Evidence for DNA-mediated nuclear compartmentalization distinct from phase separation

McSwiggen, David; Hansen, Anders S.; Teves, Sheila S; Marie-Nelly, Hervé; Hao, Yvonne; Heckert, Alec; Vyas, Dinesh; Dugast-Darzacq, Claire; Tjian, Robert; Darzacq, Xavier

doi:10.7554/elife.47098

Cited by 258 publications

(283 citation statements)

References 51 publications

(91 reference statements)

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“…2B, C), demonstrating that this treatment does not interfere with direct binding to DNA. This observation is consistent with results from human cells, in which hexanediol does not disrupt nonspecific DNA binding of RNA polymerase II (51).…”

Section: Rnap Clustering Is Mediated By Protein-protein Interactionssupporting

confidence: 91%

“…Despite this exciting progress, it remains challenging to study phase separation in any organism but particularly in bacteria. Indeed, while the appearance of fluorescent foci in cells is promising, this alone is not compelling evidence for LLPS because foci can form through many alternative mechanisms (51). So additional tools for interrogating putative condensates are urgently needed.…”

Section: Single-molecule Tracking As a Tool For Examining Llps In Vivomentioning

confidence: 99%

“…Fortunately, single-molecule tracking has recently emerged as a powerful tool for examining LLPS in vivo (51,111,112). Indeed, the strongest evidence for bacterial condensates to date comes from single-molecule tracking of proteins in polar microdomains in Caulobacter (113).…”

Section: Single-molecule Tracking As a Tool For Examining Llps In Vivomentioning

confidence: 99%

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Clusters of bacterial RNA polymerase are biomolecular condensates that assemble through liquid-liquid phase separation

Ladouceur

Parmar

Biedzinski

et al. 2020

Preprint

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Once described as mere "bags of enzymes", bacterial cells are in fact highly organized, with many macromolecules exhibiting non-uniform localization patterns. Yet the physical and biochemical mechanisms that govern this spatial heterogeneity remain largely unknown. Here, we identify liquid-liquid phase separation (LLPS) as a mechanism for organizing clusters of RNA polymerase (RNAP) in E. coli. Using fluorescence imaging, we show that RNAP quickly transitions from a dispersed to clustered localization pattern as cells enter log phase in nutrientrich media. RNAP clusters are sensitive to hexanediol, a chemical that dissolves liquid-like compartments in eukaryotic cells. In addition, we find that the transcription antitermination factor NusA forms droplets in vitro and in vivo, suggesting that it may nucleate RNAP clusters. Finally, we use single-molecule tracking to characterize the dynamics of cluster components.Our results indicate that RNAP and NusA molecules move inside clusters, with mobilities faster than a DNA locus but slower than bulk diffusion through the nucleoid. We conclude that RNAP clusters are biomolecular condensates that assemble through LLPS. This work provides direct evidence for LLPS in bacteria and suggests that this process serves as a universal mechanism for intracellular organization across the tree of life. SignificanceBacterial cells are small and were long thought to have little to no internal structure. However, advances in microscopy have revealed that bacteria do indeed contain subcellular compartments. But how these compartments form has remained a mystery. Recent progress in larger, more complex eukaryotic cells has identified a novel mechanism for intracellular organization known as liquid-liquid phase separation. This process causes certain types of molecules to concentrate within distinct compartments inside the cell. Here, we demonstrate that the same process also occurs in bacteria. This work, together with a growing body of literature, suggests that liquidliquid phase separation is a universal mechanism for intracellular organization that extends across the tree of life.

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Section: Rnap Clustering Is Mediated By Protein-protein Interactionssupporting

confidence: 91%

Section: Single-molecule Tracking As a Tool For Examining Llps In Vivomentioning

confidence: 99%

See 1 more Smart Citation

Clusters of bacterial RNA polymerase are biomolecular condensates that assemble through liquid-liquid phase separation

Ladouceur

Parmar

Biedzinski

et al. 2020

Preprint

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“…However, it was recently demonstrated that HSV-1 VRCs are not disrupted by treatment with 1,6-hexanediol, which is a feature of many other cellular BMCs. Moreover, the authors report that the recruitment of RNA polymerase II (RNAP II) to VRCs and its movement within VRCs are best explained by a model other than LLPS [105]. However, the role of LLPS in biological processes can be subtle and complex.…”

Section: Biophysical Processes In Replication Compartment Formationmentioning

confidence: 99%

Replication Compartments of DNA Viruses in the Nucleus: Location, Location, Location

