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.

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

2020 ◽  
Author(s):  
Shan Lu ◽  
Qiaozhen Ye ◽  
Digvijay Singh ◽  
Elizabeth Villa ◽  
Don W. Cleveland ◽  
...  
Keyword(s):  
Phase Separation ◽  
Rna Binding ◽  
M Protein ◽  
Rna Packaging ◽  
Dimerization Domain ◽  
Virion Assembly ◽  
N Protein ◽  
Intrinsically Disordered ◽  
Intrinsically Disordered Regions ◽  
C Terminus

The 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 a centrally located 40 amino acid intrinsically disordered domain drives phase separation of N protein when bound to RNA, with the morphology of the resulting condensates affected by inclusion in the RNA of the putative SARS-CoV-2 packaging signal. The SARS-CoV-2 M protein, normally embedded in the virion membrane with its C-terminus extending into the virion core, independently induces N protein phase separation that is dependent on the N protein's C-terminal dimerization domain and disordered region. Three-component mixtures of N+M+RNA form condensates with mutually exclusive compartments containing N+M or N+RNA, including spherical 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 N protein with both the viral genomic RNA and the SARS-CoV-2 M protein facilitates RNA packaging and virion assembly.

scholarly journals Rpd3L and Hda1 histone deacetylases facilitate repair of broken forks by promoting sister chromatid cohesion

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Pedro Ortega ◽  
Belén Gómez-González ◽  
Andrés Aguilera
Keyword(s):  
Sister Chromatid ◽  
Histone Deacetylases ◽  
Genome Stability ◽  
Genome Instability ◽  
Sister Chromatid Cohesion ◽  
Histone Deacetylation ◽  
Strand Breaks ◽  
Chromatid Cohesion ◽  
Dna Strands

AbstractGenome stability involves accurate replication and DNA repair. Broken replication forks, such as those encountering a nick, lead to double strand breaks (DSBs), which are preferentially repaired by sister-chromatid recombination (SCR). To decipher the role of chromatin in eukaryotic DSB repair, here we analyze a collection of yeast chromatin-modifying mutants using a previously developed system for the molecular analysis of repair of replication-born DSBs by SCR based on a mini-HO site. We confirm the candidates through FLP-based systems based on a mutated version of the FLP flipase that causes nicks on either the leading or lagging DNA strands. We demonstrate that Rpd3L and Hda1 histone deacetylase (HDAC) complexes contribute to the repair of replication-born DSBs by facilitating cohesin loading, with no effect on other types of homology-dependent repair, thus preventing genome instability. We conclude that histone deacetylation favors general sister chromatid cohesion as a necessary step in SCR.

scholarly journals Localization and organization of protein factors involved in chromosome inheritance in Dictyostelium discoideum

2007 ◽  
Vol 388 (4) ◽  
pp. 355-365 ◽  
Author(s):  
Markus Kaller ◽  
Balint Földesi ◽  
Wolfgang Nellen
Keyword(s):  
Origin Recognition Complex ◽  
Protein Targeting ◽  
Rna Binding ◽  
Binding Activity ◽  
Centromeric Heterochromatin ◽  
Heterochromatin Protein 1 ◽  
Dna And Rna ◽  
C Terminus ◽  
Chromo Shadow Domain

Abstract Heterochromatin protein 1 (HP1) proteins are highly conserved heterochromatin components required for genomic integrity. We have previously shown that the two HP1 isoforms expressed in Dictyostelium, HcpA and HcpB, are mainly localized to (peri-)centromeric heterochromatin and have largely overlapping functions. However, they cause distinct phenotypes when overexpressed. We show here that these isoforms display quantitative differences in dimerization behavior. Dimerization preference, as well as the mutant phenotype in overexpression strains, depends on the C-terminus containing the hinge and chromo shadow domains. Both Hcp proteins are targeted to distinct subnuclear regions by different chromo shadow domain-dependent and -independent mechanisms. In addition, both proteins bind to DNA and RNA in vitro and binding is independent of the chromo shadow domain. Thus, this DNA and/or RNA binding activity may contribute to protein targeting. To further characterize heterochromatin, we cloned the Dictyostelium homolog of the origin recognition complex subunit 2 (OrcB). OrcB localizes to distinct subnuclear foci that were also targeted by HcpA. In addition, it is associated with the centrosome throughout the cell cycle. The results indicate that, similar to Orc2 homologs from other organisms, it is required for different processes in chromosome inheritance.

scholarly journals Polymerase pausing induced by sequence-specific RNA binding protein drives heterochromatin assembly

