Single-Molecule Characterization of the Dynamics and Regulation of Endogenous Transcription Factor Hubs

Author: Yoshida, Shawn R.

Year: 2027

Degree: Dissertation (Ph.D.)

Advisor: Chong, Shasha

Committee Members: Guttman, Mitchell; Shan, Shu-ou; Wei, Lu; Chong, Shasha

Option: Biochemistry and Molecular Biophysics

DOI: 10.7907/hv43-8h80

Abstract

DNA-binding transcription factors (TFs) regulate eukaryotic gene expression through intrinsically disordered low-complexity domains (LCDs) that drive the formation of local, high-concentration hubs at target genomic loci via dynamic, multivalent LCD-LCD interactions. These hubs are critical to activate transcription of target genes; despite this, many of their physical properties and regulatory mechanisms remain unexplored at endogenous expression levels. This is largely due to the time-consuming nature of gene editing and the necessity for super-resolution microscopy methods that can resolve sub-diffraction-limit endogenous TF hubs compared to overexpression, which offers a faster alternative that also produces larger, micron-scale droplets that do not require specialized microscopy. However, a growing body of evidence suggests that the exquisite spatiotemporal coordination of endogenous TF hubs is poorly recapitulated by overexpression studies. Thus, here, we address these gaps by combining CRISPR/Cas9-mediated endogenous fluorescent labeling with single-molecule and super-resolution imaging methods including photoactivated localization microscopy (PALM), single-particle tracking (SPT), and stimulated emission depletion microscopy (STED), to characterize the biophysical properties and regulation mechanisms of our model oncogenic fusion TF, EWS::FLI1, at unprecedented resolution in Ewing sarcoma cells. We first establish that sample fixation with paraformaldehyde can differentially affect the appearance of certain LCD-driven droplets, artificially increasing their size and number in some cases and diminishing it in others. The spatial distribution of EWS::FLI1 does not appear to be sensitive to paraformaldehyde fixation. We then used PALM and showed that endogenous EWS::FLI1 forms sub-diffraction-limit hubs of mean diameter around 100 nm, and extracted the energetics and kinetics of hub formation, showing that they are not liquid-liquid phase-separated droplets, rather, they behave as supersaturation-driven nucleating clusters. We next showed that EWS::FLI1 hub formation is a neomorphic property of the fusion and is not directly conferred by either of its parental proteins. We also investigated numerous axes of EWS::FLI1 hub regulation using a combination of PALM, SPT, STED, and confocal microscopy. We showed that during mitosis, hubs dissolve and EWS::FLI1 remains weakly enriched on mitotic chromatin, that nascent RNA modulates EWS::FLI1-hub binding kinetics without altering hub dimensions, that the post-translational modification O-GlcNAc can make hubs more fluid and reduce EWS::FLI1 retention in hubs, and that EWS::FLI1 preferentially associates with initiating serine-5 phosphorylated RNA Polymerase II. We also showed that treatment with small molecules LY2835219 or trabectedin dissolves and mislocalizes EWS::FLI1 hubs, respectively, suppressing transcription of EWS::FLI1 target genes, highlighting their therapeutic potential. Together, this work provides a physically grounded, single-molecule view into the formation, regulation, and function of oncogenic TF hubs, and establishes an imaging-based framework broadly applicable to the study of other proteins involved in transcriptional control and disease.

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