CHARACTERIZATION OF THE FUNCTIONAL-ROLE OF E-BOX ELEMENTS FOR THE TRANSCRIPTIONAL ACTIVITY OF RAT ACETYLCHOLINE-RECEPTOR EPSILON-SUBUNIT AND GAMMA-SUBUNIT GENE PROMOTERS IN PRIMARY MUSCLE-CELL CULTURES

The expression of gamma and epsilon subunits of the acetylcholine rece ptor from mammalian skeletal muscle is regulated independently during myogenic differentiation and innervation. Genomic DNA fragments contai ning 5'-flanking sequences of the epsilon-subunit and gamma-subunit ge nes were characterised by a series of 5' deletions fused to the chlora mphenicol-acetyltransferase gene and transiently expressed by transfec tion of primary cultures of rat muscle cells and non-muscle cells. A 6 .3-kb epsilon-subunit fragment can be reduced to yield a 270-bp fragme nt that confers 5-10-times higher expression levels in muscle cells co mpared to in non-muscle cells. The region composed of nucleotides -185 to -128 increases the transcriptional activity moderately while the 1 4-bp palindrome containing a single E box at nucleotides -88 to -83 ma y interact with the promoter but has no enhancer properties in muscle cells. From a 1.1-kb genomic fragment of the gamma-subunit gene, 167 b p were sufficient for muscle-specific expression. Two promoter-proxima l E-box elements enhance promoter activity in muscle and mediate trans activation by myogenic factors. Myogenin and myf5 were much more effic ient than MRF4 or MyoD1 which exerted only little transactivation. Cot ransfection experiments show that increased expression of Id in primar y muscle cells inhibits chloramphenicol-acetyltransferase expression m ediated by the gamma-subunit gene promoter and support the view that m yogenic factors play an important role in the transcriptional regulati on of the gamma-subunit gene.