From the
In this study, we performed an analysis of the neuronal
nicotinic acetylcholine receptor
Nicotinic acetylcholine receptors (nAchRs)
A site-specific Sp1 binding site mutation was
introduced by recombinant polymerase chain reaction into
To prepare a reporter construct, in
which the GA motif within the
To prepare a
promoter construct (4XSp1-TATA-luc) containing multiple copies of the
EMSA was performed with
We showed previously that the activities of
The
transcription factor AP2 has been shown to be widely expressed in
neural crest cell lineages during mouse embryogenesis
(32) .
Because the PC12 line is derived from cells arising from neural crest
and because we identified an AP2 binding motif in the
In this and a previous report
(17) we identified cis
and trans elements important for nAchR
Neither C1 nor C2 was completely supershifted by anti-Sp1
antibody. The inefficient recognition of C1 and C2 by anti-Sp1 antibody
is not likely to result from species-specific differences in epitopes,
because the peptide used to prepare the polyclonal antibody is
perfectly conserved between rats and humans. A more likely possibility
is that C1 and C2 contain additional Sp1-related proteins that are very
similar in size to Sp1. This idea is supported by the discovery of a
family of differentially expressed genes encoding Sp1 and the related
proteins, Sp2, Sp3, and Sp4
(18, 27, 28) . These
proteins bind to DNA with similar specificities and affinities. The
molecular weights of Sp1, Sp3, and Sp4 are also very similar, and thus
complexes formed by these proteins on DNA are not readily
distinguishable by electrophoresis
(18, 27) . Sp1, Sp2,
and Sp3 are is considered to be ubiquitously expressed
(18, 35) , although the level of Sp1 expression varies
at least 100-fold in different tissues
(35) . In contrast,
expression of Sp4 is much more limited; Sp4 RNA is abundant in brain
but cannot be detected in several other tissues
(18) .
Promoters for a number of other neural restricted ligand-gated ion
channel genes have been characterized, and comparison of these to the
Although appropriate expression
of SCG10 and type II sodium channel genes is critically dependent on
interaction of distant silencer elements with repressors, the
expression of brain-enriched factors that interact with common Sp1
binding motifs ( e.g. GA motif) raises the possibility that the
minimal promoters of neural restricted genes that contain these motifs
contribute to tissue-specific expression. One such example of a
brain-enriched factor, noted above, is the Sp1-related factor, Sp4
(18) . Another example is a factor that interacts with the
promoter of the tissue plasminogen activator gene. The tissue
plasminogen activator gene is abundantly expressed in brain and is
controlled by a promoter that is more active in the presence of
brain-derived nuclear extracts than in those obtained from liver or
kidney. Analysis of tissue-specific elements in the tissue plasminogen
activator promoter revealed GC boxes that bind both an Sp1-like factor
and a brain-enriched factor
(19) . If
We thank John Incardona and Dr. Terry Rosenberry for
providing S2 cells and a Lipofectin transfection method for S2 cells,
and we thank Dr. R. Tjian for pPacSp1.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
3 subunit gene promoter region,
-238/+47, to identify cis and trans elements that are
important for basal activity in PC12 cells. Sequence analyses of the
3 promoter and footprint assays revealed an Sp1 binding site
between -79 and -57 (termed the
3 GA motif) and an AP2
binding site between -30 and -7. Using mobility shift
analysis, we found that PC12 cell extracts contain proteins that
specifically bind to the
3 GA motif and are immunologically
related to Sp1. Mutation of the
3 GA motif, which prevented
binding of Sp1, resulted in a 75% decrease in promoter activity.
Mutation of the AP2 site resulted in only a minor loss of promoter
activity, which is consistent with the lack of AP2 binding activity in
PC12 extracts. In Drosophila Schneider line 2 (S2) cell
cotransfection assays, Sp1 activated the
3 promoter in a GA
motif-dependent manner. Furthermore, multimerization of the GA motif
upstream of the
-globin TATA box conferred Sp1 responsiveness. Our
results indicate that Sp1 can activate transcription through direct
interaction with the
3 GA motif and that this motif plays a major
role in
3 promoter basal activity in PC12 cells.
