Shared molecular consequences of epigenetic machinery disruption in neuronal progenitors

The Mendelian disorders of the epigenetic machinery (MDEMs) are an emerging cause of intellectual disability and growth abnormalities. Hippocampal neurons are a consistently affected cell type in these disorders. To investigate the effects of epigenetic machinery (EM) disruption in a disease-relevant context, we established a murine hippocampal neurodifferentiation model enabling systemic knockout (KO) of multiple EM factors. We then interrogated each EM-KO using Oxford Nanopore DNA methylation (DNAm) sequencing and RNA sequencing. Disruption of Kmt2a and Dnmt1 produced strikingly convergent gene expression changes. However, while Dnmt1-KO induced extensive DNAm alterations, KO of Kmt2a had little effect on methylation. Loss of either EM factor led to premature neuronal differentiation, partially explaining the transcriptional convergence. The transcription factor GSX1 emerged as a potential regulatory node, with altered methylation and expression in Kmt2a-KO cells, and ∼20% of its known targets were differentially expressed. Extending our methylation analysis to 46 EM genes, we found that loss of DNA methyltransferases caused the strongest DNAm changes, whereas other EM-KOs induced milder-to-no effects. Clustering of EM factors based on promoter DNAm revealed two EM subgroups enriched for interactions with the DNAm machinery. Allele-specific analysis identified a shared differentially methylated region (DMR) at Zic4 across the entire group of EM-KOs, and Zic4 overexpression appears to maintain the NPC state. Taken together, our results reveal both gene-specific and convergent effects across diverse EM KOs and provide new insight into the molecular etiology of the MDEMs.