Abstract
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.
Highlights
Controlling the local concentration of molecules within cells is fundamental to living organisms, with membrane-bound organelles serving as the prototypic mechanism
Despite initial indications that replication compartments (RCs) exhibit many of the macroscopic hallmarks of LLPS, we find that recruitment of Polymerase II (Pol II) and other DNA-binding proteins to RCs is achieved through a distinct compartmentalization mechanism
Because we were most interested in the early stages of lytic infection when Pol II is actively recruited to the RC, we focused our experiments on the period between 3 hours post infection, when RCs begin to emerge, and 6 hpi when infected cells begin to display significant cytopathic effects (Figure 1—video 1 and 2)
Summary
Controlling the local concentration of molecules within cells is fundamental to living organisms, with membrane-bound organelles serving as the prototypic mechanism. A number of studies suggest that many proteins have the ability to spontaneously form separated liquid phases in vitro (Banani et al, 2017), and recent work highlights the possibility that similar liquid compartments may occur in vivo (Courchaine et al, 2016; Bracha et al, 2018) Such liquid-liquid demixing (liquid-liquid phase separation, LLPS) has been proposed to be a common mechanism in sequestering specific macromolecules within a compartment, or in increasing their local concentration and thereby facilitating molecular interactions. Formation of these structures is thought to be predominantly driven by multivalent interactions mediated through intrinsically disordered regions (IDRs), or via modular binding motifs, RNA, or DNA (Banani et al, 2017). Our findings reveal that nonspecific binding can play a key role in the recruitment and retention of Pol II during infection, and more generally in the repertoire of distinct mechanisms a cell might employ to generate membraneless compartments
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