(
)
are excitatory ligand-gated ion channels encoded by a
family of at least 15 differentially expressed genes
(1, 2, 3, 4) . These genes encode
subunits that can be assembled into different heteromeric subtypes,
depending on the particular cell-restricted pattern of subunit
expression and undefined intracellular rules of permissive
subunit-subunit interactions
(5) . The nAchR subunit genes
4,
3, and
5 are clustered over an
60-kilobase
region of the vertebrate genome
(6, 7, 8) . The
clustered organization may reflect a mechanism for coordinating cell
type and temporal expression of these genes as has been demonstrated
for the vertebrate globin genes
(9) . Indeed, these clustered
genes are coexpressed in many ganglionic neuronal cell populations and
the chromaffin tumor line PC12
(6, 7, 10, 11, 12) . In the
central nervous system, however, the expression patterns of these genes
overlap to some extent but are not entirely concordant
(7, 13, 14, 15, 16) . This
suggests that within the cluster, gene-specific cis elements also play
a role in cell type-specific expression of these genes. We are
interested in investigating the mechanisms that coordinately regulate
nAchR genes to permit the assembly of different neural restricted
subtype heteromers and are focusing on transcriptional regulation of
the clustered genes. Because of the clustered organization, the first
question we addressed was whether individual promoters are associated
with these genes. Analysis of RNA isolated from PC12 cells and
sympathetic ganglia indicated that the
3 gene initiates
transcription at multiple sites positioned within the
4/
3
intergenic region. Transient transfection analysis in these cells
indicated a promoter that maps to the
3 start site region
(17) . In this report, we have investigated the
3 promoter
to identify important cis elements and interacting trans factors in
PC12 cells. An Sp1 binding site (referred to as a GA motif) positioned
near the start site region was found to play a major role for
3
promoter activity in these cells. In contrast, an AP2 binding site
positioned within the start site region had only a small effect on
reporter activity. The expression of brain-enriched regulatory proteins
that interact with Sp1 binding motifs
(18, 19) raises
the possibility that the
3 GA motif plays a role in establishing
the precise cell-specific pattern of
3 expression in
vivo.
Luciferase Reporter Plasmids
The 3 promoter
5` deletion constructs used in this study were prepared with the
ExoIII deletion kit from Promega. The resulting 5` boundaries
of these deletion constructs were determined by dideoxy sequence
analysis
(20) .
3
reporter, -1607/+47-luc, to generate mutSp1-luc. A mutagenic
oligonucleotide carrying base substitutions 5`-GGT CCG ATG CAT ATG CCA
CGA CGG GCG AAC GTC C-3` (residues -78 to -44) was used in
a reaction with a downstream primer positioned within the luciferase
gene to generate a 385-bp fragment. The second mutagenic
oligonucleotide, 5`-TCG TGG CAT ATG CAT CGG ACC ACG GTC TCC-3`
(residues -57 to -87), was used in a separate polymerase
chain reaction with an upstream primer to generate an 874-bp fragment.
Products of the two reactions were gel-purified, heat-denatured, and
then annealed by the overlapping complementary 5` ends. The 3` ends
were extended, and then products were amplified using upstream and
downstream primers. Amplified DNA was cut with Bpu1102I and
XhoI and then used in a ligation reaction to replace the
Bpu1102I/ XhoI fragment of the
-1607/+47-luc vector. Similarly, a site-specific AP2
mutation was introduced into -1607/+47-luc with the
following mutagenic oligonucleotides: upper, 5`-CGC CTC CAT GCA TTA CAG
CTG CTT GCA GCG CTT G-3` (residues -30 to +4); lower, 5`-GCA
GCT GTA ATG CAT GGA GGC GGG GAG GCC-3` (residues -8 to
-38). The resulting construct is called mutAP2-luc. To introduce
the Sp1 and AP2 double mutation (mutSp1/AP2-luc), an AP2 mutation was
introduced into the mutSp1-luc construct using the same mutagenic
oligonucleotides described above. Underlined residues above indicate
base change sites. All constructs carrying polymerase chain
reaction-generated mutations were sequenced across the
-238/+47 region to ensure introduction of base changes at
only the intended positions.
