Two-parameter mobility assessments discriminate diverse regulatory factor behaviors in chromatin
Enzymatic probes of chromatin structure reveal accessible versus inaccessible chromatin states, while super-resolution microscopy reveals a continuum of chromatin compaction states. Using single-molecule tracking to characterize two parameters of histone H2B movements, we resolved five types of chromatin mobility domains displaying distinct subnuclear localizations. Heterochromatin constituents correlated with the lowest mobility chromatin, whereas transcription factors varied widely with regard to their respective mobility with low or highly mobile 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. Non-specific DNA and nucleosome binding accounted for most of the low mobility of FOXA1. Our analysis shows how the parameters of the mobility of chromatin-bound factors, but not their diffusion behaviors or residence times within chromatin, distinguish functional characteristics of different chromatin interacting proteins.