Role of Intronic E- and N-box Motifs in the Transcriptional Induction of the Acetylcholinesterase Gene during Myogenic Differentiation (2001)

In this study, we examined whether an intronic N-box motif is involved in the expression of acetylcholinesterase (AChE) during myogenesis. We determined that AChE transcripts are barely detectable in cultured myoblasts and that their levels increase dramatically in myotubes. Nuclear run-on assays revealed that this increase was accompanied by a parallel induction in the transcriptional activity of the AChE gene. These changes in transcription were also observed in transfection experiments using AChE promoter-reporter gene constructs. Mutation of the intronic N-box at position +755 base pairs (bp) reduced by more than 70% expression of the reporter gene in myotubes. Disruption of an adjacent E-box, at position +767 bp, also reduced expression of the reporter gene following myogenic differentiation. Co-transfection experiments using AChE promoter-reporter gene constructs and a myogenin expression vector showed that expression of this regulatory factor increased expression of the reporter gene in myotubes. Although the AChE promoter contains multiple E-boxes, mutation of this intronic one was sufficient to prevent the myogenin-induced increase in reporter gene expression. Together, these results indicate that changes in AChE gene transcription occur during myogenesis and highlight the contribution of the intronic N- and E-box motifs in the developmental regulation of theAChE gene in skeletal muscle. In this study, we examined whether an intronic N-box motif is involved in the expression of acetylcholinesterase (AChE) during myogenesis. We determined that AChE transcripts are barely detectable in cultured myoblasts and that their levels increase dramatically in myotubes. Nuclear run-on assays revealed that this increase was accompanied by a parallel induction in the transcriptional activity of the AChE gene. These changes in transcription were also observed in transfection experiments using AChE promoter-reporter gene constructs. Mutation of the intronic N-box at position +755 base pairs (bp) reduced by more than 70% expression of the reporter gene in myotubes. Disruption of an adjacent E-box, at position +767 bp, also reduced expression of the reporter gene following myogenic differentiation. Co-transfection experiments using AChE promoter-reporter gene constructs and a myogenin expression vector showed that expression of this regulatory factor increased expression of the reporter gene in myotubes. Although the AChE promoter contains multiple E-boxes, mutation of this intronic one was sufficient to prevent the myogenin-induced increase in reporter gene expression. Together, these results indicate that changes in AChE gene transcription occur during myogenesis and highlight the contribution of the intronic N- and E-box motifs in the developmental regulation of theAChE gene in skeletal muscle. acetylcholinesterase GA-binding protein reverse transcription-polymerase chain reaction base pair(s) kilobase(s) N-box-containing rat AChE promoter phosphate-buffered saline tibialis anterior chloramphenicol acetyltransferase dithiothreitol phenylmethylsulfonyl fluoride electrophoretic mobility shift assay Acetylcholinesterase (AChE)1 is widely recognized as an essential component of cholinergic synapses. In both the central and peripheral nervous systems, it is responsible for the hydrolysis of acetylcholine released from nerve terminals thereby ensuring the efficiency of synaptic transmission. Although a single gene encodes AChE, several molecular forms can be generated as a result of alternative splicing and distinct post-translational processing (for reviews, see Refs. 1Massoulié J. Pezzementi L. Bon S. Krejci E. Vallette F.-M. Prog. Neurobiol. 1993; 41: 31-91Crossref PubMed Scopus (1032) Google Scholar, 2Taylor P. Radic Z. Ann. Rev. Pharmacol. Toxicol. 