XIST Expression And X-chromosome Inactivation(XCI) In Pigs

Female mammals silence one of two X chromosomes to achieve dosage compensation for X-linked gene products with the only one X chromosome in males.This phenomenon is called X chromosome inactivation(XCI).XCI is mainly triggered by the expression of noncoding RNA,Xist,which is in the X-inactivation centre.Two different modes of XCI are included:the imprinted XCI mode,which silences the paternally derived X chromosome,and the random XCI mode,which silences either the maternally derived or paternally derived X chromosome.Imprinting XCI occurs in early embryos and extra-embryonic tissues in mice and cattle,whereas only random XCI occurs in humans,horses and rabbits during development.XCI is a series of epigenetic modifications that reflect the state of reprogramming.Therefore,XCI status is an important criterion to judge the quality of embryonic stem cells(ESCs),induced pluripotent stem cells(iPSCs)and somatic cell nuclear transfer(SCNT)embryos.The ESCs/iPSCs that contain two active X chromosomes are considered to be the ground state.Some cells in the cloned embryos developed abnormal XCI,affecting the expression of X-linked genes,leading to developmental abnormalities in cloned animals.By reducing Xist expression in the early cloned embryos of mice,the cloning efficiency could be improved significantly.The same problem of low cloning efficiency also exists for pigs.Compared to the mouse model,little is known about XCI status during the porcine development.To date,no ground state pig iPSCs or ESCs have been established that are capable of germline transmission.A better understanding of porcine XIST would facilitate researches in porcine iPSCs,ESCs and pig SCNT.In order to facilitate the study of porcine XIST,the complete sequence of porcine XIST RNA was identified by homology alignment,PCR amplification,3' RACE and 5' RACE.A conserved indel was found between Rongchang pig and Duroc XIST RNA by alignment XIST of different breeds.This indel can be used to distinguish the expression of XIST from either the maternal or paternally X chromosome.Two parental XIST alleles expression in early female preimplantation embryos and hybrid placentas indicated that the pig silences either the maternal or paternal X chromosome.What's more,the hypomethylation of XIST in gametes indicated that there was no methylated modification in gametes before fertilization.In addition,analysis of RNA sequencing dates of E20 female hybrid placentas also supported the random XCI mode.Therefore,both the expression of XIST and X-linked genes suggested that the pig only use the random XCI mode.To find out the XCI dynamic processes in porcine embryo development,H3K27me3 immunofluorescence staining and XIST differential methylated region(DMR)methylation analysis were performed.The results showed that there were abnormal XCI cells in pig cloned embryos,but the proportion of abnormal XCI cells was significantly lower than that of mice(5.1%-7.6%VS 20.0%-51.7%).In addition,the proportion of accomplished XCI cells in porcine blastocyst stage was lower than that in mice(23%VS 80%),indicating that the porcine embryo complete XCI in later development.Compared to the in vitro fertilization(IVF)embryos,SCNT embryos retained the epigenetic memory of somatic cells in XIST methylation.Porcine iPSCs can be classified into 2 major classes based on XCI status.The class I cells in 2i/LIF medium,resemble naive state mESCs,possessing 2 active X chromosomes(Xa)with no XIST expression or H3K27me3 mark,and class ? cells in KOSR/bFGF medium,resemble primed mESCs,containing one Xa and one Xi with XIST expression and H3K27me3 mark.Overall,pig used only random XCI mode during development through analysis of XIST and X-linked genes expression.There was a proportion of aberrant XCI cells in porcine SCNT embryo and the SCNT embryos retained epigenetic memory of somatic cells.Pig iPSCs can be classified into 2 types:one type of cells resemble naive mESCs while the other type of cells resemble primed mESCs retaining one inactive X.