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首页> 外文期刊>Cellular Physiology and Biochemistry >Dynamic Methylation Changes of DNA and H3K4 by RG108 Improve Epigenetic Reprogramming of Somatic Cell Nuclear Transfer Embryos in Pigs
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Dynamic Methylation Changes of DNA and H3K4 by RG108 Improve Epigenetic Reprogramming of Somatic Cell Nuclear Transfer Embryos in Pigs

机译：RG108对DNA和H3K4的动态甲基化作用改善了猪体细胞核移植胚胎的表观遗传重编程

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Background/Aims DNA methylation and histone modifications are essential epigenetic marks that can significantly affect the mammalian somatic cell nuclear transfer (SCNT) embryo development. However, the mechanisms by which the DNA methylation affects the epigenetic reprogramming have not been fully elucidated. Methods In our study, we used quantitative polymerase chain reaction (qPCR), Western blotting, immunofluorescence staining (IF) and sodium bisulfite genomic sequencing to examine the effects of RG108, a DNA methyltransferase inhibitor (DNMTi), on the dynamic pattern of DNA methylation and histone modifications in porcine SCNT embryos and investigate the mechanism by which the epigenome status of donor cells’ affects SCNT embryos development and the crosstalk between epigenetic signals. Results Our results showed that active DNA demethylation was enhanced by the significantly improving expression levels of TET1, TET2, TET3 and 5hmC, and passive DNA demethylation was promoted by the remarkably inhibitory expression levels of DNMT1, DNMT3A and 5mC in embryos constructed from the fetal fibroblasts (FFs) treated with RG108 (RG-SCNT embryos) compared to the levels in embryos from control FFs (FF-SCNT embryos). The signal intensity of histone H3 lysine 4 trimethylation (H3K4me3) and histone H3 lysine 9 acetylation (H3K9Ac) was significantly increased and the expression levels of H3K4 methyltransferases were more than 2-fold higher expression in RG-SCNT embryos. RG-SCNT embryos had significantly higher cleavage and blastocyst rates (69.3±1.4%, and 24.72±2.3%, respectively) than FF-SCNT embryos (60.1±2.4% and 18.38±1.9%, respectively). Conclusion Dynamic changes in DNA methylation caused by RG108 result in dynamic alterations in the patterns of H3K4me3, H3K9Ac and histone H3 lysine 9 trimethylation (H3K9me3), which leads to the activation of embryonic genome and epigenetic modification enzymes associated with H3K4 methylation, and contributes to reconstructing normal epigenetic modifications and improving the developmental efficiency of porcine SCNT embryos.
机译：背景/目的DNA甲基化和组蛋白修饰是必不可少的表观遗传标记，可以显着影响哺乳动物体细胞核移植（SCNT）胚胎的发育。但是，尚未完全阐明DNA甲基化影响表观遗传重编程的机制。方法在我们的研究中，我们使用了定量聚合酶链反应（qPCR），蛋白质印迹法，免疫荧光染色（IF）和亚硫酸氢钠基因组测序来研究DNA甲基转移酶抑制剂（DNMTi）RG108对DNA甲基化动力学模式的影响。猪SCNT胚胎中的蛋白和组蛋白修饰，并研究供体细胞的表观基因组状态影响SCNT胚胎发育和表观遗传信号之间的串扰的机制。结果我们的结果表明，通过显着提高TET1，TET2，TET3和5hmC的表达水平，可以增强主动DNA的去甲基化作用，而通过抑制由胎儿成纤维细胞构建的胚胎中DNMT1，DNMT3A和5mC的显着表达水平，可以促进被动DNA的去甲基化作用。与对照FFs（FF-SCNT胚胎）的胚胎水平比较，用RG108（RG-SCNT胚胎）处理的（FFs）。组蛋白H3赖氨酸4三甲基化（H3K4me3）和组蛋白H3赖氨酸9乙酰化（H3K9Ac）的信号强度显着增加，并且H3K4甲基转移酶的表达水平在RG-SCNT胚胎中高2倍以上。与FF-SCNT胚胎（分别为60.1±2.4％和18.38±1.9％）相比，RG-SCNT胚胎的卵裂和胚泡率显着更高（分别为69.3±1.4％和24.72±2.3％）。结论RG108引起的DNA甲基化的动态变化导致H3K4me3，H3K9Ac和组蛋白H3赖氨酸9三甲基化（H3K9me3）模式的动态变化，从而导致胚胎基因组和与H3K4甲基化相关的表观遗传修饰酶的激活。重建正常的表观遗传修饰并提高猪SCNT胚胎的发育效率。

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