The best characterized function of acetylcholinesterase (AChE,
EC 3.1.1.7) (
)is that of terminating neurotransmission by
catalyzing the hydrolysis of acetylcholine at cholinergic synapses in
the central and peripheral nervous systems. AChE is expressed in a
variety of tissues including muscle, nerve, and hematopoietic cells. It
exists in different molecular forms that arise as a consequence of
splicing of alternative exons encoding distinct carboxyl termini. This
gives rise to the different structural forms of AChE which include
soluble monomers, dimers, and tetramers, glycophospholipid-linked
monomers and dimers, and heteromeric associations of catalytic subunits
with structural subunits. The different molecular species are
selectively expressed in various tissues. For example, the
glycophospholipid-linked species in mammals is largely confined to the
hematopoietic system, while a heteromeric species containing tetramers
of catalytic subunits linked to a collagen-like tail unit is mainly
localized in synapses of the neuromuscular junction.
Previous
studies using the murine muscle cell line C2C12 showed that AChE
activity and the number of nicotinic acetylcholine receptors increase
during muscle differentiation. Differentiation at this stage is
characterized by withdrawal from the cell cycle, elongation, and fusion
of myoblasts to give rise to multinucleated myotubes. The increases in
receptor expression and mRNA reflect an increase in
transcription(1, 2, 3, 4, 5, 6) .
Interestingly for Ache, the transcriptional rate is unchanged
during the transition from myoblasts to myotubes, and stabilization of
the transcript is responsible for the enhanced expression(7) .
Four muscle-specific transcription factors, encoded by a family of
genes, myoD(8) , myogenin(9, 10) , MRF4(11, 12) , and myf5(13) , have been identified which, when
individually introduced into non-myogenic cells, convert them to
myoblasts. These factors share amino acid identity within a basic
domain that mediates DNA binding to the E-box consensus DNA sequence
CANNTG, and an adjacent helix-loop-helix domain that allows
dimerization. They were shown to activate skeletal muscle-specific
genes including the
,
,
, and
subunits of the
nicotinic acetylcholine receptor
(nAChR)(4, 5, 6, 14, 15) ,
muscle creatine kinase(16, 17) , and myosin light
chain (18) in transient transfections using fused
promoter-reporter genes. They are necessary for skeletal
muscle-specific expression and for the transcriptional regulation of
the AChR subunits in response to electrical activity and denervation
and for developmentally specific expression in transgenic
mice(19, 20, 21, 22, 23) .
Contrary to these genes, Ache is transcribed in myoblasts
at the same rate as in myotubes. Here, we investigate whether the Ache gene is activated in an earlier stage of commitment of
stem cells into the muscle lineage. We employ myogenic factors to
induce myoblast formation, and we examine the regulation of
transcription of Ache during this early commitment to the
muscle phenotype. We find that Ache is actively transcribed in
the fibroblastic cell line C3H10T1/2 and is not transcriptionally
activated by myogenic transcription factors. Also, we observe that
these factors are not sufficient to induce the stabilization of Ache mRNA. Analysis of the Ache promoter elements
shows that the promoter activity depends on the presence of a GC-rich
region at bp -95 to -59 that contains binding sites for the
transcription factors Sp1 and Egr-1.
EXPERIMENTAL PROCEDURES
Cell Culture
C3H10T1/2 (10T1/2), a mouse
fibroblastic cell line (obtained from the American Type Cell
Collection) and a clone of 10T1/2 fibroblasts that constitutively
expresses myogenin, C3H10TFL2-3B (10TFL2-3, kindly provided
by Dr Eric Olson, The University of Texas M.D. Anderson Cancer Center,
Houston, TX, and described in Brennan et al.(24) ),
were maintained in Dulbecco's modified Eagle's medium
supplemented with 20% heat-inactivated fetal bovine serum. C2C12 cells,
a mouse muscle cell line (obtained from the American Type Cell
Collection), were cultured in Dulbecco's modified Eagle's
medium supplemented with 20% fetal calf serum and 0.5% chick embryo
extract (Life Technologies, Inc.). Differentiation of C2C12 and
10TFL2-3 cells was induced by switching the medium to
Dulbecco's modified Eagle's medium supplemented with 2%
horse serum or to Dulbecco's modified Eagle's medium
supplemented with 2% horse serum and 0.5% fetal bovine serum,
respectively. Cells were grown in a 10% CO
humidified
atmosphere at 37 °C.
