Uncovering the Acetylation Sites of Dnmt3L to Regulate Protein Stability and Potency of Differentiation in Embryonic Stem Cells

The epigenetic status, a key mechanism regulating the cellular identity and differentiation potential of pluripotent stem cells (PSCs), dynamically responds to the culture environment, affecting the safe and effective use of PSCs for basic research and therapeutic applications. However, it remains unclear about key mediator(s) for representing the epigenetic signatures of PSCs under distinct culture conditions. Here, we investigated the role of DNA methyltransferase 3-Like (Dnmt3L) in modulating the DNA methylation and differentiation potential of mouse embryonic stem cells (mESCs). Unlike other de novo DNA methyltransferases, Dnmt3L exhibited a uniquely dynamic expression pattern during prolonged 2i-LIF cultures, marked by a rapid post-transcriptional upregulation that sensitively reflected changes in the extracellular environment. Through mass spectrometry, we identified acetylation at lysine-residues K238 and K412 as key factors controlling Dnmt3L protein stability. These modifications critically affected the expression of genes associated with naïve pluripotency and differentiation, especially within germline, neural, and cardiac lineages through locus-specific DNA methylation. Our findings highlight Dnmt3L as a key regulator of epigenetic stability at developmentally critical loci in mESCs, acting as a dynamic responder to changes in the extracellular culture environment. Thus, understanding the regulation mechanism of Dnmt3L could provide insight into the onset of epigenetic aberrations and suggest the optimal culture conditions to preserve epigenetic integrity of ESCs, which have significant implications for regenerative medicine.