Mbd3/NuRD Regulates Self-renewal And Apoptosis Of PKCi-mESCs

Mouse embryonic stem cells(mESCs)could be divided into Na(?)ve and Primed states.Na(?)ve mESCs are derived from inner cell mass(ICM)of preimplantation balstocsts,they have characteristics of proliferation indefinitely in vitro,self-renewal,multilineage differentiation and germline transmission.Pluripotency of Na(?)ve ESCs are regulated by multiple signaling pathways,and protein kinase C(PKC)plays an important role in it,inhibition of PKC(PKCi)is sufficient to maintain the self-renewal of mESCs and rat ESCs(r ESCs),however,the Na(?)ve(pluropotency and transcriptome)characteristics of mESCs in the presence of PKCi are not fully understood.The nucleosome remodeling and deacethylase(NuRD),as an epigenetic regulatory complex,but the correlation of NuRD complex with PKCi is still unknown.Methyl-Cp G-binding domain 3(Mbd3)is one of the component of the NuRD complex,it regulates gene transcription to maintain mESCs stability by binding to promoters of genes to mediate formation of heterochromatin and transcriptional silencing,but the mechanism by which Mbd3/NuRD regulates mESCs pluripotency is still unclear.In addition,there are few studies on Mbd3/NuRD regulating mESCs apoptosis.In this study,we invesigate(1)the characteristics of pluripotency and transcriptome of Na(?)ve mESCs derived from the PKCi(G(?)6983)(PKCi-mESCs);(2)the correlation between PKCi and NuRD complex and molecular mechanisms of PKCi-mESCs pluripotency regulated by Mbd3/NuRD;(3)the function of Mbd3/NuRD during PKCi-mESCs cell apoptosis.The results were as follows:1 PKCi-mESCs have the characteristics of Na(?)ve state ESCsmESCs derived from ICM of blastocysts from C57BL/6J preimplantation mouse with PKCi and traditional 2i L medium(LIF,PD0325901(inhibitor of MEK signaling pathway)and CHIR99021(inhibitor of GSK signaling pathway))were used as objects,the expression of pluripotency genes were analysied by q PCR and immunofluorescence,the differentiation potential in vitro of PKCi-mESCs were performed by embryoid assay,germline transmission and RNA-Seq assay were performed.Results showed that,PKCi-mESCs presented “Domed-shaped” morphology;PKCi-mESCs highly expressed core pluripotent markers Nanog,Oct4 and Sox2,Na(?)ve markers Klf4,Rex1,Nrob1 and Fgf4,but lowly expressed Primed marker Fgf5;PKCi-mESCs expressed core TFs NANOG,OCT4,SOC2 and Na(?)ve TFs FGF4,KLF4 and REX1 but not Primed TF FGF5,with high modification of H3K4me3 and low modification of H3K27me3;had good differentiation potential in vitro;chimeric mouses were sucessfully obtained when PKCi-mESCs were injected into the blastocysts of ICR mice,and one chimera founder produced homozygous offspring,Results above suggested that PKCi-mESCs were Na(?)ve mESCs.RNA-Seq results showed that there were 3794 differentially expressed genes(DEGs)between 2i L-mESCs and PKCi-mESCs,among them,2010 DEGs were up-regulated and1784 DEGs were down-regulated.GO functional analysis revealed that the DEGs were mainly enriched in cellular component,molecular function and biological process,cellular component were mainly enriched in plasma membrane and extracellular component,molecular function were mainly enriched in transcription factor activity,sequence-specific DNA binding,biological process were mainly enriched in development process,cell differentiation,cell process,system development and cell development;PKCi-mESCs highly expressed core TF Oct4,Na(?)ve markers Klf17,Dnmt3 l and Rexo1,pluripotency genes c-Myc and Lin28 a,2i L-mESCs highly expressed core TFs Sox2 and Nanog,Na(?)ve markers Fgf4,Klf4 and Tbx3;KEGG enrichment analysis showed that there were 1093 DEGs involved in 288 signaling pathways,especially,ERK and AKT signaling pathways were significantly enriched,compared with PKCi removal group,PKCi significantly decreased the ration of p-ERK/ERK and increased the ratio of p-AKT/AKT.Results indicated that PKCi(G(?)6983)supported derivation of Na(?)ve mESCs from blastocysts of C57BL/6J,with different transcriptome characteristics with 2i L-mESCs,ERK and AKT signaling pathways were involved in PKCi-mESCs self-renewal.2 Mbd3/NuRD regulates PKCi-mESCs