Stage-specific Kdm6b-transcription factor partnerships orchestrate temporal precision during stepwise motor neuron differentiation [CutRun_CutTag]

Histone modifiers instruct cellular differentiation, yet how they achieve temporal precision remains enigmatic. Here we demonstrate that the H3K27 demethylase Kdm6b acts as a master epigenetic conductor and achieves timed gene activation by sequentially partnering with stage-specific transcription factors (TFs) in motor neuron (MN) differentiation. Genome-wide profiling revealed Kdm6b occupancy progressively shifted from proximal promoters to distal enhancers, along with increasing regulatory elements during maturation. At occupied sites, Kdm6b triggered rapid H3K27me3 loss with concurrent H3K27ac/H3K4me1 gain, establishing activation-competent chromatin. By integrating developmental TFs and histone modification landscapes, Kdm6b builds up the dynamic epigenetic choreography to precisely regulate MN developmental programs from early MN fate specification, intermediate cell differentiation and growth to later maturation. The ordered expression of developmental genes was compromised by stage-specific Kdm6b inhibition. Our work resolves how a single epigenetic regulator achieves temporal precision in driving stepwise MN development, with broad implications for neurodevelopment and diseases. Overall design: Cut&Run-seq of Kdm6b, Neurod1 and Cux1/2, and Cut&Tag-seq of H3K27me3, H3K27ac, H3K4me1 and H3K4me3 within in vitro differentiated motor neurons from mouse embryonic stem cells at stages D4, D4H8, D5 and D6.