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Abstract

We have isolated and analysed the 5' flanking region of the rat acetylcholine receptor (AChR) beta subunit gene and determined regulatory elements that confer muscle specificity. Deletion mapping revealed a minimal TATA-box-less promoter region containing an initiator motif. An 85-bp fragment has been shown to promote high muscle-specific expression of a chloramphenicol acetyltransferase (CAT) reporter construct upon transfection in primary muscle cells. This sequence can be functionally 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 cause the loss of transcriptional activity of the basal promoter fragment. The enhancer activity depends on the presence of both E boxes that cooperate in a synergistic fashion. We therefore conclude that the control 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. Cotransfection experiments in NIH3T3 cells demonstrate that the rat AChR beta subunit gene can be transactivated by myogenic factors displaying a preference for myogenin, as well as MRF4 and myf5 compared to a clearly weaker responsiveness to MyoD1.