Analysis of binding and activating functions of the chick muscle acetylcholine receptor gamma-subunit upstream sequence.

1. The skeletal muscle acetylcholine receptor comprises several subunits whose coordinated expression during myogenesis is probably controlled by cis elements in the individual subunit genes. We have previously analyzed promoter regions of the alpha and delta genes (Wang et al., 1988, 1990); to gain further insight into receptor regulation, we have now studied the promoter of the chick muscle gamma-subunit gene. 2. This analysis was faciliated by the close upstream proximity of the coding region of the delta-subunit gene and the consequent brevity (740 bp) of the untranslated linker connecting the two genes (Nef et al., 1984). Nuclease protection and primer extension analysis revealed that transcription of the gamma-subunit gene starts at position 56 upstream of the translational initiation site. 3. Nested deletions of the promoter region were employed to identify functionally important elements. A 360-bp sequence (-324 to +36) was found to activate transcription, in a position- and orientation-independent manner, during myotube formation. This sequence comprises 5 M-CAT (Nikovits et al., 1986) similarities and contains, at positions -52/-47 and -33/-28, two CANNTG (Lassar et al., 1989) motifs. 4. Binding experiments were performed by means of gel retardation, gel shift competition, and footprint analysis. The CANNTG motifs were found to bind MyoD and myogenin fusion proteins and to interact with proteins in nuclear extracts from cultured myotubes. 5. Point mutations in the CANNTG motifs revealed that these elements are crucial for full promoter activity in myotubes and essential in fibroblasts cotransfected with a myogenin expression vector. 6. We conclude that the activity of the gamma-subunit gene is determined largely by E boxes, which in vivo are likely to be activated by MyoD family proteins; in addition, other transactivators such as the M-CAT binding protein presumably play a role. Both CANNTG elements and M-CAT motifs are also present in the alpha- and delta-subunit enhancer and may therefore account for the coordinate expression of the three subunits during muscle differentiation.