1994; 34: 281-320Crossref PubMed Scopus (597) Google Scholar, 3Legay C. Microsc. Res. Tech. 2000; 49: 56-72Crossref PubMed Scopus (77) Google Scholar). It has been previously suggested that this polymorphism allows for the expression of AChE catalytic subunits in different cell types and at various subcellular locations where specific molecular forms may perform site-specific functions (4Toutant J.P. Massoulié J. Kenney A.J. Turner A.J. Mammalian Ectoenzymes.in: Elsevier, Amsterdam1987: 289-328Google Scholar).In skeletal muscle, AChE expression is known to be markedly influenced by the state of differentiation and innervation of the muscle fibers. Numerous studies have indeed reported dramatic changes in the expression of the enzyme during myo- and synapto-genesis as well as in mature muscles following alterations in their normal levels of neuromuscular activity and/or in their basic supply of trophic factors. Given that AChE is an excellent marker of synaptic differentiation, several groups have recently begun to decipher the molecular basis underlying these changes in AChE expression. For example, increases in AChE mRNA expression have previously been reported during myogenic differentiation (5Fuentes M.E. Taylor P. Neuron. 1993; 10: 679-687Abstract Full Text PDF PubMed Scopus (62) Google Scholar, 6Jbilo O. L'Hermite Y. Talesa V. Toutant J.P. Chatonnet A. Eur. J. Biochem. 1994; 225: 115-124Crossref PubMed Scopus (42) Google Scholar, 7Grubic Z. Komel R. Walker W.F. Miranda A.F. Neuron. 1995; 14: 317-327Abstract Full Text PDF PubMed Scopus (47) Google Scholar, 8Legay C. Huchet M. Massoulié J. Changeux J.P. Eur. J. Neurosci. 1995; 7: 1803-1809Crossref PubMed Scopus (59) Google Scholar, 9Luo Z.D. Wang Y. Werlen G. Camp S. Chien K.R. Taylor P. Mol. Pharmacol. 1999; 56: 886-894Crossref PubMed Scopus (30) Google Scholar). Moreover, activity-induced changes in AChE transcript levels have been observed in both cultured myotubes as well as in skeletal muscle in vivo (10Cresnar B. Crne-Finderle N. Breskvar K. Sketelj J. J. Neurosci. Res. 1994; 38: 294-299Crossref PubMed Scopus (42) Google Scholar, 11Michel R.N. Vu C.Q. Tetzlaff W. Jasmin B.J. J. Cell Biol. 1994; 127: 1061-1069Crossref PubMed Scopus (80) Google Scholar, 12Sveistrup H. Chan R. Jasmin B.J. Am. J. Physiol. 1995; 269: C856-C862Crossref PubMed Google Scholar, 13Boudreau-Larivière C. Chan R.Y.Y. Jasmin B.J. J. Neurochem. 2000; 74: 2250-2258Crossref PubMed Scopus (29) Google Scholar, 14Rossi S.G. Vasquez A.E. Rotundo R.L. J. Neurosci. 2000; 20: 919-928Crossref PubMed Google Scholar).Despite these recent advances in our understanding of some of the biosynthetic events regulating AChE expression in skeletal muscle, our knowledge of the specific molecular mechanisms that account for these changes in mRNA expression remains largely unknown. Nonetheless, alterations in the stability of pre-synthesized AChE transcripts have been suggested to account for at least a portion of the changes in mRNA levels seen during myogenic differentiation (5Fuentes M.E. Taylor P. Neuron. 1993; 10: 679-687Abstract Full Text PDF PubMed Scopus (62) Google Scholar, 9Luo Z.D. Wang Y. Werlen G. Camp S. Chien K.R. Taylor P. Mol. Pharmacol. 1999; 56: 886-894Crossref PubMed Scopus (30) Google Scholar) and following muscle denervation (13Boudreau-Larivière C. Chan R.Y.Y. Jasmin B.J. J. Neurochem. 2000; 74: 2250-2258Crossref PubMed Scopus (29) Google Scholar). On the other hand, the contribution of transcriptional regulatory mechanisms has also been documented in several studies that examined expression of the AChE gene by nuclear run-on assays (13Boudreau-Larivière C. Chan R.Y.Y. Jasmin B.J. J. Neurochem. 2000; 74: 2250-2258Crossref PubMed Scopus (29) Google Scholar, 15Rimer M. Randall W.R. Biochem. Biophys. Res. Commun. 1999; 260: 251-255Crossref PubMed Scopus (8) Google Scholar) and promoter analyses (16Mutero A. Camp S. Taylor P. J. Biol. Chem. 1995; 270: 1866-1872Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 17Chan R.Y. Boudreau-Larivière C. Angus L.M. Mankal F.A. Jasmin B.J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4627-4632Crossref PubMed Scopus (79) Google Scholar, 18Culetto E. Combes D. Fedon Y. Roig A. Toutant J.P. Arpagaus M. J. Mol. Biol. 1999; 290: 951-966Crossref PubMed Scopus (38) Google Scholar). Together, these studies indicate therefore, that both transcriptional control mechanisms as well as post-transcriptional events contribute to the regulation of AChE mRNA levels in skeletal muscle cells.In this context, we have recently begun to examine the mechanisms underlying the preferential accumulation of AChE transcripts within the postsynaptic sarcoplasm of muscle fibers (see Refs. 11Michel R.N. Vu C.Q. Tetzlaff W. Jasmin B.J. J. Cell Biol. 1994; 127: 1061-1069Crossref PubMed Scopus (80) Google Scholar, 17Chan R.Y. Boudreau-Larivière C. Angus L.M. Mankal F.A. Jasmin B.J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4627-4632Crossref PubMed Scopus (79) Google Scholar, 19Jasmin B.J. Lee R.K. Rotundo R.L. Neuron. 