Role of E and CArG Boxes in Developmental Regulation of Muscle Glycogen Phosphorylase Promoter during Myogenesis

Muscle glycogen phosphorylase (MGP) transcript and protein levels increase during skeletal muscle development in tandem with the products of other muscle genes responsible for glucose and glycogen metabolism. Previous studies demonstrated that a 269 bp region 5' to exon 1 of MGP is sufficient for developmental regulation in the C2C12 myogenic cell line (Froman et al., 1994). This genomic region (-209 to +60) contains four consensus E box motifs, a CArG-like sequence, and a GC-rich domain. Native MGP transcripts were not detected in pluripotent CH310T1/2 fibroblasts, but low levels of MGP mRNA were measured in CH310T1/2 cells that were stably transfected with MyoD. Three of the E box motifs in the MGP proximal promoter interacted with C2C12 nuclear proteins. However, cotransfection of the MGP promoter with myogenic regulatory factors, including MyoD and myogenin, produced less than 2-fold activation compared with 20-fold activation of the desmin promoter. Mutational analyses of the MGP promoter demonstrated that increased expression in C2C12 myotubes did not require any of the E box motifs or the CArG-like element. A small region (-76 to -68) upstream of GC-rich domain (-64 to -51) significantly reduced promoter activities in both myoblasts and myotubes. The functional studies suggest that MGP is developmentally regulated during myogenesis by alternative pathways that utilize unidentified regulatory elements or ancillary factors.