Resolving multiple epigenetic barriers to pig SCNT-mediated reprogramming during embryonic genome activation [BiSulfite-seq]

Pig cloning by somatic cell nuclear transfer (SCNT) frequently undergoes incomplete epigenetic remodeling during the maternal-to-zygotic transition, which leads to a significant embryonic loss before implantation. Here, we generated the first genome-wide landscapes of histone methylation in pig SCNT embryos. Excessive H3K9me3 and H3K27me3, but not H3K4me3, were observed in the genomic regions with unfaithful embryonic genome activation and donor cell-specific gene silencing. A combination of H3K9 demethylase KDM4A and GSK126, an inhibitor of H3K27me3 writer, could remove these epigenetic barriers and restore the global transcriptome in SCNT embryos. More importantly, TDG was defined as a pig-specific epigenetic regulator for nuclear reprogramming, which was not reactivated by H3K9me3 and H3K27me3 removal. Both combined treatment and transient TDG overexpression could promote DNA demethylation and enhance the blastocyst forming rates of SCNT embryos, which offers valuable methods to increase the cloning efficiency of genome-edited pigs for agricultural and biomedical purposes. Overall design: Examination of DNA methylation levels in pig 4-cell SCNT embryos and pig fetal fibroblasts.

通过体细胞核移植（somatic cell nuclear transfer, SCNT）制备的猪克隆胚胎，在母源-合子转换过程中常出现表观遗传重编程不完全的缺陷，进而引发着床前胚胎的大量丢失。本研究首次构建了猪SCNT胚胎的全基因组组蛋白甲基化图谱。在胚胎基因组激活异常以及供体细胞特异性基因沉默的基因组区域中，可观测到过量积累的H3K9me3与H3K27me3修饰，但未检测到H3K4me3的异常富集。联合使用H3K9去甲基化酶KDM4A与H3K27me3写入酶抑制剂GSK126，能够清除这些表观遗传障碍，恢复SCNT胚胎的全局转录组表达谱。更为关键的是，本研究鉴定出TDG是猪特异性的细胞核重编程表观遗传调控因子，其表达无法通过去除H3K9me3与H3K27me3修饰得以重新激活。联合处理与瞬时过表达TDG均可促进DNA去甲基化过程，并提升SCNT胚胎的囊胚形成率，为提升农业与生物医学领域用基因组编辑猪的克隆效率提供了极具应用价值的技术策略。实验设计概述：检测猪4细胞期SCNT胚胎与猪胎儿成纤维细胞的DNA甲基化水平。