1993; 11: 467-477Abstract Full Text PDF PubMed Scopus (90) Google Scholar). In these studies, we showed that the synaptic accumulation of AChE transcripts results, at least partially, from the local transcriptional activation of the AChE gene (17Chan R.Y. Boudreau-Larivière C. Angus L.M. Mankal F.A. Jasmin B.J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4627-4632Crossref PubMed Scopus (79) Google Scholar). By mutation/deletion analysis, we further demonstrated the key role of the first intron in regulating both the muscle-specific expression of the AChEgene as well as its preferential synaptic expression. In particular, our studies have shown the contribution of an intronic N-box motif and of the ets-related transcription factor GABP (see Refs.20Koike S. Schaeffer L. Changeux J.P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10624-10628Crossref PubMed Scopus (92) Google Scholar, 21Duclert A. Savatier N. Schaeffer L. Changeux J.-P. J. Biol. Chem. 1996; 217: 17433-17438Abstract Full Text Full Text PDF Scopus (89) Google Scholar, 22Fromm L. Burden S.J. Genes Dev. 1998; 12: 3074-3083Crossref PubMed Scopus (106) Google Scholar, 23Schaeffer L. Duclert N. Huchet-Dymanus M. Changeux J.-P. EMBO. 1998; 17: 3078-3090Crossref PubMed Scopus (135) Google Scholar, 24Gramolini A.O. Angus L.M. Schaeffer L. Burton E.A. Tinsley J.M. Davies K.E. Changeux J.P. Jasmin B.J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3223-3227Crossref PubMed Scopus (119) Google Scholar) in the synaptic regulation of the AChE gene in muscle fibers (17Chan R.Y. Boudreau-Larivière C. Angus L.M. Mankal F.A. Jasmin B.J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4627-4632Crossref PubMed Scopus (79) Google Scholar). Given the key role of this intronic N-box in the regulation of AChE in adult skeletal muscle, we sought in the present study to determine whether this DNA element also participates in the control of the AChE gene during myogenic differentiation. In addition, we also examined whether other sites, located within the first intron, are also important in controlling AChE expression by focusing on an adjacent E-box motif.DISCUSSIONMyogenic differentiation is a developmentally regulated process characterized by a series of coordinated biochemical and morphological changes accompanied by the fusion of mononucleated myoblasts into multinucleated myotubes. Together with cytoskeletal and contractile proteins, expression of several synaptic proteins, including AChR, N-CAM, utrophin, and AChE, is also enhanced to varying degrees during myogenesis. In this context, previous studies have shown that, in the case of AChR (34Buonanno A. Merlie J.P. J. Biol. Chem. 1986; 261: 11452-11455Abstract Full Text PDF PubMed Google Scholar, 35Evans S. Goldman D. Heinemann S. Patrick J. J. Biol. Chem. 1987; 262: 4911-4916Abstract Full Text PDF PubMed Google Scholar, 36Baldwin T.J. Burden S.J. J. Cell Biol. 1988; 107: 2271-2279Crossref PubMed Scopus (37) Google Scholar, 37Fontaine B. Changeux J.P. J. Cell Biol. 1989; 108: 1025-1037Crossref PubMed Scopus (107) Google Scholar) and utrophin (38Gramolini A.O. Jasmin B.J. Nucleic Acids Res. 1999; 27: 3603-3609Crossref PubMed Scopus (40) Google Scholar), the increased expression of mRNAs can partially be attributed to an increase in the rate of transcription of these genes. Here, we show that transcription of theAChE gene is also increased during the early phases of myogenic differentiation. In fact, it appears that during the initial stages of muscle cell development, the rate of transcription of theAChE gene correlates well with the pattern of mRNA expression (compare undifferentiated myoblasts, confluent myoblasts, and 2-day-old myotubes in Figs. 1 and 2). In agreement with our findings, Rossi et al. (39Rossi S.G. Vazquez A. Rotundo R.L. Soc. Neurosci. Abstr. 