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1.
Genes Cells ; 29(7): 567-583, 2024 Jul.
Article in English | MEDLINE | ID: mdl-38837646

ABSTRACT

Chromatin condensation state is the key for retrieving genetic information. High-mobility group protein (HMG) proteins exhibit DNA-binding and bending activities, playing an important role in the regulation of chromatin structure. We have shown that nucleosomes tightly packaged into heterochromatin undergo considerable dynamic histone H2A-H2B maintenance via the direct interaction between HP1/Swi6 and facilitate chromatin transcription (FACT), which is composed of the Spt16/Pob3 heterodimer and Nhp6. In this study, we analyzed the role of Nhp6, an HMG box protein, in the FACT at heterochromatin. Pob3 mutant strains showed derepressed heterochromatin-dependent gene silencing, whereas Nhp6 mutant strains did not show significant defects in chromatin regulation or gene expression, suggesting that these two modules play different roles in chromatin regulation. We expressed a protein fusing Nhp6 to the C-terminus of Pob3, which mimics the multicellular FACT component Ssrp1. The chromatin-binding activity of FACT increased with the number of Nhp6 fused to Pob3, and the heterochromatin formation rate was promoted more strongly. Furthermore, we demonstrated that this promotion of heterochromatinization inhibited the heterochromatic variegation caused by epe1+ disruption. Heterochromatic variegation can be observed in a variety of regulatory steps; however, when it is caused by fluctuations in chromatin arrangement, it can be eliminated through the strong recruitment of the FACT complex.


Subject(s)
Heterochromatin , Schizosaccharomyces pombe Proteins , Schizosaccharomyces , Heterochromatin/metabolism , Heterochromatin/genetics , Schizosaccharomyces/genetics , Schizosaccharomyces/metabolism , Schizosaccharomyces pombe Proteins/metabolism , Schizosaccharomyces pombe Proteins/genetics , Gene Expression Regulation, Fungal , Epigenesis, Genetic , Gene Silencing , High Mobility Group Proteins/metabolism , High Mobility Group Proteins/genetics
2.
Open Biol ; 14(1): 230379, 2024 Jan.
Article in English | MEDLINE | ID: mdl-38166399

ABSTRACT

Wee1 is a cell cycle regulator that phosphorylates Cdk1/Cdc2 and inhibits G2/M transition. Loss of Wee1 in fission yeast results in an early onset of mitosis. Interestingly, we found that cells lacking Wee1 require the functional spindle checkpoint for their viability. Genetic analysis indicated that the requirement is not attributable to the early onset of mitosis. Live-cell imaging revealed that some kinetochores are not attached or bioriented in the wee1 mutant. Furthermore, Mad2, a component of the spindle checkpoint known to recognize unattached kinetochores, accumulates in the vicinity of the spindle, representing activation of the spindle checkpoint in the mutant. It appears that the wee1 mutant cannot maintain stable kinetochore-microtubule attachment, and relies on the delay imposed by the spindle checkpoint for establishing biorientation of kinetochores. This study revealed a role of Wee1 in ensuring accurate segregation of chromosomes during mitosis, and thus provided a basis for a new principle of cancer treatment with Wee1 inhibitors.


Subject(s)
Schizosaccharomyces pombe Proteins , Schizosaccharomyces , Kinetochores/metabolism , Schizosaccharomyces/genetics , Schizosaccharomyces/metabolism , Spindle Apparatus/metabolism , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Microtubules/metabolism , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Mitosis , Protein-Tyrosine Kinases/genetics , Protein-Tyrosine Kinases/metabolism , Schizosaccharomyces pombe Proteins/genetics , Schizosaccharomyces pombe Proteins/metabolism
3.
Commun Biol ; 5(1): 78, 2022 01 20.
Article in English | MEDLINE | ID: mdl-35058555

ABSTRACT

DNA transfection is an important technology in life sciences, wherein nuclear entry of DNA is necessary to express exogenous DNA. Non-viral vectors and their transfection reagents are useful as safe transfection tools. However, they have no effect on the transfection of non-proliferating cells, the reason for which is not well understood. This study elucidates the mechanism through which transfected DNA enters the nucleus for gene expression. To monitor the behavior of transfected DNA, we introduce plasmid bearing lacO repeats and RFP-coding sequences into cells expressing GFP-LacI and observe plasmid behavior and RFP expression in living cells. RFP expression appears only after mitosis. Electron microscopy reveals that plasmids are wrapped with nuclear envelope (NE)‒like membranes or associated with chromosomes at telophase. The depletion of BAF, which is involved in NE reformation, delays plasmid RFP expression. These results suggest that transfected DNA is incorporated into the nucleus during NE reformation at telophase.


