©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Promoter Elements of the Mouse Acetylcholinesterase Gene
TRANSCRIPTIONAL REGULATION DURING MUSCLE DIFFERENTIATION (*)

(Received for publication, September 15, 1994; and in revised form, November 2, 1994 )

Annick Mutero Shelley Camp Palmer Taylor

From the Department of Pharmacology, University of California, San Diego, La Jolla, California 92093-0636

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The increase in acetylcholinesterase expression during muscle differentiation from myoblasts to myotubes was shown previously to reflect primarily a greater stability of the messenger RNA (mRNA). Here, we investigate the regulation of the acetylcholinesterase gene during early determination of the muscle phenotype. (i) We employ myogenic transcription factors to transform non-muscle cells into myoblasts in order to assess the role of the myogenic transcription factors in this regulation. (ii) We analyze the Ache promoter region by deletion analysis, point mutagenesis, and gel mobility shift assays. The myogenic transcription factors do not accelerate transcription of the Ache gene in spite of the presence of E-boxes at -335 base pairs from the start of transcription and in the first intron, and they are not able to trigger stabilization of the Ache mRNA when constitutively expressed in 10T1/2 fibroblasts. A GC-rich region (at -105 to -59 base pairs from the start of transcription) containing overlapping binding sites for the transcription factors Sp1 and Egr-1 is essential for promoter activity. Mutation of the Sp1 sites dramatically reduces the promoter activity while mutation of the Egr-1 sites has little effect. Sp1 and Egr-1 compete for binding to overlapping sites and an increase in Egr-1 decreases the expression of the Ache gene.


INTRODUCTION

The best characterized function of acetylcholinesterase (AChE, EC 3.1.1.7) (^1)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 alpha, , , 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(2) 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 beta-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 beta-galactosidase was cotransfected as an internal control for transfection efficiency. 10T1/2 and 10TFL2-3 cells were transiently transfected using Ca(3)(PO(4))(2) precipitation(27) . Briefly, cells were seeded at 10^6 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(3)(PO(4))(2) 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 beta-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 times 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 times 10^6 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 alpha-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 times 10^5 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 beta-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 beta-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. alpha-Tubulin was included to normalize for RNA input (Fig. 3A). Data obtained from the densitometric analysis, and normalized for alpha-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 alpha-tubulin were used. A, representative autoradiogram. B, quantification of the signals was performed by densitometric analysis. Densities were normalized with respect to alpha-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 beta-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-beta-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 (box) and C () wild-type constructs and Egr-1B (circle), Egr-1C (Delta), and Sp1ABC (circle) 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 beta-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 alpha-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 alpha-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, (^2)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.


FOOTNOTES

*
This work was supported by fellowships from the International Brain Research Organization and Conseil Regional Provence Alpes Côte d'Azur (to A. M.) and United States Public Health Service GM 18360 (to P. T.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) L06620 [GenBank]and L37723[GenBank].

(^1)
The abbreviations used are: AChE, acetylcholinesterase; nAChR, nicotinic acetylcholine receptor; bp, base pair(s); kb, kilobase(s).

(^2)
A. Mutero, unpublished data.


ACKNOWLEDGEMENTS

We thank Dr. Eric Olson for providing us with the 10TLF2-3 cell line.


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