KMT2D-deficiency destabilizes lineage progression in immature neural progenitors

Neurodevelopment is driven by the tightly regulated integration of chromatin shaping, transcription, and proliferation. Although KMT2D is known to catalyze activating H3K4 methylation marks at promoters and enhancers, its role in regulating early fate decisions of neural progenitors remains poorly defined. Here, we characterized these early events using single-cell multiome sequencing of human induced pluripotent stem cell (hiPSC)-derived cerebral organoids to simultaneously profile chromatin and transcriptional states in KMT2D knockout (KMT2D^-/-) cells. We found that core lineage transcription factors such as PAX6, NEUROD4, and OLIG1 activated prematurely but failed to stabilize, reflecting disrupted chromatin-transcription coordination at a stage when progenitors must balance self-renewal and fate initiation. Live-cell imaging using cell cycle reporters revealed that KMT2D^-/- progenitors accumulate in G1 phase more rapidly within 24-hours of neural induction. Further, neural progenitors derived from KMT2D haploinsufficient hiPSCs displayed similar, but less pronounced, disruptions in lineage mark expression, supporting a dose-dependent role of KMT2D in neurodevelopmental disorders. Altogether, our findings indicate that KMT2D plays a critical role in ensuring stable lineage resolution and supporting robust developmental trajectories during early stages of neurodevelopment.