRNA (lanes 2 and
6); C, probes digested in the presence of a sense
cRNA control, yielding protected fragments of 358 (Probe 1, lane
3) and 328 (Probe 2, lane 7) bases; P,
protected fragments using 7.5 µg of bovine adrenal medulla
poly(A)+ mRNA (lanes 4 and 8).
The sizes of several RNA fragments used for calibration of the gel are
indicated to the left of each panel, whereas the sizes of
the protected fragments are to the right.
5 Subunit Promoter--
A series of
constructs was generated to determine the regions of the
5 subunit
promoter that contributed to its maximal activity (Fig.
3). These constructs were introduced into
SHSY-5Y and chromaffin cells, a neuroblastoma cell line and a primary
cell culture, respectively, that express the
5 subunit endogenously
(7, 28). In SHSY-5Y cells, the construct containing 111 bp of
5
promoter sequence plus 155 bp of 5'-noncoding region (p111
5LUC)
showed the maximal activity. This construct covered all Sp1 sites
already mentioned. A shorter construct (p65
5LUC) with only two Sp1
sites showed a 50% decrease in promoter activity, whereas further
5'-deletions that removed all the Sp1 sites (p39
5LUC, p+7
5LUC,
and p+56
5LUC) were inactive. In chromaffin cells, results were
similar, suggesting that sequences in the minimal promoter, between 111 and 39 bp upstream of the start site of transcription, appear to be
critical for basal transcription of the
5 subunit gene in transient
transfection assays. Reporter constructs larger than p111
5LUC did
not show significant changes in relative luciferase activity when
expressed in chromaffin cells. However, in SHSY-5Y cells, a small but
constant decrease was observed from one construct to the next larger
one, being maximal with p752
5LUC, which showed a marked decrease in activity (36%). The largest construct tested (p1412
5LUC), however, exhibited increased activity (73%). Therefore, in SHSY-5Y but not
chromaffin cells, elements predominantly located between -600 and
-750 with respect to the transcription initiation site have a negative
effect on
5 promoter activity.
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Fig. 3.
Deletion map analysis of
5 gene promoter activity. SHSY-5Y and
chromaffin cells were transfected with each of the plasmids (named
p
5LUC with the number of promoter base pairs included in the
construct) containing the luciferase reporter under the control of the
different fragments of the
5 subunit promoter and
pCH110/
-galactosidase as a transfection efficiency control. Promoter
activity was normalized to values obtained with the p163
5LUC
construct. The means ± S.E. (error bars) are given for
two or three individual experiments carried out in triplicate. A scheme
of the sequence (nucleotides -1409 to +155) of the bovine
5 subunit
gene promoter and of each plasmid used in the deletion studies is shown
on the left, including four putative GC-rich regulatory
elements.
5 promoter with the SV40 viral promoter,
present in the vector pGL2-Control, and the nAChR
7 and
4 subunit
promoters (Fig. 4). These subunits are
also expressed in chromaffin and SHSY-5Y cells (7, 8, 28), probably
forming part of the same (
4) or different (
7) nAChR subtype in
which
5 is present. In addition, the
4 subunit gene is located in the same gene cluster as the
5 subunit gene (11, 12), as indicated
previously. In chromaffin cells, the maximal nAChR subunit promoter
activity corresponded to the
5 subunit, which accounted for about
one-half of the activity shown by the SV40 promoter and was 10- and
2-fold higher that the activities of the
4 and
7 promoters,
respectively. By contrast, in SHSY-5Y cells, the activities of the
three subunit promoters were similar and about one-fifth the activity
of the SV40 promoter. Therefore, it appears that some cell-specific
differences exist among the three nAChR subunit promoters, which in any
case are weaker than the SV40 viral promoter.
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Fig. 4.
Comparison of the 5
subunit promoter with the SV40 viral promoter and the nAChR
7 and
4 subunit
promoters. SHSY-5Y and chromaffin cells were transfected with
p111
5LUC, p199
7LUC (see Ref. 50), p(19)
4 (a deletion construct
giving the maximal activity for the bovine
4 promoter (L. M. Valor,
A. Campos-Caro, C. Carrasco-Serrano, M. Criado, S. Viniegra, J. J. Ballesta, unpublished results)), and pGL2-Control to compare the
activity of the
5,
7, and
4 promoters relative to the SV40
viral promoter. Promoter activity was normalized to values obtained
with the pGL2-Control plasmid. Data are expressed as described in the
legend to Fig. 3.
