Identification and characterization of cis-acting elements that regulate expression of the glycogen synthase-2 (GYS-2) gene

Program: Abstracts - Orals, Featured Poster Presentations, and Posters
Session: MON 758-775-Beta Cells, Glucose Control & Complications
Monday, June 17, 2013: 1:45 PM-3:45 PM
Expo Halls ABC (Moscone Center)

Poster Board MON-761
Komsan Anyamaneeratch*1, Pinnara Rojvirat1 and Sarawut Jitrapakdee2
1Mahidol university, Bangkok, Thailand, 2Mahidol University, Bangkok, Thailand
Glycogen synthase is a key glycogenic enzyme which converts glucose to glycogen in skeletal muscle and liver. Two isozymes of glycogen synthase, namely, the glycogen synthase-1 (GYS-1) and glycogen synthase-2 (GYS-2) are expressed in skeletal muscle and liver, respectively. GYS-2 is post-translationally regulated by reversible phosphorylation/dephosphorylation. However, little is known whether GYS2 expression is transcriptionally regulated especially during nutritional and hormonal alterations. As a prelude to understand transcriptional regulation of the GYS-2 gene, the 1.5 kb 5’-flanking sequence of GYS-2 gene was cloned and sequenced. The GYS-2 promoter contains one CCAAT box locating within the first 100 nucleotides upstream of the transcription start site. To examine whether this GYS2 gene promoter (-1424/+167) contains full functional regulatory elements, it was ligated upstream of the luciferase reporter gene. Transient transfections of the GYS2-luciferase reporter construct into HepG2 indicated that this promoter fragment can drive expression of the luciferase reporter gene 5-fold higher than the cells transfected with the empty vector, indicating that this promoter contains full basal regulatory sequences. Truncation of the GYS-2 promoter fragment from -1424 to -814 resulted in a significant increase in the reporter gene activity, suggesting that the repressor element(s) is located between these regions. On the other hand, deletion of nucleotides from - 813 to -198 resulted in a marked decrease of luciferase activity, suggesting that an activator(s) sequence is located within this region. Bioinformatic analysis of the 1564 nucleotides of GYS-2 regulatory sequence using PROMO database identified several putative binding sites for general transcription factors such as NF-Y, USF1 and USF2, the liver-enriched transcription factor, HNF-3b and HNF4α, and the insulin-mediated transcription factors, i.e. SREBP-1c and FOXO1. Furthermore, a putative binding site for transcription factor which is implicated in energy homeostasis including CCAAT/enhancer binding proteins (C/EBPs) has also been identified.

Nothing to Disclose: KA, PR, SJ

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Sources of Research Support: Faculty of Science, Mahidol University