scholarly journals Phase separation and DAXX redistribution contribute to LANA nuclear body and KSHV genome dynamics during latency and reactivation

2021 ◽  
Vol 17 (1) ◽  
pp. e1009231
Author(s):  
Olga Vladimirova ◽  
Alessandra De Leo ◽  
Zhong Deng ◽  
Andreas Wiedmer ◽  
James Hayden ◽  
...  
Keyword(s):  
Phase Separation ◽  
Nuclear Body ◽  
Lytic Replication ◽  
Nuclear Bodies ◽  
Nuclear Antigen ◽  
Morphological Transformation ◽  
Chromosome Conformation ◽  
Lytic Reactivation ◽  
Morphological Transitions ◽  
Nuclear Structures

Liquid-liquid phase separation (LLPS) can drive formation of diverse and essential macromolecular structures, including those specified by viruses. Kaposi’s Sarcoma-Associated Herpesvirus (KSHV) genomes associate with the viral encoded Latency-Associated Nuclear Antigen (LANA) to form stable nuclear bodies (NBs) during latent infection. Here, we show that LANA-NB formation and KSHV genome conformation involves LLPS. Using LLPS disrupting solvents, we show that LANA-NBs are partially disrupted, while DAXX and PML foci are highly resistant. LLPS disruption altered the LANA-dependent KSHV chromosome conformation but did not stimulate lytic reactivation. We found that LANA-NBs undergo major morphological transformation during KSHV lytic reactivation to form LANA-associated replication compartments encompassing KSHV DNA. DAXX colocalizes with the LANA-NBs during latency but is evicted from the LANA-associated lytic replication compartments. These findings indicate the LANA-NBs are dynamic super-molecular nuclear structures that partly depend on LLPS and undergo morphological transitions corresponding the different modes of viral replication.

scholarly journals Chromatin Network Retards Droplet Coalescence

2021 ◽  
Author(s):  
Yifeng Qi ◽  
Bin Zhang
Keyword(s):  
Phase Separation ◽  
Cell Types ◽  
Nuclear Body ◽  
Free Energy Calculations ◽  
Nuclear Bodies ◽  
Chromosome Conformation ◽  
Chromatin Interactions ◽  
Dynamical Simulations ◽  
Correlated Motion ◽  
Kinetics Of

AbstractNuclear bodies are membraneless condensates that may form via liquid-liquid phase separation. The viscoelastic chromatin network could impact their stability and may hold the key for understanding experimental observations that defy predictions of classical theories. However, quantitative studies on the role of the chromatin network in phase separation have remained challenging. Using a diploid human genome model parameterized with chromosome conformation capture (Hi-C) data, we studied the thermodynamics and kinetics of droplet formation inside the nucleus. Dynamical simulations predicted the formation of multiple droplets for protein particles that experience specific interactions with nucleolus-associated domains (NADs). Coarsening dynamics, surface tension, and coalescence kinetics of the simulated droplets are all in quantitative agreements with experimental measurements for nucleoli. Free energy calculations further supported that a two-droplet state, which is often observed for nucleoli seen in somatic cells, is metastable and separated from the single-droplet state with an entropic barrier. Our study suggests that protein-chromatin interactions facilitate the nucleation of droplets, but hinders their coarsening due to the correlated motion between droplets and the chromatin network: as droplets coalesce, the chromatin network becomes increasingly constrained. Therefore, protein-chromatin interactions arrest phase separation in multi-droplet states and may drive the variation of nuclear body numbers across cell types.

scholarly journals Nuclear body phase separation drives telomere clustering in ALT cancer cells

2020 ◽  
Vol 31 (18) ◽  
pp. 2048-2056 ◽  
Author(s):  
Huaiying Zhang ◽  
Rongwei Zhao ◽  
Jason Tones ◽  
Michel Liu ◽  
Robert L. Dilley ◽  
...  
Keyword(s):  
Dna Repair ◽  
Phase Separation ◽  
Cancer Cells ◽  
Nuclear Body ◽  
Promyelocytic Leukemia ◽  
Nuclear Bodies ◽  
Telomere Elongation ◽  
Alternative Lengthening ◽  
Induce Phase Separation ◽  
Induce Phase

