Kdm6b-mediated epigenetic coordination of temporal precision during motor neuron differentiation [CutRun_CutTag]

Histone modifiers are crucial for instructing multiple-stage cellular differentiation, yet the mechanisms underlying their temporal precision remain enigmatic. Here, we demonstrate that the H3K27 demethylase Kdm6b acts as an epigenetic regulator, coordinating stepwise motor neuron (MN) differentiation through sequential partnerships with stage-specific transcription factors (TFs). Genome-wide profiling reveals a progressive gain in Kdm6b occupancy, especially at distal regulatory elements, as differentiation proceeds. Kdm6b dynamically shapes chromatin landscapes by coordinating H3K27me3 removal with H3K27ac and H3K4me1 acquisition, thereby enabling timed gene activation from MN specification to maturation. Stage-specific inhibition of Kdm6b compromised the ordered expression of developmental genes. Mechanistically, Kdm6b interacts with temporal TFs over time to ensure precise transcriptional control and MN differentiation. Our work elucidates how a single epigenetic regulator achieves temporal fidelity in driving stepwise MN development, and exemplifies a mechanistic insight into epigenetic regulation in determining developmental timing, with broad implications for understanding neurodevelopment and related diseases.