Identification Of Key Genes And Their Functions In Early Embryonic Development Based On Single-cell Transcriptome And Genome-Wide Bisulfite Sequencing In Yaks

As an important livestock in alpine grazing area,yak has low reproductive efficiency,low development rate of oocytes and embryos in vitro,few quality breeding stock and yak has not been successfully cloned,limit the development of plateau animal husbandry.Early embryo production in vitro and somatic cell cloning technology have a great significance in livestock breeding.Therefore,screening and identificated the key regulatory genes of preimplantation embryo development and further study the molecular mechanisms affecting preimplantation embryo development have important theoretical significance and production value on revealing the law of embryo development,improving the level of in vitro embryo production and the efficiency of somatic cell cloning.Extensive epigenetic reprogramming occurs during preimplantation development.However,the effects of DNA methylation on preimplantation embryonic development in yaks are still unknown,and how the epigenetic reprogramming contributes to transcriptional regulatory networks is still unclear.Recently,with the emergence of single-cell RNA-Seq and single-cell BS-seq sequencing techniques,detailed studies of epigenetic state and phenotypic associations have been made possible.Thus,this study aims to explore the gene regulatory network and dynamic changes of DNA methylation in yak oocytes and preimplantation embryos in vitro by single-cell RNA-Seq and single-cell BS-seq,reveal the DNA methylation reprogramming of preimplantation embryos,and identify key candidate genes which cause developmental abnormalities in embryos and cloned embryos.By using real-time quantitative PCR,Western Blot,immunofluorescence staining,microinjection,somatic cell nuclear transfer,Co-Immunoprecipitation,Bisulfite sequencing,overexpression and interference to further explore the molecular mechanism of key developmental genes,all these together improving the propagation rate of excellent yak individuals and lay a theoretical foundation for protecting the excellent germplasm resources of yak.The results are as follows:1.In this study,the most comprehensive single cell transcriptome map of yak embryo development stage was constructed by single cell RNA-Seq analysis of 5 developmental stages of yak MⅡ oocyte,2-cell,4-cell,8-cell and morula.The activation period of embryonic genome was determined,and the specific candidate gene regulatory networks were screened for each stage.A total of 44 to 7588 differentially expressed genes were detected between any two consecutive developmental stages.GO enrichment analysis was related to the developmental process.KEGG was enriched in ubiquitin-mediated proteolysis,actin cytoskeleton regulation,MAPK signaling pathway,Hippo signaling pathway,Wnt signaling pathway,purine metabolism and other pathways.31 conserved maternal central genes and 39 conserved zygote central genes,including SIN3 A,PRC1,KIF15,HDAC1/2 and HSPD1,were identified by cross-species transcriptomic analysis between yak,human,bovine and mouse,respectively.Finally,real-time quantitative PCR was used to verify the differential genes identified,and the results proved reliable.2.In this study,the whole genome DNA methylation map of yak embryo at the developmental stage was constructed by single cell BS-seq analysis of the embryos at four developmental stages: 2-cell,4-cell,8-cell and morula.The DNA methylation level was high at the 2-cell stage,demethylation began at the 4-cell stage,and the methylation level decreased rapidly,and reached the lowest point at the 8-cell stage.The DNA methylation level recovered to the hypermethylation level at the morula stage,the results is similar to the CGI methylation levels.Different functional regions showed different levels of DNA methylation.Cluster analysis of differential Cp G sites showed that the methylation of a large number of Cp G sites was remained at low levels during pre-implantation embryonic development.In this study,we analyzed the dynamic changes of genome-wide DNA methylation during early embryonic development.Approximately 106,000 differentially methylated regions were identified in embryonic genome activation(EGA)phase.GO analyzed showed differentially methylated genes were enriched in ion,protein and ATP binding,ribonucleotide and nucleotide binding,and cation and metal ion transport activities.KEGG was enriched in metabolic pathway,PI3K-AKT,MAPK signaling pathway,ubiquitin-mediated proteolhydrolysis,Hippo signaling pathway,and Wnt signaling pathway.The BS-seq method was used to verify the methylation of the identified genes,and the results proved to be credible.By combining single-cell methylation and transcriptome data,this study found a negative correlation between promoter methylation levels and the expression of corresponding genes.The level of gene expression in the gene body was positively correlated with its corresponding degree of DNA methylation modification.The proportion of methylated genes was higher than that of unmethylated genes in