The Regulation Of KDM4D And KDM4E In Bovine IVF Embryonic Development And SCNT-mediated Nuclear Reprogramming

Somatic cell nuclear transfer(SCNT)can reprogram terminally differentiated cells into the totipotent state,which can be used to generate full-term organisms from these cells.Therefore,animal production by means of SCNT has great potential applications in therapeutic cloning,disease models,regenerative biomedicine,livestock breeding,and species preservation.However,the cloning efficiency is extremely low in most mammalian species,which is a major limitation to the practical use of the SCNT technique.High rates of embryonic losses,stillbirths,perinatal mortality and postnatal abnormity have been typical outcomes.Thus far,aberrant epigenetic reprogramming of the donor genome by ooplasma is believed to be the main reason for these developmental defects of SCNT embryos.Previous studies have observed the global hypermethylation of histone H3 lysine 9 tri-(H3K9me3)and di-methylation(H3K9me2)in bovine SCNT embryos during zygotic genome activation(ZGA).We thus speculate that persistent H3K9me3 and H3K9me2 are barriers to SCNT-mediated reprogramming.In this study,through comparative transcriptome and quantitative PCR(qPCR)analysis,two bovine H3K9 demethylases,lysine-specific demethylase 4D(KDM4D)and 4E(KDM4E),exhibited insufficient expression in SCNT embryos at the ZGA stage.By performing loss-of-function experiments on in vitro fertilized(IVF)embryos,and gain-of-function experiments on SCNT embryos,we confirmed that KDM4 D and KDM4 E were essential for active H3K9 demethylation and nuclear reprogramming during ZGA.The main works and results are as follows:1.Through comparative transcriptome and qPCR analysis,the transcriptional patterns of KDM4 D and KDM4 E at each stage of IVF and SCNT embryos were investigated.The results showed that both genes exhibited the burst of expression in these two embryos during ZGA,and the transcriptional level of KDM4 E was higher than KDM4 D.In addition,KDM4 D and KDM4 E expression was lower in SCNT embryos than in IVF embryos.2.By performing siRNA microinjection,the functions of KDM4 D and KDM4 E for IVF embryonic development were dissected.Simultaneous knockdown of both genes could impede H3K9 demethylation during IVF ZGA,suppress the transcriptional activation of satellite I and some zygotic genes,and impair the developmental competence of IVF embryos.For example,the proportion of embryos reaching the blastocyst stage and blastocyst cell number were reduced,while apoptotic cells in blastocyst were increased.Moreover,only knockdown of KDM4 E,but not KDM4 D,could obtain the similar defects,indicating that KDM4 E plays a more crucial role during embryonic development.3.By performing KDM4 D and KDM4 E mRNA microinjection,either wild-type KDM4 D or KDM4 E overexpression could similarly eliminate H3K9 methylation during SCNT ZGA,reactivate the transcriptional level of satellite I and some zygotic genes,and improve the developmental competence of SCNT embryos.For example,the proportion of embryos reaching the blastocyst stage and inner cell mass cell number were increased,and the birth rate of cloned cattle was also promoted.However,the catalytic defective mutants of KDM4 D and KDM4 E could not improve SCNT embryonic development in vitro.4.From the RNA sequencing data,we found 6,984 differentially expressed genes(DEGs)between eight-cell IVF and SCNT embryos.Among these,1,893 genes,which were downregulated in SCNT embryos,could be reactivated by KDM4 E overexpression.These DEGs were enriched in the biological processes,such as signal transduction,embryo development,cell cycle,transcriptional regulation,and were enriched in the cell pathways related to tight junction,pluripotency regulation of stem cells,ubiquitin mediated proteolysis and Hippo signaling pathway.Simultaneously,2,057 genes,which were upregulated in SCNT embryos,were restored by KDM4 E overexpression.These DEGs were enriched in the biological processes,such as oxidation-reduction,protein methylation,response to DNA damage stimulus,and were enriched in the cell pathways related to oxidative phosphorylation,protein and RNA degradation,as well as RNA transcription.In the current study,on the one hand,we correlate H3K9 demethylation during ZGA with the high expression levels of KDM4 D and KDM4 E in IVF embryos.These findings provide new evidence of epigenetic modulation by the KDM4 family in embryonic development.On the other hand,we define KDM4 D and KDM4 E as intrinsic defective factors that are related to aberrant H3K9 methylation barriers to nuclear reprogramming,and thus provide a more precise and effective approach to improving the SCNT technique in transgenic cattle production.