The Mechanism Of ESRRB In The Conversion Of Porcine Induced Pluripotent Stem Cells(piPSCs)to Trophoblast Stem Cells(TSCs)

Due to the similarities in anatomy,physiology and genetics,as a unique mammalian model,pigs play a vital role in human regenerative medicine.Pig-derived organs may solve the problem of the shortage of human organs for transplantation worldwide in the future.Based on this,pig induced pluripotent stem cells(piPSCs)have received extensive attention from scientists and have become the focus of research on accelerating the process of animal genetics and breeding and constructing disease models.However,the current research on piPSCs faces many problems: due to the lack of chimeric ability of piPSCs,germline chimeric animals cannot be formed.Therefore,understanding the gene regulatory network and molecular mechanisms that regulate the pluripotency and self-renewal of piPSCs is essential to solve these problems,and is of great significance for future pre-clinical research based on piPSCs.As a key pluripotent transcription factor,Estrogen Related Receptor Beta(ESRRB)plays a specific role in biological processes such as early embryonic development,pluripotency maintenance and primordial germ cell differentiation.Therefore,studying the mechanism of ESRRB in piPSCs is of great significance to deeply understand the regulation mode of pluripotent regulatory network for piPSCs and improve the chimeric ability of piPSCs.In this study,we analyzed the expression profile of ESRRB in various PSCs and its effect on the pluripotency and differentiation ability of piPSCs,and revealed the regulatory mechanism of ESRRB/OCT4/SOX2 as the core to regulate the transformation of piPSCs into trophoblast stem cells(TSCs).The test results are as follows:1.In this study,the expression profile of ESRRB was detected in early embryos.The results showed that ESRRB was expressed from 8-cell stage to blastocyst stage,and could be expressed in Inner cell mass(ICM)and Trophoderm(TE)at the same time.In this study,ESRRB was used as a candidate gene to regulate na?ve PSCs.Then explore the function of ESRRB in piPSCs.2.Firstly,we constructed ESRRB overexpressed piPSCs lines(ESRRB-piPSCs).The results showed that ESRRB-piPSCs were weakly AP positive,and the ability of embryoid bodies formation and differentiation was weakened.The subsequent embryo chimerism test showed that ESRRB improved the ability of piPSCs to chimera to ICM and TE at the same time.This result echoes that ESRRB can regulate the upregulation of TSCs molecular markers.3.RNA-seq results showed that trophoblast related molecular markers CDX2,GATA3,KRT8 and KRT18 were significantly up-regulated,indicating that cells began to transform into trophoblast.Double luciferase showed that ESRRB could directly bind to the promoter regions of CDX2 and KRT8.Then the protein structure of ESRRB was analyzed,and the protein domain deletion vectors of ESRRB were designed: N terminus and C terminus.Compared with WT group,N terminus group had similar characteristics,indicating that ESRRB mainly plays a role through N terminus structure.4.In order to further explore which pluripotent transcription factors were involved in this process,this study constructed a co-expression system of ESRRB and OCT4/SOX2.The results showed that the simultaneous presence of ESRRB and OCT4 could activate the expression of CDX2,KRT8 and KRT18,and the expression of CDX2,KRT8 and KRT18 would be significantly reduced in the absence of either of them.This result speculates that OCT4 and ESRRB may work together to activate CDX2,KRT8 and KRT18.By analyzing the IP-MS data of ESRRB and OCT4,it was found that 108 proteins could bind together.These genes include KRT8,LIN28 B,HMGA1,CTCF,CBX3,TRIM24,WDR43,SNW1,POLR2,TLN1,MAPK1 and HMGN1 transcription factors and DNA binding and transcription proteins.In-depth research found that the effect of KRT8 on the activation of CDX2 by ESRRB is not significant,although it binds to the CDX2 promoter region.Subsequently,Ch IP-seq data confirmed that ESRRB and OCT4 could directly bind to the genomic region of CDX2 and jointly activate CDX2.5.The detection of histone methylation modification of ESRRB piPSCs showed that the overall level of H3K36me2 changed most significantly.The results of q RT-PCR showed that KDM4 A and KDM4 B did not change significantly in ESRRB-piPSCs group compared with CON-piPSCs.KDM4 C was significantly increased in ESRRB-piPSCs group.This result suggests that ESRRB may regulate the expression of H3K36me2 by regulating KDM4 C.Then we tested whether ESRRB may directly regulate KDM4 C through Ch IP-seq.IGV results showed that ESRRB could bind directly to the intron region of KDM4 C,while OCT4 could bind to the intergenic region of KDM4 C.This result showed that both ESRRB and OCT4 could directly regulate KDM4 C.The addition of KDM4 inhibitor can significantly improve the AP positive rate of ESRRB-piPSCs,indicating that KDM4 C can participate in the function of ESRRB in regulating CDX2.Then KDM4 C was supplemented in CON-piPSCs.The results showed that compared with the control group,the AP staining results of KDM4C-piPSCs showed that the clones showed weak positive or even negative results after overexpression,and the expression of CDX2 was significantly up-regulated.This result shows that ESRRB could regulate KDM4 C to activate CDX2 to a certain extent.In conclusion,this study revealed that ESRRB as the core,recruited SOX2,KRT8 and OCT4 and formed a complex to act on the promoter regions of CDX2,KRT8 and KRT18 and regulate the transformation of piPSCs to TSCs.In addition,we also demonstrated that ESRRB can regulate the state of cells by regulating KDM4 C,an H3K36me3 demethylase.This transcriptional activation mechanism provides a theoretical basis for exploring the role of ESRRB in piPSCs,and enriches the understanding of the field of epigenetic activation mechanism.