| Imprinted gene cluster of Igf2-H19 plays an important role in regulating the development of fetus and placenta.Previous studies showed that DNA methylation,histone modification,and non-coding RNA are involved in the establishment,maintenance,and expression of imprinting genes.In this study,CRISPR/dCas9 technology were used to modify DNA methylation in the third and fourth differentially methylated regions(m3 and m4)in the imprinting control region(ICR)of the Igf2-H19 gene cluster in mouse embryonic stem cells(mESCs),and the synergistic regulation of DNA methylation and histone modification on imprinted gene expression were investigated.1.DNA methylation at Igf2-H19 locus in mouse embryonic stem cell linesMouse blastocysts were cultured in a 2i/L culture system,and 9m ESCs lines were isolated and established.The m ESC clones showed a dome-like shape with positive alkaline phosphatase staining and normal chromosome number;The cells expressed the marker multipotent genes Nanog,Oct4 and Sox2,had strong proliferation ability and the potential of differentiation into tridermal lineages;DNA methylation sequencing results showed that DNA methylation levels in the m3 and m4 regions of the Igf2-H19 gene cluster were lower in female mESCs than those in male mESCs.Then the DNA methylation of the m3 and m4 in the Igf2-H19 gene cluster was targeted by CRISPR/dCas9-Dnmt3 a,respectively.The results showed that the targeting modification of m3 region could increase the DNA methylation level in this region,but the expression of Igf2 and H19 did not change;The targeting modification of m4 region also increased the DNA methylation in this region,meanwhile,Igf2 expression were increased,and H19 expression were decreased;After joint modification of m3 and m4 regions,the DNA methylation level in the corresponding regions increased,Igf2 expression were increased,and H19 expression were decreased.Compared with the effects of DNA demethylation drug 5-Aza-d C treatment,the DNA methylation level in the m4 region was decreased,Igf2 expression was decreased,and H19 expression was increased when cells were treated by the optimal concentration of 20?M.The above results indicated that the changes of DNA methylation in the m4 region played an important role in regulation of Igf2 and H19 expression.2.Synergistic effects of DNA methylation and histone modification on the H19 ICRTo further explore the synergistic effect of DNA methylation and histone modification on regulation of Igf2-H19 locus,the si RNA technique was used to interfere H3K9me3 methyltransferase Setdb1 in female m ESC.Immunofluorescence and ChIP-qPCR results showed that both H3K9me3 modification and DNA methylation levels in m3 and m4 regions was significantly reduced after si RNA interference,meanwhile,H19 expression was increased,and Igf2 expression was not changed.In addition,this study analyzed the changes of histone modifications after targeting DNA methylation by using CRISPR/dCas9-Dnmt3 a.The results of ChIP-qPCR showed that H3K9me3 was slightly increased in m3 and m4 regions after the increase of DNA methylation,but H3K4me3 had no change.The decrease of DNA methylation by 5-Aza-dC caused no change both in H3K4me3 and H3K9me3 modifications,in addition,the enrichment of TET3 in m3 and m4 regions did not change after the interference of Setdb1 or treatment by 5-Aza-dC.In conclusion,DNA methylation levels in the m3 and m4 regions of the Igf2-H19 gene cluster were lower in female cells than those in male m ESCs,the CRISPR/dCas9-Dnmt3 a system could specifically increase DNA methylation.The change of DNA methylation in the m4 region affect the expression of Igf2 and H19,the DNA methylation levels in m3 and m4 regions decreased with the decrease of H3K9me3,while the expression of H19 were increased;however,H3K9me3 modification did not change with DNA methylation strictly.These results suggest that DNA methylation and histone modification synergistically regulate the expression of imprinted gene Igf2. |
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