Single-molecule tracking reveals two low-mobility states for transcriptional regulators in the nucleus

KAUSTUBH WAGH; STAVREVA, DIANA A.; RIKKE A.M. JENSEN; VILLE PAAKINAHO; GREGORY FETTWEIS; R. LOUIS SCHILTZ; SUSANNE MANDRUP; DIEGO M PRESMAN; ARPITA UPADHYAYA; GORDON L. HAGER*

Cold Spring Harbor, NY

Congreso; Single Biomolecules - CSHL Meeting; 2022

CSHL

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 (SMT), coupled with machine learning, we examined the mobility of multiple transcription factors and coregulators. Our results indicate that steroid hormone receptors, as well as other transcriptional coregulators, architectural proteins, and remodelers, all display two distinct low-mobility states. Ligand activation results in a dramatic increase in the proportion of TFs in both low-mobility states. Strikingly, histone H2B also exhibits the same two low-mobility states and examination of several different cell lines demonstrates the universality of these states for both TFs and H2B. Our results indicate that both low-mobility states are intimately coupled with mobile chromatin. 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. Individual H2B and TF molecules can dynamically switch between the two low-mobility states. 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