3 promoter was replaced with SV40
21-bp repeats, the Sp1 footprint region in -1607/+47-luc was
substituted with an SV40 promoter Sp1 footprint region by polymerase
chain reaction-based mutagenesis. Specifically, ACG CAG GAG ACC GTG GTC
CGA CGC CCC TCC CAC GAC GGG CGA A (residues -92 to -50 of
3 promoter) were substituted with CCG CGG CCC CTA ACT CCG CCC ATC
CCG CCC CTA ACT CCG CCC A, which contains two 21-bp repeats (one of
which is an 18 out of 21 bp match) of the SV40 promoter. The mutagenic
primers were the following: upper, 5`-CTC CGC CCA TCC CGC CCC TAA CTC
CGC CCA CGT CCC GCC TCG GC-3`; lower, 5`-GGG GCG GGA TGG GCG GAG TTA
GGC CCC GCG GCG ATG CCT CGC C-3`. The upstream and downstream primers
were the same as those used to prepare mutSp1-luc.
3 GA motif positioned upstream of the
-globin TATA box, two
53-mer oligonucleotides that contain four copies of the -75 to
-65 portion of the Sp1 footprint region within the
3 gene
were prepared and annealed: upper, 5`-CCG ACG CCC CTC CCA CGC CCC TCC
CAC GCC CCC TCC CAC GCC CTC CCA CGT AC-3`; lower, 5`-GTG GGA GGG GCG
TGG GAG GGG CGT GGG AGG GGC GTG GGA GGG GCG TCG GGT AC-3`. The
resulting double-stranded oligonucleotide was inserted into a
luciferase reporter, 20 bp upstream of the
-globin TATA box.
Cell Culture
PC12 cells were grown using
conditions previously described
(17) . Schneider line 2 (S2)
cells were grown at 26 °C in Shields and Sang M3 insect medium
(Sigma) containing 10% fetal bovine serum (HyClone) that was first
heat-inactivated at 56 °C for 30 min.
Transfection
PC12 cells were transfected by
electroporation as described previously
(17) . S2 cells were
transfected with Lipofectin reagent (Life Technologies, Inc.). S2 cells
were pelleted and washed twice with M3 medium. 1 10
cells were then resuspended in 0.5 ml of M3 medium. For each
transfection, 30 µl of Lipofectin and 25 µg of plasmid DNA were
aliquoted separately into tubes containing 1 ml of M3 medium.
Lipofectin and DNA in M3 medium were then mixed and immediately added
to resuspended S2 cells. After 2-4 h of incubation at room
temperature, cells were precipitated and washed twice with M3 medium.
Cells were then resuspended in 10 ml of complete M3 medium, plated, and
kept at 26 °C. After 48 h, cells were precipitated and washed twice
with phosphate-buffered saline. Cell extracts were prepared using
luciferase cell lysis reagent (Promega). Luciferase assays,
-galactosidase assays, and protein assays were performed as
described previously
(17) .
DNase I Footprinting
Footprinting assays were
performed as described previously
(17) with 1-6 footprint
units of either Sp1 or AP2 protein (Promega).
Electrophoretic Mobility Shift Assay (EMSA)
PC12
cell extracts were prepared essentially as described
(21) .
Cells were grown to near confluence, harvested by centrifugation, and
washed twice with phosphate-buffered saline. All of the following steps
were performed at 4 °C. The cells were resuspended in four packed
cell volumes of 10 m
M Tris-HCl (pH 7.9), 1 m
M EDTA,
and 5 m
M dithiothreitol. After 20 min, the cells were lysed by
homogenization in a Dounce homogenizer with eight strokes using a
``B'' pestle. Four packed cell volumes of 50 m
M Tris-HCl (pH 7.9), 10 m
M MgCl, 2 m
M dithiothreitol, 25% sucrose, 50% glycerol were added, and the
suspension was gently mixed. With continued gentle stirring, 1 packed
cell volume of saturated (NH
)
SO
was
added dropwise. The lysate was gently stirred for an additional 30 min,
followed by centrifugation at 45,000 rpm in a Beckman L7-55 rotor
for 3 h. Protein was precipitated from the supernatant by the addition
of ammonium sulfate (0.3 g/ml) and neutralized with 1
M NaOH
(0.1 ml/10 g of solid (NH
)
SO
). The
precipitate was resuspended (5% of the volume of the high speed
supernatant) in 25 m
M HEPES (pH 7.9), 100 m
M KCl, 12
m
M MgCl
, 0.5 m
M EDTA, 2 m
M dithiothreitol, 17% glycerol and dialyzed against resuspension
buffer for 8-12 h. The dialyzed material was then aliquoted and
stored at -80 °C until needed.
