In Vivo Dissection of the Intrinsically Disordered Receptor Domain of Tim23

Background: Gene expression in eukaryotic cells can be governed by histone variants, which replace replication-coupled histones, conferring unique chromatin properties. MacroH2A1 is a histone H2A variant containing a domain highly similar to H2A and a large non-histone (macro) domain. MacroH2A1, in turn, is present in two alternatively exon-spliced isoforms: macroH2A1.1 and macroH2A1.2, which regulate cell plasticity and proliferation in a remarkably distinct manner. The N-terminal and the C-terminal tails of H2A histones stem from the nucleosome core structure and can be target sites for several post-translational modifications (PTMs). MacroH2A1.1 and macroH2A1.2 isoforms differ only in a few amino acids and their ability to bind NAD-derived metabolites, a property allegedly conferring their different functions in vivo. Some of the modifications on the macroH2A1 variant have been identified, such as phosphorylation (T129, S138) and methylation (K18, K123, K239). However, no study to our knowledge has analyzed extensively, and in parallel, the PTM pattern of macroH2A1.1 and macroH2A1.2 in the same experimental setting, which could facilitate the understanding of their distinct biological functions in health and disease. Methods: We used a mass spectrometry-based approach to identify the sites for phosphorylation, acetylation, and methylation in green fluorescent protein (GFP)-tagged macroH2A1.1 and macroH2A1.2 expressed in human hepatoma cells. The impact of selected PTMs on macroH2A1.1 and macroH2A1.2 structure and function are demonstrated using computational analyses. Results: We identified K7 as a new acetylation site in both macroH2A1 isoforms. Quantitative comparison of histone marks between the two isoforms revealed significant differences in the levels of phosphorylated T129 and S170. Our computational analysis provided evidence that the phosphorylation status in the intrinsically disordered linker region in macroH2A1 isoforms might represent a key regulatory element contributing to their distinct biological responses. Conclusions: Taken together, our results report different PTMs on the two macroH2A1 splicing isoforms as responsible for their distinct features and distribution in the cell.

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The remarkable diversity of plant PEPC (phosphoenolpyruvate carboxylase): recent insights into the physiological functions and post-translational controls of non-photosynthetic PEPCs

Biochemical Journal ◽

10.1042/bj20110078 ◽

2011 ◽

Vol 436 (1) ◽

pp. 15-34 ◽

Cited By ~ 167

Author(s):

Brendan O'Leary ◽

Joonho Park ◽

William C. Plaxton

Keyword(s):

Phosphoenolpyruvate Carboxylase ◽

Tricarboxylic Acid Cycle ◽

Critical Role ◽

Subcellular Location ◽

Serine Phosphorylation ◽

Regulatory Subunit ◽

Physiological Functions ◽

Intrinsically Disordered ◽

Class 1

PEPC [PEP (phosphoenolpyruvate) carboxylase] is a tightly controlled enzyme located at the core of plant C-metabolism that catalyses the irreversible β-carboxylation of PEP to form oxaloacetate and Pi. The critical role of PEPC in assimilating atmospheric CO2 during C4 and Crassulacean acid metabolism photosynthesis has been studied extensively. PEPC also fulfils a broad spectrum of non-photosynthetic functions, particularly the anaplerotic replenishment of tricarboxylic acid cycle intermediates consumed during biosynthesis and nitrogen assimilation. An impressive array of strategies has evolved to co-ordinate in vivo PEPC activity with cellular demands for C4–C6 carboxylic acids. To achieve its diverse roles and complex regulation, PEPC belongs to a small multigene family encoding several closely related PTPCs (plant-type PEPCs), along with a distantly related BTPC (bacterial-type PEPC). PTPC genes encode ~110-kDa polypeptides containing conserved serine-phosphorylation and lysine-mono-ubiquitination sites, and typically exist as homotetrameric Class-1 PEPCs. In contrast, BTPC genes encode larger ~117-kDa polypeptides owing to a unique intrinsically disordered domain that mediates BTPC's tight interaction with co-expressed PTPC subunits. This association results in the formation of unusual ~900-kDa Class-2 PEPC hetero-octameric complexes that are desensitized to allosteric effectors. BTPC is a catalytic and regulatory subunit of Class-2 PEPC that is subject to multi-site regulatory phosphorylation in vivo. The interaction between divergent PEPC polypeptides within Class-2 PEPCs adds another layer of complexity to the evolution, physiological functions and metabolic control of this essential CO2-fixing plant enzyme. The present review summarizes exciting developments concerning the functions, post-translational controls and subcellular location of plant PTPC and BTPC isoenzymes.