A chemical dimerization approach is developed to induce phase separation of APB nuclear bodies involved in telomere elongation in alternative lengthening of telomeres (ALT) cancer cells. It reveals that ALT telomere-associated promyelocytic leukemia nuclear body (APB) fusion leads to telomere clustering to provide templates for homology-directed telomere synthesis, an ability that is decoupled from APB function in enriching DNA repair factors.

scholarly journals EBNA3C Coactivation with EBNA2 Requires a SUMO Homology Domain

2004 ◽  
Vol 78 (1) ◽  
pp. 367-377 ◽  
Author(s):  
Adam Rosendorff ◽  
Diego Illanes ◽  
Gregory David ◽  
Jeffrey Lin ◽  
Elliott Kieff ◽  
...  
Keyword(s):  
Amino Acids ◽  
Gene Activation ◽  
Point Mutations ◽  
Nuclear Body ◽  
Nuclear Bodies ◽  
Nuclear Antigen ◽  
Barr Virus ◽  
Epstein Barr ◽  
Necessary And Sufficient ◽  
Regulated Promoters

ABSTRACT Epstein-Barr virus (EBV) nuclear antigen 3C (EBNA3C) is critical for EBV immortalization of infected B lymphocytes and can coactivate the EBV LMP1 promoter with EBNA2. EBNA3C amino acids 365 to 545 are necessary and sufficient for coactivation and are required for SUMO-1 and SUMO-3 interaction. We found that EBNA3C but not EBNA3CΔ343-545 colocalized with SUMO-1 in nuclear bodies and was modified by SUMO-2, SUMO-3, and SUMO-1. EBNA3C amino acids 545 to 628 and amino acids 30 to 365 were also required for EBNA3C sumolation and nuclear body localization but were dispensable for coactivation, indicating that EBNA3C sumolation is not required for coactivation. Furthermore, EBNA3C amino acids 476 to 992 potently coactivated with EBNA2 but EBNA3C amino acids 516 to 922 lacked activity, indicating that amino acids 476 to 515 are critical for coactivation. EBNA3C amino acids 476 to 515 include DDDVIEV507-513, which are similar to SUMO-1 EEDVIEV84-90. EBNA3C m1 and m2 point mutations, DDD507-509 mutated to AAA and DVIEVID509-513 mutated to AVIAVIA, respectively, diminished SUMO-1 and SUMO-3 interaction in directed yeast two-hybrid and glutathione S-transferase pulldown assays. Furthermore, EBNA3C m1 and m2 did not coactivate the LMP1 promoter with EBNA2. Overexpression of wild-type SUMO-1, SUMO-3, and the SUMO-conjugating enzyme UBC9 coactivated the LMP1 promoter with EBNA2. Since EBNA2 activation is dependent on p300/CBP, the possible effect of EBNA3C on p300-mediated transcription was assayed. EBNA3C potentiated transcription of p300 fused to a heterologous DNA binding domain, whereas EBNA3C m1 and m2 did not. All of these data are consistent with a model in which EBNA3C upregulates EBNA2-mediated gene activation by binding to a sumolated repressor and inhibiting repressive effects on p300/CBP and other transcription factor(s) at EBNA2-regulated promoters.

scholarly journals KSHV Episome Tethering Sites on Host Chromosomes: Regulation of Latency-Lytic Switch by the ChAHP complex

