Dynamic Regulation of Endogenous Transcription Factor Hubs at Single‐Molecule Resolution

Eukaryotic transcription factors (TFs) form local, high-concentration hubs at specific genomic loci through dynamic, multivalent interactions mediated by their low-complexity domains, a behavior essential for transcriptional activation. Characterizing the dynamics and regulation of TF hubs requires high-resolution imaging of TFs in their native cellular environment; however, such biophysical characterization remains limited. Here, we combine CRISPR/Cas9-mediated genome editing and single-molecule microscopy to investigate the behaviors of endogenous oncogenic fusion TF EWS::FLI1 in Ewing sarcoma cells. EWS::FLI1 forms sub-diffraction-limit hubs with dissolution mechanisms that prevent macroscopic liquid-liquid phase separation. Hub formation is a neomorphic behavior of EWS::FLI1 not conferred by its parental proteins, EWSR1 and FLI1. During mitosis, EWS::FLI1 hubs dissolve, but EWS::FLI1 molecules continue to dynamically associate with mitotic chromosomes, consistent with a mitotic bookmarking role. Nascent RNA destabilizes EWS::FLI1 hubs without affecting their dimensions. Finally, LY2835219 and trabectedin, compounds previously reported to affect EWS::FLI1 function, disrupt and mislocalize endogenous EWS::FLI1 hubs, respectively, highlighting their therapeutic potential for treating Ewing sarcoma. Together, our results reveal new insights into the assembly and regulation of endogenous EWS::FLI1 hubs at an unprecedented resolution and establish a framework for biophysical and functional characterization of other TF hubs in the future.