P-end-labeled double-stranded oligonucleotides that were
incubated with PC12 cell extract or either Sp1 or AP2 protein in EMSA
buffer (15 m
M HEPES (pH 7.1), 60 m
M KCl, 1.2 m
M dithiothreitol, 7.2 m
M MgCl
, 0.3 m
M EDTA, 10% glycerol, 0.1 mg/ml poly(dI
dC)). For
oligonucleotide competition analysis, a 150-600-fold molar excess
of competitor oligonucleotides was also added to the mixture. After 30
min at 30 °C, the mixture was chilled on ice for 5 min before
analysis on polyacrylamide gels. For some EMSAs, 1-3 µl of
anti-Sp1 or anti-AP2 antibody (Santa Cruz Biotechnology, Inc.) was
added to the mixture. The reaction was then incubated at 4 °C for 2
h. DNA-protein complexes were fractionated on 5% polyacrylamide gels in
1
Tris-glycine buffer (50 m
M Tris base, 380 m
M glycine, and 2 m
M EDTA) at 4 °C. Free probe and
DNA-protein complexes were visualized by autoradiography using Kodak
XAR-5 film.
3 promoter
constructs -1607/+47-luc and -238/+47-luc did not
differ significantly when the constructs were transfected into PC12
cells
(17) . Luciferase activity of each of these reporters was
20% of that obtained from the SV40 promoter. Because the
-238/+47 contains the entire
3 multistart site region,
these results together suggest that
3 promoter elements important
for activity in PC12 cells are positioned within this G + C-rich
fragment. To determine the position of these elements we quantitated
the reporter activity of a set of deletions spanning the
-238/+47 segment of the
3 gene and upstream region. As
shown in Fig. 1, deletion to -77 caused only a small
decrease in reporter activity. However, a large decrease (80% of
-238/+47) in promoter activity was observed in a reporter in
which sequences upstream of -36 were absent. Deletion of
sequences to +30 resulted in background reporter activity. These
results indicate the presence of an important cis element within
segment -77/-36.
Figure 1:
Analysis of
3 promoter deletions. Deletion constructs are shown relative to
the -238/+47 portion of the
3 upstream region. The
dashed line indicates the transcription initiation
region. Relative luciferase activities were obtained after correction
for transfection efficiency with a co-transfected RSV-
gal plasmid.
Error bars represent mean ± the range from
duplicate transfections.
Transcription Factors Sp1 and AP2 Bind to the
Positioned within segment -77/+30 are
sequence motifs that are either perfect or single base mismatches to
the consensus binding sites for transcription factors Sp1
(22) and AP2
(23) . To determine whether or not any of
these consensus sequences were genuine Sp1 or AP2 binding sites, we
performed footprint analyses using pure Sp1 and AP2 proteins,
respectively (Promega). As shown in Fig. 2, these proteins produced
single nonoverlapping footprints near the multistart site region. The
strong footprint produced by Sp1 was positioned between -79 and
-57, which is adjacent to the 5` boundary of the start site
region. Inspection of the Sp1-protected segment revealed a core
sequence, 5`-CCCCTCCC-3`, that is a perfect match to the previously
determined Sp1 consensus binding motif
(24) . Moreover, this
motif is reminiscent of an inverted GA box, which was previously
characterized as an Sp1 binding motif in various promoters such as JC
virus early promoter
(25) and insulin-like growth
factor-binding protein-2 promoter
(26) . Thus, we refer to this
Sp1 binding site as an 3
Promoter
3 GA motif. AP2 protected a segment,
-30/-7, positioned within the transcription start site
region (Fig. 2 B). This footprint included a core sequence with
a single base mismatch to the AP2 consensus site derived from analysis
of the SV40 promoter
(23) .