2021 ◽  
Author(s):  
Ashish Kumar ◽  
Yuanzhi Lyu ◽  
Yuichi Yanagihashi ◽  
Chanikarn Chantarasrivong ◽  
Vladimir Majerciak ◽  
...  
Keyword(s):  
Dna Binding ◽  
Cell Nucleus ◽  
Latent Infection ◽  
Dna Binding Protein ◽  
Lytic Replication ◽  
Nuclear Antigen ◽  
Host Chromosome ◽  
Lncrna Expression ◽  
Lytic Reactivation ◽  
Genomic Regions

Abstract Kaposi’s sarcoma-associated herpesvirus (KSHV) establishes a latent infection in the cell nucleus, but where KSHV episomal genomes are tethered and the mechanisms underlying KSHV lytic reactivation are unclear. Here, we study the nuclear microenvironment of KSHV episomes and show that the KSHV latency-lytic replication switch is regulated via viral long non-coding (lnc)RNA-CHD4 (chromodomain helicase DNA binding protein 4) interaction. KSHV episomes localize with a CHD4 complex, ChAHP, at epigenetically active genomic regions and tethers frequently near centromeric regions of host chromosomes. The ChAHP complex also occupies the 5’-region of a highly-inducible lncRNAs and terminal repeats of KSHV genome with latency-associated nuclear antigen (LANA). Viral lncRNA binding competes with CHD4 DNA binding, and KSHV reactivation is accompanied by the detachment of KSHV episomes from host chromosome docking sites We propose a model in which elevated lncRNA expression determines the KSHV latency-lytic decision by regulating LANA/ChAHP DNA binding at inducible viral enhancers.

scholarly journals Regulation of KSHV Latency and Lytic Reactivation

Viruses ◽  
2020 ◽  
Vol 12 (9) ◽  
pp. 1034
Author(s):  
Grant Broussard ◽  
Blossom Damania
Keyword(s):  
Life Cycle ◽  
Kaposi's Sarcoma ◽  
Primary Effusion Lymphoma ◽  
Kaposi’S Sarcoma ◽  
Viral Life Cycle ◽  
Lytic Replication ◽  
Nuclear Antigen ◽  
Host Immune System ◽  
Transcription Activator ◽  
Lytic Reactivation

Kaposi’s sarcoma-associated herpesvirus (KSHV) is associated with three malignancies— Kaposi’s sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castleman’s disease (MCD). Central to the pathogenesis of these diseases is the KSHV viral life cycle, which is composed of a quiescent latent phase and a replicative lytic phase. While the establishment of latency enables persistent KSHV infection and evasion of the host immune system, lytic replication is essential for the dissemination of the virus between hosts and within the host itself. The transition between these phases, known as lytic reactivation, is controlled by a complex set of environmental, host, and viral factors. The effects of these various factors converge on the regulation of two KSHV proteins whose functions facilitate each phase of the viral life cycle—latency-associated nuclear antigen (LANA) and the master switch of KSHV reactivation, replication and transcription activator (RTA). This review presents the current understanding of how the transition between the phases of the KSHV life cycle is regulated, how the various phases contribute to KSHV pathogenesis, and how the viral life cycle can be exploited as a therapeutic target.

scholarly journals Clastosome: A Subtype of Nuclear Body Enriched in 19S and 20S Proteasomes, Ubiquitin, and Protein Substrates of Proteasome

2002 ◽  
Vol 13 (8) ◽  
pp. 2771-2782 ◽  
Author(s):  
Miguel Lafarga ◽  
Maria Teresa Berciano ◽  
Emma Pena ◽  
Isabel Mayo ◽  
Jose G. Castaño ◽  
...  
Keyword(s):  
Proteasome Inhibitors ◽  
Cell Types ◽  
Nuclear Body ◽  
Nuclear Bodies ◽  
26S Proteasome ◽  
High Concentration ◽  
Protein Substrates ◽  
Adenovirus E1a ◽  
Ubiquitin Proteasome ◽  
Nuclear Structures

