Zhai Yanhui, Zhang Zhiren, Yu Hao, Su Li, Yao Gang, Ma Xiaoling, Li Qi, An Xinglan, Zhang Sheng, Li Ziyi
First Hospital, Jilin University, Changchun, China.
College of Veterinary Medicine, Jilin University, Changchun, China.
Cell Physiol Biochem. 2018;50(4):1376-1397. doi: 10.1159/000494598. Epub 2018 Oct 24.
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.
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.
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).
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.
背景/目的：DNA甲基化和组蛋白修饰是重要的表观遗传标记，可显著影响哺乳动物体细胞核移植（SCNT）胚胎的发育。然而，DNA甲基化影响表观遗传重编程的机制尚未完全阐明。
在我们的研究中，我们使用定量聚合酶链反应（qPCR）、蛋白质免疫印迹法、免疫荧光染色（IF）和亚硫酸氢盐基因组测序来检测DNA甲基转移酶抑制剂（DNMTi）RG108对猪SCNT胚胎中DNA甲基化和组蛋白修饰动态模式的影响，并研究供体细胞的表观基因组状态影响SCNT胚胎发育的机制以及表观遗传信号之间的相互作用。
我们的结果表明，与对照胎儿成纤维细胞（FF）构建的胚胎（FF-SCNT胚胎）相比，用RG108处理的胎儿成纤维细胞（FF）构建的胚胎（RG-SCNT胚胎）中，TET1、TET2、TET3和5hmC的表达水平显著提高，增强了主动DNA去甲基化，而DNMT1、DNMT3A和5mC的表达水平显著抑制，促进了被动DNA去甲基化。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）的信号强度显著增加，并且RG-SCNT胚胎中H3K4甲基转移酶的表达水平高出2倍以上。RG-SCNT胚胎的卵裂率和囊胚率（分别为69.3±1.4%和24.72±2.3%）显著高于FF-SCNT胚胎（分别为60.1±2.4%和18.38±1.9%）。
RG108引起的DNA甲基化动态变化导致H3K4me3、H3K9Ac和组蛋白H3赖氨酸9三甲基化（H3K9me3）模式的动态改变，从而激活胚胎基因组和与H3K4甲基化相关的表观遗传修饰酶，并有助于重建正常的表观遗传修饰，提高猪SCNT胚胎的发育效率。
