2022 Vol. 13, No. 2
PD0325901(MEK1/2 inhibitor) in 2i/L medium causes rapid loss of DNA methylation at the paternal differentially DNA methylation regions (DMRs) in androgenetic haploid ESCs (AG-haESCs), probably due to inhibition of Dnmts. Meanwhile, H3K4me3 deposition at paternal DMRs prevents de novo methylation of DMRs. Replacement of PD0325901 with SRC inhibitor (CGP77675, termed a2i/L), however, enables long-term maintenance of DNA methylation at paternal DMRs in AG-haESCs generated through a two-step protocol based on a2i/L (TSa2i/L). CGP77675 enhances the protein level of DNA methylation-related genes and promotes H3K9me3 deposition at paternal DMRs. Moreover, AGhaESCs with hypermethylated H19-DMR sustain enhanced cell proliferation, further promoting paternal-DMR methylation maintenance. Together, TSa2i/L ensures epigenetic integrity of paternal DMRs in AG-haESCs that can be efficiently used as sperm replacement.