Plasmid Construction and Site-specific
Mutagenesis
Several restriction fragments covering 1 kb upstream
of the transcription initiation site, exon 1, and intron 1 to the
beginning of exon 2 were cloned into pXP-1 and pXP-2 vectors which
contain the luciferase gene as a reporter by the procedure reported
previously(25) . Mutations in the E-box element and potential
binding sites for Sp1 and Egr-1 were generated by the method of Kunkel et al.(26) using the 590-bp HindIII-SstI restriction fragment corresponding to
the Ache promoter sequences present in the construct C (Fig. 1), as a template. Mutations were checked by sequencing of
the entire restriction fragment before cloning into the luciferase
reporter plasmid. The construct pX1.720 (4) containing promoter
sequences of the nAChR
-subunit fused to luciferase was used as a
control.
Figure 1:
Promoter elements
and deletion analysis of the acetylcholinesterase gene. The 5`
noncoding sequence spanning from -867 to +1575 bp containing
promoter sequences, the first exon, and the first intron of the mouse Ache gene, was fused to the luciferase reporter gene in the
pXP2 vector (construct A). Constructs B, C, D, E, and F were generated
from A by deletion of the intronic region and nested deletions from its
5` end. The promoter of the
-subunit of the nAChR was
assayed in parallel as a positive control of muscle-specific
activation. Promoter activities were estimated by luciferase values
normalized to
-galactosidase and are reported here by the ratio to
the activity of the longest Ache construct (A) or of
the
-AChR construct in 10T1/2 cells. The activity of
construct A is 5.1 ± 1.8 times greater than the
-AChR construct in 10T1/2 cells. Means and S.E. of three experiments. No
myogenic transactivation of the Ache promoter is observed in
10TFL2-3 cells. Promoter activity of Ache is retained in
the region -105 to +129 bp. Consensus sites for
transcription factors in this region are
shown.
Cell Transfections
Typically, reporter gene
plasmids (15 µg) were transfected into cells on 100-mm plates. For
constructs with different sizes than construct C (6.5 kb), quantities
were adjusted to yield the same number of picomoles of expressing
plasmid per transfection, and plasmid of the empty vector were added to
correct for differences in amount of DNA. For transactivation
experiments, unless otherwise mentioned, 5 µg of the expression
plasmid of the respective transcription factor were cotransfected:
myogenin (kindly provided by Dr. Eric Olson, M. D. Anderson Hospital), myoD, myf5 (a gift from Dr. H. H. Arnold, Technishe
Universität Braunschweig), and MRF4 (a
gift from Dr. S. Konieczny, Purdue University). cDNAs were in the
pEMSVscribe expression vector(8) . Egr-1 and WT1 cDNAs were in
a Rous sarcoma virus-long terminal repeat expression vector; Sp1 (a
gift from Dr. Jeff Saffer, Pacific Northwest Laboratories, Richland,
WA) was in pSV2A101 under the control of the SV40 early promoter. In
all experiments, 5 µg of cytomegalovirus-lacZ containing the
cytomegalovirus promoter linked to
-galactosidase was
cotransfected as an internal control for transfection efficiency.
10T1/2 and 10TFL2-3 cells were transiently transfected using
Ca
(PO
)
precipitation(27) .
Briefly, cells were seeded at 10
cells/10-cm plate the day
before and were refed with fresh medium 3 h prior to transfection. The
DNAs were combined and added to the cells as a
Ca
(PO
)
precipitate. Four hours
after transfection, cells were submitted to a 15% glycerol shock for 1
min, washed twice with 5 ml of phosphate-buffered saline, pH 7.4, once
with 5 ml of Dulbecco's modified Eagle's medium plus 2%
horse serum and 0.5% fetal bovine serum (differentiation medium), and
incubated overnight. Cells were refed 24 h later. Luciferase activities
were determined 48 h after transfection and normalized to
-galactosidase activities.
Electrophoretic Mobility Shift Assays
Nuclear
extracts were prepared as described by Schreiber et
al.(28) . Protein concentrations in the extracts were
estimated using BCA reagent (Pierce). The probes used were NarI-SmaI restriction fragments (-105 and
-60 bp) excised from construct D (Fig. 1) or from an
equivalent construction bearing mutations at the sites Sp1 A and Sp1 B.
These fragments were labeled by end-filling using the Klenow fragment
of DNA polymerase I in the presence of [
P]dCTP.
