earticle

영어

β-O-linked N-acetylglucosamine(O-GlcNAc) is dynamic post-translational modification in nucleus and cytosol and it is regulated by O-GlcNAc transferase(OGT) and O-GlcNAcase. Many proteins, such as transcription factors, enzymes, cytoskeletal proteins, ribosomal proteins and chaperones have been identified to be modified with O-GlcNAc. O-GlcNAc modification modifies at their Ser/Thr residues and affects their functions. The hexosamine biosynthesis pathway takes about 5% of glucose in total glucose flux, and it’s final product is UDP-GlcNAc, which is source of O-GlcNAc modification. O-GlcNAc modification is reported to involve in skeletal muscle metabolism and development process. We studied whether O-GlcNAc modification is involved in differentiation in myoblast C2C12 cell. We observed that total O-GlcNAc modification level was dynamically changed during myogenesis. After treatment NButGT, we observed that myogenin expression level decreased. Using RT PCR, we found that mRNA level of myogenin decrease after treatment NButGT. And we found that the promoter activity of myogenin decreases after NButGT using reporter gene assay. For finding the promoter region affected O -GlcNAc , we did reporter gene assay using shorter length promoter and found at least 169 base pair upstream region of myogenin is affected by O-GlcNAc. And using this region, we found that Mef2c protein can be important transcription factor regulated by O-GlcNAc using avidin-biotin complex DNA binding assay. We confirm that Mef2c is O-GlcNAcylated using immunoprecipitation and we found several O-GlcNAcylated sites on Mef2c using mass spectrometry. Based on the result, we made the Mef2c point mutants converted from Serine, Threonine of O-GlcNAcylated sites to Alanine. Additionally, we found O-GlcNAc modification could regulate DNA binding affinity and transcriptional complex formation.