scholarly journals Clr4SUV39H1 and Bdf2BRD4 ubiquitination mediate transcriptional silencing via heterochromatic phase transitions

2021 ◽  
Author(s):  
Hyun-Soo Kim ◽  
Benjamin Roche ◽  
Sonali Bhattacharjee ◽  
Leila Todeschini ◽  
An-Yun Chang ◽  
...  
Keyword(s):  
Small Rnas ◽  
Genome Stability ◽  
Transcriptional Silencing ◽  
Centromeric Heterochromatin ◽  
Deubiquitinating Enzyme ◽  
Dynamic Activity ◽  
Intrinsically Disordered ◽  
Ubiquitin Conjugating Enzyme ◽  
Liquid Liquid Phase Separation

Heterochromatin has crucial roles in genome stability by silencing repetitive DNA and by suppressing recombination. In the fission yeast Schizosaccharomyces pombe, Clr4 methylates histone H3 lysine 9 (H3K9) to maintain centromeric heterochromatin during the cell cycle, guided by RNA interference (RNAi). Clr4 interacts with a specialized ubiquitination complex CLRC, which is recruited in part by small RNA and the RNAi transcriptional silencing complex (RITS) but otherwise, the role of CLRC is unknown. Here we show that the E2 ubiquitin conjugating enzyme Ubc4 interacts with CLRC and targets Clr4 for mono-ubiquitination at an intrinsically disordered region. Mono-ubiquitination is essential for the transition from co-transcriptional (H3K9me2) to transcriptional (H3K9me3) gene silencing by transiently releasing Clr4 from centromeric heterochromatin, and is reversed by the deubiquitinating enzyme Ubp3. Addition of ubiquitin changes the liquid-liquid phase separation (LLPS) of Clr4 to enhance its dynamic activity. Ubc4-CLRC also poly-ubiquitinates Bdf2, the homologue of BET family double bromodomain protein BRD4 for degradation, which otherwise accumulates at centromeres to promote transcription and production of small RNAs.

Protein phosphatase-1: dual activity regulation by Inhibitor-2

2020 ◽  
Vol 48 (5) ◽  
pp. 2229-2240
Author(s):  
Sarah Lemaire ◽  
Mathieu Bollen
Keyword(s):  
Protein Phosphatase ◽  
Indirect Evidence ◽  
Intrinsically Disordered Protein ◽  
Competitive Inhibitor ◽  
Protein Phosphatase 1 ◽  
Published Data ◽  
Dynamic Activity ◽  
Cellular Processes ◽  
Intrinsically Disordered

Inhibitor-2 (I2) ranks amongst the most ancient regulators of protein phosphatase-1 (PP1). It is a small, intrinsically disordered protein that was originally discovered as a potent inhibitor of PP1. However, later investigations also characterized I2 as an activator of PP1 as well as a chaperone for PP1 folding. Numerous studies disclosed the importance of I2 for diverse cellular processes but did not describe a unifying molecular principle of PP1 regulation. We have re-analyzed the literature on I2 in the light of current insights of PP1 structure and regulation. Extensive biochemical data, largely ignored in the recent I2 literature, provide substantial indirect evidence for a role of I2 as a loader of active-site metals. In addition, I2 appears to function as a competitive inhibitor of PP1 in higher eukaryotes. The published data also demonstrate that several segments of I2 that remain unstructured in the PP1 : I2 complex are in fact essential for PP1 regulation. Together, the available data identify I2 as a dynamic activity-modulator of PP1.

Quadruplex Folding of DNA Promotes the Condensation of Linker Histones via Liquid-Liquid Phase Separation

2020 ◽  
Author(s):  
Masahiro Mimura ◽  
Shunsuke Tomita ◽  
Yoichi Shinkai ◽  
Kentaro Shiraki ◽  
Ryoji Kurita
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Electrostatic Interactions ◽  
Droplet Formation ◽  
Liquid Phase Separation ◽  
Intrinsically Disordered ◽  
G Quadruplex ◽  
Dna Quadruplex ◽  
Liquid Liquid Phase Separation