Charman

Weitzman

2020

Viruses

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DNA viruses that replicate in the nucleus encompass a range of ubiquitous and clinically important viruses, from acute pathogens to persistent tumor viruses. These viruses must co-opt nuclear processes for the benefit of the virus, whilst evading host processes that would otherwise attenuate viral replication. Accordingly, DNA viruses induce the formation of membraneless assemblies termed viral replication compartments (VRCs). These compartments facilitate the spatial organization of viral processes and regulate virus-host interactions. Here, we review advances in our understanding of VRCs. We cover their initiation and formation, their function as the sites of viral processes, and aspects of their composition and organization. In doing so, we highlight ongoing and emerging areas of research highly pertinent to our understanding of nuclear-replicating DNA viruses.Viruses 2020, 12, 151 2 of 20 The Initiation and Formation of Replication CompartmentsDNA viruses that replicate in the nucleus exhibit a diverse array of strategies to complete their replication cycle and ultimately produce progeny virions. Despite their many differences, the strategies employed by DNA viruses to initiate productive infection and establish VRCs share many features in common. Indeed, it is likely that all DNA viruses that replicate in the nucleus form VRCs. Following penetration of the host cell and the transport of viral capsids/cores, viral DNA genomes enter the nucleus, where they begin a program of temporally regulated gene expression that initiates productive infection [1,2]. Early gene products include viral proteins required to manipulate the host-cell environment and replicate the viral genome. Viral replication proteins interact with the viral genome and initiate genome replication leading to VRC formation, which is often in concert with certain co-opted host proteins. However, these viruses must overcome several challenges to form VRCs successfully. Firstly, incoming genomes must avoid cellular intrinsic antiviral defenses and homeostatic regulatory pathways such as elements of the DNA damage response (DDR) that respond to the presence of foreign DNA and act to suppress viral gene expression [3][4][5][6][7][8][9][10]. Secondly, VRCs typically form at specific sites within the nucleus, suggesting that viral genomes need to be targeted to these sites in order to initiate VRC formation [6,11,12]. Furthermore, it has been hypothesized that the formation and growth of VRCs may require manipulation of the nuclear environment and the re-organization of host chromatin to overcome the spatial restraints of the nucleus [11][12][13][14][15]. In this section, we review key features of VRC initiation and highlight some major challenges to VRC formation. Interplay between Viral Replication Compartment Formation and Promyelocytic Leukemia Protein Nuclear BodiesA common theme amongst many nuclear-replicating DNA viruses is initiation of VRCs at sites in close proximity to promyelocytic leukemia protein (PML) nuclear bodies (NBs). Since the dis...

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“…Notably, during latency key lytic promoters like RTA contain both the activating H3K4me3 and the repressive H3K27me3 histone marks, representing a 'poised' state [53]. At later stages of infection, KSHV, EBV and HSV-1 genomes have been shown to be largely nucleosome free, consistent with histones not being packaged in virions [33,35,54,55]. At later stages of infection, KSHV, EBV and HSV-1 genomes have been shown to be largely nucleosome free, consistent with histones not being packaged in virions [33,35,54,55].…”

Section: Discussionmentioning

confidence: 99%

RNA decay during gammaherpesvirus infection reduces RNA polymerase II occupancy of host promoters but spares viral promoters

et al. 2020

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In mammalian cells, widespread acceleration of cytoplasmic mRNA degradation is linked to impaired RNA polymerase II (Pol II) transcription. This mRNA decay-induced transcriptional repression occurs during infection with gammaherpesviruses including Kaposi's sarcomaassociated herpesvirus (KSHV) and murine gammaherpesvirus 68 (MHV68), which encode an mRNA endonuclease that initiates widespread RNA decay. Here, we show that MHV68induced mRNA decay leads to a genome-wide reduction of Pol II occupancy at mammalian promoters. This reduced Pol II occupancy is accompanied by down-regulation of multiple Pol II subunits and TFIIB in the nucleus of infected cells, as revealed by mass spectrometrybased global measurements of protein abundance. Viral genes, despite the fact that they require Pol II for transcription, escape transcriptional repression. Protection is not governed by viral promoter sequences; instead, location on the viral genome is both necessary and sufficient to escape the transcriptional repression effects of mRNA decay. We propose a model in which the ability to escape from transcriptional repression is linked to the localization of viral DNA within replication compartments, providing a means for these viruses to counteract decay-induced transcript loss. Author summaryWhile transcription and messenger RNA (mRNA) decay are often considered to be the unlinked beginning and end of gene expression, recent data indicate that alterations to either stage can impact the other. Here we study this connection in the context of lytic gammaherpesvirus infection, which accelerates mRNA degradation through the expression of the viral endonuclease muSOX. We show that RNA polymerase II promoter occupancy is broadly reduced across mammalian promoters in response to infection-induced mRNA PLOS Pathogens | https://doi.

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Evidence for DNA-mediated nuclear compartmentalization distinct from phase separation

Cited by 258 publications

References 51 publications

Clusters of bacterial RNA polymerase are biomolecular condensates that assemble through liquid-liquid phase separation

Clusters of bacterial RNA polymerase are biomolecular condensates that assemble through liquid-liquid phase separation

Replication Compartments of DNA Viruses in the Nucleus: Location, Location, Location

RNA decay during gammaherpesvirus infection reduces RNA polymerase II occupancy of host promoters but spares viral promoters

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