E3 Ligase WWP1 Regulates SOX2 Protein Stability

Owing to the self-renewal and pluripotent features,human embryonic stem cells(hESCs)possess great potential in both basic research and clinical application in the fields of developmental biology and regenerative medicine.A set of publications have reported that key transcription factors OCT4,SOX2,and NANOG play critical roles in cell fate determination of hESCs,but currently studies regarding the function and regulatory mechanisms of post-translational modifications on the above three factors are relatively less.To seek for specific E3 ubiquitin ligases of OCT4,SOX2,and NANOG,exogenous WWP1 or WWP2 was enforced to expression in HEK 293 FT cells along with OCT4,SOX2,and NANOG,respectively.We find that co-expression of WWP1 reduced the protein level of SOX2 drastically in a dosage-dependent manner,which could be abrogated by lysosome inhibitor treatment,while exerted no impact on OCT4 and NANOG.In comparison to this,co-expression of WWP2 only led to OCT4 protein level decrease.Later,we demonstrated that WWP1 protein interacted with SOX2 protein directly through GST-pulldown and Co-IP,and catalyzed ubiquitin modification on SOX2.Furthermore,cysteine at 890 residues(C890)is responsible for the E3 ubiquitin ligase activity.Subsequently,CRISPR/Cas9 method was utilized to knock out the 24 th exon of WWP1 gene due to the existence of genetic codon for C890.Intriguingly,WWP1 deficiency increased the expression levels of neural development-related genes in hESCs according to transcriptome analysis.In line with this,ectoderm marker genes,PAX6 and SOX1,were up-regulated upon WWP1 deficiency.However,SOX2 protein stability and expression level declined in WWP1 deficiency hESCs compared to its wild-type counterpart,which is opposite to the results obtained in in vitro experiments.Next,more studies are required to be performed to decipher this puzzle.Taken together,the current study reveals that WWP1 is the specific E3 ubiquitin ligase of SOX2,and WWP1 may promote SOX2 protein turnover through ubiquitin-lysosome pathway.In addition,WWP1 may participate in self-renewal maintenance of hESCs by suppress the expression of ectoderm marker genes.Thus,our study provides new insights into the regulatory networks in terms of protein ubiquitination in hESCs.