2000; 26: 1090Google Scholar) and R. L. Rotundo 2R. L. Rotundo, personal communication. have also reported recently an increase in the expression of the AChE gene that parallels the induction in AChE mRNA at early stages of chick muscle cell development. Thus, as originally suggested by Merlie and Sanes (40Merlie J.P. Sanes J.R. Nature. 1985; 317: 66-68Crossref PubMed Scopus (243) Google Scholar) as well as by Klarsfeld (41Klarsfeld A. Biochimie ( Paris ). 1987; 69: 433-437Crossref PubMed Scopus (5) Google Scholar), expression of genes encoding key synaptic proteins in muscle may indeed be coordinately regulated during myogenic differentiation.Together with the recent data obtained by Rossi et al. (39Rossi S.G. Vazquez A. Rotundo R.L. Soc. Neurosci. Abstr. 2000; 26: 1090Google Scholar) and Rotundo,2 our results appear in contrast to the earlier findings of Taylor and colleagues (5Fuentes M.E. Taylor P. Neuron. 1993; 10: 679-687Abstract Full Text PDF PubMed Scopus (62) Google Scholar, 9Luo Z.D. Wang Y. Werlen G. Camp S. Chien K.R. Taylor P. Mol. Pharmacol. 1999; 56: 886-894Crossref PubMed Scopus (30) Google Scholar). In their previous work, these investigators failed to detect an increase in the transcriptional activity of the AChE gene, thereby concluding that post-transcriptional mechanisms operating at the level of mRNA stability accounted for the increased AChE expression seen during myogenic differentiation. However, this discrepancy may be reconciled if we consider that, in their experimental approach, Taylor and co-workers focused on more advanced stages of myogenic differentiation during which maturational events are likely taking place. In support of this, it is important to note that we (present study) and Rossi et al. (39Rossi S.G. Vazquez A. Rotundo R.L. Soc. Neurosci. Abstr. 2000; 26: 1090Google Scholar) and Rotundo2 all observed a decrease in the transcriptional activity of theAChE gene in older myotubes, an observation entirely consistent with the results of Taylor and colleagues who also detected a slight reduction in the expression of the AChE gene in fully differentiated myotubes (5Fuentes M.E. Taylor P. Neuron. 1993; 10: 679-687Abstract Full Text PDF PubMed Scopus (62) Google Scholar). Taken together, these results indicate therefore that transcriptional mechanisms participate in the regulation of AChE mRNA expression during myogenic differentiation but only at the earliest stages.As stated above, the increase in AChE mRNA expression that occurred in confluent myoblasts and 2-day-old myotubes can be solely explained by an increase in the transcriptional activity of the AChEgene. This increase in transcription appeared transient, because it returned toward control levels in older myotubes. At that stage however, AChE mRNA levels were further increased indicating, therefore, that post-transcriptional mechanisms likely account for this sustained increase. In this context, Taylor and colleagues have also implied an important contribution for post-transcriptional mechanisms during the later stages of muscle differentiation (5Fuentes M.E. Taylor P. Neuron. 1993; 10: 679-687Abstract Full Text PDF PubMed Scopus (62) Google Scholar, 9Luo Z.D. Wang Y. Werlen G. Camp S. Chien K.R. Taylor P. Mol. Pharmacol. 1999; 56: 886-894Crossref PubMed Scopus (30) Google Scholar). In agreement with this view, it is relevant to note that recent studies performed in our laboratory have also illustrated the key role of the AChE 3′-untranslated repeat and of distinct RNA-binding proteins in regulating the stability of AChE transcripts in mature myotubes (42Bélanger G. Chan R.Y.Y. Jasmin B.J. Soc. Neurosci. Abstr. 