Sp1 but Not AP2 Binding Activity Is Detectable in PC12
Cells
The zinc-finger protein, Sp1, has been implicated as an
important trans-acting factor for numerous genes
(24) . Recent
studies indicate that Sp1 is one of several members of a differentially
expressed gene family
(18, 27, 28) . Therefore,
we were interested in determining whether PC12 cells express Sp1 or
Sp1-like proteins that bind the 3 GA motif. We prepared PC12 cell
extracts to perform EMSA with an oligonucleotide probe
(Fig. 3 A) containing the entire Sp1 footprinted region
(Sp1
). As shown in Fig. 3 B, two major
protein complexes (C1 and C2) were formed on Sp1
. C1
had the same mobility as the complex formed by recombinant Sp1 protein
(Fig. 3 C). These complexes were specific because
unlabeled Sp1
inhibited complex formation, but an
oligonucleotide in which the
3 GA motif was mutated
(mutSp1
) did not. The formation of two major
complexes, C1 and C2, on an Sp1 binding site is similar to
Sp1-containing complexes reported by others
(26, 29, 30, 31) .
Figure 3:
Detection of GA motif binding activity in
PC12 cell extracts. A, the middle strand represents a portion
of the 3 promoter, which contains the GA motif
( overline). The upper and lower sequences
represent Sp1
probe and mutant Sp1
oligonucleotide (mutSp1
), respectively, used in
EMSAs presented in B and C. Substituted bases of
mutSp1
are indicated by asterisks.
B, PC12 cell extracts were incubated with Sp1
probe in the absence or presence of increasing amounts of
unlabeled double-stranded competitors. Lane 1, probe
alone; lanes 2-8, 8 µg of PC12 cell
extract; lanes 3-5, 150-, 300-, and 600-fold
molar excess of Sp1
competitor; lanes 6-8, 150-, 300-, and 600-fold molar excess of
mutSp1
competitor. Major complexes are indicated as
C1 and C2. C, Sp1
probe was
incubated with either 48 ( lanes 2-4) or 8
µg ( lanes 8-10) of PC12 cell extract
( CE) or 1 footprint unit of human Sp1 protein ( lanes 6-7) in the presence of 0.1 µg ( lanes 3, 7, and 9), 0.2 µg ( lane 4), or 0.3 µg ( lane 10) of anti-Sp1
polyclonal antibody ( Ab Sp1). As discussed under
``Results'', lower molecular weight complexes indicated by
asterisk in B and C may represent
degradation products of C1 and/or C2 or uncharacterized intact
proteins.
To determine
whether these complexes contained Sp1 and/or Sp1-related proteins,
anti-Sp1 antibody was incubated with the extract and probe mixture.
Addition of anti-Sp1 antibody reduced the intensity of both C1 and C2
and resulted in the appearance of a larger complex that did not enter
the gel, suggesting that Sp1 is present in C1 and C2. (Fig.
3 C, lanes 1- 4). The inability to
completely supershift C1 and C2 could result from insufficient anti-Sp1
antibody or the presence of other Sp1-related molecules in addition to
Sp1 in the complexes. To distinguish between these two possibilities,
we adjusted the amount of extract so that the level of C1 formed would
be similar to the level of the complex containing recombinant Sp1. As
shown in Fig. 3 C ( lanes 5-10),
0.1 µg of the antibody completely supershifted the complex formed
by pure Sp1, but only a partial supershift of C1 and C2 was observed
with 0.3 µg of the antibody. Therefore, C1 and C2 are likely to
contain an additional Sp1-related protein(s). We also noted the
presence of additional higher mobility complexes (Fig. 3, B and C, asterisks) that were specifically
competed by unlabeled Sp1but were not supershifted
(Fig. 3 C, asterisk); therefore, these represent
either degradation products of Sp1 that lack the epitope recognized by
anti-Sp1 antibody or distinct proteins unrelated to Sp1.
