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

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.

Comprehensive histochemical profiles of histone modification in male germline cells during meiosis and spermiogenesis: Comparison of young and aged testes in mice

“…anti-H3K4me2 rabbit polyclonal 1/500 Millipore (07-030) [13][14][15] anti-H3K4me3 rabbit polyclonal 1/500 Abcam (ab8580) [16][17][18] anti-H3K9me3 rabbit polyclonal 1/500 Abcam (ab8898) [19][20][21] anti-H3K27ac rabbit polyclonal 1/500 Abcam (ab4729) [22][23][24] anti-H3K27me2 rabbit polyclonal 1/500 Abcam (ab24684) [25,26] anti-H3K27me3 rabbit polyclonal 1/500 Millipore (07-449) [24,[27][28][29] anti-H3K79me2 rabbit polyclonal 1/500 Abcam (ab3594) [30][31][32] anti-H3K79me3 rabbit polyclonal 1/500 Abcam (ab2621) [33][34][35] anti-SCP3 mouse monoclonal 1/500 Abcam (ab97672) [36][37][38] anti-α-Tubulin (S2 Fig) mouse monoclonal 1/500 Sigma-Aldrich (T6199) [39,40] All antibodies are commercially available, and their specificity has been reported in previous studies.…”

Sperm-borne small RNAs regulate α-tubulin acetylation and epigenetic modification of early bovine somatic cell nuclear transfer embryos

“…Taking bovine as an example, large offspring syndrome (LOS) is the most common abnormal development, resulting in a severe loss of the cloned offspring in perinatal period (Smith, Suzuki et al 2012). Numerous studies have reported that the incomplete epigenetic reprogramming of nuclear donor cells is a main cause for low embryonic development efficiency and abnormality of cloned offspring (Akagi, Geshi et al 2013, Tao, Zhang et al 2017. The common epigenetic reprogramming occurring during early embryonic development includes DNA methylation, histone modification, and chromatin remodeling (Betthauser, Pfister-Genskow et al 2006).…”

Applications of omics and nanotechnology to improve pig embryo production in vitro

“…In addition, MM‐102 increased blastocyst formation rates and total cell numbers, contributing to the production of cloned porcine embryos (Zhang et al, ). Tao et al () observed that the selective inhibitor EPZ00477 (EPZ) of a histone H3K79 methyltransferase, DOT1L, restored H3K79me2 levels to those observed in IVF embryos, increased the blastocyst rate of porcine SCNT embryos, and elevated expression of genes associate with pluripotency. Similarly, co‐treatment with TSA and BIX‐01294, an inhibitor of G9a histone methyltransferase that reduces H3K9me2 levels in somatic cells, corrected abnormal H3K9me2 and 5mC (DNA methylation) levels in 4‐cell stage and trophectoderm lineage of SCNT blastocysts and increased the expression of genes related to lineage differentiation when compared to in vivo‐ and in vitro‐fertilized embryos (Cao et al, ).…”

“…In addition, MM-102 increased blastocyst formation rates and total cell numbers, contributing to the production of cloned porcine embryos (Zhang et al, 2018). Tao et al (2017) observed that the selective inhibitor EPZ00477 (EPZ) of a histone H3K79 methyltransferase, DOT1L, restored H3K79me2 levels to those observed in IVF embryos, increased the blastocyst rate of porcine SCNT embryos, and elevated expression of genes associate with pluripotency.…”