A Sp1 oligonucleotide (Promega) with the sequence
5`-ATTCGATCGGGGCGGGGCGAGC-3` was used as an unlabeled competing
oligonucleotide. Antibodies against Sp1 and Egr-1 and blocking peptides
were from Santa Cruz Biotechnology Inc.Typically, each reaction
contained 5 µg of nuclear extract, the antibodies or unlabeled
competitor oligonucleotide where specified, 5 µg of bovine serum
albumin, 4 µg of poly(d(I-C)) in 12% glycerol, 12 mM Hepes, pH 7.9, 4 mM Tris, pH 7.9. After a 10-min
incubation on ice, 20,000 cpm of the
P radiolabeled probe
were added. The mixture was then incubated for 15 min at 30 °C. The
complexes were resolved from free DNA by electrophoresis on a
pre-electrophoresed 6% acylamide gel in 1
TGE (Tris
glycine-EDTA pH 8.5) buffer at 4 °C.
Nuclear Run-on Transcription
Nuclear run-on
transcription assays were performed essentially as described
previously(7) . Briefly, nuclei were allowed to terminate
transcription in the presence of [
P]UTP and the
RNA was subsequently isolated. Typically, 2
10
cpm
of radiolabeled mRNA were hybridized to slot blots onto which 5 µg
of plasmid inserts or phage DNA had been immobilized. Single-stranded
M13 DNAs containing a 2.1-kb AChE insert and corresponding to separate
coding and noncoding strands were used. Background hybridization was
estimated using M13 single-stranded DNA without insert. A 2.1-kb AChE
cDNA insert was excised from pBluescript with an EcoRI-KpnI cleavage (construct 19-3). A 1.8-kb EcoRI restriction fragment containing the cDNA for the mouse nAChR
-subunit was removed from the pSP65 vector (clone
obtained from Drs. Jim Boulter and Steve Heinemann, Salk Institute, La
Jolla, Ca). A mouse
-tubulin 1.4-kb insert was cut out of
pBluescript with PstI. EcoRI fragments corresponding
to a 1.5-kb myogenin cDNA were removed from pEMSV-myo8. A 1.8-kb EcoRI fragment corresponding to the myoD cDNA was
isolated from pEMSVscribe.
RNase Protection
The templates used for in
vitro transcription are: for Ache, a cDNA fragment
corresponding to exons 4 and 6, subcloned in a pBluescript SKII plasmid
and linearized with XhoI; for the AChR
-subunit,
a cDNA fragment subcloned in pSP65 linearized by XhoI; and for myoD, a cDNA fragment subcloned in a pBluescript SKII and
linearized with NarI. The U1 antisense RNA probe was
transcribed from a human cDNA cloned in pSP65 and linearized with HindIII(29) .Protection experiments were performed
essentially as described in Ausubel et al.(30) .
Hybridization of 40 µg of total RNA with 5
10
cpm of the probe was performed at 60 °C overnight. RNase A
(10 µg/ml) and RNase T1 (2 µg/ml) were used for the digestions.
Forty µg of tRNA were included in the assays to assess nonspecific
hybridization.
RESULTS
Sequence Analysis of the Promoter Region
The
sequence of the Ache promoter region spanning from bp
-416 to +129 was previously reported(25) . Here, we
have extended the sequence from bp +129 to 1575 (submitted to
GenBank). Analysis of consensus sites for transcription factors
revealed the presence in the promoter region of one E-box consensus
site at -335 bp, a GC-rich region at bp -95 to -59
containing Sp1 and Egr-1 sites, a AP2 site directly upstream of the
start of transcription. Four additional E-boxes, two Sp1 sites, and an
AP2 site were also identified in the first intron between exons 1 and 2 (Fig. 1).
