Dennis_1997_DNA.Cell.Biol_16_1099

Expression of the nicotinic acetylcholine receptor (AChR) is transcriptionally regulated during the development of vertebrate striated muscle. To better define regulatory elements involved in this process, site-directed mutations were made in the gene's 86 bp muscle specific enhancer. Transient expression assays in skeletal muscle C2C12 cells indicated that all three E-boxes, plus a novel sequence outside the E-boxes, are necessary for full activity of the AChR gene in myotubes. Gel mobility shift assays demonstrated that mutations in the non-E-box sequence disrupted the formation of two DNA/protein complexes while not affecting myoD binding. Methylation interference footprinting confirmed that the complexes form at nucleotides within the mutated region, and also include part of the central E-box. UV crosslinking of nuclear proteins to a DNA probe identified five proteins of 125, 81, 55, 42, and 35 kDa that bind to this region; with the 125 kDa protein being differentially bound in U.V. crosslink assays during the transition from myoblasts to myotubes. These data suggest that interactions between this DNA element and the five proteins contribute to the transcriptional control of the AChR alpha-subunit gene expression during the differentiation of skeletal muscle.