Ccq1p and the Condensin Proteins Cut3p and Cut14p Prevent Telomere Entanglements in the Fission Yeast Schizosaccharomyces pombe

ABSTRACT The Schizosaccharomyces pombe telomere-associated protein Ccq1p has previously been shown to participate in telomerase recruitment, heterochromatin formation, and suppression of checkpoint activation. Here we characterize a critical role for Ccq1p in mitotic transit. We show that mitotic cells lacking Ccq1p lose minichromosomes at high frequencies but that conditional knockdown of Ccq1p expression results in telomere bridging within one cell cycle. Elevating Ccq1p expression resolves the telomere entanglements caused by decreased Taz1p activity. Ccq1p affects telomere resolution in the absence of changes in telomere size, indicating a role for Ccq1p that is independent of telomere length regulation. Using affinity purification, we identify the condensin proteins Cut3p and Cut14p as candidate Ccq1p interactors in this activity. Condensin loss-of-function disrupts Ccq1p telomeric localization and normal intertelomere clustering, while condensin overexpression relieves the chromosome segregation defects associated with conditional Ccq1p knockdown. These data suggest that Ccq1p and condensins collaborate to mediate resolution of telomeres in mitosis and regulate intertelomeric clustering during interphase.

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LIA4Encodes a Chromoshadow Domain Protein Required for Genomewide DNA Rearrangements in Tetrahymena thermophila

Eukaryotic Cell ◽

10.1128/ec.00125-14 ◽

2014 ◽

Vol 13 (10) ◽

pp. 1300-1311 ◽

Cited By ~ 7

Author(s):

Scott A. Horrell ◽

Douglas L. Chalker

Keyword(s):

Critical Role ◽

Dna Rearrangement ◽

Rnai Machinery ◽

Content Type ◽

Heterochromatin Formation ◽

H3k27 Methylation ◽

Dna Elimination ◽

Somatic Genome ◽

Subsequent Elimination ◽

Elimination Pathway

ABSTRACTExtensive DNA elimination occurs as part of macronuclear differentiation duringTetrahymenasexual reproduction. The identification of sequences to excise is guided by a specialized RNA interference (RNAi) machinery that targets the methylation of histone H3 lysine 9 (K9) and K27 on chromatin associated with these internal eliminated sequences (IESs). This modified chromatin is reorganized into heterochromatic subnuclear foci, which is a hallmark of their subsequent elimination. Here, we demonstrate that Lia4, a chromoshadow domain-containing protein, is an essential component in this DNA elimination pathway.LIA4knockout (ΔLIA4) lines fail to excise IESs from their developing somatic genome and arrest at a late stage of conjugation. Lia4 acts after RNAi-guided heterochromatin formation, as both H3K9 and H3K27 methylation are established. Nevertheless, withoutLIA4, these cells fail to form the heterochromatic foci associated with DNA rearrangement, and Lia4 accumulates in the foci, indicating that Lia4 plays a key role in their structure. These data indicate a critical role for Lia4 in organizing the nucleus duringTetrahymenamacronuclear differentiation.

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The Catalytic-Dependent and -Independent Roles of Lsd1 and Lsd2 Lysine Demethylases in Heterochromatin Formation in Schizosaccharomyces pombe

Cells ◽

10.3390/cells9040955 ◽

2020 ◽

Vol 9 (4) ◽

pp. 955

Author(s):

Bahjat F. Marayati ◽

James F. Tucker ◽

David A. De La Cerda ◽

Tien-Chi Hou ◽

Rong Chen ◽

...

Keyword(s):

