Evidence for DNA-mediated nuclear compartmentalization distinct from phase separation.

RNA Polymerase II (Pol II) and transcription factors form concentrated hubs in cells via multivalent protein-protein interactions, often mediated by proteins with intrinsically disordered regions. During Herpes Simplex Virus infection, viral replication compartments (RCs) efficiently enrich host Pol II into membraneless domains, reminiscent of liquid-liquid phase separation. Despite sharing several properties with phase-separated condensates, we show that RCs operate via a distinct mechanism wherein unrestricted nonspecific protein-DNA interactions efficiently outcompete host chromatin, profoundly influencing the way DNA-binding proteins explore RCs. We find that the viral genome remains largely nucleosome-free, and this increase in accessibility allows Pol II and other DNA-binding proteins to repeatedly visit nearby DNA binding sites. This anisotropic behavior creates local accumulations of protein factors despite their unrestricted diffusion across RC boundaries. Our results reveal underappreciated consequences of nonspecific DNA binding in shaping gene activity, and suggest additional roles for chromatin in modulating nuclear function and organization

Extracting fast subpopulations from fragmentary live cell single-particle trajectories

Stroboscopic photoactivated single particle tracking (spaSPT) relies on stochasticlabeling to isolate the paths of individual fluorophores, and can provide information about the behavior of biological macromolecules in their native cellular environment. Existing spaSPT modalities generate large numbers of short trajectories, each representing a fragment of an individual emitter's path. When interpreting this data through the lens of diffusion models, it is essential to account for the fragmentary nature of trajectories, experimental biases arising from the imaging geometry, and our ignorance about the correct underlying diffusion model. In this thesis, we describe several methods for interpretation of spaSPT data that provide estimates about the number and characteristics of mobility states in spaSPT data while accounting for known experimental artifacts. We explore the uses and limitations of these models on simulated and experimental datasets.In the final chapter, we apply these methods to study the competitive chromatin binding in the type II nuclear receptors (T2NRs). T2NRs are a class of ligand-activated transcription factors that require heterodimerization with a common factor, the retinoid X receptor (RXR), to bind chromatin and regulate target genes. Because all T2NRs must dimerize with a common pool of RXR, competition between individual T2NRs may limit access to the bound state. This mechanism has been proposed to underlie the oncogenic inactivation of wildtype retinoic acid receptor alpha (RARA), a prototypical T2NR, in the presence of RARA fusion proteins that occur in acute promyelocytic leukemia (APL). Such fusion proteins may compete RXR away from the wildtype RARA, impairing its ability to regulate target genes. Here, we use spaSPT to directly measure the effects of RARA fusion proteins on the chromatin binding of endogenously tagged RARA and RXR. Using the tools developed in the previous chapters, we find that RARA fusion proteins act as stronger competitors for dimerization with RXR than wildtype T2NRs, and are also seemingly exempt from a novel autoregulatory mechanism that controls the concentration of wildtype RARA. Together, these results provide new insights into the interdependence of T2NR gene regulation and the manner by which it is disrupted in disease

Evidence for DNA-mediated nuclear compartmentalization distinct from phase separation.

RNA Polymerase II (Pol II) and transcription factors form concentrated hubs in cells via multivalent protein-protein interactions, often mediated by proteins with intrinsically disordered regions. During Herpes Simplex Virus infection, viral replication compartments (RCs) efficiently enrich host Pol II into membraneless domains, reminiscent of liquid-liquid phase separation. Despite sharing several properties with phase-separated condensates, we show that RCs operate via a distinct mechanism wherein unrestricted nonspecific protein-DNA interactions efficiently outcompete host chromatin, profoundly influencing the way DNA-binding proteins explore RCs. We find that the viral genome remains largely nucleosome-free, and this increase in accessibility allows Pol II and other DNA-binding proteins to repeatedly visit nearby DNA binding sites. This anisotropic behavior creates local accumulations of protein factors despite their unrestricted diffusion across RC boundaries. Our results reveal underappreciated consequences of nonspecific DNA binding in shaping gene activity, and suggest additional roles for chromatin in modulating nuclear function and organization