DNA Methylation Patterns Analysis And Epigenetic Marker Mining For Mouse Nuclear Cloning Embryo Reprogramming

Somatic cell nuclear transfer(SCNT)also known as somatic cell cloning,is a commonly used technique in the field of animal cell engineering to study cell reprogramming.It can reprogram terminally differentiated cells into cells with pluripotent state and develop into homogenous individual offsprings.Although SCNT has the advantages of being safe and obtaining pluripotent cells in cell reprogramming,the efficiency of reprogramming in most animals is still very low,and most cloned embryos arrest during early development.Overcoming epigenetic barriers is currently the primary strategy for improving reprogramming efficiency.The key histone methylation H3K9me3 and H3K27me3 have been clearly labeled as the reprogramming apparent barriers in previous studies,while there are few reports on DNA methylation molecular markers.It is urgent to conduct excavation through genome-wide analysis.In this study,we analyzed DNA methylation profiles of cloned embryos with different developmental fate and feritilized embryos.We found that cloned embryos showed increased methylation in global demethylation wave compared to feritilizedembryos.Then we further identified three abnormal methylation patterns in cloned embryos,including rDMRs? pDMRs and dDMRs.In addition,we found that the promoter region?first intron and 3'UTR are the major genomic functional regions with DMR in the analysis of differential methylation regions.Surprisingly,we also observed that the shorter the differential methylation regions are,the more significant on DNA methylation level change.The higher methylation level of the cloned embryos leads to early zygotic genomic genes,pluripotency maintenance factors and key transcription factors not being properly activated,which is an important reason for the failure of cloned embryos reprogramming.After that,we combined the transcriptome RNA-seq data for comprehensive analysis to identify gene clusters with high DNA methylation level and down-regulation of gene expression in cloned embryos.The low expression of these genes would cause the early development of cloned embryos to deviate from the normal track.Further,we identified a series of key reprogramming barrier factors by constructing a weighted gene co-expression network,including Sp110,Sp140,Pcgf5,Zfp207,Ubtfl1,Serbp1,Dppa2,Yy1,Prps1,Rrn3,Obox6,Kpna2,Elovl6,Trim13,Katna1,etc.Dppa2,Obox6,Pcgf5,Yy1 and Rrn3 have been proved to be the key genes in early embryonic development and play an important role in promoting early embryonic development.In addition,we have found that Dnmt1,Dnmt3 a and Dnmt3 b are elevated in SCNT embryos in cell reprogramming,whereas in the Tet family,only Tet1 expression levels decreased,suggesting that hypermethylation of cloned embryos may be caused by high expression of the Dnmt family and lowexpression of Tet1.This study comprehensively analyzed the abnormal patterns of whole-genome DNA methylation in cloned embryos,and provided theoretical support for overcoming the DNA methylation epigenetic barrier in reprogramming and improving the efficiency of reprogramming.It is helpful for the clinical application of somatic cell nuclear transfer.