2 ADJACENT E-BOX ELEMENTS AND A M-CAT BOX ARE INVOLVED IN THE MUSCLE-SPECIFIC REGULATION OF THE RAT ACETYLCHOLINE-RECEPTOR BETA-SUBUNIT GENE

We have isolated and analysed the 5' flanking region of the rat acetyl choline receptor (AChR) beta subunit gene and determined regulatory el ements that confer muscle specificity. Deletion mapping revealed a min imal TATA-box-less promoter region containing an initiator motif. An 8 5-bp fragment has been shown to promote high muscle-specific expressio n of a chloramphenicol acetyltransferase (CAT) reporter construct upon transfection in primary muscle cells. This sequence can be functional ly dissected in a basal muscle-specific promoter element carrying a M- CAT box that is flanked at the 5' end by an enhancer element with two binding sites for myogenic factors. Point mutations in the M-CAT box c ause the loss of transcriptional activity of the basal promoter fragme nt. The enhancer activity depends on the presence of both E boxes that cooperate in a synergistic fashion. We therefore conclude that the co ntrol of muscle-specific and developmental expression of the rat AChR beta subunit gene requires both regulatory elements, the M-CAT box and two adjacent E boxes, located in close proximity to each other. Cotra nsfection experiments in NIH3T3 cells demonstrate that the rat AChR be ta subunit gene can be transactivated by myogenic factors displaying a preference for myogenin, as well as MRF4 and myf5 compared to a clear ly weaker responsiveness to MyoD1.