Effects Of KDM6A And KDM6B On The H3K27me3 Level And Developmental Potential Of Porcine Nuclear Transfer Embryos

Somatic cell nuclear transfer(SCNT)plays an important role in animal breeding,biomedicine,xenotransplantation,stem cell and reprogramming mechanism research.After nuclear transplantation is activated,somatic cells will undergo a full range of reprogramming,so that the implanted somatic cell nucleus can normally perform zygote gene activation(ZGA).A large number of studies have shown that abnormal epigenetic modification is the main obstacle to SCNT reprogramming.The abnormal epigenetic modifications reported in the SCNT reprogramming process include DNA methylation,histone methylation,and acetylation.Among them,histone methylation modification plays an important role in somatic cell reprogramming and early embryo development.Recent studies have shown that H3K27me3 is an important epigenetic barrier to SCNT reprogramming in mice and cattle.Compared with in vitro fertilization(IVF)embryos,the mouse SCNT embryos are before the 2 cell stage,and the bovine SCNT embryos are at the 2 to 8 cell stage,and the genome shows an overall abnormally high H3K27me3 modification.In the early embryonic development stage of mice,over-expression of kdm6a can significantly improve the development rate of SCNT embryos;by knocking down kdm6b,kdm6a can be increased,promote ZGA,and further improve the development rate of SCNT embryos.In the early embryonic development stage of cattle,the over-expression of KDM6A significantly improved the reprogramming efficiency of SCNT,increased the development rate of SCNT embryos.Preliminary studies in this laboratory have shown that there may be abnormally high H3K27me3 modification in early cloned embryos of pigs.Treatment with a small molecule inhibitor GSK126 reduces the level of H3K27me3 modification in cells and embryos,and the rate of cloned blastocysts increased in the treatment group.However,this inhibitor is highly toxic and will cause impaired reprogramming and affect embryonic development.Therefore,the purpose of this study is to further evaluate whether there is abnormally high H3K27me3 modification in pig early cloned embryos,and whether it can reduce abnormally high H3K27me3 modification in pig early cloned embryos by directly regulating the levels of enzymes(KDM6A and KDM6B)established by H3K27me3 Improve the development efficiency of pig SCNT embryos.In order to answer the above questions,this study first examined the H3K27me3 modification pattern of porcine IVF and SCNT early embryos,and the results showed that SCNT embryos had significant H3K27me3 abnormal enrichment at the 2 and 4 cell stages.On this basis,by injecting KDM6A mRNA 5-6 h after activation of SCNT embryos,KDM6A was overexpressed in early SCNT embryos.The results showed that overexpression of KDM6A can reduce the high H3K27me3 modification of SCNT embryos 2 and 4 cell stages,and significantly improve SCNT embryo development rate.Studies have shown that knock down kdm6b in mice can increase kdm6a and further increase the development rate of SCNT embryos.In order to verify whether it is possible to increase the growth rate of SCNT embryos by knocking down with kdm6b in pigs,the specific injection of siKDM6B 5-6 h after SCNT embryo activation interferes with KDM6B.High H3K27me3 modification,and will lead to a significant reduction in the development rate of cloned embryos.In summary,this research got the following conclusions:1.SCNT embryos at the 2 and 4 cell stage H3K27me3 modification level was significantly higher than IVF embryos.2.Overexpression of KDM6A can effectively reduce the high H3K27me3 modification of SCNT embryo 2 and 4 cell stages,and improve the development rate of pig SCNT embryos.3.Knockdown KDM6B could not reduce the high H3K27me3 modification of SCNT embryo 2 and 4 cell stage,and it would affect the pig SCNT embryo development rate.