scholarly journals Formation of nuclear condensates by the Mediator complex subunit Med15 in mammalian cells

BMC Biology ◽  
2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Yuanyuan Shi ◽  
Jian Chen ◽  
Wei-Jie Zeng ◽  
Miao Li ◽  
Wenxue Zhao ◽  
...  
Keyword(s):  
Phase Separation ◽  
Transcriptional Activation ◽  
Mammalian Cells ◽  
Gene Activation ◽  
Mediator Complex ◽  
Intrinsically Disordered ◽  
Dual Specificity ◽  
Proliferation And Differentiation ◽  
A Subunit ◽  
Endogenous Proteins

Abstract Background The Mediator complex is an evolutionarily conserved multi-subunit protein complex that plays major roles in transcriptional activation and is essential for cell growth, proliferation, and differentiation. Recent studies revealed that some Mediator subunits formed nuclear condensates that may facilitate enhancer-promoter interactions and gene activation. The assembly, regulation, and functions of these nuclear condensates remain to be further understood. Results We found that Med15, a subunit in the tail module of the Mediator complex, formed nuclear condensates through a novel mechanism. Nuclear foci of Med15 were detected by both immunostaining of endogenous proteins and live cell imaging. Like Med1 foci and many other biomolecular condensates, Med15 foci were sensitive to 1, 6-Hexanediol and showed rapid recovery during fluorescence recovery after photobleaching. Interestingly, overexpressing DYRK3, a dual-specificity kinase that controls the phase transition of membraneless organelles, appeared to disrupt Med1 foci and Med15 foci. We identified two regions that are required to form Med15 nuclear condensates: the glutamine-rich intrinsically disordered region (IDR) and a short downstream hydrophobic motif. The optodroplet assay revealed that both the IDR and the C-terminal region of Med15 contributed to intracellular phase separation. Conclusions We identified that the Mediator complex subunit Med15 formed nuclear condensates and characterized their features in living cells. Our work suggests that Med15 plays a role in the assembly of transcription coactivator condensates in the nucleus and identifies Med15 regions that contribute to phase separation.

scholarly journals ENL initiates multivalent phase separation of the super elongation complex (SEC) in controlling rapid transcriptional activation

2020 ◽  
Vol 6 (14) ◽  
pp. eaay4858 ◽  
Author(s):  
Chenghao Guo ◽  
Zhuanzhuan Che ◽  
Junjie Yue ◽  
Peng Xie ◽  
Shaohua Hao ◽  
...  
Keyword(s):  
Phase Separation ◽  
Rna Polymerase Ii ◽  
Transcriptional Activation ◽  
Liquid Droplet ◽  
Critical Role ◽  
Elongation Factor ◽  
Elongation Complex ◽  
Intrinsically Disordered ◽  
Super Elongation Complex

Release of paused RNA polymerase II (Pol II) requires incorporation of the positive transcription elongation factor b (P-TEFb) into the super elongation complex (SEC), thus resulting in rapid yet synchronous transcriptional activation. However, the mechanism underlying dynamic transition of P-TEFb from inactive to active state remains unclear. Here, we found that the SEC components are able to compartmentalize and concentrate P-TEFb via liquid-liquid phase separation from the soluble inactive HEXIM1 containing the P-TEFb complex. Specifically, ENL or its intrinsically disordered region is sufficient to initiate the liquid droplet formation of SEC. AFF4 functions together with ENL in fluidizing SEC droplets. SEC droplets are fast and dynamically formed upon serum exposure and required for rapid transcriptional induction. We also found that the fusion of ENL with MLL can boost SEC phase separation. In summary, our results suggest a critical role of multivalent phase separation of SEC in controlling transcriptional pause release.

scholarly journals Liquid-liquid phase separation of light-inducible transcription factors increases transcription activation in mammalian cells and mice

2021 ◽  
Vol 7 (1) ◽  
pp. eabd3568
Author(s):  
Nils Schneider ◽  
Franz-Georg Wieland ◽  
Deqiang Kong ◽  
Alexandra A. M. Fischer ◽  
Maximilian Hörner ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Transcription Factors ◽  
Phase Separation ◽  
Liquid Phase ◽  
Mammalian Cells ◽  
Liquid Phase Separation ◽  
Intrinsically Disordered ◽  
Intrinsically Disordered Regions ◽  
Gene Switches ◽  
Liquid Liquid Phase Separation

