| BRG1 is the core catalytic subunit of the chromatin remodeling complex SWI/SNF family,which has helicase and ATPase activities,and plays an important role in opening chromatin,changing local nucleosome localization,regulating mammalian cell gene transcription(activation or inhibition),and somatic cell reprogramming.At present,the research on the role of BRG1 in somatic cell nuclear transfer is still blank.For this reason,this study used porcine fetal fibroblasts as donor cells,and the effect of regulating BRG1 expression on the expression of genes related to epigenetic modification of donor cells and the opening degree of chromatin was first analyzed.Then somatic cell nuclear transfer was carried out to explore the effect of BRG1 overexpression in donor cells on the development of pig nuclear transfer embryos.The results lay a foundation for clarifying the regulatory effect of BRG1 on the epigenetic modification of donor cells and further improving the efficiency of porcine somatic cell nuclear transfer.The results are as follows:1.Effects of regulating BRG1 expression on the level of epigenetic modification and chromatin opening of porcine fetal fibroblastsImmunofluorescence and q RT-PCR methods were used to analyze the expression of epigenetic modification related genes.The immunofluorescence results showed that in the serum starvation treatment group,overexpression of BRG1 resulted in increase in the expression levels of 5-m C and H3K14 ac,while decrease in the expression level of H3K9me3.The q RT-PCR results showed that overexpressing BRG1 significantly increased the expression of DNMT1 and HAT1(P<0.05),while the expression of SVU39H1,SUV39H2,and SETDB1decreased(P<0.05).In order to explore the effect of BRG1 overexpression in donor cells on the openness of chromatin and the level of gene transcription,first immunofluorescence was used to detect HP1α expression,then ATAC-seq and RNA-seq methods were performed to detect the openness of chromatin and the gene expression.Immunofluorescence results showed that overexpression of BRG1 reduced HP1α expression in fetal fibroblasts.Analysis of ATAC-seq results showed that overexpression of BRG1 promoted higher degree of openness in SMARCA4,NANOG,SOX2,MAP2K6,and HIF1 A genes.RNAseq results showed that overexpression of BRG1 was highly correlated with genes related to neuronal production,development and autophagy,and differentially upregulated genes were enriched in signaling pathways such as PI3K-AKT.2.Effects of regulating BRG1 expression in donor cells on the development of porcine somatic cell nuclear transfer embryosFirstly,the expression patterns of BRG1 in porcine oocytes matured in vitro,parthenogenetic activation and early embryos of somatic cell nuclear transfer were analyzed,and the effects of overexpression of BRG1 gene in donor cells on the development of early embryos of porcine nuclear transfer and the expression of related genes were discussed.The results of q RT-PCR and immunofluorescence showed that BRG1 was expressed at all stages of in vitro maturation of pig oocytes and parthenogenetic embryos.In nuclear transfer embryos,compared with the negative control group,BRG1 expression was significantly increased at the 2-cell and 4-cell stages in the experimental group(P<0.05).Overexpression of BRG1 gene significantly increased the blastocyst rate of pig nuclear transfer embryos(17.83% vs 27.33%,P<0.05),and significantly increased the expression of NANOG,SOX2,EIF3 A,TFIIA genes(P<0.05).The above results showed that BRG1 enhanced the opening degree of SMARCA4,NANOG,SOX2,MAP2K6,HIF1 A genes,and up-regulated BRG1 expression in donor cells could improve the early development ability of porcine somatic cell nuclear transfer embryos. |
