The Effects Of Cycloleucine On The Chromatin Accessibility Of Porcine Fibroblast And The Subsequent Development Of Nuclear Transfer Embryos

The efficiency of somatic cell nuclear transfer(SCNT)is closely related to the degree of donor cell reprogramming.The epigenetic modification of donor cells before implantation affects the structural state of chromatin,and then affects nuclear reprogramming.Some studies have found that RNA m6 A methylation plays an important role in somatic cell reprogramming and can regulator gene transcription and chromatin accessibility,but the role of this modification in pig somatic cell nuclear transfer is not clear.Therefore,this study preliminarily revealed the role of m6 A modification in regulating the accessibility of chromatin in porcine fetal fibroblasts(PFFs)and the development of cloned embryos constructed from PFFs by exploring the effect of cycloleucine(CL),an RNA methylation inhibitor,on the development of porcine fetal fibroblasts(PFFs)chromatin and subsequent nuclear transfer embryos.The main results of the study are as follows:1.The effect of PFFs on cell chromatin accessibility by CL culture.Firstly,different concentrations of CL were used to treat PFFs for different times.Through methods such as cell morphology observation,CCK-8 cell proliferation activity detection,and q RT-PCR,it was found that when PFFs were cultured in 10 mmol/L CL for 48 hours,the cell morphology was normal,and there was no significant difference in proliferation activity compared to the control group(P>0.05),and the expression of the anti apoptotic gene Bcl-2 in the cells was significantly upregulated(P<0.05).Therefore,it was determined that 10 mmol/L CL for 48 hours was the appropriate treatment concentration and time.The results of micrococcal nuclease shearing experiment showed that CL cultured PFFs promoted the accessibility of cell chromatin;And the expression level of heterochromatin labeled HP1α gene in cells was significantly decreased(P<0.05).Secondly,the reason why CL affects the accessibility of chromatin in PFFs cells was analyzed.It was found that the overall level of m6 A methylation in PFFs cells cultured in CL was significantly downregulated(P<0.05),and the expression levels of m6 A methyltransferase genes METTL3 and WTAP in cells were significantly downregulated(P<0.05),the expression of demethylase FTO was significantly upregulated(P<0.05),and the expression of the readers YTHDC1 and YTHDF2 were significantly downregulated(P<0.05);Further detection of the expression level of the downstream histone methyltransferase SETDB1 gene and the H3K9me3 modification level in the m6A-YTHDC1 mechanism revealed a significant downregulation of SETDB1 gene expression and H3K9me3 modification level in PFFs cultured by CL(P<0.05).2.Explored the effect of CL treatment of PFFs on the development of subsequent nuclear transfer embryos.In this experiment,PFFs cultured by CL were used as donor cells for nuclear transfer.It was found that the blastocyst development rate of SCNT embryos was significantly higher than that of control embryos(P<0.05);RT-q PCR was used to detect the expression of marker genes and blastocyst pluripotency genes at the activation stage of zygote genome.It was found that the expression of zygote activating genes e IF3 A and TFIIA,and the expression of pluripotency genes Nanog and Oct-4at the blastocyst stage of SCNT embryos in CL group were significantly higher than those in the control group(P<0.05);The results of immunofluorescence staining analysis showed that the m6 A and H3K9me3 levels of SCNT embryos in the CL group were significantly lower than those in the negative control at the 2-cell,4-cell,and 8-cell stages(P<0.05).The above results show that:(1)PFFs cultured by the appropriate concentration of CL are conducive to promoting the accessibility of chromatin.The reason is that CL downregulates the level of m6 A modification in cells.By participating in the m6A-YTHDC1 mechanism in cells,YTHDC1 reduces the recruitment of downstream SETDB1 methyltransferase,and reduces the level of histone H3K9me3 modification,thereby promoting the accessibility of chromatin in cells;(2)CL culture of donor cell PFFs is conducive to improving the developmental potential of cloned embryos,and to a certain extent overcome the abnormal H3K9me3 obstacle in SCNT embryos.