Pioneer transcription factors (PTFs) possess the unique capability to access closed chromatin regions and initiate cell fate changes, yet the underlying mechanisms remain elusive. Here, we characterized the single-molecule dynamics of PTFs targeting chromatin in living cells, revealing a notable ‘confined target search’ mechanism. PTFs such as FOXA1, FOXA2, SOX2, OCT4 and KLF4 sampled chromatin more frequently than non-PTF MYC, alternating between fast free diffusion in the nucleus and slower confined diffusion within mesoscale zones. Super-resolved microscopy showed closed chromatin organized as mesoscale nucleosome-dense domains, confining FOXA2 diffusion locally and enriching its binding. We pinpointed specific histone-interacting disordered regions, distinct from DNA-binding domains, crucial for confined target search kinetics and pioneer activity within closed chromatin. Fusion to other factors enhanced pioneer activity. Kinetic simulations suggested that transient confinement could increase target association rate by shortening search time and binding repeatedly. Our findings illuminate how PTFs recognize and exploit closed chromatin organization to access targets, revealing a pivotal aspect of gene regulation.

先锋转录因子（PTF）具有进入封闭染色质区域并启动细胞命运改变的独特能力，但其潜在机制仍然难以捉摸。在这里，我们对活细胞中靶向染色质的 PTF 的单分子动力学进行了表征，揭示了一种值得注意的“有限目标搜索”机制。 FOXA1、FOXA2、SOX2、OCT4 和 KLF4 等 PTF 比非 PTF MYC 更频繁地采样染色质，在细胞核中的快速自由扩散和中尺度区域内较慢的受限扩散之间交替。超分辨显微镜显示闭合染色质组织为中尺度核小体致密结构域，局部限制 FOXA2 扩散并丰富其结合。我们查明了与 DNA 结合域不同的特定组蛋白相互作用无序区域，这对于封闭染色质内的有限目标搜索动力学和先锋活动至关重要。与其他因素的融合增强了先锋活动。动力学模拟表明，瞬时限制可以通过缩短搜索时间和重复结合来提高目标关联率。我们的研究结果阐明了 PTF 如何识别和利用封闭染色质组织来访问目标，揭示了基因调控的关键方面。