Mixed-Lineage Leukemia 1 Inhibition Enhances the Differentiation Potential of Bovine Embryonic Stem Cells by Increasing H3K4 Mono-Methylation at Active Promoters. (BS-seq)
Mixed-lineage leukemia 1 (MLL1) introduces 1-, 2- and 3-methylation into histone H3K4 through the evolutionarily conserved set domain. In this study, bovine embryonic stem cells (bESCs, known as bESCs-F7) were established from in vitro-fertilized (IVF) embryos via Wnt signaling inhibition; however, their contribution to the endoderm in vivo is limited. To improve the quality of bESCs, MM-102, an inhibitor of MLL1, was applied to the culture. The results showed that MLL1 inhibition along with GSK3 and MAP2K inhibition (3i) at the embryonic stage did not affect bESCs' establishment and pluripotency. MLL1 inhibition improved the pluripotency and differentiation potential of bESCs via the up-regulation of stem cell signaling pathways such as PI3K-Akt and WNT. MLL1 inhibition decreased H3K4me1 modification at the promoters and altered the distribution of DNA methylation in bESCs. In summary, MLL1 inhibition gives bESCs better pluripotency, and its application may provide high-quality pluripotent stem cells for domestic animals. Overall design: In this study, a CTFR-bESC culture system was applied to establish bovine- ESCs from MM-102 (50 µM)-, 2i (0.5 µM PD0325901 and 3 µM CHIR99021)- and 3i (2i plus 30 µM MM-102)-treated bovine IVF embryos. When embryos formed blastocysts at 7–8 days, the blastocysts, removed from the zona pellucida, were plated to establish bESCs using mouse fetal fibroblast (MEF) feeder cells. The obtained bESC cell lines were named bESCs-102, bESCs-2i and bESCs-3i. Given that MM-102 treatment of embryos altered the pluripotency of the established bESCsone involved the addition of 5 µM of MM-102 to CTFR in long-term cultures, and in the other, bESCs were cultured for 7 days with the addition of 50 µM MM-102, and then switched back to CTFR. The derived cell lines were named bESCs-102-5 and bESCs-102-50