Resolving multiple epigenetic barriers to pig SCNT-mediated reprogramming during embryonic genome activation [RNA-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: RNA-seq profiles of pig four- to eight-cell SCNT embryos and pig fetal fibroblasts.

利用体细胞核移植（somatic cell nuclear transfer, SCNT）制备克隆猪时，胚胎常在母源-合子转换阶段出现表观遗传重编程不完全的问题，进而导致着床前胚胎大量丢失。本研究首次绘制了猪SCNT胚胎的全基因组组蛋白甲基化图谱。研究发现，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，可检测到过量的组蛋白H3赖氨酸9三甲基化（H3K9me3）与组蛋白H3赖氨酸27三甲基化（H3K27me3），但未出现组蛋白H3赖氨酸4三甲基化（H3K4me3）的异常富集。联合使用H3K9去甲基化酶KDM4A与H3K27me3修饰酶抑制剂GSK126，可清除上述表观遗传障碍，并恢复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是猪特异性的核重编程表观遗传调控因子，且无法通过去除H3K9me3与H3K27me3使其重新激活。联合处理与TDG瞬时过表达均可促进DNA去甲基化，并提升SCNT胚胎的囊胚形成率，为提升农业与生物医药领域基因组编辑克隆猪的制备效率提供了极具价值的技术手段。实验设计：猪4-8细胞期SCNT胚胎与猪胎儿成纤维细胞的RNA测序（RNA-seq）图谱。