<p>Liquid-liquid phase separation (LLPS) of proteins and DNA has recently emerged as a possible mechanism underlying the dynamic organization of chromatin. We herein report the role of DNA quadruplex folding in liquid droplet formation via LLPS induced by interactions between DNA and linker histone H1 (H1), a key regulator of chromatin organization. Fluidity measurements inside the droplets and binding assays using G-quadruplex-selective probes demonstrated that quadruplex DNA structures, such as the G-quadruplex and i-motif, promote droplet formation with H1 and decrease molecular motility within droplets. The dissolution of the droplets in the presence of additives indicated that in addition to electrostatic interactions between the DNA and the intrinsically disordered region of H1, π-π stacking between quadruplex DNAs could potentially drive droplet formation. Given that DNA quadruplex structures are well documented in heterochromatin regions, it is imperative to understand the role of DNA quadruplex folding in the context of intranuclear LLPS.<b></b></p>

scholarly journals Quadruplex Folding of DNA Promotes the Condensation of Linker Histones via Liquid-Liquid Phase Separation

2020 ◽  
Author(s):  
Masahiro Mimura ◽  
Shunsuke Tomita ◽  
Yoichi Shinkai ◽  
Kentaro Shiraki ◽  
Ryoji Kurita
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Electrostatic Interactions ◽  
Droplet Formation ◽  
Liquid Phase Separation ◽  
Intrinsically Disordered ◽  
G Quadruplex ◽  
Dna Quadruplex ◽  
Liquid Liquid Phase Separation

<p>Liquid-liquid phase separation (LLPS) of proteins and DNA has recently emerged as a possible mechanism underlying the dynamic organization of chromatin. We herein report the role of DNA quadruplex folding in liquid droplet formation via LLPS induced by interactions between DNA and linker histone H1 (H1), a key regulator of chromatin organization. Fluidity measurements inside the droplets and binding assays using G-quadruplex-selective probes demonstrated that quadruplex DNA structures, such as the G-quadruplex and i-motif, promote droplet formation with H1 and decrease molecular motility within droplets. The dissolution of the droplets in the presence of additives indicated that in addition to electrostatic interactions between the DNA and the intrinsically disordered region of H1, π-π stacking between quadruplex DNAs could potentially drive droplet formation. Given that DNA quadruplex structures are well documented in heterochromatin regions, it is imperative to understand the role of DNA quadruplex folding in the context of intranuclear LLPS.<b></b></p>

scholarly journals ATP regulates TDP-43 pathogenesis by specifically binding to an inhibitory component of a delicate network controlling LLPS

2020 ◽  
Author(s):  
Dang Mei ◽  
Liangzhong Lim ◽  
Jian Kang ◽  
Jianxing Song
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Neuron Death ◽  
Physiological Conditions ◽  
Intrinsically Disordered ◽  
Physicochemical Features ◽  
First Time ◽  
Inhibitory Interactions ◽  
Liquid Liquid Phase Separation

ABSTRACTVery recently, liquid-liquid phase separation (LLPS) of cytoplasmic TDP-43 independent of forming stress granule (SG) has been decoded to initiate the neuron death for ALS. Mysteriously neurons maintain ATP concentrations of ∼3 mM, but whether ATP modulates TDP-43 LLPS remains completely unknown. Here we characterized the effects of ATP on LLPS of TDP-43 PLD and its six mutants by DIC and NMR. For the first time, the results revealed: 1) ATP does induce and subsequently dissolve LLPS of TDP-43 PLD. 2) ATP achieves the modulation all by specifically binding Arg mostly saturated at 1:100. 3) LLPS of TDP-43 PLD and its exaggeration into aggregation are controlled by a delicate network composed of both attractive and inhibitory interactions, thus rationalizing the susceptibility of TDP-43 PLD to various ALS-causing mutations. Our studies together establish that ATP specifically binds Arg in intrinsically-disordered domains even not RGG-/R-rich, implying that ATP might be a universal regulator for most, if not all, R-containing intrinsically-disordered domains by altering their physicochemical features, conformations, dynamics, LLPS and assembly. Under physiological conditions, TDP-43 even in neuronal cytoplasm is highly bound with ATP and thus inhibited for its toxic LLPS, highlighting a central role of ATP in TDP-43 pathogenesis and aging.