5 promoter constructs already tested in chromaffin and SHSY-5Y
cells were also transfected into COS cells in an attempt to find
elements that would confer cell specificity. Although this cell line
does not express nAChRs, the pattern of promoter activity (Fig.
5) was similar to the one found in
chromaffin cells. Thus, p111
5LUC showed the maximal activity. A
shorter construct (p65
5LUC) with only two Sp1 sites showed a 44%
decrease in promoter activity, whereas further 5'-deletions that
removed all the Sp1 sites (p39
5LUC, p+7
5LUC, and p+56
5LUC)
were inactive. Constructs larger than p111
5LUC did not show
significant changes in luciferase activity relative to this construct.
Interestingly, whereas in SHSY-5Y and chromaffin cells, the
5
promoter was clearly weaker than the SV40 promoter, present in the
pGL2-Control vector, in COS cells, it was ~2-fold stronger than the
viral promoter. Therefore, no cell-specific elements were found in the
5 promoter region analyzed.
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Fig. 5.
Activity of 5
promoter constructs in COS cells. Each of the
5
promoter-luciferase constructs previously analyzed in SHSY-5Y and
chromaffin cells (see Fig. 3) was transfected into COS cells. Promoter
activity was normalized to values obtained with the p163
5LUC
construct. Data are expressed as described in the legend to Fig.
3.
40, two of them
representing perfect Sp1 consensus sites and another two with one
mismatch, were chosen for mutagenic analysis. Single mutations of the
four GC boxes indicated that all of them contribute to the activity of
the
5 gene, but in a different way, depending on the type of
transfected cell (Fig. 6). Thus, when the
most promoter-proximal of the GC boxes was altered (site 1, Fig.
6A),
5 promoter activity in SHSY-5Y cells decreased to
~50% of that observed for the parent construct (p111
5LUC),
whereas single mutations of the other three had no effect (Fig.
6B). The double mutant of sites 1 and 2 produced an activity
close to the obtained for site 1, whereas the simultaneous mutation of
sites 3 and 4 did not have any effect, further suggesting that sites
2-4, alone or in pairs, are not required for optimal promoter
activity. Surprisingly, activity was almost abolished when all four GC
boxes were altered simultaneously, which may indicate that sites 2-4,
together with site 1, do integrate a whole synergistic mechanism
required for basal promoter activity. By contrast, in chromaffin cells,
mutation of any of the GC boxes produced a similar decrease in promoter activity (50-69%), and double mutants showed a further decline. Finally, as happened with SHSY-5Y cells, the simultaneous mutation of
the four GC motifs produced the maximal decrease in activity.
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Fig. 6.
Sites 1-4 are functional elements required
for 5 subunit gene expression.
A, the proximal region of the
5 subunit promoter
(nucleotides -111 to -40) is depicted with the putative regulatory
elements underlined. This region contains putative binding
sites for transcription factor Sp1 (boxed). Several
nucleotides of each potential element were mutated as indicated below
the sequence to yield constructs analyzed in transfection experiments
(B). B, the name of each mutant construct
indicates the element(s) that have been altered (also crossed
out in the scheme). Plasmids were transfected into SHSY-5Y and
chromaffin cells, and activities were measured. Luciferase activity was
normalized to values obtained with the p111
5LUC construct. Data are
expressed as described in the legend to Fig. 3.
27 of the
5 Promoter by EMSA--
DNA fragments carrying
wild-type promoter region
111 to
27 were labeled and incubated with
nuclear extracts from chromaffin and SHSY-5Y cells (Fig.
7). Several retarded bands were observed in both cases (Fig. 7 A, lanes 2 and
4). Some of them were common to both extracts (
and
).
