The Functions And Mechanisms Of The Wwp2 Catalyzed Oct4 Ubiquitination And Ash2l In Reprogramming And Pluripotency Maintenance

Pluripotent stem cells could serve as an ideal in vitro model for drug screening and cell therapy based on their indefinite proliferative capacity and pluripotent potential,exhibiting a wide range of application prospects in regenerative medicine.However,it is essential to decipher the molecular mechanisms regulating the pluripotent cells maintenance,differentiation and somatic cell reprogramming prior to their application in clinical therapy.It has been indicated that transcription factors,epigenetics and signaling pathways together take part in controlling pluripotent cell status.Among them,key transcription factor Oct4 is vital for cell fate determination.We previously showed that Wwp2-catalyzed ubiquitinated Oct4 proteins are subject to degradation through 26 S proteasomes during the differentiation process of mouse embryonic stem cells(mESCs)and 5 lysine residues of Oct4 were identified as ubiquitin-conjugation sites using mass spectrometry analysis.Herein,we report disruption of W wp2-catalyzed ocT4 ubiquitination is dispensable for mESC self-renewal but leads to a reprogramming efficiency 10 times higher than the wild type Oct4 in mouse embryonic fibroblasts(MEFs)combined with Sox2 and Klf4.Mechanistically,the mutations of the ubiquitination sites improve the Oct4 protein stability and enhance Ash2 l isoform b-mediated histone 3 lysine 4 methylation.Furthermore,we identify that Ash2l-b is the down-stream gene of Oct4,eliciting a drastic increase in the reprogramming efficiency and playing supression roles during differentiation processes towards endoderm,while Ash2l-a,as another isoform of Ash2 l,functions in promoting the differentiation.In summary,we propose a more efficient and rapid method for somatic cell reprogramming by disruption of Wwp2-catalyzed Oct4 ubiquitination,and find distinct functions of the two transcripts of Ash2 l in pluripotency establishment and differentiation processes,providing insight into the regulatory networks in pluripotency maintenance,exit and establishment.