Patterns Of Histone H4K20 Methylation Modification In Early Mouse Embryos

Mammalian somatic cell nuclear transfer(SCNT)is the only reproductive technology that can use a single donor somatic cell nucleus to produce cloned animals.Although its applications in basic biology,industry and medicine are very extensive,the birth rate of cloned animals is usually much lower than other reproductive technologies,such as IVF.This low efficiency can be attributed to abnormal epigenetic modifications such as DNA methylation,histone modifications,histone variants,and X chromosome inactivation.Histone H4K20 methylation modification is a type of histone modification.Its trimethylation modification(H4K20me3)is consistent with H3K9me3 and H3K27me3,which have been identified as disorders of epigenetic reprogramming.They are all heterochromatin markers.At present,the dynamic pattern of histone H4K20 methylation modification during mouse preimplantation embryo development,especially in ZGA stage fertilized eggs and SCNT embryos is still unclear.We use mouse fertilized egg embryos and early SCNT embryos as research materials.First,the method of immunofluorescence was used to explore the methylation modification pattern of histone H4K20 in early mouse embryos.The results show that the different methylation of histone H4K20 presents different modifications in oocytes and preimplantation embryos.H4K20me1,H4K20me3 modifications always exist from oocytes to preimplantation embryos,which indicates that H4K20me1,H4K20me3 modifications promote the pre-implantation embryo development in a specific way;H4K20me2 modification exists in the female pronucleus of the fertilized egg,but does not exist or very weakly in the 2-cell,4-cell,8-cell,and it rises significantly in the later stages.The results showed that histone H4K20 methylation was differentially modified in oocytes and preimplantation embryos.In order to further explore the relationship between histone H4K20-related methyltransferase and demethylase gene expression levels and histone apparent modification,we used RT-PCR to analyze the expression levels of the H4K20 methyltransferase genes SETD8,Suv4-20h1,Suv4-20h2 and H4K20 demethylase genes PHF8 and Rad23 b,and the results showed that both H4K20 methyltransferase genes and H4K20 demethylase genes were highly expressed during the ZGA period.In order to further explore the dynamic pattern of H4K20 methylation modification during the reprogramming process of pronuclear fertilized eggs and SCNT early embryos,we performed immunofluorescence staining on fertilized eggs and SCNT embryos of different pronuclear stages.The results showed that H4K20me1 was modified in fertilized eggs.It persists in the female pronucleus and the male pronucleus of the fertilized egg and presents two different chromatin characteristics;The H4K20me2 and H4K20me3 modifications present asymmetric histone modifications during the PN1-PN5 stage of the pronucleus of the fertilized egg.They are only modified in the female pronucleus,but not in the male pronucleus.We divided the reconstructed cloned embryos into five stages: NT-0 h,NT-2 h,NT-7 h,NT-27 h,and NT-2cell according to their developmental status,and explored the methylation modification of histone H4K20.The results showed that it is compatible with different prokaryotic stages of fertilization.Compared with egg embryos,histone H4K20me2 has no methylation modification in NT-27 h cloned embryos,while histone H4K20me3 also has no methylation modification in NT-2cell cloned embryos.It is speculated that the modification of histone H4K20me2/3 may be epigenetic disorders of nuclear transfer reprogramming in vivo.In summary,our research found that the three types of H4K20 methylation modifications have different dynamic patterns in early mouse embryos;H4K20-related methyltransferase and demethylase genes are all highly expressed during the ZGA period;at the same time compared with normal fertilized eggs,cloned embryos in pseudo-pronucleus stage have different dynamic patterns of histone H4K20 methylation modification,but the mechanism still needs further study.These studies will provide a theoretical basis for further revealing the obstacles of SCNT reorganization and improving cloning efficiency.