Tyrosine Phosphorylation Modification Regulates The Transcriptional Activity Of Sox2

Sox2 is a key factor in self-renewal and pluripotency of embryonic stem cells(ESCs).Sox2,together with Oct4 and Nanog,forms the core transcriptional network that is essential for early embryogenesis and plays a central role in self-renewal and pluripotency of embryonic stem cells,as well as their differentiation into specific lineages.By promoting pluripotency gene expression and repressing the expression of lineage differentiation related genes,Sox2Z as well as Oct4 and Nanog,functions to keep self-renewal and pluripotent state of ESCs.It is reported that oct4,Sox2 and Nanog have various post-translational modifications such as phosphorylation,SUMOylation,O-GlcNAcylation,which would regulate their functions in ESCs.Previous reports have shown that Sox2 is phosphorylated in serine and threonine residues,which plays critical roles in regulating Sox2 functions.Whether Sox2 also undergoes tyrosine phosphorylation and whether this phosphorylation regulates Sox2 functions remain unknown.In this study,we explored the regulation of Sox2 by tyrosine phosphorylation.We first performed an Alanine scanning on all the Serine,Threonine,Tyrosine,Lysine and Arginine residues across Sox2 whole sequence and characterized their effects on reprogramming.In total,we generated 98 Sox2 mutants covering 107 sites,among which 23 mutants were defective in the somatic reprogramming assay.These 23 residues except Y279,are all located in the HMG domain of Sox2 and presumably mediate Sox2 interaction with its cognate DNA.We further performed detailed characterization on Sox2 residue Y279 and hypothesized this site would undergo phosphorylation,given it is located in the TAD domain of Sox2 and Y279A mutant displayed reduced efficiency in reprogramming assay.We demonstrated that Sox2 can indeed be phosphorylated at Y279(as well as Y127)by the protein tyrosine kinase Pyk2 and this phosphorylation is important for Sox2 functions in self-renewal of ESCs and in reprogramming.In addition,we characterized how phosphorylation affects Sox2 function and found that Sox2 Y279F mutation does not change the stability and subcellular localization of Sox2,does not affect its interaction with Oct4 or Nanog.Instead,Y279F mutation decreases Sox2 transcriptional activity for endogenous Sox2 target genes and reduces its interaction with Tet2.Taken all the findings together,we demonstrated that Sox2 is phosphorylated by Pyk2 at Y127 and Y279,which is important in self-renewal and pluripotency of mESCs.Sox2 phosphorylation at Y279 promotes its interaction with Tet2 to regulate somatic cellreprogramming.We propose the following model for Sox2 Y279 phosphoryltion in somatic cell reprogramming.Sox2 binds to promoter regions of its target genes,whichare supposedly highly methylated.Tet enzymes,mediated by Sox2,are recruited to these promoters and start active DNA demethylation.During this process,phosphorylation of Sox2 at Y279 strongly enhances its interaction with Tets and therefore boosts active DNA demethylation process to facilitate somatic cell reprogramming.