DOT1L inhibitor improves early development of porcine somatic cell nuclear transfer embryos

Incomplete epigenetic reprogramming of the genome of donor cells causes poor early and full-term developmental efficiency of somatic cell nuclear transfer (SCNT) embryos. Previous research indicate that inhibition of the histone H3 K79 methyltransferase DOT1L, using a selective pharmacological inhibitor EPZ004777 (EPZ), significantly improved reprogramming efficiency during the generation of mouse induced pluripotent stem cells. However, the roles of DOT1L in porcine nuclear transfer-mediated cellular reprogramming are not yet known. Here we showed that DOT1L inhibition via 0.5 nM EPZ treatment for 12 or 24 h significantly enhanced the blastocyst rate of SCNT embryos and dramatically reduced the level of H3K79me2 during SCNT 1-cell embryonic development. Additionally, H3K79me2 level in the EPZ-treated SCNT embryos was similar to that in in vitro fertilized embryos, suggesting that DOT1L-mediated H3K79me2 is a reprogramming barrier to early development of porcine SCNT embryos. qRT-PCR analysis further demonstrated that DOT1L inactivation did not change the expression levels of DOT1L itself but increased the expression levels of POU5F1, LIN28, SOX2, CDX2 and GATA4 associated with pluripotency and early cell differentiation. In conclusion, DOT1L inhibitor improved early developmental efficiency of porcine SCNT embryos probably via inducing the increased expression of genes important for pluripotency and lineage specification.供体细胞基因组的表观遗传重编程不完全，会导致体细胞核移植（somatic cell nuclear transfer, SCNT）胚胎的早期及足月发育效率低下。既往研究表明，使用选择性药理学抑制剂EPZ004777（简称EPZ）抑制组蛋白H3 K79甲基转移酶DOT1L，可显著提升小鼠诱导多能干细胞生成过程中的重编程效率。然而，DOT1L在猪细胞核移植介导的细胞重编程中的作用仍未明确。本研究发现，采用0.5 nM的EPZ处理12或24小时以抑制DOT1L，可显著提升体细胞核移植胚胎的囊胚率，并在猪体细胞核移植1细胞期胚胎发育过程中大幅降低H3K79me2的水平。此外，经EPZ处理的体细胞核移植胚胎中H3K79me2水平与体外受精胚胎相近，这提示DOT1L介导的H3K79me2是猪体细胞核移植胚胎早期发育的重编程障碍。实时定量PCR（quantitative real-time PCR, qRT-PCR）分析进一步证实，DOT1L失活并不会改变DOT1L自身的表达水平，却会上调与多能性及早期细胞分化相关的POU5F1、LIN28、SOX2、CDX2及GATA4的表达量。综上，DOT1L抑制剂或可通过上调多能性及谱系特化相关重要基因的表达，提升猪体细胞核移植胚胎的早期发育效率。