Gene Expression ◽

Gene Silencing ◽

Schizosaccharomyces Pombe ◽

Cell Fate ◽

Critical Role ◽

Heterochromatin Formation ◽

Growth Defects ◽

Histone Modifiers ◽

Demethylase Activity ◽

Organismal Development

In eukaryotes, heterochromatin plays a critical role in organismal development and cell fate acquisition, through regulating gene expression. The evolutionarily conserved lysine-specific demethylases, Lsd1 and Lsd2, remove mono- and dimethylation on histone H3, serving complex roles in gene expression. In the fission yeast Schizosaccharomyces pombe, null mutations of Lsd1 and Lsd2 result in either severe growth defects or inviability, while catalytic inactivation causes minimal defects, indicating that Lsd1 and Lsd2 have essential functions beyond their known demethylase activity. Here, we show that catalytic mutants of Lsd1 or Lsd2 partially assemble functional heterochromatin at centromeres in RNAi-deficient cells, while the C-terminal truncated alleles of Lsd1 or Lsd2 exacerbate heterochromatin formation at all major heterochromatic regions, suggesting that Lsd1 and Lsd2 repress heterochromatic transcripts through mechanisms both dependent on and independent of their catalytic activities. Lsd1 and Lsd2 are also involved in the establishment and maintenance of heterochromatin. At constitutive heterochromatic regions, Lsd1 and Lsd2 regulate one another and cooperate with other histone modifiers, including the class II HDAC Clr3 and the Sirtuin family protein Sir2 for gene silencing, but not with the class I HDAC Clr6. Our findings explore the roles of lysine-specific demethylases in epigenetic gene silencing at heterochromatic regions.

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Importance of Branched-Chain Amino Acid Utilization in Francisella Intracellular Adaptation

Infection and Immunity ◽

10.1128/iai.02579-14 ◽

2014 ◽

Vol 83 (1) ◽

pp. 173-183 ◽

Cited By ~ 23

Author(s):

Gael Gesbert ◽

Elodie Ramond ◽

Fabiola Tros ◽

Julien Dairou ◽

Eric Frapy ◽

...

Keyword(s):

Amino Acid ◽

Critical Role ◽

Cell Types ◽

Host Cells ◽

Metabolic Adaptation ◽

Amino Acid Transporters ◽

Loss Of Function ◽

Content Type ◽

Branched Chain Amino Acid ◽

Branched Chain

Intracellular bacterial pathogens have adapted their metabolism to optimally utilize the nutrients available in infected host cells. We recently reported the identification of an asparagine transporter required specifically for cytosolic multiplication ofFrancisella. In the present work, we characterized a new member of the major super family (MSF) of transporters, involved in isoleucine uptake. We show that this transporter (here designated IleP) plays a critical role in intracellular metabolic adaptation ofFrancisella. Inactivation of IleP severely impaired intracellularF. tularensissubsp.novicidamultiplication in all cell types tested and reduced bacterial virulence in the mouse model. To further establish the importance of theilePgene inF. tularensispathogenesis, we constructed a chromosomal deletion mutant ofileP(ΔFTL_1803) in theF. tularensissubsp.holarcticalive vaccine strain (LVS). Inactivation of IleP in theF. tularensisLVS provoked comparable intracellular growth defects, confirming the critical role of this transporter in isoleucine uptake. The data presented establish, for the first time, the importance of isoleucine utilization for efficient phagosomal escape and cytosolic multiplication ofFrancisellaand suggest that virulentF. tularensissubspecies have lost their branched-chain amino acid biosynthetic pathways and rely exclusively on dedicated uptake systems. This loss of function is likely to reflect an evolution toward a predominantly intracellular life style of the pathogen. Amino acid transporters should be thus considered major players in the adaptation of intracellular pathogens.

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Pdc1 Functions in the Assembly of P Bodies in Schizosaccharomyces pombe

Molecular and Cellular Biology ◽

10.1128/mcb.01583-12 ◽

2013 ◽

Vol 33 (6) ◽

pp. 1244-1253 ◽

Cited By ~ 18

Author(s):

Chun-Yu Wang ◽

Wen-Ling Chen ◽

Shao-Win Wang

Keyword(s):

Schizosaccharomyces Pombe ◽

Fission Yeast ◽

Affinity Purification ◽

Coiled Coil ◽

Body Protein ◽

Content Type ◽

Body Formation ◽

P Bodies ◽

C Terminus ◽

Decapping Enzymes

P bodies are cytoplasmic RNA granules containing the Dcp1-Dcp2 decapping enzymes where mRNA decay can occur. Here, we describe the characterization of P bodies in the fission yeastSchizosaccharomyces pombe. Most information on the property and function of P bodies stems from studies in the distantly related budding yeastSaccharomyces cerevisiae, and Edc3 was identified as a scaffold protein required for P-body assembly. However, we found that, unlike inS. cerevisiae, fission yeast Edc3 was dispensable for P-body formation. Pdc1, a novel partner of the fission yeast decapping enzyme, with a limited similarity to plant Edc4/Varicose that is required for the assembly of P bodies, was identified (tandem affinity purification–matrix-assisted laser desorption ionization tandem mass spectrometry [TAP-MALDI MS/MS]). Pdc1 interacts with Dcp2 through its C terminus and contains a coiled-coil region for self-interaction to mediate P-body formation. In line with the model that Pdc1 cross-bridges different proteins, additional interactions can be demonstrated with components such as Edc3 and Ste13. Although Pdc1 is not required for the interaction between Dcp1 and Dcp2, our data suggest that Pdc1 acts as a functional homologue of Edc4, a third component of the decapping enzymes that is thought to be absent from fungi. Together, these results highlight the diverse P-body protein compositions between different species and might help to provide insight into their evolutionary paths.

