BIX-01294 increases pig cloning efficiency by improving epigenetic reprogramming of somatic cell nuclei

Abstract

Accumulating evidence suggests that faulty epigenetic reprogramming leads to the abnormal development of cloned embryos and results in the low success rates observed in all mammals produced through somatic cell nuclear transfer (SCNT). The aberrant methylation status of H3K9me and H3K9me2 has been reported in cloned mouse embryos. To explore the role of H3K9me2 and H3K9me in the porcine somatic cell nuclear reprogramming, BIX-01294, known as a specific inhibitor of G9A (histone-lysine methyltransferase of H3K9), was used to treat the nuclear-transferred (NT) oocytes for 14-16 h after activation. The results showed that the developmental competence of porcine SCNT embryos was significantly enhanced both in vitro (blastocyst rate 16.4% vs 23.2%, P!0.05) and in vivo (cloning rate 1.59% vs 2.96%) after 50 nm BIX-01294 treatment. BIX-01294 treatment significantly decreased the levels of H3K9me2 and H3K9me at the 2-and 4-cell stages, which are associated with embryo genetic activation, and increased the transcriptional expression of the pluripotency genes SOX2, NANOG and OCT4 in cloned blastocysts. Furthermore, the histone acetylation levels of H3K9, H4K8 and H4K12 in cloned embryos were decreased after BIX-01294 treatment. However, co-treatment of activated NT oocytes with BIX-01294 and Scriptaid rescued donor nuclear chromatin from decreased histone acetylation of H4K8 that resulted from exposure to BIX-01294 only and consequently improved the preimplantation development of SCNT embryos (blastocyst formation rates of 23.7% vs 21.5%). These results indicated that treatment with BIX-01294 enhanced the developmental competence of porcine SCNT embryos through improvements in epigenetic reprogramming and gene expression.

TSA and BIX-01294 Induced Normal DNA and Histone Methylation and Increased Protein Expression in Porcine Somatic Cell Nuclear Transfer Embryos

“…Two recent studies demonstrated that knockdown or BIX-01294-mediated inactivation of histone methyltransferas G9a that catalyzes histone H3K9 dimethylation could significantly improve the cloning efficiency in mouse and pig[20,21]. To investigate whether combined treatment of TSA and BIX-01294 synergistically improved the early developmental competence of porcine cloned embryos, we first examined the effect of BIX-01294 treatment alone on the early developmental efficiency of cloned embryos under our experimental conditions.…”

“…For example, histone hyperacetylation induced by HDACs inhibitor TSA could significantly improve the full-term developmental rate in pig[25]. Decreasing the H3K9me2 level induced by BIX-01294 treatment also could elevate the porcine cloning efficiency [21]. However, the early and full-term developmental efficiency of SCNT embryos is still low compared with naturally fertilized embryos.…”

“…In the present study, we observed that 1 μM BIX-01294 treatment alone promoted the rate of blastocyst formation of porcine cloned embryos. However, a recent study reported that 5 nM and 50 nM BIX-01294 treatment significantly improved in vitro development of porcine somatic cloned embryos[21]. These specific reasons underlying the discrepancy need to be further explored in future.…”

“…Recent studies demonstrate that histone demethylase, either KDM4A or KDM4B overexpression-mediated reduction in H3K9me3 level dramatically improves the overall mouse cloning efficiency [18,19]. Meanwhile, G9a knockdown by RNAi or the reduction of H3K9me2 level through a small molecule inhibitor BIX-01294-mediated inhibition of G9A activity significantly promotes the cloning efficiency in mouse [20] and pig [21]. However, the molecular mechanisms underlying the enhanced cloning efficiency in pig via modulating the status of histone acetylation and methylation remain largely unknown.…”

Down-Regulation of H3K4me3 by MM-102 Facilitates Epigenetic Reprogramming of Porcine Somatic Cell Nuclear Transfer Embryos

“…H3K9 and H3K4 methylation also show abnormal expression levels in porcine SCNT embryos [14]. The down-regulation of H3K9 methylation by BIX-01294 could improve epigenetic reprogramming of porcine SCNT embryos [15], but there have been very few studies about whether the down-regulation of H3K4 methylation can play a similar role during porcine SCNT embryo development.…”