3 promoter,
we next investigated whether PC12 cells express proteins that bind this
motif. We radiolabeled an oligonucleotide (AP2
probe)
corresponding to the AP2 footprint region and incubated it with pure
AP2 in the absence or presence of either unlabeled competitor
AP2
or a mutated AP2
(mutAP2
) oligonucleotide (Fig. 4 A). As
shown in Fig. 4 B, formation of AP2 complexes was almost
completely inhibited by incubation with excess unlabeled AP2
oligonucleotides but not with an equivalent amount of
mutAP2
oligonucleotides. These oligonucleotides were
then used to determine whether we could detect AP2 binding activity in
PC12 cell extracts. Unexpectedly, we did not detect AP2 immunoreactive
binding activity to the AP2 motif in these extracts.
Figure 4:
AP2
protein is not detectable in PC12 cell extracts. A, middle strand represents a portion of the 3 promoter that contains
the AP2 motif ( overline). The upper and lower sequences represent AP2
probe and mutant
AP2
oligonucleotide (mutAP2
),
respectively, used in EMSAs presented in B and C.
Substituted bases of mutAP2
are indicated by
asterisks. B, EMSAs were performed with
AP2
probe and AP2 protein in the absence or presence
of increasing amounts of indicated competitors. Lane 1, probe alone; lanes 2-8, 0.5
footprint units of AP2 protein; lanes 3-5,
150-, 300-, and 600-fold molar excess of unlabeled AP2
competitor; lanes 6-8, 150-, 300-, and
600-fold molar excess of unlabeled mutAP2
competitor.
C, AP2
probe was incubated with either AP2
protein (1 footprint unit) or PC12 cell extract (48 µg) in the
presence of 0.1 ( lanes 3 and 5) or 0.3
µg ( lane 6) of anti-AP2 antibody. The
asterisk indicates a very low abundance of uncharacterized
complexes that migrate much faster than the AP2-containing
complexes.
Transfection Analysis of GA and AP2 Motif Mutations in
the
To determine whether the 3 Promoter
3 GA motif
constitutes an important cis element in the
3 promoter, we
prepared mutated
3 reporters in which wild type GA motif sequences
were changed to those of mutSp1
(Fig. 3 A). As shown in Fig. 5, this mutated
construct (mutSp1-luc) directed luciferase activity that was 25% of
that obtained from the wild type construct (-1607/+47-luc).
This result, which is consistent with deletion analysis (Fig. 1),
demonstrates that the GA motif is critical for
3 basal activity in
PC12 cells. We also replaced the Sp1 footprinted region of
3 with
two copies of the SV40 early promoter 21-bp repeats, which contain
multiple Sp1-binding GC boxes. Transfection of this construct into
cells resulted in an
2-fold increase in promoter activity
(Fig. 5, SV40-Sp1-luc), presumably because more Sp1 can bind to
the promoter. Thus,
3 promoter function may be rate-limited by the
level of GA motif binding activity in PC12 cells.
Figure 5:
Transfection analysis of GA and AP2 motif
mutations. PC12 cells were transfected with 10 µg of luciferase
reporters driven by either 3 wild type promoter
(-1607/+47-luc) or reporter constructs containing mutated GA
motif (mutSp1), mutated AP2 site (mutAP2), or both (mutSp1/AP2).
SV40-Sp1 represents a reporter construct in which the
3 Sp1
footprint region was substituted with the 21-bp repeat region of the
SV40 promoter. no insert, pGL2 basic vector. PC12
cell extracts were prepared 2 days after electroporation. Relative
luciferase activities were obtained as described in the legend to Fig.
1. Error bars represent mean ± the range from
duplicate experiments.
Given the data
presented in Figs. 1 and 4, a mutation within the AP2 binding site
would not be expected to substantially alter 3 promoter activity.