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Mutations in fbiD (Rv2983) as a Novel Determinant of Resistance to Pretomanid and Delamanid in Mycobacterium tuberculosis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01948-20 ◽

2020 ◽

Vol 65 (1) ◽

pp. e01948-20

Author(s):

Dalin Rifat ◽

Si-Yang Li ◽

Thomas Ioerger ◽

Keshav Shah ◽

Jean-Philippe Lanoix ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Clinical Evidence ◽

Clinical Samples ◽

Loss Of Function ◽

Wild Type ◽

Content Type ◽

Solid Media ◽

High Level ◽

Level Resistance

ABSTRACTThe nitroimidazole prodrugs delamanid and pretomanid comprise one of only two new antimicrobial classes approved to treat tuberculosis (TB) in 50 years. Prior in vitro studies suggest a relatively low barrier to nitroimidazole resistance in Mycobacterium tuberculosis, but clinical evidence is limited to date. We selected pretomanid-resistant M. tuberculosis mutants in two mouse models of TB using a range of pretomanid doses. The frequency of spontaneous resistance was approximately 10−5 CFU. Whole-genome sequencing of 161 resistant isolates from 47 mice revealed 99 unique mutations, of which 91% occurred in 1 of 5 genes previously associated with nitroimidazole activation and resistance, namely, fbiC (56%), fbiA (15%), ddn (12%), fgd (4%), and fbiB (4%). Nearly all mutations were unique to a single mouse and not previously identified. The remaining 9% of resistant mutants harbored mutations in Rv2983 (fbiD), a gene not previously associated with nitroimidazole resistance but recently shown to be a guanylyltransferase necessary for cofactor F420 synthesis. Most mutants exhibited high-level resistance to pretomanid and delamanid, although Rv2983 and fbiB mutants exhibited high-level pretomanid resistance but relatively small changes in delamanid susceptibility. Complementing an Rv2983 mutant with wild-type Rv2983 restored susceptibility to pretomanid and delamanid. By quantifying intracellular F420 and its precursor Fo in overexpressing and loss-of-function mutants, we provide further evidence that Rv2983 is necessary for F420 biosynthesis. Finally, Rv2983 mutants and other F420H2-deficient mutants displayed hypersusceptibility to some antibiotics and to concentrations of malachite green found in solid media used to isolate and propagate mycobacteria from clinical samples.

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Rif1 regulates telomere length through conserved HEAT repeats

Nucleic Acids Research ◽

10.1093/nar/gkab206 ◽

2021 ◽

Vol 49 (7) ◽

pp. 3967-3980

Author(s):

Calla B Shubin ◽

Rini Mayangsari ◽

Ariel D Swett ◽

Carol W Greider

Keyword(s):

Dna Binding ◽

Telomere Length ◽

Functional Role ◽

Conserved Residues ◽

Binding Interface ◽

Binding Function ◽

Length Regulation ◽

Telomere Length Regulation ◽

Telomere Regulation ◽

Conserved Amino Acids

AbstractIn budding yeast, Rif1 negatively regulates telomere length, but the mechanism of this regulation has remained elusive. Previous work identified several functional domains of Rif1, but none of these has been shown to mediate telomere length. To define Rif1 domains responsible for telomere regulation, we localized truncations of Rif1 to a single specific telomere and measured telomere length of that telomere compared to bulk telomeres. We found that a domain in the N-terminus containing HEAT repeats, Rif1177–996, was sufficient for length regulation when tethered to the telomere. Charged residues in this region were previously proposed to mediate DNA binding. We found that mutation of these residues disrupted telomere length regulation even when Rif1 was tethered to the telomere. Mutation of other conserved residues in this region, which were not predicted to interact with DNA, also disrupted telomere length maintenance, while mutation of conserved residues distal to this region did not. Our data suggest that conserved amino acids in the region from 436 to 577 play a functional role in telomere length regulation, which is separate from their proposed DNA binding function. We propose that the Rif1 HEAT repeats region represents a protein-protein binding interface that mediates telomere length regulation.

