KMT2D-deficiency destabilizes lineage progression in immature neural progenitors

Neurodevelopment is driven by integrated regulation of chromatin shaping, transcription, and proliferative timing. Although KMT2D is known to catalyze H3K4 methylation at promoters and enhancers to activate lineage genes, its role in regulating fate specification within neural progenitor populations remains poorly defined. We performed single-cell multiome sequencing on human induced pluripotent stem cell (hiPSC)-derived cerebral organoids to simultaneously profile chromatin and transcriptional states in KMT2D-deficient cells. We found that lineage transcription factors such as PAX6, NEUROD4, and OLIG1 showed premature activation accompanied by disrupted temporal coordination between chromatin accessibility and transcription, indicating unstable regulation that fails to sustain the expression of several lineage-specific genes. Live-cell imaging using FUCCI reporters revealed that KMT2D-deficient progenitors accumulate in G1 phase more rapidly within 24-hours of neural induction. Further, KS1 patient-derived neural progenitors showed similar, yet less pronounced, disruptions in lineage mark expression over time. Our findings suggest that loss of KMT2D perturbs the coordination of chromatin accessibility, gene expression, and cell cycle timing in neural progenitors at the onset of lineage acquisition, potentially initiating generational instability that impairs lineage resolution and alters differentiation trajectories over time. Overall design: Multiome sequencing was performed on cerebral organoids derived from WTC11 (isogenic control) and KMT2D-/- human induced pluripotent stem cells (hiPSCs). Organoids were cultured for two months and sampled at two time points (Day 51 and Day 74), pooled by size, and dissociated to single-cell suspensions. Nuclei isolated from these suspensions underwent simultaneous single-nucleus ATAC-seq and single-cell RNA-seq using the Chromium Next GEM Multiome platform (10X Genomics). The variable investigated was differences between genotypes (WTC11 vs. KMT2D-/-) focused on chromatin accessibility and gene expression to understand neural progenitor cell fate and regulatory dynamics.