Nuclear bodies represent a heterogeneous class of nuclear structures. Herein, we describe that a subset of nuclear bodies is highly enriched in components of the ubiquitin–proteasome pathway of proteolysis. We coined the term clastosome (from the Greekklastos, broken and soma, body) to refer to this type of nuclear body. Clastosomes contain a high concentration of 1) ubiquitin conjugates, 2) the proteolytically active 20S core and the 19S regulatory complexes of the 26S proteasome, and 3) protein substrates of the proteasome. Although detected in a variety of cell types, clastosomes are scarce under normal conditions; however, they become more abundant when proteasomal activity is stimulated. In contrast, clastosomes disappear when cells are treated with proteasome inhibitors. Protein substrates of the proteasome that are found concentrated in clastosomes include the short-lived transcription factors c-Fos and c-Jun, adenovirus E1A proteins, and the PML protein. We propose that clastosomes are sites where proteolysis of a variety of protein substrates is taking place.

scholarly journals Phosphorothioate Antisense Oligonucleotides Induce the Formation of Nuclear Bodies

1998 ◽  
Vol 9 (5) ◽  
pp. 1007-1023 ◽  
Author(s):  
Peter Lorenz ◽  
Brenda F. Baker ◽  
C. Frank Bennett ◽  
David L. Spector
Keyword(s):  
Antisense Oligonucleotides ◽  
De Novo ◽  
Regulation Of Gene Expression ◽  
Nuclear Body ◽  
Nuclear Bodies ◽  
Phosphorothioate Oligodeoxynucleotides ◽  
Nuclear Structures ◽  
In Cells

Antisense oligonucleotides are powerful tools for the in vivo regulation of gene expression. We have characterized the intracellular distribution of fluorescently tagged phosphorothioate oligodeoxynucleotides (PS-ONs) at high resolution under conditions in which PS-ONs have the potential to display antisense activity. Under these conditions PS-ONs predominantly localized to the cell nucleus where they accumulated in 20–30 bright spherical foci designated phosphorothioate bodies (PS bodies), which were set against a diffuse nucleoplasmic population excluding nucleoli. PS bodies are nuclear structures that formed in cells after PS-ON delivery by transfection agents or microinjection but were observed irrespectively of antisense activity or sequence. Ultrastructurally, PS bodies corresponded to electron-dense structures of 150–300 nm diameter and resembled nuclear bodies that were found with lower frequency in cells lacking PS-ONs. The environment of a living cell was required for the de novo formation of PS bodies, which occurred within minutes after the introduction of PS-ONs. PS bodies were stable entities that underwent noticeable reorganization only during mitosis. Upon exit from mitosis, PS bodies were assembled de novo from diffuse PS-ON pools in the daughter nuclei. In situ fractionation demonstrated an association of PS-ONs with the nuclear matrix. Taken together, our data provide evidence for the formation of a nuclear body in cells after introduction of phosphorothioate oligodeoxynucleotides.

scholarly journals Chromatin network retards nucleoli coalescence

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Yifeng Qi ◽  
Bin Zhang
Keyword(s):  
Phase Separation ◽  
Quantitative Agreement ◽  
Free Energy Calculations ◽  
Nuclear Bodies ◽  
Chromosome Conformation ◽  
Chromatin Interactions ◽  
Quantitative Studies ◽  
Dynamical Simulations ◽  
Genome Model ◽  
Kinetics Of

AbstractNuclear bodies are membraneless condensates that may form via liquid-liquid phase separation. The viscoelastic chromatin network could impact their stability and may hold the key for understanding experimental observations that defy predictions of classical theories. However, quantitative studies on the role of the chromatin network in phase separation have remained challenging. Using a diploid human genome model parameterized with chromosome conformation capture (Hi-C) data, we study the thermodynamics and kinetics of nucleoli formation. Dynamical simulations predict the formation of multiple droplets for nucleolar particles that experience specific interactions with nucleolus-associated domains (NADs). Coarsening dynamics, surface tension, and coalescence kinetics of the simulated droplets are all in quantitative agreement with experimental measurements for nucleoli. Free energy calculations further support that a two-droplet state, often observed for nucleoli in somatic cells, is metastable and separated from the single-droplet state with an entropic barrier. Our study suggests that nucleoli-chromatin interactions facilitate droplets’ nucleation but hinder their coarsening due to the coupled motion between droplets and the chromatin network: as droplets coalesce, the chromatin network becomes increasingly constrained. Therefore, the chromatin network supports a nucleation and arrest mechanism to stabilize the multi-droplet state for nucleoli and possibly for other nuclear bodies.