2000; 26: 1089Google Scholar). Together, these results indicate, therefore, that during the early stages of myogenic differentiation, transcriptional regulatory mechanisms play a predominant role in the control of AChE expression whereas the contribution of post-transcriptional events becomes more important at later stages. The concerted effects of these various mechanisms are likely important for the rapid increase in AChE expression in developing muscle cells and to ensure that this induction is well maintained in mature myotubes as to prepare them for the arrival of exploratory motor axons.Recently, a DNA element termed an N-box motif was identified (20Koike S. Schaeffer L. Changeux J.P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10624-10628Crossref PubMed Scopus (92) Google Scholar) and shown to be critical for directing the preferential synaptic expression of genes encoding the acetylcholine receptor δ- (20Koike S. Schaeffer L. Changeux J.P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10624-10628Crossref PubMed Scopus (92) Google Scholar, 22Fromm L. Burden S.J. Genes Dev. 1998; 12: 3074-3083Crossref PubMed Scopus (106) Google Scholar) and ε- (21Duclert A. Savatier N. Schaeffer L. Changeux J.-P. J. Biol. Chem. 1996; 217: 17433-17438Abstract Full Text Full Text PDF Scopus (89) Google Scholar) subunits as well as the utrophin (24Gramolini A.O. Angus L.M. Schaeffer L. Burton E.A. Tinsley J.M. Davies K.E. Changeux J.P. Jasmin B.J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3223-3227Crossref PubMed Scopus (119) Google Scholar) and AChE(17Chan R.Y. Boudreau-Larivière C. Angus L.M. Mankal F.A. Jasmin B.J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4627-4632Crossref PubMed Scopus (79) Google Scholar) genes. Interestingly, in the case of the AChE gene, we noted that, although four N-boxes are located within 800 bp of the initiator element (two in the promoter region and two in the first intron; see Ref. 17Chan R.Y. Boudreau-Larivière C. Angus L.M. Mankal F.A. Jasmin B.J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4627-4632Crossref PubMed Scopus (79) Google Scholar), the one located in the first intron at position +755 bp was critical for controlling the synapse-specific expression of the reporter gene. Given our results showing the transcriptional induction of the AChE gene during myogenic differentiation, we therefore became interested in determining whether this N-box also participated in the regulation of AChE expression in developing muscle cells. Our transfection experiments showed, in agreement with our nuclear run-on data, that indeed expression of the AChE gene is increased during myogenic differentiation. Furthermore, these experiments revealed the key role played by the intronic N-box in controlling expression of the AChE gene during differentiation of muscle cells. To our knowledge, this is the first report demonstrating the importance of the N-box motif during myogenic differentiation.Several recent studies have shown that the ets-related transcription factors GABP α and β can bind to the N-box motif to transactivate genes encoding synaptic proteins (17Chan R.Y. Boudreau-Larivière C. Angus L.M. Mankal F.A. Jasmin B.J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4627-4632Crossref PubMed Scopus (79) Google Scholar, 22Fromm L. Burden S.J. Genes Dev. 1998; 12: 3074-3083Crossref PubMed Scopus (106) Google Scholar, 23Schaeffer L. Duclert N. Huchet-Dymanus M. Changeux J.-P. EMBO. 1998; 17: 3078-3090Crossref PubMed Scopus (135) Google Scholar, 24Gramolini A.O. Angus L.M. Schaeffer L. Burton E.A. Tinsley J.M. Davies K.E. Changeux J.P. Jasmin B.J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3223-3227Crossref PubMed Scopus (119) Google Scholar, 43Khurana T.S. Rosmarin A.G. Shang J. Krag T.O. Das S. Gammeltoft S. Mol. Biol. Cell. 1999; 10: 2075-2086Crossref PubMed Scopus (104) Google Scholar). Therefore, we sought to determine whether expression of GABP was affected during myogenic differentiation. Electrophoretic mobility shift assays revealed that GABP-binding activity to the N-box was slightly increased in nuclear extracts from myotubes versusmyoblasts, and this was confirmed by Western blot analysis. Although the increase appears rather modest, it is important to note that the transactivation potential of GABP is also influenced by its phosphorylation status with stronger transcriptional activation occurring in the absence of an increase in DNA binding activity (22Fromm L. Burden S.J. Genes Dev. 1998; 12: 3074-3083Crossref PubMed Scopus (106) Google Scholar). These results, together with the promoter analysis, further highlight the importance of the trans-acting element GABP and its corresponding cis-acting element, i.e. the N-box motif, in the expression of AChE during myogenic differentiation.The ets-related transcription factors often cooperate with other transcription factors (for reviews, see Refs. 44Wasylyk B. Hahn S.L. Giovane A. FEBS Lett. 1993; 211: 7-18Google Scholar, 45Janknecht R. Nordheim A. Biochim. Biophys. Acta. 1993; 1155: 346-356Crossref PubMed Scopus (206) Google Scholar), including AP-1 (46Wasylyk B. Wasylyk C. Flores P. Begue A. Leprince D. Stehelin D. Nature. 