Identification of two regulatory binding sites which confer myotube specific expression of the mono-ADP-ribosyltransferase ART1 gene

Using C2C12 and C3H-10T 1/2 cells as models of myogenesis, we found that ART1 expression was restricted to myotube formation. We identified a fragment spanning the gene 1.3 kb upstream of the transcriptional start site as the functional promoter of the ART1 gene. This region contains an E box and an A/T-rich element, two conserved binding sites for transcription factors found in the promoters of most skeletal muscle specific genes. Mutating the DNA consensus sequence of either the E box or the A/T-rich element resulted in a nearly complete loss of ART1 promoter inducibility, indicating a cooperative role of the transcription factors binding to those sites. Gel mobility shift analyses carried out with nuclear extracts from C2C12 and C3H-10T 1/2 cells revealed binding of myogenin to the E box and MEF-2 to the A/T-rich element, the binding being restricted to C2C12 and C3H-10T 1/2 myotubes.Here we describe the molecular mechanism underlying the regulation of the ART1 gene expression in skeletal muscle cells. The differentiation-dependent upregulation of ART1 mRNA is induced by the binding of myogenin to an E box and of MEF-2 to an A/T-rich element in the proximal promoter region of the ART1 gene. Thus the transcriptional regulation involves molecular mechanisms similar to those used to activate muscle-specific genes.Mono ADP-ribosytransferases (ARTs) are an important class of enzymes that catalyse the transfer of the ADP-ribose from NAD+ to a specific amino acid residue in the target protein [1,2]. This reaction has been originally identified as the pathogenic mechanism of bacterial toxins, including cholera, pertussis and diphtheria toxin [3,4]. There is increasing evidence that endogenous ARTs also play important roles in higher animals and human [5-7]. So far, the family of mammalian ARTs comprises five members (ART1-5) [8]. They are all ectoenzymes, anchored in the outer leaflet of the plasma membrane via a glycosylphosphatidylinositol-tail with the exception of AR