The Role Of Pcgf6 On Early Embryo Development In Zebrafish

ObjectiveIn recent years,more and more studies have found that epigenetic regulation plays an important role in biological individual development.Among them,Polycomb group(PcG)proteins,a group of evolutionarily conserved protein complexes,are thought to be epigenetic inhibitors of development-related genes.PcG-mediated gene silencing involves two protein complexes:PRC1,mediate H2AK119ub1,and PRC2,mediate H3K27me3.The molecular structure of PRC 1 is very complex.The PCGF family,interacting with the RING1A/B protein,has 6 members(PCGF1 to PCGF6),so the molecular mechanism of PRC 1-mediated gene silencing is complicated.cPRC1 protein complex includes PCGF2 or PCGF4.The cPRC1 complex can be further classified into specific subgroups based on the interaction between PCGF2/4 and CBX(CBX2/4/6/7/8)or PHC(PHC1/2/3).ncPRC1 interacts with PCGF1,PCGF3,PCGF5 or PCGF6 and RYBP(RING1 and YY1 binding proteins)or YAF2(YY1 related factor 2),but not with CBX2/4/6/7/8 and PHC 1/2/3.In this research,we study the function of Pcgf6,which is highly expressed in ESCs.So far there are no articles reporting on the epigenetic regulation of Pcgf6 on ESCs,so this article will explore the regulatory role of Pcgf6 on ESCs through various methods to improve the physiological function of the Pcgf family,in order to provide more research for the development and differentiation of ESCs direction.MethodsIn vivo,we use zebrafish as a model.Through microinjection of Pcgf6 Morpholino and Pcgf6 mRNA,in situ hybridization,PCR,ChIP and other methods,we study the role of Pcgf6 in the early stage of zebrafish development.In vitro,we use P19 cell line as an ESCs model,and construct Pcgf6 knockdown model.We also use PCR,ChIP to study the role of Pcgf6 in P19 cell line.ResultsWe find that Pcgf6 is higher expressed before 75%-epiboly,and then the expression decreased.This is the time when embryonic stem cells start to differentiate into the three germ layers cells.After overexpression of Pcgf6,embryonic development is significantly delayed,and the expression of transcription factors pou5fl,klf4,and sox2 which are associated with pluripotency of zebrafish embryonic stem cells,increased.The occupancy of H3K27me3 and H2AK119ub1,related to transcriptional suppression,at pou5fl,klf4,and sox2 promoter region decreased,the occupancy of H3K4me3,related to transcriptional activation,increased.In addition,we use P19 cells as a model in vitro.Knocking down Pcgf6 leads to same results as in vivo.Therefore,we believe that Pcgf6 is very important for maintaining the self-renewal of embryonic stem cells(ESCs)and plays a critical role in activating of pluripotency related genes.Conclusions1.Pcgf6 regulates the early development of zebrafish;2.Pcgf6 has transcriptional activation effects on zebrafish pou5f1,klf4,sox2;3.Pcgf6 is involved in maintaining the maintenance of pluripotency in mouse P19 cells,and also has transcriptional activation,activating oct4,klf4,and sox2.