1990; 346: 191-193Crossref PubMed Scopus (415) Google Scholar), or with cofactors such as the CREB-binding protein also known as CBP/p300 (47Bannert N. Avots A. Baier M. Serfling E. Kurth R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1541-1546Crossref PubMed Scopus (58) Google Scholar), to exert their effects. Becauseets-related factors, including GABP, may also possess a conserved domain with homology to basic helix-loop-helix transcription factors such as myogenic factors (48Seth A. Papas T.S. Oncogene. 1990; 5: 1761-1767PubMed Google Scholar), it appeared possible that GABP could in fact interact with myogenic factors to regulate expression of the AChE gene. This view appeared particularly attractive given the presence of an E-box in the immediate vicinity of the N-box motif at position +755 bp in the first intron of the AChE gene. Promoter analysis showed that indeed this E-box is crucial in regulating expression of the AChE gene during myogenic differentiation, because its mutation reduced by more than 90% expression of the reporter gene in transfected myotubes. Consistent with these findings, we further showed by co-transfection of AChE promoter-reporter gene constructs and a myogenin expression vector, that myogenin increases the expression of luciferase in myotubes. Remarkably, this effect was dependent upon a single E-box, because mutation of the intronic E-box prevented the myogenin-induced increase in reporter gene expression despite the presence of numerous E-boxes the promoter region of the AChE gene (see Ref. 17Chan R.Y. Boudreau-Larivière C. Angus L.M. Mankal F.A. Jasmin B.J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4627-4632Crossref PubMed Scopus (79) Google Scholar). Our findings are therefore in agreement with the effects of myogenic factors on expression of genes in muscle cells (for reviews, see Refs. M. Biochem. Soc. 1996; PubMed Scopus (37) Google Scholar, B. Dev. 1998; PubMed Scopus Google Scholar, Rev. Dev. Biol. 1998; 14: PubMed Scopus Google Scholar). In addition, our in muscles also showed that this E-box a critical role in regulating the level of expression of theAChE gene in Taken together with our previous study (17Chan R.Y. Boudreau-Larivière C. Angus L.M. Mankal F.A. Jasmin B.J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4627-4632Crossref PubMed Scopus (79) Google Scholar), these data the importance of located within the first intron of the AChE gene in the control of its expression in skeletal muscle (see also Z.D. Camp S. A. Taylor P. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). Acetylcholinesterase (AChE)1 is widely recognized as an essential component of cholinergic synapses. In both the central and peripheral nervous systems, it is responsible for the hydrolysis of acetylcholine released from nerve terminals thereby ensuring the efficiency of synaptic transmission. Although a single gene encodes AChE, several molecular forms can be generated as a result of alternative splicing and distinct post-translational processing (for reviews, see Refs. 1Massoulié J. Pezzementi L. Bon S. Krejci E. Vallette F.-M. Prog. Neurobiol. 1993; 41: 31-91Crossref PubMed Scopus (1032) Google Scholar, 2Taylor P. Radic Z. Ann. Rev. Pharmacol. Toxicol. 