Dissecting the Functional Consequences of De Novo DNA Methylation Dynamics in Human Motor Neuron Differentiation and Physiology

The somatic DNA methylation (DNAme) landscape is established early in development but remains highly dynamic within focal regions that overlap with gene regulatory elements. The significance of these dynamic changes, particularly in the central nervous system, remains unresolved. Here, we utilize a powerful human embryonic stem cell differentiation model for the generation of motor neurons (MNs) in combination with genetic mutations in the de novo DNAme machinery. We quantitatively dissect the role of DNAme in directing somatic cell fate with high-resolution genome-wide bisulfite-, bulk-, and single-cell-RNA sequencing. We find defects in neuralization and MN differentiation in DNMT3A knockouts (KO) that can be rescued by the targeting of DNAme to key developmental loci using catalytically inactive dCas9. We also find decreased dendritic arborization and altered electrophysiological properties in DNMT3A KO MNs. Our work provides a list of DNMT3A-regulated targets and a mechanistic link between de novo DNAme, cellular differentiation, and human MN function. [Display omitted] •DNMT3A KO alters the fate of ESC-derived neural cells by modulating transcription factor expression•Single-cell RNA-seq reveals induction of floor plate cells in DNMT3A KO cultures•Targeted DNA methylation editing of PAX6/ARX gene regulatory elements rescues differentiation defects•Lack of DNMT3A impairs morphology and physiological activity of MNs Kiskinis and colleagues demonstrate that DNA methylation dynamics play a central role in the differentiation of human pluripotent stem cells toward highly specialized motor neurons. Through a combination of molecular and functional analysis they identify key transcriptional mediators of these effects and link DNA methylation to neuronal patterning and function.