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

Abstract

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.

Loss of DNA methyltransferase activity in primed human ES cells triggers increased cell-cell variability and transcriptional repression

“…We have previously generated knockout human ES cell lines for the three catalytically active DNA methyltransferases (Liao et al, 2015). With the advance of singlecell technologies, we wanted to explore the effects of these knockouts within individual cells to better understand how the subtle changes in the undifferentiated state translate to substantial disruptions upon exit from pluripotency (Ziller et al, 2018). Using our scRNA-seq approach, we observed a global increase in cellular and gene expression variation for all DNMT mutants.…”

“…Three catalytically active DNA methyltransferases (DNMTs) are responsible for maintenance (DNMT1) and de novo DNA methylation (DNMT3A/3B) in mammals, and all three are essential for normal development (Smith and Meissner, 2013). DNA methylation by DNMT3A/3B plays a particularly important role during development and ES cell differentiation (Gifford et al, 2013;Ziller et al, 2018), and both catalytically active enzymes are highly expressed in undifferentiated cells. Bulk experiments have shown a limited global impact of DNMT3A/3B knockout on the global DNA methylation landscape in human ES cells (Liao et al, 2015).…”

De novoDNA methylation controls neuronal maturation during adult hippocampal neurogenesis

“…Previous studies analyzed the role of de novo DNA methyltransferases in Dnmt3a-deficient embryonic stem cells during neuronal differentiation in culture (Wu et al, 2010(Wu et al, , 2012Ziller et al, 2018). To compare our results with these studies, we first investigated whether inducible deletion of de novo DNA methyltransferases in NSPCs influenced their proliferation and differentiation potential in vitro.…”

“…De novo DNA methyltransferases are crucial for embryonic and postnatal brain development and memory formation in adulthood (Gulmez Karaca et al, 2020;Odell et al, 2020;Wu et al, 2010), but their specific role during adult neurogenesis has been as yet unknown. Previous studies suggested that, in neural precursor cells derived from Dnmt3a-deficient embryonic stem cells, astrogliogenesis was increased at the expense of neurogenesis (Wu et al, 2010(Wu et al, , 2012Ziller et al, 2018). However, due to the lack of inducible Dnmt3a knock-out models it remained unknown whether a neurogenic fate of neural precursor cells is pre-defined by DNA methylation patterns established during development or whether it is specified by further de novo DNA methylation in the course of terminal neuronal differentiation.…”

Nucleocytoplasmic Proteomic Analysis Uncovers eRF1 and Nonsense Mediated Decay as Modifiers of ALS C9orf72 Toxicity

“…4a). To determine whether our findings on ETF1 redistribution hold significance in a more physiological disease model system, we used established protocols to differentiate human spinal motor neurons (MNs) 47 from iPSCs derived from three individual ALS patients with a C9orf72 repeat expansion and from three healthy controls ( Supplementary Fig. 4b and Supplementary Table 3).…”