Deletion Analysis of the Promoter Region
We
generated a nested set of deletion constructs encompassing the Ache promoter region and ligated them to a luciferase reporter
gene in plasmids pXP1 and pXP2(31) . A construct containing
promoter sequence of the nAChR
-subunit was included as a
positive control of muscle-specifically activated promoter. These
constructs were transiently transfected in the C3H10T1/2 mouse
fibroblastic cell line (10T1/2) and in the cell line C3H10TFL2-3B
(10TFL2-3; a 10T1/2 line stably transfected with myogenin) to
assess a possible effect of myogenin on transcription from the Ache promoter. Luciferase activities were measured 48 h later and
normalized to
-galactosidase activities. The activity of the Ache promoter-reporter fusion gene compared to that of the nAChR
-subunit promoter was reproducibly about 5-fold
higher in the 10T1/2 cells (Fig. 2), and equivalent or lower in
the 10TFL2-3 cells. Activities of the deletion constructs are
shown as a percent of activity of the longest Ache construct (Fig. 1). Construct A which contains the longest promoter
sequence had similar activity in the two cell lines 10T1/2 and
10TFL2-3, while transcription from the
-AChR promoter was 5-7-fold greater in the myogenin expressing
cell line 10TFL2-3, than in the 10T1/2 cells. This indicates that
the E-boxes present in the promoter region and in the first intron are
not functional in the Ache system. Consistently, deletion of
the regions containing the E-boxes in constructs C and D had no
significant effect. Deletion of the first intron did not alter the
promoter activity, arguing that the intronic region is devoid of
essential regulatory elements. In fact, a 1.5-2-fold increase of
the promoter activity is observed when constructs C and D are compared
to constructs A and B in the 10T1/2 cell line. Deletions in the 5`
portion of the promoter region show that construct D (-105 to
+129 bp) retains most of the promoter activity, whereas deletion
of the region from -105 to -59 bp (construct E) results in
an almost complete loss of the promoter activity (Fig. 1).
Hence, this region of 47 bp is likely to contain the important
regulatory elements.
Figure 2:
Transactivation of Ache and
-AChR promoters by transient expression of myogenin.
Constructs containing the promoter regions of AChE (-867 to
+1575 bp) and AChR
-subunit fused to the luciferase reporter
gene were transiently cotransfected with increasing amounts of myogenin
expression vector in 10T1/2 mouse fibroblasts. Luciferase activities
were measured 48 h after transfection and normalized to
-galactosidase activities. Activation ratios relative to the
activity without cotransfection of myogenin were calculated. Means
± S.E. of three experiments are
plotted.
Transient Transactivation with Myogenic Transcription
Factors
To test whether the Ache promoter could be
transactivated by muscle-specific myogenic factors, we transiently
coexpressed construct A (Fig. 1) with increasing amounts of myoD, myogenin, MRF4, and myf5 expression
plasmids in the 10T1/2 fibroblastic cell line. Slight transactivation
of the Ache promoter was observed, but only at the highest
concentrations of the plasmids expressing the myogenic factors (as
shown for myogenin in Fig. 2). In contrast, the promoter of the
-subunit of AChR gene,
-AChR, fused to the
luciferase reporter was readily transactivated at far lower
concentrations of myogenic factors. These results indicate that the
myogenic factors exert minimal influence on the Ache promoter
in what is presumed to be concentrations found in situ.
Transcription Rates of Ache and
AChR
To
compare further the expression of the two genes, we examined by nuclear
run-on assays the transcription rate of the Ache gene in
10T1/2, differentiated 10TFL2-3 cells, C2C12 myoblasts, and C2C12
myotubes. Transcription rates of
-AChR, myogenin, and myoD were assayed in parallel.
-Tubulin was included to
normalize for RNA input (Fig. 3A). Data obtained from
the densitometric analysis, and normalized for
-tubulin densities
are shown in Fig. 3B. The Ache gene appears
transcriptionally active in all the cell lines examined and shows
comparable transcription rates, whereas transcription of the
-AChR gene is undetectable in 10T1/2, active in the
myogenin-expressing derivative of 10T1/2 cells (10TFL2-3), very
low in C2C12 myoblasts, and increased by about 4-fold in the
differentiated C2C12 myotubes compared to C2C12 myoblasts. These
relative transcription rates for Ache and
-AChR in 10T1/2 and 10TFL2-3B cells correlate with the promoter
activities observed using the luciferase reporter gene. To insure that
the majority of hybridization for the Ache mRNA was due to the
sense strand transcribed by RNA polymerase II, we compared the sense
and antisense DNA strands of the Ache coding sequences (not
shown). More than 95% of the signal was found to be due to
hybridization with the antisense strand DNA. Therefore, we conclude
that the Ache promoter is transcribed at similar rates in the
various differentiated and undifferentiated cells and is not
transcriptionally activated appreciably either at the myogenic stage of
determination yielding a myoblast, or during further differentiation in
which myotubes are formed. We also examined the transcription rate of
the Ache gene in NIH3T3 fibroblasts to insure that
transcription of Ache gene is not unique to 10T1/2 cells.
Virtually the same rate of transcription of the Ache gene was
observed in these cells (not shown).
