Role of intronic E- and N-box motifs in the transcriptional induction of the acetylcholinesterase gene during myogenic differentiation

In this study, we examined whether an intronic N-box motif is involved in t he expression of acetylchollinesterase (AChE) during myogenesis, We determi ned that AChE transcripts are barely detectable in cultured myoblasts and t hat their levels increase dramatically in myotubes, Nuclear run-on assays r evealed that this increase was accompanied by a parallel induction in the t ranscriptional activity of the AChE gene, These changes in transcription we re also observed in transfection experiments using AChE promoter-reporter g ene 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 expressi on of the reporter gene following myogenic differentiation. Co-transfection experiments using AChE promoter-reporter gene constructs and a myogenin ex pression vector showed that expression of this regulatory factor increased expression of the reporter gene in myotubes. Although the AChE promoter con tains multiple E-boxes, mutation of this intronic one was sufficient to pre vent the myogenin-induced increase in reporter gene expression. Together, t hese results indicate that changes in AChE gene transcription occur during myogenesis and highlight the contribution of the intronic Nand E-box motifs in the developmental regulation of the AChE gene in skeletal muscle.