The Study Of H3K27me3 Modification And Transcription Abnormality Of Bovine Early Cloned Embryos

Through somatic cell nuclear transfer(SCNT),terminal differentiated cells can be reprogrammed to the totipotency state and generate a complete individual.This advance enable SCNT technology to be widely used in animal breeding,endangered animal protection,disease model construction,biological medicine and human therapeutic cloning biological medicine,and endangered species protection.However,cloned embryos often show developmental arrest of different stages,which leads to low birth rate of cloned animals;even development to the end,the born cloned animals often show multiple developmental defects,these factors become the main obstacle to the widespread application of SCNT technology.Incomplete dedifferentiation and reprogramming is considered to be one of the main reasons for the low efficiency of SCNT reprogramming.In this research,the repressive histone modified-H3K27me3 of early cloned embryos and transcription abnormality of zygotic genome activation stage were studied respectively in bovine.H3K27me3 was identified as the obstacle of bovine SCNT reprogramming,and we also revealed that transcriptional memory inherited from donor cells existed in bovine cloned embryos.The main research contents and results are as follows:(1)Through immunofluorescence staining,the abnormally high proportion of H3K27me3 modification was determined in bovine early SCNT embryos.Subsequently,we attempted to modify the abnormal modification of H3K27me3 in SCNT embryos by inhibiting the activity of H3K27me3 methyltransferase-EZH2,inhibiting the expression level of EZH2,or overexpressing H3K27me3 demethylase-KDM6 A.Treatment of donor cells with the EZH2-selective inhibitor GSK343 could significantly reduce the expression level of H3K27me3 in donor cells.However,since GSK343 could induce cell autophagy and reduce cell viability,the development ability of bovine cloned embryos is ultimately affected.Inhibition of H3K27me3 methylase-EZH2 interfered the genomic stability of early embryos,induce cell apoptosis,lead to the developmental arrest of early cloned embryos,and ultimately reduced the blastocyst rate.Neither method could effectively improve the efficiency of bovine SCNT reprogramming.However,the overexpression of KDM6 A in the reconstructed embryo could significantly reduce the expression level of H3K27me3 modification in the cloned embryo,promote the blastocyst formation of the cloned embryo,and then improve the efficiency of SCNT reprogramming.(2)In order to further reveal the molecular mechanism of KDM6 A overexpression improving the efficiency of bovine SCNT reprogramming,RNA-seq was performed.The results showed that KDM6 A overexpression could promote the transcription level of multiple functional groups including cell adhesion,cell metabolism and X linked genes in cloned embryos.Of those genes,MBD3L2(methyl-CpG-binding domain protein 3-like 2)gene,which is related with embryonic genome activation,showed transcriptional insufficiency in mice,human and bovine early cloned embryos,and KDM6 A expression could restore the transcription defect of MBD3L2 in cloned embryos,the results also confirmed MBD3L2 gene was required for the preimplantation development of bovine cloned embryos.(3)For transcriptional abnormality during zygote genome activation of bovine cloned embryos,RNA-seq analysis results indicated that,compared with IVF embryos,there were 5,169 up-regulated genes and 8,653 down-regulated genes in cloned embryos.The analysis results of Gene Ontology(GO)showed that,5169 up-regulated genes were mainly enriched in multiple biological processes such as “RNA processing”,“translation”,“ribosome biosynthesis”,“translation initiation”,and “RNA methylation”;8653 down-regulated genes were enriched in multiple biological processes such as “intracellular signal transduction”,“cell development”,“cell proliferation”,“embryonic development”,and “reproductive process”.In short,“cell signal transduction” and “embryonic development” related genes existed transcriptional insufficiency,meanwhile,“translation” related genes existed excessive transcription.(4)Through the joint analysis of the transcriptome datas of donor cells,SCNT embryos and IVF embryos,we confirmed the existence of transcriptional memory inherited from donor cells in early cloned embryos.These abnormal transcription memory was called somatic memory,including 2108 active-memory genes and 5609 silent-memory genes.GO analysis showed that,active-memory genes were mainly involved in “translation”,“transcription”,and “RNA processing” while the silent-memory genes were mainly enriched in multiple biological processes such as “cell signal transduction”,“sexual reproduction”,“reproductive process”,and “embryonic development”.This result revealed that the high activation of genes related to “transcription” and “translation”and in donor cells was left into SCNT embryos;meanwhile,the transcription of genes which were involved in “signal transduction”,“sexual reproduction”,and “embryonic development” had not been effectively activated,and the expression level of these genes was significantly lower than that in IVF embryos.Active memory may be the result of incomplete dedifferentiation while silent memory may be the manifestation of incomplete reprogramming,which may be one of the important reasons for the low efficiency of bovine SCNT reprogramming.(5)Finally,we evaluated the effect of KDM5 B overexpression on transcription pattern during zygotic activation in bovine cloned embryos.RNA-seq analysis results show that KDM5 B overexpression could effectively correct the abnormal transcription profile in bovine SCNT embryos which inherited from the donor cells,not only reduced the expression level of active-memory genes,but also improved the transcriptional level of silent-memory genes,especially restored the transcription defects of genes which related with “cell signal transduction” and “embryonic development”,and ultimately greatly improved the efficiency of bovine SCNTreprogramming.