SIRT2 Downregulates The Expression Of PDGFR? And Facilitates The Differentiation Of CG4 Cells

BACKGROUNDSIRT2(Silent information regulator 2)is one of the members of the Sirtuin family,which is an NAD~+-dependent protein deacetylase,expressed in large quantities in the central nervous system,especially in oligodentroglia cells.SIRT2 is mainly localized in the cytoplasm,but it has the ability of transfering from the cytoplasm to the nucleus along with the cell cycle change to perform biological functions.In the current study,it is known that SIRT2 participates and regulates cell aging and metabolism,inflammatory response,genome stability,cell cycle and cell differentiation.In our previous study,we found that SIRT2promoted the differentiation of CG4 cells.PURPOSEAlthough we found that SIRT2 increased neurite outgrowth and MBP expression in CG4 cells and promoted the differentiation of CG4 cells,the detail role of SIRT2 in oligodendrocyte differentiation is unclear.In this study,we used undifferentiated and differentiated CG4 cells,then identified genes that interact with SIRT2 during CG4 cells differentiation by the assay of chromatin immunoprecipitation.Through subsequent experiments,we will clarify the mechanism of the interaction and verify the effects of SIRT2 on the target gene,involving in the differentiation and proliferation of CG4 cells.METHODSIRT2-pEGFP-c2 and pEGFP-c2 plasmids were transfected into CG4 cells then cultured CG4 cells under the conditions of GM(growth medium)and DM(differentiation medium)respectively,and we observed the phenomenon of SIRT2 transfer into the nucleus.Designing primers for promoter regions CPG island of related genes then select relevant antibodies for ChIP experiment to idenfity genes that interact with SIRT2 protein,and further study the mechanism of interaction.Meanwhile,in SIRT2 over-expresstion and SIRT2 knocked-down CG4 cells,we perform a DNA methylation assay on the gene to investigate the effect of SIRT2 on the DNA methylation and histone acetylation.Subsequently,we determined the effect of SIRT2 on the proliferation and differentiation of CG4 cells through cell proliferation assay.RESULTTransfection of HEK293 cells with SIRT2-pEGFP-c2 plasmid is consistent with previous studies that SIRT2 is located in cytoplasm.The pEGFP-c2-SIRT2 plasmid were transfected into CG4 cells then we cultured CG4 cells in growth and differentiation media respectively.We observed that the SIRT2 was transferred into the nucleus under the CG4 cells cultured in the differentiation medium,but it still localized in the cytoplasm under growth medium.Through performing nuclear-cytoplasm separation experiment,the results of Western blot further validate this conclusion.We then designed primers for the promoter region CPG island of the relevant genes.When CG4 cells were in a differentiated state,we used the antibody of SIRT2 for ChIP experiments and found that SIRT2 binds to the promoter of PDGFR?in the differentiation medium but not in grouth medium.Western blot experiments showed that SIRT2 overexpression inhibited the expression of PDGFR?in CG4 cells.Then we performed ChIP experiments using antibodies such as acetylated protein and Histone3K27.When SIRT2 was overexpressed,the level of acetylated protein in the promoter region of PDGFR?was decreased and we also found that the activity of Histone3K27 was enhanced.Histone3 K27 is a marker of inhibition of transcriptional activation,this indicated that SIRT2inhibits PDGFR?expression by deacetylating histones of the PDGFR?promoter region.Subsequently,we respectively extracted the genomic DNA in CG4 cells with over-expresstion and knocked-down of SIRT2for methylation assay.By analyzing the sequencing results,we found that SIRT2 over-expression significantly increased DNA methylation in the PDGFR?promoter region in CG4 cells,compared with SIRT2 knocked-down.Knockdown of PDGFR?by siRNA and over-expression of SIRT2 both inhibited the proliferation of CG4 cells,while knockdown of SIRT2 by siRNA decreased the proliferation of CG4 cells.CONCLUSION1.When CG4 cells initiate differentiation,SIRT2 is partially transferred from the cytoplasm into the nucleus and binds to the promoter region of PDGFR?.2.In the process of CG4 cell differentiation,SIRT2 inhibits the expression of PDGFR?by deacetylating and/or methylating PDGFR?promoter.3.Over-expression of SIRT2 and knock-down of PDGFR?inhibited the proliferation of CG4 cells,however,it is beneficial to the differentiation of CG4 cells.4.SIRT2 may facilitate the differentiation of CG4 cells via repression of PDGFR?expression.