The Mechanism Of H4L20me3 On The Developmental Potential Of Cloned Porcine Embryos

The low cloning efficiency greatly limits the application of somatic cell nuclear transfer in pig breeding.O ne of the reasons is that the epigenetic modification from somatic cells can not be fully reprogrammed by egg cytoplasm.H4K20me3 is an repressive histone modification,which has been reported to be incompletely demethylated before zygotic genome activation(ZGA)in cloned porcine embryo.But insufficient data are available regarding the mechanism of H4K20me3 during pig cloned embryos developments.We designed a series of experiments to explore the role of H4K20me3 during the development of cloned embryos:First,by Immunofluorescence and C h IP-seq,we determined the abnormal enrichment of H4K20me3 in pig cloned embryos.Then,we used A-196 inhibitor to treat cloned embryos,and detected the removing of abnormal H4K20me3 enrichment and the improvement of cloned embryo development.Moreover,biological events like ZGA,cell cycle and telomere lengthening were also explored in fertilized embryos,and cloned emrbyos with and without A-196 treatment.The results in details are as follows:1)Immunofluorescence results showed that H4K20me3 was highly enriched in cloned embryos before and during ZGA.C h IP-seq showed that H4K20me3 was highly enriched in reprogramming-resistent genomic regions(RRRs)and memory-perserved genomic regions(MPRs).These findings indicat that H4K20me3 is one of the reprogramming barriers for pig cloned embryos.2)A-196 treatment could significantly improvethe developmental potential of cloned embryos in vitro.The blastocyst developmental rates were increased from 17.12±3.33%to 30.34±5.61%.3)Comparative analysis by RNA-seq and quantative PCR showed that the improvement of cloned embryo development was unrelated to ZGA.4)Mmunofluorescent and 5-Ethynyl-2’-deoxyuridine(Ed U)showed a S-phase cell cycle block in cloned porcine embryos.When 81.00 ± 2.05% of IVF embryos enter phase G2,only 60.33 ± 3.09% of cloned embryos enter phase G2.A-196 treatment could rescue the S-phase block and restore the cell cycle profile close to that of IVF embryos.5)The telomere length of A-196-treated cloned embryos was 70% longer than that in control cloned embryos.Together,our research suggested that H4K20me3 is one of the reprogramming barriers for pig cloned embryos.O vercoming abnormal H4K20me3 enrichments effectively improved the development potential of pig cloned embryos.This research is of great significance in exploring the somatic cell reprogramming mechanism and improving the application of cloning technology in pig breeding.