The Function And Mechanism Of USP21 Regulate Mouse Embryonic Stem Cell Self-renewal And Reprogram

Embryonic stem cells(ESCs)are a type of pluripotent stem cells,they are derived from inner cell mass(ICM).ESCs has the potential of unlimited proliferation and also can dif-ferentiate into almost all kinds of cells.Based on these features,ESCs have extremely im-portant applications in the basic research and clinical applications.Key transcriptional factors of ESCs,such as Oct4,Sox2 and Nanog,play a crucial role in the maintenance of self-renewal and pluripotency.In order to maintain ESCs self-renewal and pluripotency,they can associate with other transcriptional factors,protein modification factors and epigenetic factors to form transcriptional regulatory networks.Ubiquitin protein modification system as a kind of dynamic modification process,can precisely regulate the pluripotency and differen-tiational potency of ESCs.Nanog is a key transcriptional factor of ESCs,it is essential for the maintenance of plu-ripotency and promotion somatic cell reprogramming.The level of Nanog is regulated by al-leles,transcriptional level and post-translational modification.Relative to the transcriptional level,little is known about the post-translational modification of Nanog.Nanog is a short-lived protein and quickly degraded by the ubiquitin-dependent peoteasome system.Pro-teasome inhibitor MG132 remarkably stabilize the protein level of Nanog.Previous study showed that the phosphorylation of Nanog can be recognized and ubiquitinated by the E3 lig-ase Fbxw8.Subsequently,the ubiquitinated Nanog can be recognized and degraded by the proteasome.These findings suggested thay the protein stability of Nanog can be regulated by the ubiquitin-dependent proteasome system(UPS).However,how to maintain the stability of Nanog in the ESCs and during the process of somatic cell reprogramming?Can its stability regulated by the deubiquiting enzymes(DUBs)?Therefore,the stability of Nanog in the maintenance of pluripotency and during the differentiation still remains to be further studied.To search for DUBs that could stabilize Nanog,we fused firefly luciferase to the C ter-minus of Nanog and used this fusion protein(Nanog-Luc)as a reporter of Nanog stability.As the fused luciferase is degraded together with the Nanog,the degradation of Nanog can be easily quantified by measuring the luciferase activity.Forty-six mammalian DUBs were screened with this reporter.We found that co-expression with USP21,but not the other DUBs,significantly increased the luciferase activity of Nanog-Luc.Further study showed that USP21 directly interacts with and deubiquitinates Nanog.We found that deletion of USP21 caused the loss of mESC morphology and promote ESCs differentiation.Conversely,overexpression of USP21 promoted mESC self-renewal under less optimal culture conditions(withdrawl of LIF).Furthermore,Usp21 depletion reduced the ability of mESCs to form teratomas.Nanog is required to attain the ground state of authentic pluripotency in the final phase of somatic cell reprogramming.Therefore,we examined whether USP21 was required for the generation of induced pluripotent stem cells(iPSCs)from MEFs with Oct4,Sox2 and Klf4.Usp21-KO MEFs displayed a significantly lower reprogramming efficiency than WT MEFs,as indicated by the reduced number of AP-positive colonies on day 12 of induction.On the other hand,overexpression of USP21 in OG2 MEFs carrying an Oct4-GFP reporter significantly in-creased the number of GFP+ iPSC colonies induced by Oct4,Sox2,Klf4 and Nanog on day 16.In contrast,CA-USP21 had no effect.Collectively,our data indicate that USP21 is a specific DUB for Nanog and plays important roles in mESC self-renewal and somatic cell repro-gramming by regulating Nanog stability.The process of maintanence of ESCs pluripotency is not only affected by external envi-ronment,but also regulated by all kinds of signaling pathways.Leukocyte Inhibitor Factor(LIF)plays an important role in the maintanence of pluripotency in ESCs.At the transcrip-tional level,the expression of USP21 in mESCs was activated by the LIF/STAT3 pathway,which was critical for the maintenance of mESC and the self-renewal of mESCs.It was re-ported that the FGF4/ERK signaling pathway is activated during the ESCs differentiation.Previouse studies showed that the activated ERK phosphorylated and promoted tanascription-al factors Klf2,Klf4 degraded by the ubiquitin-depedent proteasome system,resulting the differentiation of ESCs.However,our study demonstrate that ERK1-induced phosphorylation of USP21 at S539 prevents its interaction with Nanog and thus negatively regulates USP21-mediated Nanog deubiquitination and stabilization,promotes ESCs differentiation.Our work reveals the mechanism of key signaling pathway in ESCs to regulate self-renewal and pluripotency from different angles.More and more studies showed that epigenetic regulation plays important role in the maintenance of self-renewal and somatic cell reprogramming.H2A ubiquitination and chro-matin compaction combine to mediate the polycomb repressive complex 1-dependent repres-sion of genes that are crucial for the maintenance of ESC identity.However,how the mon-oubiquitination of H2A is regulated in ESCs still remains to be further studied.We demon-strated that USP21 is recruited to chromatin by Nanog to deubiquitinate H2A and facilitate Nanog-mediated gene expression in mESCs.In summary,we identified USP21 as a specific deubiquitinating enzyme for the pluripo-tency factor Nanog.USP21 plays a critical role in maintaining the self-renewal of mESCs by stabilizing Nanog and facilitating the chromatin remodelling that favors Nanog-mediated gene transcription.Depletion of USP21 leads to Nanog degradation and mESC differentiation.The regulation of Nanog by USP21 is controlled by both LIF/STAT3 and FGF/ERK signalling pathway:the expression of USP21 is regulated by LIF/STAT3 signalling and the function of USP21 is regulated by ERKs.Our study suggests a regulatory mechanism by which extrinsic signals regulates the fate of mESCs via the deubiquitination of pluripotency factor.In addition,USP21 is recruited to gene promoters by Nanog to deubiquitinate histone H2A at K119 and thus facilitates Nanog-mediated gene expression.The dynamic reversibility of the ubiquitin modification and the recent development of general proteasome inhibitors and specific block-ers and activators of ligase functions pave the way for the future manipulation of the UPS in ESCs.In particular,this approach may lead to more efficient reprogramming of somatic cells or provide new therapies targeting cancer stem cells.