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Eukaryotic clamp loaders and unloaders in the maintenance of genome stability

Experimental & Molecular Medicine ◽

10.1038/s12276-020-00533-3 ◽

2020 ◽

Vol 52 (12) ◽

pp. 1948-1958

Author(s):

Kyoo-young Lee ◽

Su Hyung Park

Keyword(s):

Dna Damage ◽

Genomic Instability ◽

Genome Stability ◽

Critical Role ◽

Nuclear Antigen ◽

Checkpoint Activation ◽

Sliding Clamp ◽

Clamp Loaders ◽

Factor C

AbstractEukaryotic sliding clamp proliferating cell nuclear antigen (PCNA) plays a critical role as a processivity factor for DNA polymerases and as a binding and acting platform for many proteins. The ring-shaped PCNA homotrimer and the DNA damage checkpoint clamp 9-1-1 are loaded onto DNA by clamp loaders. PCNA can be loaded by the pentameric replication factor C (RFC) complex and the CTF18-RFC-like complex (RLC) in vitro. In cells, each complex loads PCNA for different purposes; RFC-loaded PCNA is essential for DNA replication, while CTF18-RLC-loaded PCNA participates in cohesion establishment and checkpoint activation. After completing its tasks, PCNA is unloaded by ATAD5 (Elg1 in yeast)-RLC. The 9-1-1 clamp is loaded at DNA damage sites by RAD17 (Rad24 in yeast)-RLC. All five RFC complex components, but none of the three large subunits of RLC, CTF18, ATAD5, or RAD17, are essential for cell survival; however, deficiency of the three RLC proteins leads to genomic instability. In this review, we describe recent findings that contribute to the understanding of the basic roles of the RFC complex and RLCs and how genomic instability due to deficiency of the three RLCs is linked to the molecular and cellular activity of RLC, particularly focusing on ATAD5 (Elg1).

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Loss of the Conserved Alveolate Kinase MAPK2 Decouples Toxoplasma Cell Growth from Cell Division

mBio ◽

10.1128/mbio.02517-20 ◽

2020 ◽

Vol 11 (6) ◽

Author(s):

Xiaoyu Hu ◽

William J. O’Shaughnessy ◽

Tsebaot G. Beraki ◽

Michael L. Reese

Keyword(s):

Toxoplasma Gondii ◽

Protein Kinases ◽

Daughter Cell ◽

Protozoan Parasite ◽

Productive Infection ◽

Centrosome Duplication ◽

Loss Of Function ◽

Content Type ◽

Mitogen Activated Protein ◽

Parasite Replication

ABSTRACT Mitogen-activated protein kinases (MAPKs) are a conserved family of protein kinases that regulate signal transduction, proliferation, and development throughout eukaryotes. The apicomplexan parasite Toxoplasma gondii expresses three MAPKs. Two of these, extracellular signal-regulated kinase 7 (ERK7) and MAPKL1, have been implicated in the regulation of conoid biogenesis and centrosome duplication, respectively. The third kinase, MAPK2, is specific to and conserved throughout the Alveolata, although its function is unknown. We used the auxin-inducible degron system to determine phenotypes associated with MAPK2 loss of function in Toxoplasma. We observed that parasites lacking MAPK2 failed to duplicate their centrosomes and therefore did not initiate daughter cell budding, which ultimately led to parasite death. MAPK2-deficient parasites initiated but did not complete DNA replication and arrested prior to mitosis. Surprisingly, the parasites continued to grow and replicate their Golgi apparatus, mitochondria, and apicoplasts. We found that the failure in centrosome duplication is distinct from the phenotype caused by the depletion of MAPKL1. As we did not observe MAPK2 localization at the centrosome at any point in the cell cycle, our data suggest that MAPK2 regulates a process at a distal site that is required for the completion of centrosome duplication and the initiation of parasite mitosis. IMPORTANCE Toxoplasma gondii is a ubiquitous intracellular protozoan parasite that can cause severe and fatal disease in immunocompromised patients and the developing fetus. Rapid parasite replication is critical for establishing a productive infection. Here, we demonstrate that a Toxoplasma protein kinase called MAPK2 is conserved throughout the Alveolata and essential for parasite replication. We found that parasites lacking MAPK2 protein were defective in the initiation of daughter cell budding and were rendered inviable. Specifically, T. gondii MAPK2 (TgMAPK2) appears to be required for centrosome replication at the basal end of the nucleus, and its loss causes arrest early in parasite division. MAPK2 is unique to the Alveolata and not found in metazoa and likely is a critical component of an essential parasite-specific signaling network.

