Multiple binding sites for myogenic regulatory factors are required for expression of the acetylcholine receptor gamma-subunit gene.

The nicotinic acetylcholine receptor (AChR) is a critical component of the mechanism responsible for signal transduction between nerve and muscle. Induction of AChR gene expression occurs during myogenesis when myoblasts exit the cell cycle and fuse to form myotubes. Induction of muscle-specific genes during differentiation is believed to be mediated through the activities of members of the MyoD family of myogenic regulatory factors. At least two of the factors, MyoD and myogenin, function as transcriptional activators. Here we present evidence that MyoD is capable of activating transcription of the mouse AChR gamma subunit gene. We have identified seven potential MyoD-binding sites (E-boxes) within the 5'-flanking DNA of the gamma subunit gene. We demonstrate that two small regions of the 5'-flanking DNA, one containing a pair of E-boxes and the other containing a single E-box, are required for gamma subunit expression in mouse muscle cells. DNase I footprinting indicates that the E-box proximal to the transcriptional initiation site is capable of interacting directly with MyoD. Furthermore, site-directed mutational analysis indicates that the single E-box is necessary for transactivation by MyoD and expression of the gamma subunit promoter region in muscle cells.