Figure 3:
Rates of transcription of Ache, AChR,
myoD, and myogenin genes. Nuclei harvested from 10T1/2,
10TFL2-3, C2C12 myoblasts (MB), and C2C12 myotubes (MT) were allowed to terminate transcription of initiated
transcripts in the presence of [
P]UTP. Labeled
RNA was purified and hybridized to slot blots containing 5 µg of
the appropriate DNA. Inserts corresponding to the cDNA sequences for
AChE, AChR
-subunit, MyoD, myogenin, and
-tubulin were used. A, representative autoradiogram. B, quantification of
the signals was performed by densitometric analysis. Densities were
normalized with respect to
-tubulin hybridization and standardized
for each gene to the value for 10TFL2-3 cells. Values shown are
means ± S.E. of three experiments.
Myogenin and AChE mRNA Stabilization
We tested
whether the constitutive expression of myogenin in the 10TFL2-3
cell line was able to induce stabilization of the Ache mRNA
upon differentiation of these cells via serum deprivation. After
3-4 days in low serum medium, 10TFL2-3 cells undergo
morphological changes in that they elongate and fuse, although not to
the same extent as C2C12 cells. Ribonuclease protection assays, carried
out with RNA isolated from nondifferentiated and differentiated
10TFL2-3 cells, show that mRNAs for muscle-specific genes as, for
example, myoD and the
-subunit of the AChR, are
virtually undetectable in the undifferentiated cells, and accumulate
during differentiation to a level comparable to that observed in C2
myotubes (Fig. 4). In contrast, while AChE mRNA accumulates via
stabilization during C2 muscle cell differentiation, this phenomenon
does not occur during 10TFL2-3 differentiation even after 9 days
in low serum medium, indicating that the regulatory factors of the
myogenic helix-loop-helix family are not able alone to trigger the
mechanism responsible for stabilization of Ache mRNA observed
in differentiated muscle cells.
Figure 4:
Ache, AChR
-subunit, and myoD mRNA levels. Ache and AChR
-subunit and myoD mRNA levels were determined in a
ribonuclease protection assay in the cell lines 10T1/2, 10TFL2-3
myoblasts, 10TFL2-3 cells differentiated for 6, 7, and 9 days, C2
myoblasts (MB), and C2 myotubes (MT). An antisense
probe for U1 was used as an internal control. tRNA was included as a
control of nonspecific hybridization.
Mutation Analysis of Sp1 and Egr-1 Sites
Deletion
analysis of the Ache promoter pointed to a region spanning
-105 to -59 bp, essential for promoter activity (Fig. 1). Sequence in this region shows a GC-rich segment
containing three consensus sites for the transcription factor Sp1
(GGGCGG) (32, 33, 34) and two consensus sites
for Egr-1 (GCGGGGGCG) which overlap Sp1 sites (Fig. 5A). To assess the role of the Sp1 and Egr-1
sites in the promoter activity, mutations were introduced in construct
C as shown in Fig. 5B in order to disrupt sequentially
the Sp1 and the Egr-1 sites. Promoter activities were compared to that
of the wild-type construct C by transfecting into 10T1/2 and
10TFL2-3 cells. Luciferase activity normalized to
-galactosidase activity is shown in Fig. 5C as a
ratio to the activity of the wild-type sequence. We find that mutations
in the Sp1 sites dramatically decrease the activity of the promoter to
15% of residual activity when all three sites are mutated; a 75%
decrease is observed for the Sp1AB mutant and a 50% decrease for the
Sp1C mutant. In contrast, mutations of the Egr-1 sites have little
effect on the promoter activity; there is no significant change upon
mutation of Egr-1B; if anything, a slight increase is observed with
Egr-1C mutation. The mutation could either facilitate the access of Sp1
to its site by virtue of insertion of two additional base pairs between
the two sites or eliminate the competition between Egr-1, being a poor
activator, and Sp1. Hence, Sp1 appears to play an important role of
activator of transcription, whereas Egr-1 might influence transcription
by competing with the binding of Sp1. Quantitatively similar reductions
were observed in the cell line 10TFL2-3 (data not shown).
Figure 5:
Mutation of promoter elements of the AChE
gene. A, nucleotide sequence of the minimal promoter region
spanning from -105 to 86 bp (relative to the primary Cap site).