differentially expressed genes,and the correlation between the methylation level of repeating elements and their corresponding expression was weak.We constructed the network map of differentially expressed genes and differentially methylated genes preliminarily and revealed the key methylation driver genes negatively correlated with the expression of differentially expressed genes and promoter methylation at EGA stage.43 genes related to yak embryonic development that may be regulated by methylation were identified.Such as UHRF1,AURKA,PLK1,TOP2 B,CENPF,NUSAP1,CDC25 B and so on.In addition,methylation levels were associated with the expression of DNA methylation related genes(TET1,TET2,TET3,DNMT3 B,and UHRF1)and imprinted genes(CD81,SDHD,IGF2,ZIM2,and MEST).These results indicated that DNA methylation may regulate the preimplantation embryo development of yak by regulating the expression of key genes.3.The differential methylated gene USP7 and the differential expressed gene HDAC1/2 associated with epigenetic modifications at EGA stage were screened.The results showed that USP7 is very important for morula to blastocyst conversion in yak,and USP7 may reduce blastocyst development rate and blastocyst quality in yak by affecting the whole DNA methylation level.By treating yak embryos with HDAC1/2 small molecule inhibitor FK228,we found that FK228 treatment group reduced in vitro blastocyst development rate and blastocyst quality of yak.Mice were used as model animals to further study the potential mechanism of USP7 affecting preimplantation embryo development.The study showed that USP7 was expressed during the early embryonic development process of mice,and USP7 protein was mainly present in the nucleus and cytoplasm.Interference with USP7 or the treatment of embryos with a small molecule inhibitor of USP7,P5091,significantly reduced the blastocyst development rate and blastocyst quality.Single-cell transcriptomic sequencing,RT-q PCR and immunofluorescence staining results showed that interference with USP7 disrupted normal lineage differentiation and key genes expression.At the same time,developmental failure caused by interference with USP7 was accompanied by severe disturbances in transcriptional activation and epigenetic modification.Western Blot and co-IP tests further revealed that USP7 may influence morula to blastocyst transition by stabilizing the target protein YAP through ubiquitin-proteasome pathway.These results suggest that USP7 may play an important role in preimplantation embryos by regulating lineage differentiation and key epigenetic modifications.4.CBX3,a differentially expressed gene associated with epigenetic modifications at the EGA stage,was highly expressed in yak morula stage and abnormally high expressed in cloned embryos.H3K9me3,as a key reprogramming barrier of cloned embryos in multiple species,was also abnormally high expressed in cloned embryos.In this study,tissue culture was used to culture yak fetal fibroblasts,and the interference vector CBX3 was constructed,q RT-PCR and Western Blot were used to verify the interference effect.Cell immunofluorescence and q RT-PCR tests showed that interference with CBX3 could significantly reduce the expression levels of H3K9me3,SUV39H1,and SUV39H2 m RNA,but interference with CBX3 had no significant effect on the m RNA expression levels of NSD1 and EZH2.The cell lines with low expression CBX3 as donor cells for somatic cell nuclear transfer,the results found that the development rate of clonal blastocyst in the low expression of CBX3 group was significantly higher than that in the control group.The results indicated that CBX3 may improve the development of yak cloned embryos by affecting the expression of H3K9me3.5.The EGA-stage differential methylation gene ZFP57 associated with epigenetic modifications was screened,and the abnormal demethylated imprinted genes in cloned embryos contained a specific recognition sites that was recognized by ZFP57 and involved in maintaining methylation levels.In this study,overexpressed ZFP57 cell lines were established which as donor cells for somatic cell nuclear transfer.We found that compared with cloned embryos,overexpressed ZFP57 embryos increased the blastocyst development rate,increased the total cell number and trophoblast cell number as well as the ratio of inner cell mass to total cell number while reducing the apoptosis rate.Meanwhile,interfered with ZFP57 reduced the development rate of IVF embryos.Moreover,it was further proved that ZFP57 can correct the abnormal hypomethylation of imprinted genes H19/IGF2,XIST,and IGF2 R on cloned embryos by bisulfite sequencing,all these suggesting that ZFP57 plays a key role in maintaining the methylation of imprinted genes.In summary,gene expression and DNA methylation in oocytes and a series of yak embryos were quantified at single-cell resolution by using methylation and transcriptome sequencing for the first time.In this study,we mapped the epigenetic reprogramming events critical to embryonic development and described in detail key genes in embryonic genome activation events and their molecular mechanisms affecting development.