2017 ◽  
Author(s):  
Jahan-Yar Parsa ◽  
Selim Boudoukha ◽  
Jordan Burke ◽  
Christina Homer ◽  
Hiten D. Madhani
Keyword(s):  
Genome Stability ◽  
Rna Binding ◽  
De Novo ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Heterochromatin Formation ◽  
Repressor Complex ◽  
Polymerase Pausing ◽  
Heterochromatin Assembly ◽  
Dependent Mechanism

Packaging of pericentromeric DNA into heterochromatin is crucial for genome stability, development and health, yet its endogenous triggers remain poorly understood1. A defining feature of pericentromeric heterochromatin is histone H3 lysine 9 methylation (H3K9me)2–4. In S. pombe, transcripts derived from the pericentromeric dg and dh repeat during S phase5–7 promote heterochromatin formation through two pathways: an RNAi-dependent mechanism involving recruitment of the Clr4 H3K9 methyltransferase complex (CLR-C) via the RITS complex8–13, and RNAi-independent mechanism involving an RNAPII-associated RNA-binding protein Seb1, the repressor complex SHREC, and RNA processing activities14–19. We show here that Seb1 promotes long-lived RNAPII pausing. Pause sites associated with sequence-specific Seb1 RNA binding events are significantly enriched in pericentromeric repeat regions and their presence correlates with the heterochromatin-triggering activities of the corresponding dg and dh DNA fragments. Remarkably, globally increasing RNAPII stalling by other means induces the formation of novel large ectopic heterochromatin domains. Such ectopic heterochromatin occurs even in cells lacking functional RITS, demonstrating that RNAPII pausing can be sufficient to trigger de novo heterochromatin independently of RNAi. These results uncover Seb1-mediated polymerase stalling as a new signal for nucleating heterochromatin assembly in repetitive DNA.

scholarly journals Multivalent interactions drive nucleosome binding and efficient chromatin deacetylation by SIRT6

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Wallace H. Liu ◽  
Jie Zheng ◽  
Jessica L. Feldman ◽  
Mark A. Klein ◽  
Vyacheslav I. Kuznetsov ◽  
...  
Keyword(s):  
Histone Deacetylases ◽  
Nucleosome Core ◽  
Intrinsically Disordered ◽  
Nucleosome Core Particles ◽  
Nucleosomal Dna ◽  
C Terminus ◽  
Intrinsic Capacity ◽  
Multivalent Interactions ◽  
H3 Acetylation ◽  
Nucleosome Binding

Abstract The protein deacetylase SIRT6 maintains cellular homeostasis through multiple pathways that include the deacetylation of histone H3 and repression of transcription. Prior work suggests that SIRT6 is associated with chromatin and can substantially reduce global levels of H3 acetylation, but how SIRT6 is able to accomplish this feat is unknown. Here, we describe an exquisitely tight interaction between SIRT6 and nucleosome core particles, in which a 2:1 enzyme:nucleosome complex assembles via asymmetric binding with distinct affinities. While both SIRT6 molecules associate with the acidic patch on the nucleosome, we find that the intrinsically disordered SIRT6 C-terminus promotes binding at the higher affinity site through recognition of nucleosomal DNA. Together, multivalent interactions couple productive binding to efficient deacetylation of histones on endogenous chromatin. Unique among histone deacetylases, SIRT6 possesses the intrinsic capacity to tightly interact with nucleosomes for efficient activity.

scholarly journals Histone Hyperacetylation in Mitosis Prevents Sister Chromatid Separation and Produces Chromosome Segregation Defects

2003 ◽  
Vol 14 (9) ◽  
pp. 3821-3833 ◽  
Author(s):  
Daniela Cimini ◽  
Marta Mattiuzzo ◽  
Liliana Torosantucci ◽  
Francesca Degrassi
Keyword(s):  
Sister Chromatid ◽  
Histone Deacetylases ◽  
Mitotic Checkpoint ◽  
Chromosome Condensation ◽  
Histone Deacetylation ◽  
Centromeric Heterochromatin ◽  
Chromatin Conformation ◽  
Mitotic Chromosomes ◽  
Heterochromatin Protein 1 ◽  
Heterochromatin Protein