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Entropy Hotspots for the Binding of Intrinsically Disordered Ligands to a Receptor Domain

Biophysical Journal ◽

10.1016/j.bpj.2020.03.026 ◽

2020 ◽

Vol 118 (10) ◽

pp. 2502-2512

Author(s):

Jie Shi ◽

Qingliang Shen ◽

Jae-Hyun Cho ◽

Wonmuk Hwang

Keyword(s):

Intrinsically Disordered ◽

Receptor Domain

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Nuclear condensates of the Polycomb protein chromobox 2 (CBX2) assemble through phase separation

Journal of Biological Chemistry ◽

10.1074/jbc.ra118.006620 ◽

2018 ◽

Vol 294 (5) ◽

pp. 1451-1463 ◽

Cited By ~ 92

Author(s):

Roubina Tatavosian ◽

Samantha Kent ◽

Kyle Brown ◽

Tingting Yao ◽

Huy Nguyen Duc ◽

...

Keyword(s):

Phase Separation ◽

Polycomb Group ◽

Site Directed Mutagenesis ◽

Developmental Defects ◽

Heterochromatin Formation ◽

Intrinsically Disordered ◽

Polycomb Protein ◽

Condensate Formation

Polycomb group (PcG) proteins repress master regulators of development and differentiation through organization of chromatin structure. Mutation and dysregulation of PcG genes cause developmental defects and cancer. PcG proteins form condensates in the cell nucleus, and these condensates are the physical sites of PcG-targeted gene silencing via formation of facultative heterochromatin. However, the physiochemical principles underlying the formation of PcG condensates remain unknown, and their determination could shed light on how these condensates compact chromatin. Using fluorescence live-cell imaging, we observed that the Polycomb repressive complex 1 (PRC1) protein chromobox 2 (CBX2), a member of the CBX protein family, undergoes phase separation to form condensates and that the CBX2 condensates exhibit liquid-like properties. Using site-directed mutagenesis, we demonstrated that the conserved residues of CBX2 within the intrinsically disordered region (IDR), which is the region for compaction of chromatin in vitro, promote the condensate formation both in vitro and in vivo. We showed that the CBX2 condensates concentrate DNA and nucleosomes. Using genetic engineering, we report that trimethylation of Lys-27 at histone H3 (H3K27me3), a marker of heterochromatin formation produced by PRC2, had minimal effects on the CBX2 condensate formation. We further demonstrated that the CBX2 condensate formation does not require CBX2–PRC1 subunits; however, the condensate formation of CBX2–PRC1 subunits depends on CBX2, suggesting a mechanism underlying the assembly of CBX2–PRC1 condensates. In summary, our results reveal that PcG condensates assemble through liquid–liquid phase separation (LLPS) and suggest that phase-separated condensates can organize PcG-bound chromatin.

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Simple biophysics underpins collective conformations of the intrinsically disordered proteins of the Nuclear Pore Complex

eLife ◽

10.7554/elife.10785 ◽

2016 ◽

Vol 5 ◽

Cited By ~ 44

Author(s):

Andrei Vovk ◽

Chad Gu ◽

Michael G Opferman ◽

Larisa E Kapinos ◽

Roderick YH Lim ◽

...

Keyword(s):

Spatial Organization ◽

Intrinsically Disordered Proteins ◽

Nucleocytoplasmic Transport ◽

Transport Proteins ◽

Disordered Proteins ◽

Biophysical Model ◽

Nuclear Pore ◽

Intrinsically Disordered

Nuclear Pore Complexes (NPCs) are key cellular transporter that control nucleocytoplasmic transport in eukaryotic cells, but its transport mechanism is still not understood. The centerpiece of NPC transport is the assembly of intrinsically disordered polypeptides, known as FG nucleoporins, lining its passageway. Their conformations and collective dynamics during transport are difficult to assess in vivo. In vitro investigations provide partially conflicting results, lending support to different models of transport, which invoke various conformational transitions of the FG nucleoporins induced by the cargo-carrying transport proteins. We show that the spatial organization of FG nucleoporin assemblies with the transport proteins can be understood within a first principles biophysical model with a minimal number of key physical variables, such as the average protein interaction strengths and spatial densities. These results address some of the outstanding controversies and suggest how molecularly divergent NPCs in different species can perform essentially the same function.

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The arrested state of processing bodies supports mRNA regulation in early development

10.1101/2021.03.16.435709 ◽

2021 ◽

Cited By ~ 1

Author(s):

M. Sankaranarayanan ◽

Ryan J. Emenecker ◽

Marcus Jahnel ◽

Irmela R. E. A. Trussina ◽

Matt Wayland ◽

...

