The Regulation Of TET3 In DNA Demethylation Of Goat Early IVF Embryonic Development And SCNT-mediated Nuclear Reprogramming

Mammalian somatic cells can be reprogrammed into a totipotent state and develop into intact individuals with the reprogramming of oocyte cytoplasm through somatic cell nuclear transfer(SCNT).SCNT is a promising technology with great value in the production of transgenic animals,protection of endangered species,breeding of livestock with superior characteristics,research on epigenetic regulation mechanisms during embryonic development and therapeutic cloning.The latest survey indicates that more than 20 mammals have been cloned successfully so far and SCNT technology has been partially commercialized in pet cloning and livestock breeding.However,low efficiency has prevented the practical use of SCNT partly owing to aberrant epigenetic reprogramming of somatic donor cell nuclei,especially with hypermethylation of genomic DNA in pre-implantation embryos.In general,abnormal DNA methylation is believed to be partly caused by shortage of DNA demethylation in early embryos.Thus,enhancement of DNA demethylation capability of early cloned embryos may be a key way to improve cloning efficiency.Reportedly,TET3 protein is the primary early-embryo demethylase that can oxidize 5-methylcytosine(5m C)to5-hydroxymethylcytosine(5hm C),but the mechanism of TET3 in DNA demethylation at goat early embryos is still unclear.In this study,we reported that TET3 is a key factor in DNA demethylation in goat preimplantation embryos through RNA interference technology,immunofluorescence staining,bisulfite sequencing,oxidative bisulfite sequencing and real-time PCR.Overexpression of TET3 in donor cells corrected abnormal DNA hypermethylation of cloned embryos and significantly enhanced in vitro and in vivo developmental rate and significantly improves goat SCNT efficiency.Through analysed the intrinsic disordered regions of DNA demethylation-related proteins,we found that DNA demethylation-related proteins belong to intrinsically disordered proteins,and they had different intrinsically disordered regions in different species.We demonstrated that this may be the reason for the different DNA demethylation pattern among species.The main contents and results for this research are as follows:1.We confirmed that TET3 is a key factor in DNA demethylation in goat early embryos,through RNA interference technology,immunofluorescence staining,bisulfite sequencing,oxidative bisulfite sequencing and real-time PCR.Immunofluorescence staining results showed that the male pronucleus didn’t undergo TET3-mediated DNA demethylation in goat zygotes,and TET3 was important in DNA demethylation at the 2-cell to 4-cell stage in goat early embryos.The DNA methylation level of 4-cell stage embryos was increased in IVF embryos which derived from TET3-knockdown oocyte.According to the bisulfite sequencing,oxidative bisulfite sequencing and real-time PCR results,knockdown of TET3 increases methylation levels of the pluripotency genes OCT4 and NANOG,and reduces NANOG expression at blastocyst stage.2.By overexpressing TET3 in goat fetal fibroblasts,we investigated the effects of TET3 overexpression on overall DNA methylation status and the DNA methylation level on specific site as well as the key pluripotency gene expression in goat somatic cells.Firstly,Basing on on the conserved sequence of TET3,we performend 5’ and 3’ c DNA end rapid amplification techniques and obtained the full length of oocytes specific TET3 coding region.Then we constructed TET3-inducible expressed vector using Tet On-3G eukaryotic expression system,and transfected goat fetal fibroblasts cells with electroporation,finally,the TET3-transgenic somatic cells were screened and TET3 was overexpressed with doxycycline induction.Immunofluorescence staining results showed that overexpression of TET3 cells can initiate genomic DNA demethylation in goat somatic.The bisulfite sequencing,oxidative bisulfite sequencing and real-time PCR results showed that overexpression of TET3 in somatic cells can reduce the promoter DNA methylation level of pluripotency gene OCT4 and NANOG,and promote the expression of these key pluripotency genes.3.We investigated the effect of TET3 overexpression in donor cells on the methylation status of SCNT embryos as well as the embryos development ability both in vitro and in vivo,by performing goat SCNT using TET3-overexpressed fetal fibroblast cells.Firstly,TET3 was overexpressed with doxycycline induction,then the TET3-overexpressed cells were used as donor cells for SCNT,the embryos development rates and embryos quality were significantly improved in vitro.Immunofluorescence staining results showed that overexpression of TET3 in somatic cells can correct hypermethylation aberrance of SCNT embryos,with SCNT mebryos has the closer DNA methylation levels to IVF embryos.Based on the bisulfite sequencing,oxidative bisulfite sequencing and real-time PCR results,overexpression of TET3 in donor cells led to decrease of DNA methylation levels in the promoter regions of OCT4 and NANOG and promoted the key pluripotency NANOG expression in blastocysts.The embros were then transferred into recipients by surgery,the pregnancy rate and birth rate of cloned goat were counted,the results showed that overexpression of TET3 in donor cells could improve the development of SCNT embryos in vivo and significantly enhance the cloning efficiency of goats.4.We used the intrinsically disordered proteins analysis software and databases to analyze the intrinsic disorder of DNA demethylation-related proteins in preimplantation embryos,the analysis results showed that DNA demethylation-related proteins belonged to intrinsically disordered proteins with TET3 had the highest tendency,and they had a lot of intrinsically disordered regions that could bind different ligands,moreover,these intrinsically disordered regions differed among species.These findings suggested that the difference of intrinsically disordered proteins may be the reason for the different DNA demethylation patterns among species in preimplantation embryos.In summary,our findings indicated the important role of TET3 in the preimplantation embryonic development of goat,which has implications for revealing the mechanism of DNA methylation reprogramming in early embryos;we also established a method to improve the efficiency of goat somatic cells nuclear transfer that has important application value in animal cloning through SCNT technology.