Single-molecule tracking reveals two low-mobility states for chromatin and transcriptional regulators within the nucleus

Transcription factors (TFs) scan the nucleus in search of their consensus binding motifs located within enhancers or promoter-proximal regions. The mechanism by which TFs navigate the complex nuclear environment to assemble the transcriptional machinery at specific genomic loci remains elusive. Using single-molecule tracking, coupled with machine learning, we examined the mobility of multiple transcription factors and coregulators. We show that chromatin (labeled by histone H2B), steroid hormone receptors, as well as other transcriptional coregulators, architectural proteins, and remodelers, all display two distinct low-mobility states. Our results indicate that both low-mobility states are intimately coupled with mobile chromatin. Ligand activation results in a dramatic increase in the proportion of steroid receptors in the lowest mobility state. Mutational analysis revealed that chromatin interactions in the lower mobility state require an intact DNA-binding domain as well as domains important for forming protein complexes with other binding partners. These domains are not necessary for engagement with the higher mobility fraction of chromatin. Importantly, these states are not spatially separated as previously believed but in fact, individual H2B and TF molecules can dynamically switch between them. Together, our results identify two unique and distinct low-mobility states of transcriptional regulators that appear to represent common pathways for transcription activation in mammalian cells.