Recombinant Sp1 produced a main retarded complex (lane 6),
coincident in position with one of those observed with nuclear extracts
(
). Larger complexes were also observed with recombinant Sp1,
probably as the result of the simultaneous binding of two or more Sp1
molecules to the same DNA fragment. Antibody supershift analysis was
employed in an attempt to identify the proteins producing the retarded
bands. One of the main complexes (
) was retarded by an anti-Sp1
antibody (arrowhead, lanes 3, 5, and 7), whereas no supershift was observed
with antibodies against several transcription factors able (Sp3, Egr-1,
and Ap-2) or not (Myc, Max, USF1, and USF2) to bind to GC boxes (data
not shown). In addition to the main band, several minor ones were also
displaced by the anti-Sp1 antibody when using chromaffin cell nuclear
extracts (compare lanes 2 and 3). They
are probably degradation products of Sp1 protein, which keep the
capacity of binding to the DNA probe and being recognized by the
antibody. The band labeled with an open circle (
) was more prominent
in SHSY-5Y extracts (lanes 4 and 5),
but we were not able to identify it. Competition experiments confirmed
the specificity and identity of the complex formed by Sp1 (Fig.
7B). Thus, an excess of unlabeled probe neutralized the
formation of all complexes (lanes 10 and 13), whereas a synthetic oligonucleotide containing an Sp1
consensus sequence abolished the formation of the Sp1 complex
(lanes 11 and 14).
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Fig. 7.
Binding of cell nuclear proteins to the
proximal region of the 5 subunit
promoter. A, the DNA fragment corresponding to region
111 to -27 was used as a gel mobility shift probe in the presence of
2 µg of crude chromaffin (CR; lanes 2 and
3) or SHSY-5Y (SH; lanes 4 and
5) cell nuclear extracts or recombinant Sp1 (0.2 footprint
units, 1 footprint unit/µl) (lanes 6 and 7).
Lane 1 is probe run in the absence of protein extracts.
Lanes 3, 5, and 7 represent
complexes obtained upon adding anti-Sp1 antibodies (Ab). A
prominent band (
) was supershifted by Sp1 antibodies
(arrowhead). Other complexes were also observed (
and
), but were not displaced by any of the antibodies tested (see
"Results"). B, the gel mobility assay was run using DNA
fragment
111 to
27 as the labeled probe and nuclear extracts from
chromaffin (lanes 9-11) and SHSY-5Y (lanes
12-14) cells. Lanes 9 and 12 were without
added competitor (Comp.;
). Competitor DNA fragment
111
to
27 (PR; lanes 10 and 13) and an
oligonucleotide with a consensus site for Sp1 (Sp1; lanes 11 and 14) were added in 100-fold excess. Lane 8 is
probe run in the absence of protein extracts. C, nuclear
proteins from chromaffin (lane 15) and SHSY-5Y (lane
16) cells and recombinant Sp1 (lane 17) were separated
by SDS-polyacrylamide gel electrophoresis on a 10% resolving gel.
Following Western blotting with Sp1 antibodies, two molecular species
of ~97 kDa were detected. N.P., nuclear protein.
5 Proximal
Promoter--
Recombinant Sp1 protein was used in DNase I footprinting
(Fig. 8) to document the preferences of
this transcription factor for the four GC-rich elements previously
characterized in transfection studies (Fig. 6). When using the same
probe of the EMSA, Sp1 protected several domains (Fig. 8,
lanes 3 and 4): the largest one
corresponded to GC boxes 1 and 2 (large filled box on the
left), whereas two additional domains were also observed, especially at
the highest Sp1 concentration (lane 4). One of them
corresponded to GC box 4, and the other overlapped the 3'-half of GC
box 3 and a downstream GC-rich element. This element was not as close
to the Sp1 consensus binding sequence as the ones previously analyzed
and, for this reason, was not included in our previous functional
characterization (Fig. 6), but, given the DNase protection results, it
was further characterized.
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Fig. 8.