The transcription start sites previously determined by primer extension
are indicated by squares, and the start sites determined by S1
nuclease protection by oval marks(25) . The major Cap
sites are shown as solid symbols and the primary start site at
base 1 is indicated by an arrow. Consensus binding sites for
transcription factors Sp1, Egr-1, and AP2 are outlined. B,
mutations were introduced in the GC-rich region of the C construct in
order to sequentially disrupt the Sp1 and Egr-1 sites and assess their
respective roles in the regulation of promoter activity. C,
construct C and the mutant constructs were transiently transfected in
the 10T1/2 cell line along with cytomegalovirus-
-galactosidase
expression plasmid to correct for variation in the transfection
efficiency. Ratios of activities (mutant over wild-type) are shown for
each mutant.
Coexpression of Transcription Factors
To examine
the influences of excess Sp1 and Egr-1 factors on the promoter
activity, we cotransfected expression vectors for Sp1, Egr-1, and the
Wilms tumor factor (WT1) with the Ache promoter. WT1 is
closely related to and binds to the same consensus site as
Egr-1(35) . We found that coexpression of Sp1 has no effect on
the promoter activity, whereas the coexpression of Egr-1 or WT1
decreases the promoter activity of the wild-type reporter gene in a
dose-dependent manner. Activities of Egr-1B and Egr-1C mutants, each of
which leaves an Egr-1 site intact, were decreased as well, while
coexpression of Egr-1 with the Sp1 ABC mutant which lacks all Sp1 sites
had no effect (Fig. 6). These results seem to indicate that
endogenous Sp1 is present at near saturating concentrations and that an
increase in Sp1 concentration does not elicit further activation. In
contrast, an increase in the Egr-1 level by transfection has a
dose-dependent inhibitory response. Competition between Sp1 and Egr-1
for adjacent sites favors Sp1 perhaps because of a higher affinity or a
higher effective concentration.
Figure 6:
Concentration dependence of Egr-1
inhibition of transcription. The A (
) and C (
) wild-type
constructs and Egr-1B (
), Egr-1C (
), and Sp1ABC (
)
mutant constructs were transiently cotransfected with increasing
amounts of Egr-1 expression plasmid in 10T1/2 and 10TFL2-3 cells.
Residual promoter activities were determined 48 h after transfection by
measuring luciferase activity in the cell extracts. Cotransfection of a
-galactosidase expression vector was used to correct for
differences in transfection efficiency. For each construct, the values
plotted correspond to the ratios of residual activity over activity in
the absence of Egr-1. The activities of the wild-type and mutant
constructs shown in Fig. 4are scaled to 100%. Values correspond
to average of three experiments.
Gel Mobility Shifts to Examine Sp1 and Egr-1
Binding
We performed electrophoretic mobility shift assays to
ascertain the Sp1 and Egr-1-like binding activities present in the cell
nuclei. We used a double stranded DNA fragment corresponding to the SmaI-NarI restriction fragment (bp -105 to
-59) which contains the Sp1 and Egr-1 sites (Fig. 7, lanes 1-8). Alternatively the same fragment from the
Sp1AB mutant was used as a probe (Fig. 7, lanes
9-16). End-labeled fragments were incubated with nuclear
extracts prepared from 10T1/2 and 10TFL2-3 cells. A major complex
was observed (lane 2) that could be supershifted with specific
antibodies against Sp1 (lane 3) but not with antibodies
against Egr-1 (lane 7) and could be competed with a unlabeled
oligonucleotide bearing a Sp1 site (lanes 5 and 6).
Formation of this complex is dramatically reduced when the Sp1AB mutant
probe is used (lane 10). The residual complex that forms can
still be supershifted by the Sp1 antibodies and presumably corresponds
to the binding to the Sp1C site (lane 11). Besides the major
complex, a faster migrating complex is observed. This complex becomes
more evident when the Sp1AB mutant probe is used (lanes 10-14 and 16). This complex can be supershifted using specific
antibodies against Egr-1 (lane 15) but not with antibodies
against Sp1 (lane 11). Moreover, it could not be competed by
the unlabeled Sp1 oligonucleotide. Blocking peptides specific for the
Sp1 (s) and Egr-1 (e) antibodies were used to assess
the specificity of the supershifts (s, lanes 4 and 12; e, lanes 8 and 16). These peptides
prevent the antibodies from supershifting the complexes (lanes 4,
8, 12, and 16). However, for some unexplained reason,
when these peptides were used, a reduction in the complex formation
with Sp1, but not with Egr-1, was observed.
