Analysis of the role of DNA methyltransferases during human embryonic stem cells differentiation

Epigenetics mechanisms are able to regulate the gene expression profile of a cell, without changing the DNA genomic sequence, by introducing chemical modifications to the chromatin structure that can alter its accessibility for the transcription machinery. Epigenetic modifications are particularly important during development, driving pluripotent stem cells (PSCs) specification into different cell lineages by promoting the expression of tissue-specific genes instead of pluripotency-associated regulators. DNA methylation, catalysed by DNA methyltransferases (DNMTs), represents one of the main studied epigenetic mechanisms; notably, DNMT3A and DNMT3B are responsible for the genomic de novo methylation in PSCs during development. Indeed, mutations in DNMTs genes can be found in several developmental defects, and disruption of DNMT3A is linked to post-natal lethality in mice models. Given these premises, we decided to produce DNMT3A knockout (KO) human embryonic stem cell (hESC) models, by harnessing a CRISPR/Cas9-based system; afterwards, we committed these genetically engineered cells towards a mesodermal differentiation, in particular paraxial mesoderm involved in myogenesis. In this preliminary study, we were unable to find any significant morphological difference between wild-type (WT) cells and DNMT3A KO models during early mesoderm differentiation. However, considering preliminary data showing a higher expression of DNMT3A compared to DNMT3B across muscle lineage differentiation, the DNMT3A KO models described in this study provide a reliable starting point for future research aimed at assessing the impact of DNMT3A ablation during hESCs mesodermal differentiation. Furthermore, the pluripotency of these models provides the possibility to study DNMT3A disruption during specification towards all primordial germ layers.