Shared transcriptional consequences of epigenetic machinery disruption in murine neuronal progenitors

The Mendelian disorders of the epigenetic machinery (MDEMs) are an emerging group of disorders which commonly involve disruption of intellect and growth. To investigate consequences of epigenetic machinery (EM) disruption during neurodevelopment, we systematically knocked out (KO) EM factors in neuronal progenitors isolated from the murine hippocampus and established a neurodifferentiation model to interrogate their functions. We then profiled gene expression and DNA methylation (DNAm) in the EM-KOs using RNA sequencing and Nanopore long-read DNA sequencing. While Dnmt1-KO induces extensive DNAm alterations, Kmt2a-KO has little effect on methylation. Nevertheless, the disruption of Kmt2a and Dnmt1 produces convergent transcriptional changes. Loss of either EM factor leads to premature neuronal differentiation, partially explaining this convergence, and MYC emerges as a shared regulatory node linked to downregulation of cell cycle programs in these cells. Extending our methylation analysis to 46 EM genes, we find that loss of DNA methyltransferases induces the strongest DNAm changes, whereas other EM-KOs have subtle or negligible effects. However, clustering of EM-KOs based on promoter DNAm levels reveals three distinct EM subgroups, of which two are enriched for interactions with the DNAm machinery. Allele-specific analysis of DNAm further identifies a single differentially methylated region shared across the 46 EM-KOs, localized to the B6J allele over the Zic4 3'UTR. Furthermore, Zic4 overexpression appears to maintain the neuronal progenitor state, suggesting functional relevance of this locus. Taken together, our results reveal both gene-specific and convergent transcriptional effects across diverse EM-KOs and provide novel insights into the downstream consequences of EM dysfunction.