In somatic cell nuclear transfer (SCNT) embryos, developmental defects first appear at the time of zygotic genome activation (ZGA), a process that is under the control of DNA and histone methylation. However, dynamics of 5-mC and 5-hmC during ZGA differ between porcine and bovine SCNT embryos, and histone methylation during ZGA in goat SCNT embryos remains poorly understood. Therefore, in the present study, we investigated the dynamic changes of 5-mC, 5-hmC, H3K4me2/3, and H3K9me3, as well as the expression of key genes related to these epigenetic modifications, during ZGA in goat cloned embryos. Compared with the IVF embryos, the 5-mC signal intensity was significantly increased at the 2- and 4-cell stage SCNT embryos, and the H3K4me3 and H3K9me3 signal intensity was significantly increased at 2- to 8-cell stage SCNT embryos, while the 5-hmC and H3K4me2 signal intensity was significantly lower at the 4- and 8-cell stage SCNT embryos. Of note, the H3K9me3 level was also significantly higher, whereas H3K4me3 signal intensity showed no statistical difference in the pronuclear stage SCNT embryos. Moreover, the expression of TET2, DNMT3B, KDM4A, SUV39H1, G9A, and SETDB1 was significantly increased, while the expression of UHRF1, PCNA, KDM4B, KDM4D, KDM5A, KDM5B, and KDM5C was significantly decreased at the 8-cell stage SCNT embryos. Our data revealed aberrant DNA and histone methylation during ZGA in goat cloned embryos. We further inferred that the abnormally higher level of 5-mC, H3K4me3, and H3K9me3 might serve as epigenetic barriers of the reprogramming and modifying these aberrant modifications might be a promising strategy to improve cloning efficiency in goat.

中文翻译：

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山羊克隆胚胎合子基因组激活过程中的异常 DNA 和组蛋白甲基化

在体细胞核移植 (SCNT) 胚胎中，发育缺陷首先出现在合子基因组激活 (ZGA) 时，该过程受 DNA 和组蛋白甲基化的控制。然而，猪和牛 SCNT 胚胎在 ZGA 期间 5-mC 和 5-hmC 的动力学不同，并且对山羊 SCNT 胚胎中 ZGA 期间的组蛋白甲基化仍然知之甚少。因此，在本研究中，我们研究了山羊克隆胚胎 ZGA 期间 5-mC、5-hmC、H3K4me2/3 和 H3K9me3 的动态变化，以及与这些表观遗传修饰相关的关键基因的表达。与IVF胚胎相比，2-4细胞期SCNT胚胎5-mC信号强度显着增加，2-8细胞期SCNT胚胎H3K4me3和H3K9me3信号强度显着增加，而 5-hmC 和 H3K4me2 信号强度在 4 和 8 细胞阶段的 SCNT 胚胎中显着降低。值得注意的是，H3K9me3 水平也显着更高，而 H3K4me3 信号强度在原核阶段 SCNT 胚胎中没有显示出统计学差异。此外，TET2、DNMT3B、KDM4A、SUV39H1、G9A和SETDB1的表达显着增加，而UHRF1、PCNA、KDM4B、KDM4D、KDM5A、KDM5B和KDM5C的表达在8细胞期SCNT胚胎中显着降低. 我们的数据揭示了山羊克隆胚胎中 ZGA 期间的异常 DNA 和组蛋白甲基化。我们进一步推断，异常高水平的 5-mC、H3K4me3 和 H3K9me3 可能作为重编程的表观遗传障碍，修改这些异常修饰可能是提高山羊克隆效率的有前途的策略。