Identification And Function Study Of Deubiquitylase Of Pluripotency Factor Nanog

Background:Nanog was first discovered in 2003 as a critical factor by Mitsui and Chambers.It expresses in embryonic stem cells(ESCs),pluripotent cells and some tumor cell lines.Previous studies have demonstrated Nanog could maintain ESCs self-renewal and pluripotency.Ubiquitylation is one of the most important post-translational modifications(PTMs)to control protein stability.The Ubiquitin-proteasome system mediates the rapidly and highly specific addition of ubiquitin to intracellular proteins and thereby contributes to the dynamic regulation of protein abundance.The ubiquitin-proteasome system is a reversible process to be strictly controlled.Deubiquitinases(DUBs)can remove ubiquitin molecules from protein substrates and affect the function of the protein,then influence cell growth,signal transduction and many cell physiological process.At present,there are about 100 DUBs coded in the human genome.FBXW8 has been reported as an E3 of Nanog to bring it to degradation by Ubiquitin-proteasome system.We believed that one or more DUBs must exist to reversely regulate the Nanog protein degradation by removing the ubiquitin chains.So far,the DUBs that regulate Nanog protein stability remains unknown.Our research mainly focused on which DUB can maintain the stability of Nanog protein levels and how it functions in the process of ESCs self-renewal.Objects:As a pluripotency transcription factor,Nanog has an important role in maintaining ESCs self-renewal and pluripotency.Its stability is essential for keeping the undifferentiated status of ESCs.There has been reported that FBXW8 as a Nanog ubiquitin ligase catalyze its ubiquitination.However,the DUBs that regulates Nanog protein stability in ESCs have not been discovered.Our purpose is to screen the DUB of Nanog and study its function in maintaining the ESCs properties.Methods and Results:Given that endogenous Nanog is difficult to be detected in a variety of cell lines,we overexpressed the ectopic Nanog and DUB plasmids in cells Through the screening,we found that USP21 could significantly stable Nanog protein.By Co-IP,we verified the interaction between Nanog and USP21,both in vivo and in vitro.Besides,We confirmed that USP21 can signsificantly extend the half-life of Nanog.We also found that USP21 specifically removed K48-linkage ubiquitin chains,protecting Nanog from the degradation by proteasome.Interestingly,.USP21 can stable Nanog protein level by its DUB activity specifically,with no effect on other transcription factor,such as Sox2,Oct4.As Nanog plays an important role in maintaining pluripotency in ESCs,we use two kinds of differentiation models to further illustrate the role of USP21 in ESCs.