Light-inducible gene switches represent a key strategy for the precise manipulation of cellular events in fundamental and applied research. However, the performance of widely used gene switches is limited due to low tissue penetrance and possible phototoxicity of the light stimulus. To overcome these limitations, we engineer optogenetic synthetic transcription factors to undergo liquid-liquid phase separation in close spatial proximity to promoters. Phase separation of constitutive and optogenetic synthetic transcription factors was achieved by incorporation of intrinsically disordered regions. Supported by a quantitative mathematical model, we demonstrate that engineered transcription factor droplets form at target promoters and increase gene expression up to fivefold. This increase in performance was observed in multiple mammalian cells lines as well as in mice following in situ transfection. The results of this work suggest that the introduction of intrinsically disordered domains is a simple yet effective means to boost synthetic transcription factor activity.

scholarly journals Phase separation of YAP reorganizes genome topology for long-term, YAP target gene expression

2018 ◽  
Author(s):  
Danfeng Cai ◽  
Daniel Feliciano ◽  
Peng Dong ◽  
Eduardo Flores ◽  
Martin Gruebele ◽  
...  
Keyword(s):  
Phase Separation ◽  
Rna Polymerase Ii ◽  
Target Gene ◽  
Gene Activation ◽  
Super Resolution ◽  
Transcriptional Responses ◽  
Chromatin Domains ◽  
Pol Ii ◽  
Intrinsically Disordered ◽  
Accessible Chromatin

Yes-associated Protein (YAP) is a transcriptional co-activator that regulates cell proliferation and survival by binding to a selective set of enhancers for potent target gene activation, but how YAP coordinates these transcriptional responses is unknown. Here, we demonstrate that YAP forms liquid-like condensates in the nucleus in response to macromolecular crowding. Formed within seconds of hyperosmotic stress, YAP condensates compartmentalized YAP’s DNA binding cofactor TEAD1 along with other YAP-related transcription co-activators, including TAZ, and subsequently induced transcription of YAP-specific proliferation genes. Super-resolution imaging using Assay for Transposase Accessible Chromatin with photoactivated localization microscopy (ATAC-PALM) revealed that YAP nuclear condensates were areas enriched in accessible chromatin domains organized as super-enhancers. Initially devoid of RNA Polymerase II (Pol II), the accessible chromatin domains later acquired Pol II, producing newly transcribed RNA. Removal of YAP’s intrinsically-disordered transcription activation domain (TAD) prevented YAP condensate formation and diminished downstream YAP signaling. Thus, dynamic changes in genome organization and gene activation during YAP reprogramming is mediated by liquid-liquid phase separation.

scholarly journals SARS CoV-2 nucleocapsid protein forms condensates with viral genomic RNA

2020 ◽  
Author(s):  
Amanda Jack ◽  
Luke S. Ferro ◽  
Michael J. Trnka ◽  
Eddie Wehri ◽  
Amrut Nadgir ◽  
...  
Keyword(s):  
Phase Separation ◽  
Mammalian Cells ◽  
Kinase Inhibitor ◽  
Viral Rna ◽  
Host Cells ◽  
Drug Candidate ◽  
N Protein ◽  
Intrinsically Disordered

AbstractThe severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection causes COVID-19, a pandemic that seriously threatens global health. SARS CoV-2 propagates by packaging its RNA genome into membrane enclosures in host cells. The packaging of the viral genome into the nascent virion is mediated by the nucleocapsid (N) protein, but the underlying mechanism remains unclear. Here, we show that the N protein forms biomolecular condensates with viral RNA both in vitro and in mammalian cells. While the N protein forms spherical assemblies with unstructured RNA, it forms mesh like-structures with viral RNA strands that contain secondary structure elements. Cross-linking mass spectrometry identified an intrinsically-disordered region that forms interactions between N proteins in condensates, and truncation of this region disrupts phase separation. By screening 1,200 FDA approved drugs in vitro, we identified a kinase inhibitor nilotinib, which affects the morphology of N condensates in vitro and disrupts phase separation of the N protein in vivo. These results indicate that the N protein compartmentalizes viral RNA in infected cells through liquid-liquid phase separation, and this process can be disrupted by a possible drug candidate.