Posttranslational modifications of core histones contribute to driving changes in chromatin conformation and compaction. Herein, we investigated the role of histone deacetylation on the mitotic process by inhibiting histone deacetylases shortly before mitosis in human primary fibroblasts. Cells entering mitosis with hyperacetylated histones displayed altered chromatin conformation associated with decreased reactivity to the anti-Ser 10 phospho H3 antibody, increased recruitment of protein phosphatase 1-δ on mitotic chromosomes, and depletion of heterochromatin protein 1 from the centromeric heterochromatin. Inhibition of histone deacetylation before mitosis produced defective chromosome condensation and impaired mitotic progression in living cells, suggesting that improper chromosome condensation may induce mitotic checkpoint activation. In situ hybridization analysis on anaphase cells demonstrated the presence of chromatin bridges, which were caused by persisting cohesion along sister chromatid arms after centromere separation. Thus, the presence of hyperacetylated chromatin during mitosis impairs proper chromosome condensation during the pre-anaphase stages, resulting in poor sister chromatid resolution. Lagging chromosomes consisting of single or paired sisters were also induced by the presence of hyperacetylated histones, indicating that the less constrained centromeric organization associated with heterochromatin protein 1 depletion may promote the attachment of kinetochores to microtubules coming from both poles.

scholarly journals Phosphorylation of the RB C-terminus regulates condensin II release from chromatin

2020 ◽  
pp. jbc.RA120.016511
Author(s):  
Seung J Kim ◽  
James I MacDonald ◽  
Frederick A. Dick
Keyword(s):  
Cell Cycle ◽  
Genome Stability ◽  
Cell Cycle Control ◽  
Cell Receptor ◽  
Receptor Activation ◽  
Biologically Relevant ◽  
C Terminus ◽  
Independent Functions ◽  
Phosphorylation Event ◽  
Rb Phosphorylation

The retinoblastoma tumour suppressor protein (RB) plays an important role in biological processes such as cell cycle control, DNA damage repair, epigenetic regulation, and genome stability. The canonical model of RB regulation is that cyclin-CDKs phosphorylate, and render RB inactive in late G1/S, promoting entry into S phase. Recently, mono-phosphorylated RB species were described to have distinct cell-cycle independent functions, suggesting that a phosphorylation code dictates diversity of RB function. However, a biologically relevant, functional role of RB phosphorylation at non-CDK sites has remained elusive. Here, we investigated S838/T841 dual phosphorylation, its upstream stimulus, and downstream functional output.  We found that mimicking T-cell receptor activation in Jurkat leukemia cells induced sequential activation of downstream kinases including p38 MAPK, and RB S838/T841 phosphorylation.  This signaling pathway disrupts RB and condensin II interaction with chromatin.  Using cells expressing a WT or S838A/T841A mutant RB fragment, we present evidence that deficiency for this phosphorylation event prevents condensin II release from chromatin.

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.

scholarly journals Disorder in a two-domain neuronal Ca2+-binding protein regulates domain stability and dynamics using ligand mimicry

2020 ◽  
Author(s):  
Lasse Staby ◽  
Katherine R. Kemplen ◽  
Amelie Stein ◽  
Michael Ploug ◽  
Jane Clarke ◽  
...  
Keyword(s):  
Intrinsically Disordered Proteins ◽  
Three Dimensional ◽  
Life Time ◽  
Disordered Proteins ◽  
Intrinsically Disordered ◽  
Order And Disorder ◽  
C Terminus ◽  
Close Relationship ◽  
Stability Dynamics ◽  
And Function

Abstract Understanding the interplay between sequence, structure and function of proteins has been complicated in recent years by the discovery of intrinsically disordered proteins (IDPs), which perform biological functions in the absence of a well-defined three-dimensional fold. Disordered protein sequences account for roughly 30% of the human proteome and in many proteins, disordered and ordered domains coexist. However, few studies have assessed how either feature affects the properties of the other. In this study, we examine the role of a disordered tail in the overall properties of the two-domain, calcium-sensing protein neuronal calcium sensor 1 (NCS-1). We show that loss of just six of the 190 residues at the flexible C-terminus is sufficient to severely affect stability, dynamics, and folding behavior of both ordered domains. We identify specific hydrophobic contacts mediated by the disordered tail that may be responsible for stabilizing the distal N-terminal domain. Moreover, sequence analyses indicate the presence of an LSL-motif in the tail that acts as a mimic of native ligands critical to the observed order–disorder communication. Removing the disordered tail leads to a shorter life-time of the ligand-bound complex likely originating from the observed destabilization. This close relationship between order and disorder may have important implications for how investigations into mixed systems are designed and opens up a novel avenue of drug targeting exploiting this type of behavior.

Sign in / Sign up

Export Citation Format

Share Document