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

ABSTRACT: 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.

ORGANISM(S): Mus musculus

PROVIDER: GSE298928 | GEO | 2026/02/02

REPOSITORIES: GEO

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Project description:The core purpose of this project is to identify the novel epigenetic factors which play a crucial role on regulating stem cells developed from embryo development to ageing through the longitudinal analysis of their transcriptome, protein interactome, and post- translational modification network. Thus, this study would not only advance our understanding of epigenetic nature of stem cells through whole life, but also provide the novel strategy to develop the next generation stem cell therapeutics. According to our previous reports, we found that DNA methylation, particularly mediated by Dnmt3L could play a common epigenetic signature for modulating the stemness potency of both embryonic and adult stem cells. To investigate the post-translational modifications of Dnmt3L protein, we performed mass spectrometry analysis of Flag-tagged Dnmt3L proteins in murine embryonic stem cells (ESCs) and then identified several acetylated lysine residues. To examine their functional roles, we manufactured the lentivirus containing wild type Dnmt3L or the acetylated lysine mutants and they established the ESC cell lines stably harboring these constructs. To focus on epigenetic mechanism for stem cell biology, we will narrow down the key acetylated lysine sites of Dnmt3L, which can characteristically regulate the epigenetic status or transcription gene ontology. To get mechanistic insights, the transcriptome and DNA methylome features of the established ESC cell lines will be analyzed. The functional significance was examined by a series of experiments for in vitro and in vivo self-renewal and differentiation assays. Finally, we would extend these results to develop the efficient ex vivo expansion protocol for adult stem cells, particularly focusing on human mesenchymal stem cells. We expect that this proposal will not only advance our understating the epigenetic landscape of stem cell population but also contribute to establish the clinically best-suitable stem cell population. In addition, this study can be appliable to get a novel conceptional advances in other biological research such as cancer biology and ageing research.

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

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