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

Abstract

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 Nand E-box motifs in the developmental regulation of the AChE gene in skeletal muscle.

Regulation of a Transcript Encoding the Proline-rich Membrane Anchor of Globular Muscle Acetylcholinesterase

“…On the other hand, the up-regulation of AChE T mRNA by myogenin or MyoD, as well as the promoter activity of the AChE gene (36), is essential to maintaining a sufficient amount of AChE T for the production of the A form of AChE. E-boxresponsive elements in the rat AChE promoter have been shown to be involved in myogenin-mediated activation (36). On the other hand, the upstream regulating elements of FIGURE 8.…”

“…Myogenin and MyoD repressed the transcription of PRiMA mRNA in cultured myotubes and reduced the proportion of G 4 AChE, whereas the transcriptional activity of the COLQ gene could be activated by overexpression of myogenin and MyoD (21,24). On the other hand, the up-regulation of AChE T mRNA by myogenin or MyoD, as well as the promoter activity of the AChE gene (36), is essential to maintaining a sufficient amount of AChE T for the production of the A form of AChE. E-boxresponsive elements in the rat AChE promoter have been shown to be involved in myogenin-mediated activation (36).…”

Expression of utrophin A mRNA correlates with the oxidative capacity of skeletal muscle fiber types and is regulated by calcineurin/NFAT signaling

“…Total muscle protein extraction and EMSAs were performed as described (10,28). The sequences of the synthetic 32 P-labeled oligonucleotides encompassing the NFAT binding site in the utrophin A promoter were (in 5Ј-3Ј orientation) gtg cat att gga aaa cag aaa aat (sense) and att ttt ctg ttt tcc aat atg cac (antisense).…”

“…Quantitative RT-PCR was carried out to determine the relative abundance of total utrophin (both A and B together), utrophin A and B separately, and MyHC transcripts in EDL and soleus single muscle fibers, in whole muscles from mice subjected to different experimental treatments, and to determine the abundance of both LacZ and CAT transcripts in transfected C2C12 muscle cells and in transduced soleus muscles. These assays were performed as described (10,17,28). Primers that selectively amplified total utrophin, utrophin A, utrophin B, S12 rRNA, MyHC I, IIa, IIx, IIb, LacZ, and CAT were designed on the basis of available sequences (16,17,29).…”

Retinoid X Receptor Signalling in the Specification of Skeletal Muscle Lineage

“…The resulting myogenic bHLH-E heterodimers bind to DNA at the consensus sequences known as an E-box (CANNTG), specific DNA motifs present at muscle gene enhancers and/or promoters, where they regulate gene expression (Sartorelli and Caretti, 2005). These genes include cytoskeletal, sarcomeric, metabolic, and cell signaling proteins (Angus et al, 2001;Gramolini and Jasmin, 1999;Kraner et al, 1999;Li and Capetanaki, 1993;Lin et al, 1991;Marsh et al, 1998;Shield et al, 1996;Simon and Burden, 1993;Wheeler et al, 1999). A requirement for the MyoD family of transcription factors in this combinatorial complex is demonstrated by the fact that the E protein homodimers bind to the same DNA sequences as the MyoD-E protein heterodimers, yet only the MyoD-E protein complex can cooperate with MEF2 factors (Naya et al, 1999).…”