Inhibition of Histone Methyltransferase G9a Hampers Early Embryonic Development through Disruption of Epigenetic Reprogramming and Transcriptomic Homeostasis

Early embryonic developmental arrest is a primary contributor to pregnancy failure. While epigenetic reprogramming is known to be essential for successful development, its specific role in mediating developmental block remains poorly understood. This study aims to elucidate the regulatory mechanisms of the histone methyltransferase G9a during early murine embryogenesis. Using in vitro fertilization (IVF), we investigated the effects of the G9a-specific inhibitor Bix-01294 (50 nM) on embryonic progression. Our results demonstrate that G9a inhibition significantly reduces the blastocyst formation rate (23.5% vs. 78.25% in the control group) and triggers a marked increase in blastocyst apoptosis. Immunofluorescence analysis revealed that Bix-01294 treatment disrupts the dynamic patterns of DNA methylation (5mC) and hydroxymethylation (5hmC) across various developmental stages, concurrently leading to a reduction in H3K9me2 levels. Furthermore, qRT-PCR data indicated that G9a inhibition causes the transcriptomic upregulation of the 5mC-to-5hmC conversion enzymes TET1 and TET3 , while significantly downregulating the de novo methyltransferases DNMT3a and DNMT3b . Single-cell transcriptome sequencing (Smart-seq2) identified a substantial number of differentially expressed genes (DEGs) in the Bix-treated group. These DEGs were primarily enriched in biological processes related to biosynthesis, the SCF ubiquitin ligase complex, and DNA-binding transcription factor activity, with the MAPK signaling pathway identified as a key affected KEGG axis. In conclusion, this study establishes that inhibiting G9a-mediated H3K9me2 modification induces an imbalance in DNA 5mC/5hmC dynamics, causes aberrant expression of epigenetic modifiers, and disrupts transcriptomic homeostasis. These defects lead to developmental arrest and increased apoptosis, providing a theoretical framework for understanding embryonic failure and optimizing in vitro embryo culture efficiency.