DNase I analysis of Sp1 interactions within
the proximal region of the 5 subunit
promoter. The sense strand of the same DNA fragment (
111 to
27) used in EMSA was end-labeled by Klenow filling. The gel-purified
fragment was incubated with 0.4 (lane 3) or 1 (lane
4) footprint unit (fpu) of Sp1 and treated with DNase
I. A control reaction (Cont.; lane 2) was
performed in a similar manner, using bovine serum albumin as the
protein component. Following digestion with DNase I, fragments were
resolved on an 8% denaturing polyacrylamide gel. A Maxam-Gilbert A/G
chemical sequencing reaction was included as a reference ladder
(lane 1). To the left is a sequence summary of
the protected domains, with numbers corresponding to the
putative regulatory elements in Fig. 6.
5 subunit promoter, EMSA experiments were performed with
recombinant Sp1, and the DNA fragment carrying wild-type promoter
region
111 to
27 and the corresponding fragment mutated at the four
original Sp1 sites were compared (Fig.
9). At low Sp1 concentration, a retarded
band was observed with the wild-type probe (lane 3). The
same band was observed in the case of the mutant probe, but it was very
faint (lane 7). A higher Sp1 concentration increased the
intensity of the retarded band with the wild-type probe and even
induced the formation of complexes of larger size (lane 4),
probably as the result of Sp1 binding to more than one site within a
single DNA molecule. In the case of the quadruple mutant, the faint
band was more intense (lane 8), but no larger complexes were
observed, suggesting that this DNA fragment still has one intact
Sp1-binding site. That this Sp1 site is the one immediately downstream
of site 3 was demonstrated by mutating it (Fig.
10A) and performing EMSA
experiments (Fig. 10, B and C). The combination
of the same wild-type probe, already used, with nuclear extracts from
chromaffin cells yielded the typical pattern in which a prominent band
(Fig. 10B, lane 2,
) was observed. The formation of this band, previously demonstrated to be antigenically related to Sp1 (Fig. 7B), was competed by the unlabeled
probe (Fig. 10B, lanes 3 and
4), but not by the same probe mutated at the five Sp1 sites
(lanes 5 and 6). Moreover, when the
mutant fragment was used as the labeled probe, the prominent Sp1 band was not observed (lane 8). Finally, recombinant Sp1 was
unable to produce retarded bands with the quintuple mutant (Fig.
10C, lanes 13 and 14),
contrary to what occurred with the quadruple mutant (Fig. 9).
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Fig. 9.
Binding of recombinant Sp1 to GC motifs
within the promoter region of the 5 gene.
Labeled wild-type DNA fragment
111 to
27 and the corresponding
fragment mutated at the four Sp1 sites previously analyzed
(Quadruple Mutant) were used as gel mobility shift probes in
the presence of 0.02 (lanes 2 and 6), 0.1 (lanes 3 and 7), and 0.5 (lanes 4 and
8) footprint units of recombinant Sp1. Lanes 1 and 5 contain probe run in the absence of protein (
).
,
Sp1 bound to a single site;
, Sp1 bound to two sites.
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Fig. 10.
Gel mobility shift assays of the fifth
Sp1-binding site. A, region -80 and -71 contains an
additional putative binding site for Sp1 (underlined and
labeled as 5 in the upper sequence).
Several nucleotides of this potential element were mutated, as
indicated in the lower sequence, in combination with the
previously mutated sites and used as probe (Quintuple
Mutant). B, labeled wild-type DNA fragment 111 to
27 and the corresponding quintuple mutant were used as gel mobility
shift probes in the presence of 2 µg of crude chromaffin cell nuclear
extracts. Competitor (Comp.) DNA fragments corresponding to
the wild type (WT; lanes 3 and 4) and
the quintuple mutant (QM; lanes 5 and
6) were added in 25- and 100-fold excesses. Lanes
1 and 7 were run in the absence of protein (
).
corresponds to the band that was supershifted by Sp1 antibodies (see
Fig. 7), whereas the band indicated by
has not been identified.
C, the same probes from B were used in the
presence of 0.05 (lanes 10 and 13) and 0.2 (lanes 11 and 14) footprint units of recombinant
Sp1. Lanes 9 and 12 contained probe run in the
absence of protein (
).