To test this idea, we mutated the AP2 binding site to generate
mutAP2-luc and compared its activity with that of the wild type
3
promoter. As shown in Fig. 5the activity of mutAP2-luc was 30%
less than the activity obtained with -1607/+47-luc. To
confirm the results of the individual mutations, we prepared an
3
reporter (mutSp1/AP2-luc) in which both the GA motif and AP2 motif were
mutated. The activity of this construct was slightly lower than that of
mutSp1-luc. These results, together, provide strong evidence for a
major contribution of the GA motif to
3 promoter activity and only
a minor, if any, contribution of the AP2 motif (Fig. 5). Because
the AP2 site is positioned within the
3 transcription start site
region, the small but reproducible decrease in
3 promoter activity
observed with mutations in the AP2 motif may have resulted from
mutation of a subset of basal elements.
Sp1 Can Transactivate the
We have shown that Sp1 and/or an Sp1-related protein can
bind to a GA motif in the 3 Promoter and a
Heterologous Promoter via Direct Interaction with
3 GA
Motifs
3 promoter. Furthermore, the GA motif
was found to be critical for
3 promoter activity, which suggests
that Sp1 transactivates the
3 promoter upon binding to this motif.
To demonstrate this directly, we performed cotransfection assays in the
Drosophila Schneider line, S2, which does not express
endogenous Sp1. Different quantities of an expression vector, pPacSp1
(33) , driving the synthesis of Sp1 RNA were introduced into
these cells together with either -1607/+47-luc or
mutSp1-luc. As shown in Fig. 6 A, mutation of the
3
GA motif reduced transactivation by Sp1, which shows that Sp1 can
modulate the
3 promoter upon binding to this motif. Activation by
coexpressed Sp1, however, was not completely abolished by mutation of
the GA motif. The residual activation may result from binding of
overexpressed Sp1 to low affinity sites within the G + C-rich
3 promoter.
Figure 6:
GA motif-dependent transactivation by Sp1.
A, Drosophila Schneider S2 cells were co-transfected
with 10 µg of either -1607/+47-luc ( open squares) or mutSp1-luc ( solid triangles), 5 µg of RSV-gal plasmid, and
indicated amounts of pPacSp1. Cell extracts were prepared 48 h later.
Luciferase activities were corrected for
-galactosidase activity.
Fold activation was calculated as the activity of each reporter in the
presence of the indicated amount of pPacSp1 divided by the activity of
the reporter without effector. B, Sp1 transactivation of a
luciferase reporter containing zero ( solid triangles) or four
copies (open squares) of the
3 Sp1 footprinted region positioned
20 bp upstream of the
-globin TATA box. Cell extracts and
luciferase activities were obtained, and -fold activation was
calculated as described in A.
To determine whether Sp1 can transactivate a
heterologous minimal promoter via the 3 GA motif, we prepared a
luciferase reporter in which four copies of the GA motif were
positioned upstream of a
-globin TATA box. The resulting construct
(4XSp1-TATA-luc) was then cotransfected into S2 cells with various
amounts of pPacSp1 effector. The results shown in Fig. 6 B indicate that the activity of 4XSp1-TATA-luc was increased in a
dose-dependent manner by coexpression of Sp1. Transactivation was
entirely dependent upon the presence of the GA motifs, because no
transactivation was observed from a
-globin TATA reporter in which
GA motifs were absent. These results demonstrate that the GA motif can
mediate transactivation by direct interaction with Sp1 within the
context of the
3 promoter and a heterologous promoter.