1994; 34: 281-320Crossref PubMed Scopus (597) Google Scholar, 3Legay C. Microsc. Res. Tech. 2000; 49: 56-72Crossref PubMed Scopus (77) Google Scholar). It has been previously suggested that this polymorphism allows for the expression of AChE catalytic subunits in different cell types and at various subcellular locations where specific molecular forms may perform site-specific functions (4Toutant J.P. Massoulié J. Kenney A.J. Turner A.J. Mammalian Ectoenzymes.in: Elsevier, Amsterdam1987: 289-328Google Scholar). In skeletal muscle, AChE expression is known to be markedly influenced by the state of differentiation and innervation of the muscle fibers. Numerous studies have indeed reported dramatic changes in the expression of the enzyme during myo- and synapto-genesis as well as in mature muscles following alterations in their normal levels of neuromuscular activity and/or in their basic supply of trophic factors. Given that AChE is an excellent marker of synaptic differentiation, several groups have recently begun to decipher the molecular basis underlying these changes in AChE expression. For example, increases in AChE mRNA expression have previously been reported during myogenic differentiation (5Fuentes M.E. Taylor P. Neuron. 1993; 10: 679-687Abstract Full Text PDF PubMed Scopus (62) Google Scholar, 6Jbilo O. L'Hermite Y. Talesa V. Toutant J.P. Chatonnet A. Eur. J. Biochem. 1994; 225: 115-124Crossref PubMed Scopus (42) Google Scholar, 7Grubic Z. Komel R. Walker W.F. Miranda A.F. Neuron. 1995; 14: 317-327Abstract Full Text PDF PubMed Scopus (47) Google Scholar, 8Legay C. Huchet M. Massoulié J. Changeux J.P. Eur. J. Neurosci. 1995; 7: 1803-1809Crossref PubMed Scopus (59) Google Scholar, 9Luo Z.D. Wang Y. Werlen G. Camp S. Chien K.R. Taylor P. Mol. Pharmacol. 1999; 56: 886-894Crossref PubMed Scopus (30) Google Scholar). Moreover, activity-induced changes in AChE transcript levels have been observed in both cultured myotubes as well as in skeletal muscle in vivo (10Cresnar B. Crne-Finderle N. Breskvar K. Sketelj J. J. Neurosci. Res. 1994; 38: 294-299Crossref PubMed Scopus (42) Google Scholar, 11Michel R.N. Vu C.Q. Tetzlaff W. Jasmin B.J. J. Cell Biol. 1994; 127: 1061-1069Crossref PubMed Scopus (80) Google Scholar, 12Sveistrup H. Chan R. Jasmin B.J. Am. J. Physiol. 1995; 269: C856-C862Crossref PubMed Google Scholar, 13Boudreau-Larivière C. Chan R.Y.Y. Jasmin B.J. J. Neurochem. 2000; 74: 2250-2258Crossref PubMed Scopus (29) Google Scholar, 14Rossi S.G. Vasquez A.E. Rotundo R.L. J. Neurosci. 2000; 20: 919-928Crossref PubMed Google Scholar). these recent advances in our understanding of some of the biosynthetic events regulating AChE expression in skeletal muscle, our knowledge of the specific molecular mechanisms that account for these changes in mRNA expression remains largely unknown. Nonetheless, alterations in the stability of pre-synthesized AChE transcripts have been suggested to account for at least a portion of the changes in mRNA levels seen during myogenic differentiation (5Fuentes M.E. Taylor P. Neuron. 1993; 10: 679-687Abstract Full Text PDF PubMed Scopus (62) Google Scholar, 9Luo Z.D. Wang Y. Werlen G. Camp S. Chien K.R. Taylor P. Mol. Pharmacol. 1999; 56: 886-894Crossref PubMed Scopus (30) Google Scholar) and following muscle denervation (13Boudreau-Larivière C. Chan R.Y.Y. Jasmin B.J. J. Neurochem. 2000; 74: 2250-2258Crossref PubMed Scopus (29) Google Scholar). On the other hand, the contribution of transcriptional regulatory mechanisms has also been documented in several studies that examined expression of the AChE gene by nuclear run-on assays (13Boudreau-Larivière C. Chan R.Y.Y. Jasmin B.J. J. Neurochem. 2000; 74: 2250-2258Crossref PubMed Scopus (29) Google Scholar, 15Rimer M. Randall W.R. Biochem. Biophys. Res. Commun. 1999; 260: 251-255Crossref PubMed Scopus (8) Google Scholar) and promoter analyses (16Mutero A. Camp S. Taylor P. J. Biol. Chem. 1995; 270: 1866-1872Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 17Chan R.Y. Boudreau-Larivière C. Angus L.M. Mankal F.A. Jasmin B.J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4627-4632Crossref PubMed Scopus (79) Google Scholar, 18Culetto E. Combes D. Fedon Y. Roig A. Toutant J.P. Arpagaus M. J. Mol. Biol. 1999; 290: 951-966Crossref PubMed Scopus (38) Google Scholar). Together, these studies indicate therefore, that both transcriptional control mechanisms as well as post-transcriptional events contribute to the regulation of AChE mRNA levels in skeletal muscle cells. In this context, we have recently begun to examine the mechanisms underlying the preferential accumulation of AChE transcripts within the postsynaptic sarcoplasm of muscle fibers (see Refs. 11Michel R.N. Vu C.Q. Tetzlaff W. Jasmin B.J. J. Cell Biol. 1994; 127: 1061-1069Crossref PubMed Scopus (80) Google Scholar, 17Chan R.Y. Boudreau-Larivière C. Angus L.M.