Transcriptional regulation of pig GYS1 gene by glycogen synthase kinase 3β (GSK3β)

Glycogen synthase kinase 3 beta (GSK3 beta) is a ubiquitous serine/threonine kinase and has important roles in glycogen metabolism biosynthesis. Studies have revealed that GSK3 beta can directly regulate the glycogen synthase activity, yet little is known about the regulation of GSK3 beta on GYS1 gene transcription. Here, we show that overexpression of GSK3 beta decreased the mRNA expression level of GYS1. Then we cloned approximately 1.5 kb of pig GYS1 gene promoter region, generated sequential deletion constructs, and evaluated their activity. A gradual increase of the promoter activity was seen with increasing length of the promoter sequence, reaching its highest activity to the sequence corresponding to nt -350 to +224, and then decreased. However, the activities of constructed promoter fragments show different responses to GSK3 beta co-transfection. By analyzing a series of GYS1 promoter reporter constructs, we have defined two crucial regions (-1488 to -539, -350 to -147) that are responsible for GSK3 beta-induced transcriptional repression. Furthermore, the ChIP results revealed that only the first and second NF-kappa B sites of GYS1 promoter could bind to p65, and overexpression of GSK3 beta induced a significant decrease in p65 binding to the second NF-kappa B binding site, suggesting that GSK3 beta may regulate expression of GYS1 gene through binding to the second rather than the first NF-kappa B site. These data suggest that the NF-kappa B plays important roles in the transcriptional activity of pig GYS1 gene regulated by GSK3 beta.