Figure 7:
Influence of Sp1 and Egr-1 binding on gel
mobility shifts. A SmaI-NarI 45-bp restriction
fragment spanning from -105 to -60 bp and containing the
Sp1 and Egr-1 sites was excised from the wild-type or the Sp1AB mutant
construct and labeled by end-filling using Klenow in the presence of
[
P]dCTP. Wild-type (w, lanes 1-8)
or the Sp1 AB mutant (m, lanes 9-16) probes were
incubated with or without 10T1/2 cell extracts. Specific antibodies for
Sp1 (S, lanes 1, 3, 4, 5, and 9) or Egr-1 (E,
lanes 1, 7, 8, 9, 15, and 16) were used to supershift the
complexes. Blocking peptides specific for the Sp1 antibody (s,
lanes 4 and 12), or the Egr-1 antibody (e, lanes 8 and 16), were used to assess the specificity of the
supershift. An unlabeled Sp1 oligonucleotide was used as a competitor (lanes 5, 6, 13, and 14).
DISCUSSION
Transcriptional Control of the AChE Gene Is Distinct
from Other Muscle-specific Genes
Contrary to other
muscle-specific genes (nicotinic AChR subunits, muscle creatine kinase,
and myosin light chain) which are transcriptionally activated during
myogenesis when myoblasts exit the cell cycle and fuse to form
myotubes, the Ache gene is actively transcribed in myoblasts
as well as in myotubes. The increase in the expression of Ache during myogenesis was shown to occur via stabilization of the
transcript(7) .In the present report, we have examined
transcription of this gene at an earlier step of the cell commitment to
muscle, using myogenic transcription factors (MyoD, myogenin, Myf-5,
and MRF4) to commit other cell types into muscle cells. We compared the
fibroblastic cell line 10T1/2 to its counterpart that constitutively
expresses myogenin, 10TFL2-3, and to the muscle cell line C2C12.
We find that the Ache gene is transcribed at a similar rate in
all cells analyzed (10T1/2 mouse fibroblasts whether or not they
express myogenin, C2C12 mouse muscle cells, and NIH3T3 fibroblasts).
The promoter of the nAChR
-subunit, used as a positive
control, is silent in 10T1/2, 3T3 fibroblasts and C2C12 myoblasts,
however, it is specifically activated in 10TFL2-3 cells (10T1/2
cells transfected with myogenin) and C2-myotubes.
Multiple E-box
motifs are commonly found in the proximal 250 bp of regulatory regions
of muscle-specific genes. Only one E-box consensus site (CANNTG) is
present in the Ache promoter at -335 bp from the primary
start of transcription. This site does not match the usual consensus
sequences defined for the myogenic factors(16) . Four
additional E-boxes are found in the sequence of the first intron. The
presence of these sites appears not to confer muscle specific activity
to the promoter, and deletion or mutation (data not shown) of these
sites did not significantly change the transcription characteristics of
the promoter. Some muscle-specific genes, such as skeletal
-actin (36) and myosin heavy chain(37) , do not contain
E-boxes within their regulatory sequences, but are nevertheless
activated in response to the myogenic factors through intermediate
muscle-specific transcription factors such as MAPF2(38) ,
MEF-2(39) , or MCAT-binding factor(40) . This does not
seem to be the case of the Ache gene.
The Ache promoter is devoid of CCAAT or TATA-box sequences (25) , a
feature often encountered for growth factor and housekeeping genes.
AChE differs from the other muscle-specific proteins in that it is also
expressed in several other tissues including neurons, muscle and
hematopoietic cells. It is probable that this more diverse tissue
distribution requires different mechanisms for tissue-specific
expression. From our observations, it seems that the gene is
ubiquitously transcribed, and that the transcript is readily degraded
in most tissues, whereas it is specifically stabilized in the tissues
where transcripts can be detected.
