IN-VIVO AND IN-VITRO ANALYSIS OF ELECTRICAL ACTIVITY-DEPENDENT EXPRESSION OF MUSCLE ACETYLCHOLINE-RECEPTOR GENES USING ADENOVIRUS

Acetylcholine receptor (AChR) genes are repressed in extrajunctional d omains of admit muscle fiber by neurally evoked electrical activity. D enervation elicits upregulation of AChR gene transcription in extrasyn aptic areas. We have used an adenovirus (Ad)-based strategy to analyze in vitro and in vivo the electrical activity-dependent transcription of the chicken AChR alpha 1 subunit gene. The luciferase gene placed u nder the control of wild-type and mutated fragments of the alpha 1 sub unit promoter was inserted in a defective Ad vector designed for the s tudy of transcriptional regulation. Animals were infected by intramusc ular injection and in vivo luciferase levels were normalized by coinfe ction with an Ad vector containing the chloramphenicol acetyltransfera se gene driven by an electrical activity-insensitive promoter. Our res ults demonstrate that although both proximal MyoD binding sites of the alpha 1 promoter are required for muscle-specific expression of the a lpha 1 gene, only one is necessary, albeit insufficient, to enhance al pha 1 promoter activity after denervation. Parallel results were obtai ned with cultured muscle cells in vitro following tetrodotoxin blockin g of spontaneous electrical activity. These results substantiate a dir ect contribution of MyoD factors in electrical activity-dependent regu lation of AChR expression and further indicate that Ad-based vectors c onstitute a powerful tool in the field of transcriptional regulation.