5LUC (see Fig. 6), in which this element was mutated
individually or in combination with the other four. In principle, this
element seems to be unable to activate transcription by itself alone
since the mutation of the other four, leaving this site intact,
decreased activity to very low levels (Fig. 6). Thus, the contribution
of this element to transcriptional activation, if any, should be expected in a cooperative manner, as it happens with the other four
previously characterized. In fact, the single mutation had an effect on
promoter activity similar to the one observed for the other single
mutants (50% of the activity of p111
5LUC). The quintuple mutant had
14% of promoter activity (relative to p111
5LUC), close to the value
obtained for the quadruple mutant (14.7%). Therefore, it seems that
this element mediates the transcriptional activation of the
5
promoter in a collective way, combined with the other GC boxes.
DISCUSSION
5 subunit is widely expressed in the
peripheral nervous system (7, 30, 31) in combination with
3,
4,
and, in some cases,
2 subunits (9, 32). Moreover, it has been
demonstrated recently that the
5 subunit combines with
4,
2,
3, and
4 subunits (33, 34) in heterologous expression systems,
modifying nAChR channel properties. Thus, some information has been
obtained regarding
5 subunit function, but nothing was known until
now about the cis-elements and trans-acting
factors that regulate its expression.
5 subunit does not contain TATA and
CAAT boxes, but it does have several GC-rich domains, a feature found
in the promoters of the
2 (35),
3 (13),
4 (20), and
7 (10,
36) subunits. Moreover, the structure of the
5 subunit promoter
appears highly organized with two direct repeats (Fig. 1B),
each containing crucial elements for promoter activity. From 5'-end
deletion analysis (Fig. 3), we determined that the region located
between nucleotides -111 and +155 was necessary for the basal promoter
activity detected in SHSY-5Y and chromaffin cells. A large loss in
promoter activity was observed when 72 bp were deleted from the 5'-end
(compare p39
5LUC with respect to the larger construct p111
7LUC)
(Fig. 3). These constructs were also transfected into COS cells (Fig.
5), which do not endogenously produce
5 subunits, and were even more
potent in luciferase activity when using a viral promoter as a
reference. Therefore, it is possible that promoter elements needed for
cell-specific expression are not included within the promoter fragments
used in this study. Nevertheless, the action of negative elements seems
evident in SHSY-5Y cells, as demonstrated by the slight but gradual
decrease in the activity of larger constructs. Elements located between -660 and -752 appear responsible for the largest decline in activity and could be involved in a silencing mechanism. It is evident that this
mechanism might not be totally effective in SHSY-5Y cells since the
largest construct (p1409
5LUC) regains activity, and in fact, SHSY-5Y
cells endogenously express
5 subunits. However, it could be of
relevance in tissues where
5 subunit expression is repressed,
although, at least in COS cells, such a mechanism does not seem to be operative.
7LUC, promoter activity was almost suppressed, thus suggesting that they play a crucial role in the transcriptional regulation of the
5 gene. Moreover, DNase I footprinting with purified Sp1 protein revealed regions of extended protection, which
contained the four Sp1-binding sites and also an additional GC-rich
element, close to site 3 (Fig. 8). This site, in contrast to the
others, which have no or one mismatch with respect to a perfect Sp1
consensus binding sequence, has two mismatches and, for this reason,
was not considered in the initial analysis of promoter activity.
However, EMSA (Fig. 10) and gene reporter experiments confirmed the
footprinting data indicating that Sp1 can also bind to this site with a
functional significance. We postulate that Sp1 contacts the DNA through
multiple interactions to form a higher order complex over the promoter
region. Interestingly, the five sites are approximately located on the
same side of the DNA, as each turn of B-DNA contains 10.5 bp (37), and
sites 2, 5, 3, and 4 are 11, 31, 42, and 62 bp apart, respectively,
from site 1. This special arrangement could generate an activation
environment that would produce more effective interactions with the
transcription apparatus, giving rise to the formation of a multiprotein
complex that would accomplish transcription activation at a higher
level (38). The individual contribution of each Sp1 element to the formation of this hypothetical higher order complex appears differently defined, depending on the cell context. Thus, in SHSY-5Y cells, the
only critical site is the most promoter-proximal, perhaps because the
possibility of physically direct interactions between Sp1 at this site
and the basal transcriptional machinery. The importance of the others
only emerges if they are simultaneously altered, suggesting that Sp1
acts synergistically as a whole on the complete region. By contrast, in
chromaffin cells, transcriptional activity seems to depend on each of
these sites, as alteration of any of them produces a decrease in
promoter function. If we assume that the formation of a higher level
complex requires several Sp1 sites, then the possibility exists that in
SHSY-5Y but not chromaffin cells, other elements can substitute for the
Sp1 sites that were gradually altered by mutagenesis. This substitution would be not possible, however, when all of the Sp1 sites are modified.