Association/dissociation Mechanisms of Intrinsically Disordered Region of Protein Beyond Conformational Selection and Induced Fit

2019 ◽  
Author(s):  
Duy Phuoc Tran ◽  
Akio Kitao
Keyword(s):  
Free Energy ◽  
Transcriptional Activation ◽  
Binding Free Energy ◽  
Energy Structure ◽  
Induced Fit ◽  
Hydrogen Bond Formation ◽  
Conformational Selection ◽  
Intrinsically Disordered ◽  
Intrinsically Disordered Region ◽  
Dissociation Mechanisms

<p>We investigate association and dissociation mechanisms of a typical intrinsically disordered region (IDR), transcriptional activation subdomain of tumor repressor protein p53 (TAD-p53) with murine double-minute clone 2 protein (MDM2). Using the combination of cycles of association and dissociation parallel cascade molecular dynamics, multiple standard MD, and Markov state model, we are successful in obtaining the lowest free energy structure of MDM2/TAD-p53 complex as the structure very close to that in crystal without prior knowledge. This method also reproduces the experimentally measured standard binding free energy, and association and dissociation rate constants solely with the accumulated MD simulation cost of 11.675 μs, in spite of the fact that actual dissociation occurs in the order of a second. Although there exist a few complex intermediates with similar free energies, TAD-p53 first binds MDM2 as the second lowest free energy intermediate dominantly (> 90% in flux), taking a form similar to one of the intermediate structures in its monomeric state. The mechanism of this step has a feature of conformational selection. In the second step, dehydration of the interface, formation of π-π stackings of the side-chains, and main-chain relaxation/hydrogen bond formation to complete α-helix take place, showing features of induced fit. In addition, dehydration (dewetting) is a key process for the final relaxation around the complex interface. These results demonstrate a more fine-grained view of the IDR association/dissociation beyond classical views of protein conformational change upon binding.</p>

scholarly journals Does liquid–liquid phase separation drive peptide folding?

2021 ◽  
Author(s):  
Dean N. Edun ◽  
Meredith R. Flanagan ◽  
Arnaldo L. Serrano
Keyword(s):  
Infrared Spectroscopy ◽  
Phase Separation ◽  
Liquid Phase ◽  
Model Membrane ◽  
Peptide Folding ◽  
Two Dimensional ◽  
Intrinsically Disordered ◽  
Intrinsically Disordered Peptide ◽  
Two Dimensional Infrared Spectroscopy ◽  
Liquid Liquid Phase Separation

Two-dimensional infrared spectroscopy reveals folding of an intrinsically disordered peptide when sequestered into a model “membrane-less” organelle.

scholarly journals G-quadruplex DNA inhibits unwinding activity but promotes liquid–liquid phase separation by the DEAD-box helicase Ded1p

2021 ◽  
Author(s):  
Jun Gao ◽  
Zhaofeng Gao ◽  
Andrea A. Putnam ◽  
Alicia K. Byrd ◽  
Sarah L. Venus ◽  
...  
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Liquid Phase Separation ◽  
Intrinsically Disordered ◽  
Dead Box ◽  
G4 Dna ◽  
G Quadruplex ◽  
The Dead ◽  
Liquid Liquid Phase Separation

G-quadruplex (G4) DNA inhibits RNA unwinding activity but promotes liquid–liquid phase separation of the DEAD-box helicase Ded1p in vitro and in cells. This highlights multifaceted effects of G4DNA on an enzyme with intrinsically disordered domains.

scholarly journals The SARS-CoV-2 nucleocapsid phosphoprotein forms mutually exclusive condensates with RNA and the membrane-associated M protein

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Shan Lu ◽  
Qiaozhen Ye ◽  
Digvijay Singh ◽  
Yong Cao ◽  
Jolene K. Diedrich ◽  
...  
Keyword(s):  
Phase Separation ◽  
Potential Role ◽  
Rna Binding ◽  
Stress Granule ◽  
M Protein ◽  
Rich Region ◽  
Virion Assembly ◽  
N Protein ◽  
Intrinsically Disordered ◽  
Intrinsically Disordered Regions

AbstractThe multifunctional nucleocapsid (N) protein in SARS-CoV-2 binds the ~30 kb viral RNA genome to aid its packaging into the 80–90 nm membrane-enveloped virion. The N protein is composed of N-terminal RNA-binding and C-terminal dimerization domains that are flanked by three intrinsically disordered regions. Here we demonstrate that the N protein’s central disordered domain drives phase separation with RNA, and that phosphorylation of an adjacent serine/arginine rich region modulates the physical properties of the resulting condensates. In cells, N forms condensates that recruit the stress granule protein G3BP1, highlighting a potential role for N in G3BP1 sequestration and stress granule inhibition. The SARS-CoV-2 membrane (M) protein independently induces N protein phase separation, and three-component mixtures of N + M + RNA form condensates with mutually exclusive compartments containing N + M or N + RNA, including annular structures in which the M protein coats the outside of an N + RNA condensate. These findings support a model in which phase separation of the SARS-CoV-2 N protein contributes both to suppression of the G3BP1-dependent host immune response and to packaging genomic RNA during virion assembly.

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