scholarly journals RNA transcription modulates phase transition-driven nuclear body assembly

2015 ◽  
Vol 112 (38) ◽  
pp. E5237-E5245 ◽  
Author(s):  
Joel Berry ◽  
Stephanie C. Weber ◽  
Nilesh Vaidya ◽  
Mikko Haataja ◽  
Clifford P. Brangwynne
Keyword(s):  
Phase Transition ◽  
Phase Separation ◽  
Liquid Phase ◽  
Ribosome Biogenesis ◽  
Ostwald Ripening ◽  
Nuclear Body ◽  
Nuclear Bodies ◽  
Separation Mechanism ◽  
Independent Manner

Nuclear bodies are RNA and protein-rich, membraneless organelles that play important roles in gene regulation. The largest and most well-known nuclear body is the nucleolus, an organelle whose primary function in ribosome biogenesis makes it key for cell growth and size homeostasis. The nucleolus and other nuclear bodies behave like liquid-phase droplets and appear to condense from the nucleoplasm by concentration-dependent phase separation. However, nucleoli actively consume chemical energy, and it is unclear how such nonequilibrium activity might impact classical liquid–liquid phase separation. Here, we combine in vivo and in vitro experiments with theory and simulation to characterize the assembly and disassembly dynamics of nucleoli in early Caenorhabditis elegans embryos. In addition to classical nucleoli that assemble at the transcriptionally active nucleolar organizing regions, we observe dozens of “extranucleolar droplets” (ENDs) that condense in the nucleoplasm in a transcription-independent manner. We show that growth of nucleoli and ENDs is consistent with a first-order phase transition in which late-stage coarsening dynamics are mediated by Brownian coalescence and, to a lesser degree, Ostwald ripening. By manipulating C. elegans cell size, we change nucleolar component concentration and confirm several key model predictions. Our results show that rRNA transcription and other nonequilibrium biological activity can modulate the effective thermodynamic parameters governing nucleolar and END assembly, but do not appear to fundamentally alter the passive phase separation mechanism.

scholarly journals Evidence for and against Liquid-Liquid Phase Separation in the Nucleus

2019 ◽  
Vol 5 (4) ◽  
pp. 50 ◽  
Author(s):  
A ◽  
Weber
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Liquid Phase Separation ◽  
Nuclear Bodies ◽  
Nucleic Acid Binding ◽  
Nuclear Compartments ◽  
Protein Nucleic Acid ◽  
Nuclear Structures ◽  
Liquid Liquid Phase Separation ◽  
Made In

Enclosed by two membranes, the nucleus itself is comprised of various membraneless compartments, including nuclear bodies and chromatin domains. These compartments play an important though still poorly understood role in gene regulation. Significant progress has been made in characterizing the dynamic behavior of nuclear compartments and liquid-liquid phase separation (LLPS) has emerged as a prominent mechanism governing their assembly. However, recent work reveals that certain nuclear structures violate key predictions of LLPS, suggesting that alternative mechanisms likely contribute to nuclear organization. Here, we review the evidence for and against LLPS for several nuclear compartments and discuss experimental strategies to identify the mechanism(s) underlying their assembly. We propose that LLPS, together with multiple modes of protein-nucleic acid binding, drive spatiotemporal organization of the nucleus and facilitate functional diversity among nuclear compartments.

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