Quadruplex Folding of DNA Promotes the Condensation of Linker Histones via Liquid-Liquid Phase Separation

2020 ◽  
Author(s):  
Masahiro Mimura ◽  
Shunsuke Tomita ◽  
Yoichi Shinkai ◽  
Kentaro Shiraki ◽  
Ryoji Kurita
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Electrostatic Interactions ◽  
Droplet Formation ◽  
Liquid Phase Separation ◽  
Intrinsically Disordered ◽  
G Quadruplex ◽  
Dna Quadruplex ◽  
Liquid Liquid Phase Separation

<p>Liquid-liquid phase separation (LLPS) of proteins and DNA has recently emerged as a possible mechanism underlying the dynamic organization of chromatin. We herein report the role of DNA quadruplex folding in liquid droplet formation via LLPS induced by interactions between DNA and linker histone H1 (H1), a key regulator of chromatin organization. Fluidity measurements inside the droplets and binding assays using G-quadruplex-selective probes demonstrated that quadruplex DNA structures, such as the G-quadruplex and i-motif, promote droplet formation with H1 and decrease molecular motility within droplets. The dissolution of the droplets in the presence of additives indicated that in addition to electrostatic interactions between the DNA and the intrinsically disordered region of H1, π-π stacking between quadruplex DNAs could potentially drive droplet formation. Given that DNA quadruplex structures are well documented in heterochromatin regions, it is imperative to understand the role of DNA quadruplex folding in the context of intranuclear LLPS.<b></b></p>

scholarly journals Separable roles for RNAi in regulation of transposable elements and viability in the fission yeast Schizosaccharomyces japonicus

2022 ◽  
Author(s):  
Elliott Chapman ◽  
Francesca Taglini ◽  
Elizabeth H Bayne
Keyword(s):  
Transposable Elements ◽  
Fission Yeast ◽  
Transcriptional Repression ◽  
Small Rnas ◽  
Transcriptional Silencing ◽  
Transcriptional Level ◽  
Genome Maintenance ◽  
Repeat Sequences ◽  
Heterochromatin Assembly

RNA interference (RNAi) is a conserved mechanism of small RNA-mediated genome regulation commonly involved in suppression of transposable elements (TEs) through both post-transcriptional silencing, and transcriptional repression via heterochromatin assembly. The fission yeast Schizosaccharomyces pombe has been extensively utilised as a model for studying RNAi pathways. However, this species is somewhat atypical in that TEs are not major targets of RNAi, and instead small RNAs correspond primarily to non-coding pericentromeric repeat sequences, reflecting a specialised role for the pathway in promoting heterochromatin assembly in these regions. In contrast, in the related fission yeast Schizosaccharomyces japonicus, sequenced small RNAs correspond primarily to TEs. This suggests there may be fundamental differences in the operation of RNAi pathways in these two related species. To investigate these differences, we probed RNAi function in S. japonicus. Unexpectedly, and in contrast to S. pombe, we found that RNAi is essential in this species. Moreover, viability of RNAi mutants can be rescued by mutations implicated in enhanching RNAi-independent heterochromatin propagation. These rescued strains retain heterochromatic marks on TE sequences, but exhibit derepression of TEs at the post-transcriptional level. Our findings indicate that S. japonicus retains the ancestral role of RNAi in facilitating suppression of TEs via both post-transcriptional silencing and heterochromatin assembly, with specifically the heterochromatin pathway being essential for viability, likely due to a function in genome maintenance. The specialised role of RNAi in heterochromatin assembly in S. pombe appears to be a derived state that emerged after the divergence of S. japonicus.

scholarly journals Quadruplex folding promotes the condensation of linker histones and DNAs via liquid-liquid phase separation

2021 ◽  
Author(s):  
Masahiro Mimura ◽  
Shunsuke Tomita ◽  
Yoichi Shinkai ◽  
Takuya Hosokai ◽  
Hiroyuki Kumeta ◽  
...  
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Electrostatic Interactions ◽  
Droplet Formation ◽  
Liquid Phase Separation ◽  
Intrinsically Disordered ◽  
G Quadruplex ◽  
Dna Quadruplex ◽  
Liquid Liquid Phase Separation

