The Role And Molecular Mechanism Of RBBP4 In Maintenance Of Mouse Embryonic Stem Cell Pluripotency

Embryonic stem(ES)cells derived from the inner cell mass(ICM)of the mammalian blastocyst have two well-known features of self-renewal and differentiation.As the best model for research in developmental biology and regenerative medicine,however,the molecular mechanisms underlying self-renewal and pluripotency are not completely understood,which has been a major challenge for us from basic research to clinical application of embryonic stem cells.The polycomb group(Pc G)proteins are a group of physically bound proteins that play a key role in embryo development,pluripotency maintenance of stem cells,and tumorigenesis.They are linked together to assemble into functionally distinct complexes belonging to two major families: the polycomb repressive complexes 1(PRC1)and PRC2.PRC1 monoubiquitylates histone H2 A at lysine 119(H2AK119ub1)via its E3 ubiquitin ligase subunits,RING1A/B.Depending on the composition,PRC1 can be further classified as canonical and non-canonical PRC1.It can also be divided into PRC1.1-PRC1.6characterized by six mutually exclusive PCGF paralog subunits(PCGF1–PCGF6).PRC2 consists of four core subunits,EZH1/2,SUZ12,EED,RBBP4/7 and several non-core subunits.Histone methyltransferase EZH1 or EZH2 can mono-,di-,and tri-methylates histone H3(H3K27me1/2/3)at lysine 27 and repress the developmentally regulated genes.PRC2 exists in two major forms: PRC2.1 and PRC2.2.PRC2.1 contains one of the three polycomb-like protein 1–3(PHF1,MTF2,and PHF19),whereas PRC2.2 is characterized by the AEBP2 and JARID2 subunits.Recent studies have reported that the core subunit of PRC2,RBBP4,is essential during early mouse embryo development.However,the exact cause of early embryonic lethality of Rbbp4 knockouts and its potential roles in maintenance of stem cells or pluripotency have not been studied.In this study,we generated Rbbp4 conditional knockout mouse ES cells using CRISPR/ Cas9-mediated ES cell targeting technology and Rbbp4 was completely removed72 h after transfection with a plasmid encoding Cre.We found that ablating Rbbp4 gene expression in ES cells results in loss of ES cell self-renewal capability and the undifferentiated state,exhibiting fatal differentiation phenotype.To explore the molecular basis of pluripotency alterations observed in Rbbp4-null ES cells,we performed gene expression analysis for Rbbp4-null ES cells by transcriptome sequencing(RNA-seq)analysis and found that loss of Rbbp4 significantly reduces the expression of ES cell core pluripotency factors and promotes mesendodermal gene expression.Analyzing the published RNA-seq datasets in the database,we found a large number of the genes deregulated in Rbbp4-null ES cells was also observed in ES cells deficient for Suz12,Ezh2,and Eed.Moreover,transcriptional changes in Rbbp4-null ES cells significantly overlapped with those seen in Oct4-,Sox2-,or Nanog-deficient ES cells.In order to further identify the genes directly regulated by RBBP4 in the impairment of ES cell pluripotency,we analyzed the target genes directly bound by RBBP4 using chromatin immunoprecipitation followed by high-throughput sequencing(Ch IP-seq)analysis.Importantly,a detailed examination of RBBP4 Ch IP-seq revealed that RBBP4 bound to the promoters of a group of genes associated with pluripotency(Oct4 and Sox2)or mesendodermal differentiation(Brachyury,Tbx2,Sox17,and Gata4).Further differentiation assays in both in vivo and in vitro showed that the forced co-expression of Oct4 and Sox2 completely rescues the pluripotency of Rbbp4-null ES cells.Moreover,to characterize the ability of each PRC2 member to support ESC identity,we generated individual or combined PRC2-deficient ES cell lines by using CRISPR/Cas9 technology,suggesting that the ES cells deficient for each of the PRC2 subunits except Rbbp4 does not affect the ES cell self-renewal and pluripotency.Proteomic studies have shown that RBBP4 tightly associates with EZH1/2,SUZ12,and EED to form the core PRC2 complex.Rbbp4 deletion did not appear to adversely affect the protein levels of the components of PRC2.But RBBP4 can repress early mesendodermal lineage genes by recruiting the PRC2 complex to the promoter region.In summary,we investigated the functional role of Rbbp4 in mouse ES cells.We demonstrated that ablating Rbbp4 gene expression in ES cells results in a loss of the undifferentiated state and the initiation of differentiation along mesendodermal lineage.Mechanistically,we revealed Rbbp4 safeguards ES cell pluripotency in two ways.On the one hand,Rbbp4 keeps the ES cells in a pluripotent state by maintaining the requisite levels of OCT4 and SOX2.On the other hand,Rbbp4 guides the PRC2 to the loci of developmental genes where they establish and maintain the repressive states of mesendoderm lineage-specific genes in ES cells.