AChE Expression in 10TFL2-3 Cells
The cell
line 10TFL2-3 constitutively expresses myogenin. In these cells,
myogenin was shown to be located in the nucleus, but not to bind DNA in
the presence of serum due to the expression of the inhibitory protein
Id that forms dimers with myogenin that are unable to bind
DNA(24) . In low-serum conditions, Id is repressed allowing
myogenin to bind to E-boxes and to activate the transcription of a
large array of muscle-specific genes and these cells terminally
differentiate into multinucleated myotubes. Indeed, during
differentiation of the 10TFL2-3 cells, we observe that the mRNA
levels for endogenous myoD and the
-subunit of the AChR
increase, via transcriptional activation, to levels comparable to that
observed in C2 cells. In contrast, transcription of Ache is
not enhanced in the presence of myogenin, and appears similar in all
cell lines analyzed. However, of these cell lines, only C2 myotubes
express AChE and show detectable Ache message. No mRNA for Ache could be detected in 10TFL2-3 cells, indicating
that the myogenic transcription factors, although able to transactivate
other muscle-specific genes, do not, or are insufficient, to mediate
stabilization of the Ache mRNA. Other transcription factors
have been identified, including members of the
MADS-box(41, 42) , homeo-domain(43) , and
winged-helix (44) families of transcription factors, that are
known or suspected to play an important role in skeletal muscle
differentiation. A role for 3`-untranslated regions of several
muscle-specific genes was also reported(45) . From these recent
studies, additional elements are necessary to express the complete
repertoire of genes that define the skeletal muscle phenotype. This may
also apply to the factors involved in the stabilization of the Ache mRNA.
Sp1/Egr-1 Competition
We find that a region
starting at -105 bp from the start of transcription retains
substantial promoter activity whereas further deletion to -59 bp
dramatically reduces promoter activity (Fig. 1). This region has
multiple GC boxes with potential Sp1 and Egr-1 binding sites that
partially overlap (Fig. 5A). Mutation analysis of these
sites reveals that the interaction of Sp1 is essential for the promoter
activity (Fig. 5C). This interaction is confirmed by
gel mobility shift assays and supershifts using a specific antibody to
Sp1 (Fig. 7). GC-rich sequences have been shown to play a
critical role in controlling the expression of housekeeping genes and
cellular oncogenes (46, 47, 48, 49) . Sp1 is a
ubiquitously expressed transcription factor and the presence of Sp1
sites around 50 bp upstream of the start of transcription has been
described in several TATA-less promoters (50, 51, 52, 53) and is thought to
direct site-specific initiation of transcription, in place of the TATA
box, by tethering the TFIID factor to DNA and stabilizing the
preinitiation complex through protein-protein interactions. Multiple
Sp1 sites may direct transcription from different start sites. Usually,
mutation of these sites was found to decrease promoter activity
dramatically(51, 52) .In contrast to the Sp1
sites, disruption of the Egr-1 sites causes no appreciable change in
the promoter activity except for perhaps a slight increase seen for the
Egr-1 C mutant (Fig. 5C). This may be due to
elimination of competition with the binding of Sp1. The notion of
competition between Sp1 and Egr-1 is supported by the observation of an
inhibitory influence of Egr-1 on the promoter activity (Fig. 6).
Binding of Egr-1 is indeed observed in this region and is supershifted
with a specific antibody (Fig. 7). This complex is less abundant
than the Sp1 complex which may reflect a lower nuclear concentration or
binding affinity. However, in the absence of the overlapping Sp1 sites
(probe from the Sp1AB mutant), the formation of the Egr-1 complex was
greatly enhanced, further substantiating the possibility of a
competition between the two factors in the cell.
Overlap of binding
sites for other factors with Sp1 sites is a feature shared in several
genes. For instance, in the chicken nAChR
-subunit
promoter, GBF is thought to interfere with the binding of Sp1 in
myoblasts and non-muscle cells(54) . Other GC-box binding
factors such as ETF or C2A were also found to bind to sites overlapping
with Sp1 sites(55, 56) .
This competition may be of
physiological importance, for instance in mediating the increase in
expression of AChE and nAChR in extrajunctional areas following
denervation(57) . Egr-1 mRNA levels, on the other hand, were
reported to increase dramatically following cell membrane
depolarization (58) and could be involved in the
down-regulation of extrajunctional AChE and AChR subunits by electrical
activity.
Role of AP2
A consensus sequence for the binding
of AP2 (59) was found immediately upstream of the start site of
transcription previously identified by primer extension and S1 nuclease
analysis (Fig. 5A)(25) . Our studies show that
AP2 has an inhibitory effect on the promoter activity, (
)although a specific response mediated through this site
remains to be established. It is possible that binding to this site
interferes with the transcription given its proximity to the main cap
site. Whether AP2 influences the transcription of the Ache gene in a qualitative, by selection of the start site, or
quantitative manner is currently being investigated.From our
studies, it appears that Ache is not transcriptionally
regulated by the E-box myogenic factors, nor do these factors seem to
be involved in the mechanism of Ache mRNA stabilization during
terminal differentiation. We find that the factor Sp1 is essential for
the activity of the promoter and that overlapping binding sites for
Egr-1 provide a potential mechanism for precise regulation of this
promoter.