Liquid-liquid phase separation (LLPS) of proteins and DNA has recently emerged as a possible mechanism underlying the dynamic organization of chromatin. We herein report the role of DNA quadruplex folding in liquid droplet formation via LLPS induced by interactions between DNA and linker histone H1 (H1), a key regulator of chromatin organization. <a>Fluidity measurements inside the droplets, binding assays using G-quadruplex-selective probes, and structural analyses based on circular dichroism demonstrated that quadruplex DNA structures, such as the G-quadruplex and i-motif, promote droplet formation with H1 and decrease molecular motility within droplets. </a><a></a><a></a><a>The dissolution of the droplets in the presence of additives and the LLPS of the DNA structural units indicated that in addition to electrostatic interactions between the DNA and the intrinsically disordered region of H1, π-π stacking between quadruplex DNAs could potentially drive droplet formation, unlike in the electrostatically driven LLPS of duplex DNA and H1. According to phase diagrams of anionic molecules with various conformations, the high LLPS ability associated with quadruplex folding arises from the formation of interfaces consisting of organized planes of guanine bases and the side surfaces with high charge density. </a>Given that DNA quadruplex structures are well documented in heterochromatin regions, it is imperative to understand the role of DNA quadruplex folding in the context of intranuclear LLPS.<br>

scholarly journals Biological Functions of the Intrinsically Disordered N-Terminal Domain of the Prion Protein: A Possible Role of Liquid–Liquid Phase Separation

Biomolecules ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 1201
Author(s):  
Stella A. Polido ◽  
Janine Kamps ◽  
Jörg Tatzelt
Keyword(s):  
Prion Protein ◽  
Tertiary Structure ◽  
Polypeptide Chain ◽  
Protein A ◽  
Interacting Proteins ◽  
Intrinsically Disordered ◽  
Terminal Domain ◽  
Protein Classes ◽  
Liquid Liquid Phase Separation

The mammalian prion protein (PrPC) is composed of a large intrinsically disordered N-terminal and a structured C-terminal domain, containing three alpha-helical regions and a short, two-stranded beta-sheet. Traditionally, the activity of a protein was linked to the ability of the polypeptide chain to adopt a stable secondary/tertiary structure. This concept has been extended when it became evident that intrinsically disordered domains (IDDs) can participate in a broad range of defined physiological activities and play a major functional role in several protein classes including transcription factors, scaffold proteins, and signaling molecules. This ability of IDDs to engage in a variety of supramolecular complexes may explain the large number of PrPC-interacting proteins described. Here, we summarize diverse physiological and pathophysiological activities that have been described for the unstructured N-terminal domain of PrPC. In particular, we focus on subdomains that have been conserved in evolution.

Dri1 mediates heterochromatin assembly via RNAi and histone deacetylation

Genetics ◽  
2021 ◽  
Author(s):  
Hyoju Ban ◽  
Wenqi Sun ◽  
Yu-hang Chen ◽  
Yong Chen ◽  
Fei Li
Keyword(s):  
Histone Deacetylases ◽  
Genome Stability ◽  
Rna Binding ◽  
Histone Deacetylation ◽  
Rnai Pathway ◽  
Chromatin Domain ◽  
Heterochromatin Protein 1 ◽  
Intrinsically Disordered ◽  
C Terminus ◽  
Heterochromatin Assembly

Abstract Heterochromatin, a transcriptionally silenced chromatin domain, is important for genome stability and gene expression. Histone 3 lysine 9 methylation (H3K9me) and histone hypoacetylation are conserved epigenetic hallmarks of heterochromatin. In fission yeast, RNA interference (RNAi) plays a key role in H3K9 methylation and heterochromatin silencing. However, how RNAi machinery and histone deacetylases (HDACs) are coordinated to ensure proper heterochromatin assembly is still unclear. Previously, we showed that Dpb4, a conserved DNA polymerase epsilon subunit, plays a key role in the recruitment of HDACs to heterochromatin during S phase. Here, we identified a novel RNA-binding protein Dri1 that interacts with Dpb4. GFP-tagged Dri1 forms distinct foci mostly in the nucleus, showing a high degree of colocalization with Swi6/Heterochromatin Protein 1. Deletion of dri1+ leads to defects in silencing, H3K9me, and heterochromatic siRNA generation. We also showed that Dri1 physically associates with heterochromatic transcripts, and is required for the recruitment of the RNA-induced transcriptional silencing (RITS) complex via interacting with the complex. Furthermore, loss of Dri1 decreases the association of the Sir2 HDAC with heterochromatin. We further demonstrated that the C-terminus of Dri1 that includes an intrinsically disordered (IDR) region and three zinc fingers is crucial for its role in silencing. Together, our evidences suggest that Dri1 facilitates heterochromatin assembly via the RNAi pathway and HDAC.

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