Enzymatic probes of chromatin structure reveal accessible versus inaccessible chromatin states, while super-resolution microscopy reveals a continuum of chromatin compaction states. Characterizing histone H2B movements by single-molecule tracking (SMT), we resolved chromatin domains ranging from low to high mobility and displaying different subnuclear localizations patterns. Heterochromatin constituents correlated with the lowest mobility chromatin, whereas transcription factors varied widely with regard to their respective mobility with low- or high-mobility chromatin. Pioneer transcription factors, which bind nucleosomes, can access the low-mobility chromatin domains, whereas weak or non-nucleosome binding factors are excluded from the domains and enriched in higher mobility domains. Nonspecific DNA and nucleosome binding accounted for most of the low mobility of strong nucleosome interactor FOXA1. Our analysis shows how the parameters of the mobility of chromatin-bound factors, but not their diffusion behaviors or SMT-residence times within chromatin, distinguish functional characteristics of different chromatin-interacting proteins.

染色质结构的酶探针揭示了可及与不可接近的染色质状态，而超分辨率显微镜则揭示了染色质压实状态的连续性。通过单分子跟踪 (SMT) 表征组蛋白 H2B 运动，我们解析了从低到高移动性的染色质域，并显示了不同的亚核定位模式。异染色质成分与迁移率最低的染色质相关，而转录因子在其各自的迁移率与低迁移率或高迁移率染色质方面差异很大。结合核小体的先锋转录因子可以进入低迁移率染色质域，而弱或非核小体结合因子被排除在域之外并在更高迁移率域中富集。非特异性 DNA 和核小体结合是强核小体相互作用物 FOXA1 的低迁移率的主要原因。我们的分析显示了染色质结合因子的流动性参数，而不是它们的扩散行为或染色质内的 SMT 驻留时间，如何区分不同染色质相互作用蛋白的功能特征。