Abstract
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.
Highlights
Since the first somatic cell cloned animal “Dolly” was born[1], somatic cell nuclear transfer technology (SCNT) has become a forefront focus of life science
DOT1L was identified as a negative regulator of somatic cell reprogramming during the generation of induced pluripotent stem cells [11], the role of DOT1L during nuclear transfer-mediated cellular reprogramming is still unknown
H3K79me2 level in SCNT pronuclear embryos treated with 0.5 nM EPZ for 12 h or 24 h was decreased and comparable to IVF embryos from 2 h to 10 h post-insemination (Fig 3C). These results demonstrate that inhibition of DOT1L activity using 0.5 nM EPZ for 12 h or 24 h can reduce the H3K79me2 level in SCNT embryos at the pronuclear stage
Summary
Since the first somatic cell cloned animal “Dolly” was born[1], somatic cell nuclear transfer technology (SCNT) has become a forefront focus of life science. Efficiency is still less than 5% [4], which is far less than full-term developmental efficiency of naturally fertilized embryos This rate greatly limits the widespread application of nuclear transfer technology. Abnormalities in DNA methylation and histone acetylation during early development of SCNT embryos have been widely reported [7]. Consistent with these reports, inhibitors of DNA methyltransferases and/or histone deacetylases could improve early and full-term developmental efficiency of SCNT embryos[8,9]. A small molecule inhibitor BIX-01294 that reduces the H3K9me levels via repressing histone methyltransferase G9a activity, significantly promoted porcine cloning efficiency[14]. We utilized a highly selective inhibitor (EPZ00477) of a histone H3K79 methyltransferase DOT1L to investigate the function and potential mechanisms of DOT1L during early development of porcine SCNT embryos
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