The Effect Of SUMOylation On Nanog In Mouse Embryonic Stem Cells

SUMOylation is a critical and ubiquitous post-translation modification pathway in eukaryocytes, SUMOylation modulates proteins'subcellular location, stability and interaction. Nanog is an embryonic specific"master gene"found in 2003, it is very important to maintaining the pluripotency of embryonic stem cells. In order to elucidate the effect of SUMOylation on Nanog expression, Real time PCR, Western blot, Dual-Luciferase Reporter assay and immunofluorescence were introduced to understand the molecular mechanism of SUMOylation regulating Nanog in F9 carcinoma cells. The results are as follows:1. In this study, we cloned -230—+50bp(relative to transcriptional start site) fragment using Nanog genomic DNA as template, and over extension PCR were performed to produce fragment -230—+50bp with Sox2/Oct4 cis element mutation. All of these fragments were inserted into pGL4.10, and positive recombinants pGL4-230 luc,pGL4-230 Soxmut luc,pGL4-230 Octmut luc and pGL4-230 Octm/Soxm luc were verified by sequencing. To verify the function of these constructs, dual-luciferase reporter assays were done in F9 and NIH3T3 cells. The results demonstrated that the Nanog promoter -230—+50bp can drive Nanog transcription efficiently, and the Sox2/Oct4 cis element is required for Nanog transcription. We also found Nanog promoter is embryo-specific, because significant high luciferase activity was detected in F9 cells than in NIH3T3 cells.2. We demonstrated that SUMOylation represses Nanog expression. RNAi knockdown or overexpression of SUMO1 and Ubc9 resulted in the difference among the whole protein SUMOylation in F9 cells. Nanog expression was detected in the level of transcription and translation by real time PCR, western blot, dual-luciferase reporter assays when F9 cell are in different status of SUMOylation. One conclusion is the low Nanog expression level with high level of SUMOylation, in contrast, high level expression of Nanog was observed in cells with low level of SUMOylation.3. Cotransfected F9 cells with pCMV-HA-SUMO1,pCMV-HA-Ubc9 and wild type Oct4 or unmodified Oct4 K118R. Real-time PCR, western blot, as well as dual-luciferase reporter assay was introduced to examine Nanog expression. We found that SUMOylation of Oct4 promotes Nanog expression. We also concluded that SUMOylated Sox2 represses Nanog expression with the same methods.4. SUMOylation modulates proteins'function always via altering their subcellular distribution or affecting the interface of protein-protein interaction. To further explain the mechanism of SUMOylation repressing Nanog, reporter constructs with red fluorescent protein and immunofluorescence staining were performed. The results showed that SUMOylation never change Oct4 distribution in F9 cells, both Oct4 and Oct4 K118R were observed in the cell nucleolus. Similar results were obtained when we studied Sox2 and Sox2 K247R subcellular localization in F9 cell.