Keyword(s):

Physical Properties ◽

Electrostatic Interactions ◽

Processing Bodies ◽

P Bodies ◽

Intrinsically Disordered ◽

Intrinsically Disordered Regions ◽

Dead Box ◽

Premature Release ◽

Liquid Liquid Phase Separation

ABSTRACTBiomolecular condensates that form via liquid-liquid phase separation can exhibit diverse physical states. Despite considerable progress, the relevance of condensate physical states forin vivobiological function remains limited. Here, we investigated the physical properties ofin vivoprocessing bodies (P bodies) and their impact on mRNA storage in matureDrosophilaoocytes. We show that the conserved DEAD-box RNA helicase Me31B forms P body condensates which adopt a less dynamic, arrested physical state. We demonstrate that structurally distinct proteins and hydrophobic and electrostatic interactions, together with RNA and intrinsically disordered regions, regulate the physical properties of P bodies. Finally, using live imaging, we show that the arrested state of P bodies is required to prevent the premature release ofbicoid(bcd) mRNA, a body axis determinant, and that P body dissolution leads tobcdrelease. Together, this work establishes a role for arrested states of biomolecular condensates in regulating cellular function in a developing organism.

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Tuning levels of low-complexity domain interactions to modulate endogenous oncogenic transcription

10.1101/2021.08.16.456551 ◽

2021 ◽

Author(s):

Shasha Chong ◽

Thomas G. W. Graham ◽

Claire Dugast-Darzacq ◽

Gina M. Dailey ◽

Xavier Darzacq ◽

...

Keyword(s):

Single Molecule ◽

Target Genes ◽

Gene Activation ◽

Low Complexity ◽

Intrinsically Disordered ◽

Human Genes ◽

Domain Interactions ◽

Bona Fide ◽

Liquid Liquid Phase Separation

Gene activation by mammalian transcription factors (TFs) requires dynamic, multivalent, and selective interactions of their intrinsically disordered low-complexity domains (LCDs), but how such interactions mediate transcription remains unclear. It has been proposed that extensive LCD-LCD interactions culminating in liquid-liquid phase separation (LLPS) of TFs is the dominant mechanism underlying transactivation. Here, we investigated how tuning the amount and localization of LCD-LCD interactions in vivo affects transcription of endogenous human genes. Quantitative single-cell and single-molecule imaging reveals that the oncogenic TF EWS/FLI1 requires a finely tuned range of LCD-LCD interactions to efficiently activate target genes. Modest or more dramatic increases in LCD-LCD interactions toward putative LLPS repress EWS/FLI1-driven transcription in patient cells. Likewise, ectopically creating LCD-LCD interactions to sequester EWS/FLI1 into a bona fide LLPS compartment, the nucleolus, inhibits EWS/FLI1-driven transcription and oncogenic transformation. Our findings reveal fundamental principles underlying LCD-mediated transcription and suggest mislocalizing specific LCD-LCD interactions as a novel therapeutic strategy for targeting disease-causing TFs.

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Transcription factor clusters regulate genes in eukaryotic cells

eLife ◽

10.7554/elife.27451 ◽

2017 ◽

Vol 6 ◽

Cited By ~ 38

Author(s):

Adam JM Wollman ◽

Sviatlana Shashkova ◽

Erik G Hedlund ◽

Rosmarie Friemann ◽

Stefan Hohmann ◽

...

Keyword(s):

Single Molecule ◽

Functional Unit ◽

Yeast Genome ◽

Gene Promoters ◽

Intrinsically Disordered ◽

Extracellular Signal ◽

Genome Model ◽

Nuclear Targets

Transcription is regulated through binding factors to gene promoters to activate or repress expression, however, the mechanisms by which factors find targets remain unclear. Using single-molecule fluorescence microscopy, we determined in vivo stoichiometry and spatiotemporal dynamics of a GFP tagged repressor, Mig1, from a paradigm signaling pathway of Saccharomyces cerevisiae. We find the repressor operates in clusters, which upon extracellular signal detection, translocate from the cytoplasm, bind to nuclear targets and turnover. Simulations of Mig1 configuration within a 3D yeast genome model combined with a promoter-specific, fluorescent translation reporter confirmed clusters are the functional unit of gene regulation. In vitro and structural analysis on reconstituted Mig1 suggests that clusters are stabilized by depletion forces between intrinsically disordered sequences. We observed similar clusters of a co-regulatory activator from a different pathway, supporting a generalized cluster model for transcription factors that reduces promoter search times through intersegment transfer while stabilizing gene expression.

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