This assumption is supported by the fact that the double mutant of
sites 3 and 4 shows only an insignificant decrease (11%) with respect
to the activity of the parent construct p111
5LUC (Fig.
6B), whereas the deletion construct p65
5LUC, which lacks the region between -111 and -66, including sites 3-5, but also other
sequences, shows a marked decrease (48%) in activity. Perhaps some
additional element present in the deleted region becomes effective in
SHSY-5Y cells when some but not all of the Sp1 sites are altered.
Whatever the mechanism, the primary conclusion of this study is that
the basal activity of the
5 subunit promoter is defined by an array
of Sp1-binding elements to which this transcription factor can bind to
activate transcription in a synergistic manner.
5 subunit promoter, which
appears strongly dependent on a ubiquitous transcription factor like
Sp1, may be regulated in a tissue-specific manner by this factor in
several ways. (a) Availability of Sp1, as levels of this
protein have been shown to vary among different tissues (39).
(b) Competition with other members of the Sp family that could bind to the same elements. For instance, Sp3 acts with Sp1 in a
concerted way to transactivate the nAChR
4 subunit (21). However, we
have not found binding of Sp3 to the
5 promoter in EMSA of
chromaffin and SHSY-5Y nuclear extracts. In other cases, Sp3 has been
shown to repress the action of Sp1 (40-42). (c) Formation of heteromeric complexes between Sp1 and other proteins. Protein p107,
a member of the retinoblastoma family of proteins, binds Sp1 and
represses Sp1-dependent transcription (43). Sp1 also interacts with the RelA subunit of transcription factor NF-
B (44)
and the cellular protein YY1 (45). (d) Action of Sp1 as a
physical link between proximal and distal promoter elements via DNA
bending (46) or looping (47).
3,
4, and
5
subunits. As has been already mentioned, these subunits compose the
predominant nAChR subtype in the peripheral nervous system (7, 9, 32)
and have their genes clustered in the genome (11, 12). Therefore, a
concerted regulation of the expression of the three subunits is
plausible. Given that Sp1 (and other members of the Sp family) seems to
play a crucial role in the regulation of the
3 (22),
4 (20, 21),
and
5 (this study) subunits, we propose that this transcription
factor, either directly or through other proteins that can regulate its
activity, may play the coupling role in the coordinated regulation of
the three subunits. Other factors, or perhaps a differential regulation of Sp1, specific to a determined neuronal cell subset, would be responsible for the independent regulation of each subunit, which also
must take place, given the dissimilar pattern of expression of
these subunits in certain places (48, 49).
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ACKNOWLEDGEMENTS |
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The excellent technical assistance of Eva Martínez and Mar Francés is appreciated.
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FOOTNOTES |
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* This work was supported in part by DGICYT Grants PB95-0690 and PM95-0110 from the Ministry of Education of Spain, Grant SC1*CT91-0666 from the Commission of the European Economic Community, and Grant GV-D-VS-20-158-96 from the Generalitat Valenciana.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.
¶ Recipient of a Consejo Superior de Investigaciones Científicas-Bancaja postdoctoral fellowship.
Predoctoral Fellow from the Generalitat Valenciana.
To whom correspondence should be addressed. Tel.: 34-965919479;
Fax: 34-965919484; E-mail: Manuel.Criado{at}umh.es.
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ABBREVIATIONS |
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The abbreviations used are: nAChRs, nicotinic acetylcholine receptors; bp, base pair(s); EMSA, electrophoretic mobility shift assay.
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REFERENCES |
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