RESUMO
The de-novo synthesis of glycogen is now known to involve a novel class of self-glucosylating protein primers. In mammalian skeletal muscle, glycogenin-1 is the protein responsible for this initiation step. Northern blot analysis revealed that glycogenin-1 gene transcription is differentially regulated in the C2C12 mouse muscle cell line. To define the regulatory elements that control expression of the glycogenin-1 gene, we have cloned and characterized the genomic structure of the human glycogenin-1 gene and its promoter region. This gene consists of seven exons and six introns, and spans over 13kb. Transcription of human glycogenin-1 is initiated at two major sites, 80 and 86bp upstream from the initiation of translation codon. Nucleotide sequence analysis of 2.1kb of the 5'-flanking region revealed the proximal promoter contains both a TATA box and two putative Sp1 binding sites located in a CpG island. There are numerous binding sites for developmental and cell-type-specific transcription factors, including AP-1, AP-2, GATA, and several potential Oct 1 binding domains. There are also nine consensus E-boxes that bind the basic helix-loop-helix family of muscle-specific transcription factors. The transcriptional activity of the glycogenin-1 gene was investigated by transient transfection of the 5'-flanking region in HepG2 cells and C2C12 myoblasts and myotubes. These results permitted the definition of a minimal 232bp promoter fragment that is responsible for basal level transcription in a cell-type-independent manner. Furthermore, we have identified a regulatory region located between -2076 and -1736 of the 5'-flanking region of the human glycogenin-1 gene that allows myotube-specific expression in C2C12 cells.