self-renewalThe levels of NuRD complex were detected by RNA-Seq,q PCR and Western blot between PKCi-mESCs and 2i L-mESCs;the mESCs treated with sh Mbd3,FUW-Mbd3,PKCi removal and knockdown of Mbd3 followed by PKCi removal were used as objects,mESCs treated with sh NC,FUW-M2 rt TA and sh NC followed by PKCi removal were used as controls,expression levels of pluripotency and differentiation genes were detected by q PCR and Western blot,cell morphology and NANOG expression were detected by immunofluorescence,and single-cell colony formation efficeiency was analysied by AP staining.Results showed that there was no significant difference in NuRD expression between 2i L-and PKCi-mESCs,but PKCi significantly decreased m RNA levels of Hdac1,Hdac2,Mbd3,Mta1,Rbap46 and Rbap48 except Chd3,and Western blot results showed that PKCi only significantly decreased the protein levels of HDAC2 and MBD3,indicating that PKCi regulates the expression of NuRD complex at different levels of RNA transcription and protein translation;knockdown of Mbd3 increased the protein levels of NANOG and OCT4 as well as the percentage of mixed colonies but decreased the percentage of differentiated colonies,promoted PKCi-mESCs self-renewal;Mbd3overexpression decreased the NANOG,OCT4 and REX1 protein levels,increased FGF5 and c Tn T protein levels,PKCi-mESCs differentiated into platted cells and were negative for NANOG,the percentage of mixed and undifferentiated colonies were decreased while percentage of differentiated colonies was increased;PKCi removal increased Mbd3 expressed and induced similar cell differentiation caused by Mbd3 overexpression,such as protein levels of NANOG and KLF4 were decreased and levels of FGF5 and c Tn T were increased,percentage of differentiated colonies was increased and percentage of minxed and undifferentiated colonies were decreased,but knockdown of Mbd3 significantly decreased the upregulation of Mbd3 expression and cell differentiation induced by PKCi removal were resversed partially,Results suggested that Mbd3/NuRD modulated PKCi-mESCs self-renewal by regulation of pluripotency and differentiation genes.3 Mbd3 is an important regulator for apoptosis in PKCi-mESCsmESCs treated with FUW-Mbd3,PKCi removal and knockdown of Mbd3 followed by PKCi removal were used as objects,mESCs treated with FUW-M2 rt TA and sh NC followed by PKCi were used as controls.Cell number was detected by cell counting,cell viability was detected by CKK8 assay,cell apoptosis rate was detected by Annexin V staining,m RNA exprexxion levels of Bax,Bcl-2,Trail,Fasl and Caspase3 were analysied by q PCR,protein levels of BAX and BCL-2 were detected by Western blot.Results showed that Mbd3 overexpression decreased cell number and viability,increased the apoptosis rate,protein level of BCL-2 was decreased,the m RNA levels of mitochondria apoptosis-related genes Bax and Bim were dramatically increased but the level of Bcl-2 was decreased,and the levels of death receptor apoptosis-related genes Trail and Fasl as well as collective effector Caspase 3 were significantly increased;similarly,when PKCi was removed from the mESCs culture medium,the Mbd3 expression level and cell apoptosis rate were significantly increased,accompanied by the decrease of the cell number and viability,simultaneously,the expression levels of Bax,Bim,Trail,Fasl and Caspase 3 were increased but the level of Bcl-2 was decreased,while knockdown of Mbd3 significantly decreased the upregulation of Mbd3 induced by PKCi removal,and cell apoptosis induced by PKCi removal was resversed by Mbd3 knockdown partially.Results above indicated that Mbd3/NuRD induced PKCi-mESCs apoptosis via mitochondria and death receptor apoptosis pathways.In summary,the research found that PKCi(G(?)6983)supported derivation of Na(?)ve mESCs from C57BL/6J strain mouse and different transcriptome characteristics with2 i L-mESCs;the negative correlation of PKCi and NuRD complex and the role of Mbd3/NuRD in PKCi-mESCs self-renewal;the role of Mbd3/NuRD in PKCi-mESCs apoptosis.This study further expanded the molecular mechanism of Na(?)ve mESCs self-renewal,and provided a theoretical basis and research methods.