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Accessing resources for service innovation – the critical role of network relationships

Journal of Service Management ◽

10.1108/josm-10-2012-0219 ◽

2014 ◽

Vol 25 (1) ◽

pp. 2-29 ◽

Cited By ~ 44

Author(s):

Helena Rusanen ◽

Aino Halinen ◽

Elina Jaakkola

Keyword(s):

Critical Role ◽

Service Innovation ◽

Innovation Process ◽

Research Strategy ◽

Future Research ◽

Theoretical Understanding ◽

Resource Access ◽

Content Type ◽

External Resources ◽

Different Types

Purpose – This paper aims to explore how companies access resources through network relationships when developing service innovations. The paper identifies the types of resource that companies seek from other actors and examines the nature of relationships and resource access strategies that can be applied to access each type of resource. Design/methodology/approach – A longitudinal, multi-case study is conducted in the field of technical business-to-business (b-to-b) services. An abductive research strategy is applied to create a new theoretical understanding of resource access. Findings – Companies seek a range of resources through different types of network relationships for service innovation. Four types of resource access strategies were identified: absorption, acquisition, sharing, and co-creation. The findings show how easily transferable resources can be accessed through weak relationships and low-intensity collaboration. Access to resources that are difficult to transfer, instead, necessitates strong relationships and high-intensity collaboration. Research limitations/implications – The findings are valid for technical b-to-b services, but should also be tested for other kinds of innovations. Future research should also study how actors integrate the resources gained through networks in the innovation process. Practical implications – Managers should note that key resources for service innovation may be accessible through a variety of actors and relationships ranging from formal arrangements to miscellaneous social contacts. To make use of tacit resources such as knowledge, firms need to engage in intensive collaboration. Originality/value – Despite attention paid to network relationships, innovation collaboration, and external resources, previous research has neither linked these issues nor studied their mutual contingencies. This paper provides a theoretical model that characterizes the service innovation resources accessible through different types of relationships and access strategies.

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Parkin interacting substrate zinc finger protein 746 is a pathological mediator in Parkinson’s disease

Brain ◽

10.1093/brain/awz172 ◽

2019 ◽

Vol 142 (8) ◽

pp. 2380-2401 ◽

Cited By ~ 9

Author(s):

Saurav Brahmachari ◽

Saebom Lee ◽

Sangjune Kim ◽

Changqing Yuan ◽

Senthilkumar S Karuppagounder ◽

...

Keyword(s):

Parkinson’S Disease ◽

Parkinson's Disease ◽

Zinc Finger ◽

Zinc Finger Protein ◽

Critical Role ◽

Trna Synthetase ◽

Loss Of Function ◽

Substrate Protein ◽

Finger Protein ◽

Sporadic Parkinson’S Disease

Abstract α-Synuclein misfolding and aggregation plays a major role in the pathogenesis of Parkinson’s disease. Although loss of function mutations in the ubiquitin ligase, parkin, cause autosomal recessive Parkinson’s disease, there is evidence that parkin is inactivated in sporadic Parkinson’s disease. Whether parkin inactivation is a driver of neurodegeneration in sporadic Parkinson’s disease or a mere spectator is unknown. Here we show that parkin in inactivated through c-Abelson kinase phosphorylation of parkin in three α-synuclein-induced models of neurodegeneration. This results in the accumulation of parkin interacting substrate protein (zinc finger protein 746) and aminoacyl tRNA synthetase complex interacting multifunctional protein 2 with increased parkin interacting substrate protein levels playing a critical role in α-synuclein-induced neurodegeneration, since knockout of parkin interacting substrate protein attenuates the degenerative process. Thus, accumulation of parkin interacting substrate protein links parkin inactivation and α-synuclein in a common pathogenic neurodegenerative pathway relevant to both sporadic and familial forms Parkinson’s disease. Thus, suppression of parkin interacting substrate protein could be a potential therapeutic strategy to halt the progression of Parkinson’s disease and related α-synucleinopathies.

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