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. 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.

组蛋白修饰因子调控细胞分化，但其如何实现时序精准性的机制仍不明朗。本研究表明，组蛋白H3K27去甲基化酶（H3K27 demethylase）Kdm6b可作为核心表观遗传调控因子，在运动神经元（Motor Neuron, MN）分化过程中，通过与不同阶段特异性转录因子（Transcription Factor, TFs）依次结合，实现基因的时序性激活。全基因组谱分析显示，在成熟过程中，Kdm6b的结合位点逐渐从近端启动子向远端增强子转移，同时调控元件数量不断增加。在其结合位点上，Kdm6b可快速介导H3K27me3去修饰，同时伴随H3K27ac与H3K4me1的富集，从而构建具备转录激活能力的染色质状态。通过整合发育相关转录因子与组蛋白修饰图谱，Kdm6b构建起动态的表观遗传调控网络，精准调控运动神经元的发育程序——从早期运动神经元命运决定、中间阶段细胞分化与增殖，直至后期成熟阶段。若在特定阶段抑制Kdm6b的功能，发育基因的有序表达将受到破坏。本研究阐明了单个表观遗传调控因子如何通过时序精准调控驱动运动神经元的逐步发育，该发现对神经发育与相关疾病研究具有广泛启示意义。本研究在小鼠胚胎干细胞体外分化得到的运动神经元的D4、D4H8、D5、D6四个阶段，开展了针对Kdm6b、Neurod1及Cux1/2的Cut&Run测序（Cut&Run-seq），以及针对H3K27me3、H3K27ac、H3K4me1与H3K4me3的Cut&Tag测序（Cut&Tag-seq）。