3 gene promoter activity in
PC12 cells. Positioned adjacent to or within the start site region are
high affinity binding sites for transcription factors Sp1 and AP2,
respectively. The Sp1 footprinted region contains an upper strand core
sequence, 5`-CCCCTCCC-3`, that is reminiscent of Sp1 binding motifs
(termed GA boxes) that are present within the promoters of several
other genes
(25) . Transient transfection analysis of deletions
and point mutations downstream of -1607 indicated that the
3
GA motif was the only major
3 cis element we could detect outside
the transcription start site region. It is possible, however, that
closely positioned positive and negative elements went undetected by
our set of deletions. Cotransfections into Schneider S2 cells
demonstrated that the
3 GA motif is clearly capable of mediating
transactivation by Sp1, because mutation of the GA motif reduced Sp1
transactivation of
3 reporters, and multimerization of the GA
motif upstream of the
-globin TATA box resulted in GA
motif-dependent and dose-dependent activation by Sp1. Analysis of the
AP2 motif indicated that its contribution to
3 promoter activity
in PC12 cells is small. Based on the analyses in PC12 cells presented
here and previously
(17) , we propose that the
4/
3
intergenic region contains a single
3 TATA-less promoter composed
of a multistart site region, a GA motif that mediates transactivation
by Sp1 and/or Sp1-related proteins, and an AP2 motif of undefined
function (Fig. 7). Further analyses will be required to determine
the role of these elements for tissue-specific expression of
3
in vivo and also the position of additional
3 cis
elements outside the -1607/+47 region.
Figure 7:
Schematic of rat nAchR 3 promoter
elements. Transcriptional orientation and relative positions of
clustered neuronal nAchR genes are indicated by large arrows. Position and partial sequence of the
3
promoter in the rat
4/
3 intergenic region are shown. Cis
elements that bind Sp1 and AP2 are overlined. Dashed line indicates transcription start site region relative
to the 5` end of cDNA, PCA48 (17).
Mobility shift
assays were used to identify proteins in PC12 cells that could bind the
3 GA motif. Using the entire Sp1 footprinted region as probe
(Sp1
), we detected the formation of two major
complexes, C1 and C2, that were specifically competed by unlabeled
Sp1
and that were both supershifted with a polyclonal
antibody raised against a human Sp1 epitope. The mobility of C1 was
indistinguishable from that formed by pure human Sp1 on
Sp1
, suggesting that C1 contains Sp1. Because Sp1 is
known to be post-translationally modified
(34) , C2 may
represent a modified form of proteins present in C1 or a degradation
product.
3 promoter reveals notable similarities
(36, 37, 38, 39, 40, 41) .
First, at least two transcription initiation sites have been found in
the promoter regions of the GABA
R
3 and
, nAchR
2 and
7, and NMDAR1 genes. For most of these promoters
initiation occurs at multiple nonadjacent sites that are positioned
within a G + C-rich region several tens of nucleotides in length.
Second, consistent with multiple start sites none of these promoters
contains sequences that would indicate a TATA box. Third, Sp1 consensus
motifs are often found within these promoter regions, although a direct
interaction of Sp1 with these motifs has been demonstrated only for
nAchR
3 and the GABA
R
3
(36) promoter.
These common features are often referred to as those of housekeeping
genes, because many ubiquitously expressed genes contain similarly
structured promoters. However, as is apparent from the genes listed
above, these are also features of neural restricted genes. This
suggests that these genes are controlled by constitutive promoters
whose activity is modulated by sequence-specific enhancer and/or
silencer binding proteins to produce restricted patterns of expression.
Indeed, evidence for this type of mechanism has been demonstrated for
the neural restricted SCG10 and type II sodium channel genes
(42, 43, 44, 45, 46) . These
genes contain housekeeping-type minimal promoters that by themselves
lack the cis information required for proper cell type-specific
activity. To achieve appropriate cell type-specific control, silencer
motifs that are positioned upstream of the promoters and that bind
factors present in SCG10-negative and type II sodium channel-negative
tissues are required. Thus, based on the structural features of the
3 promoter it is expected that additional cis elements, such as
silencers, influence
3 promoter activity to restrict its in
vivo pattern of transcription.
3 promoter activity
can be modulated by the level of Sp1 factors, as is indicated here by
substitution of the GA motif with the SV40 promoter 21-bp repeats and
cotransfection assays in S2 cells, then increased neuronal expression
of brain-enriched factors that bind Sp1 motifs may modulate
cell-specific transcription of the
3 gene. Given the apparent
diversity of brain-enriched regulatory proteins that interact with Sp1
binding motifs and the low level of Sp1 expression in the brain
relative to other tissues
(35) it will be important to
determine the neuronal transcription factors that interact with the
3 GA motif and perhaps play a role in establishing the precise
pattern of
3 expression in vivo.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.