Preliminary Study On The Role Of KDM4A In The Early Embryonic Development Of Mice By Using RNA-Seq Technology

Pre-implantation mouse embryonic development is a complex and delicate process,which includes many key events,such as zygotic genome activation(ZGA),embryo compaction and the first cell differentiation(that is,the first fate determination).In these processes,a number of transcription factors and epigenetic factors play an important role.Exploring the mechanism of these key regulatory factors in the process of embryonic development is the basis for us to understand the complexity and systematicness of the development.However,the main determinants of ZGA during early mouse embryogenesis are still controversial.Through high-throughput sequencing techniques,our laboratory found that KDM4 D and KDM4 E regulate lysine 9 dimethyl and trimethylation modified erasure and embryonic development of histone H3 in bovine IVF embryos during ZGA,and affect somatic reprogramming in SCNT embryos.KDM4A(JMJD2A),as a lysine demethylase,can also remove dimethyl and trimethylation from lysine 9 of histone H3,thus maintaining transcriptional activity.But so far,the physiological role of KDM4 A in normal development has not been studied in detail.In this study,bioinformatics analysis was carried out on the high-throughput sequencing data of KDM4 A knockout and wild type mouse embryos,and microinjection technique was used to interfere with the expression of KDM4 A gene in mouse fertilized eggs,so as to study the biological function of KDM4 A in mouse early embryo development.The results are as follows:(1)The differential genes and alternative splicing of RNA-Seq data of KDM4 A knockout and wild type mice were analyzed by bioinformatics method.the results showed that there were significant differences in gene expression and splicing patterns between KDM4 A knockout and wild type mouse embryos,and these genes had significant differences in biological functions.These differences may be due to the fact that KDM4 A regulates the expression of genes related to histone modification and splicing factors.(2)The expression pattern of KDM4 A gene during mouse early embryonic development was determined by quantitative experiment.(3)The effect of knockout KDM4 A gene on mouse embryonic development was studied by interference fragment microinjection experiment.The results showed that interfering with KDM4 A gene expression did not affect the cleavage rate of 2-cell stage embryos,but the blastocyst rate of early embryos decreased,which proved that it could promote the development of early embryos.(4)The expression of HSP70.1,Zscan4 d and other zygotic genes in mouse 2-cell stage embryos interfering with KDM4 A was quantitatively verified.The results showed that KDM4 A was involved in the activation of some mouse zygotic genes.(5)Immunofluorescence staining and Western Blot assay were used to detect the modification of H3K9me3 in 2-cell mouse embryos with knockout KDM4 A gene,and the genes of DDX39,Hnrnpu,Prpf38 a and Rbm39 splicing factors were detected quantitatively.The results showed that KDM4 A could regulate the development of early mouse embryos by removing the modification of H3K9me3 in 2-cell stage and affecting the expression of splicing factor-related genes.In summary,this paper studied the biological function of lysine demethylase KDM4 A in early mouse embryonic development,and verified that KDM4 A regulates the subsequent development of mouse embryos by participating in the removal of H3K9me3 modification of 2-cell mouse embryos and affecting the expression of splicing factor-related genes.