Subject(s)
Cell Nucleus/physiology , DNA/genetics , Gene Expression Regulation/physiology , Membrane Proteins/metabolism , Nuclear Proteins/metabolism , Plasmids/genetics , Biological Transport , Cell Line, Tumor , Humans , Membrane Proteins/genetics , Mutation , Nuclear Proteins/genetics , Single-Cell Analysis , Telophase , Transfection
4.
Nucleic Acids Res ; 48(16): 8977-8992, 2020 09 18.
Article in English | MEDLINE | ID: mdl-32710633

ABSTRACT

The protein kinase Gcn2 is a central transducer of nutritional stress signaling important for stress adaptation by normal cells and the survival of cancer cells. In response to nutrient deprivation, Gcn2 phosphorylates eIF2α, thereby repressing general translation while enhancing translation of specific mRNAs with upstream ORFs (uORFs) situated in their 5'-leader regions. Here we performed genome-wide measurements of mRNA translation during histidine starvation in fission yeast Schizosaccharomyces pombe. Polysome analyses were combined with microarray measurements to identify gene transcripts whose translation was up-regulated in response to the stress in a Gcn2-dependent manner. We determined that translation is reprogrammed to enhance RNA metabolism and chromatin regulation and repress ribosome synthesis. Interestingly, translation of intron-containing mRNAs was up-regulated. The products of the regulated genes include additional eIF2α kinase Hri2 amplifying the stress signaling and Gcn5 histone acetyl transferase and transcription factors, together altering genome-wide transcription. Unique dipeptide-coding uORFs and nucleotide motifs, such as '5'-UGA(C/G)GG-3', are found in 5' leader regions of regulated genes and shown to be responsible for translational control.


Subject(s)
Nucleotide Motifs , Protein Serine-Threonine Kinases/metabolism , RNA, Messenger/metabolism , Schizosaccharomyces pombe Proteins/metabolism , Schizosaccharomyces/enzymology , Acetyltransferases/metabolism , Gene Expression Regulation, Fungal , Histidine/metabolism , Open Reading Frames , Protein Processing, Post-Translational , Schizosaccharomyces/genetics , eIF-2 Kinase/metabolism
5.
Protein J ; 39(2): 174-181, 2020 04.
Article in English | MEDLINE | ID: mdl-32140970

ABSTRACT

In eukaryotes, chromosome ends (telomeres) are tethered to the inner nuclear membrane. During the early stages of meiosis, telomeres move along the nuclear membrane and gather near the spindle-pole body, resulting in a bouquet-like arrangement of chromosomes. This chromosomal configuration appears to be widely conserved among eukaryotes, and is assumed to play an important role in the normal progression of meiosis, by mediating the proper pairing of homologous chromosomes. In fission yeast, the Bqt1-Bqt2 protein complex plays a key role in tethering the telomere to the inner nuclear membrane. However, the structural details of the complex required to clarify how telomeres are gathered near the spindle-pole body remain enigmatic. Previously, we devised a preparation procedure for the Schizosaccharomyces japonicus Bqt1-Bqt2 complex, in which a SUMO tag was fused to the N-terminus of the Bqt1 protein. This allowed us to purify the Bqt1-Bqt2 complex from the soluble fraction. In the present study, we found that a maltose-binding protein homolog, Athe_0614, served as a better fusion partner than the SUMO protein, resulting in the marked increase in the solubility of the Bqt1-Bqt2 complex. The Athe_0614 fusion partner may open up new avenues for X-ray crystallographic analyses of the structure of the Bqt1-Bqt2 complex.


Subject(s)
Bacterial Proteins/metabolism , Firmicutes/metabolism , Maltose-Binding Proteins/metabolism , Telomere-Binding Proteins/metabolism , Caldicellulosiruptor , Meiosis , Recombinant Fusion Proteins/metabolism , Telomere/metabolism
6.
Nat Commun ; 10(1): 5598, 2019 12 06.
Article in English | MEDLINE | ID: mdl-31811152

ABSTRACT

Pairing of homologous chromosomes in meiosis is essential for sexual reproduction. We have previously demonstrated that the fission yeast sme2 RNA, a meiosis-specific long noncoding RNA (lncRNA), accumulates at the sme2 chromosomal loci and mediates their robust pairing in meiosis. However, the mechanisms underlying lncRNA-mediated homologous pairing have remained elusive. In this study, we identify conserved RNA-binding proteins that are required for robust pairing of homologous chromosomes. These proteins accumulate mainly at the sme2 and two other chromosomal loci together with meiosis-specific lncRNAs transcribed from these loci. Remarkably, the chromosomal accumulation of these lncRNA-protein complexes is required for robust pairing. Moreover, the lncRNA-protein complexes exhibit phase separation properties, since 1,6-hexanediol treatment reversibly disassembled these complexes and disrupted the pairing of associated loci. We propose that lncRNA-protein complexes assembled at specific chromosomal loci mediate recognition and subsequent pairing of homologous chromosomes.


Subject(s)
Chromosome Pairing/physiology , Chromosomes, Fungal/metabolism , Meiosis/physiology , RNA-Binding Proteins/metabolism , Schizosaccharomyces/genetics , Cell Cycle Proteins/metabolism , Chromosomal Proteins, Non-Histone/metabolism , RNA, Long Noncoding/metabolism , Schizosaccharomyces/physiology , Schizosaccharomyces pombe Proteins/metabolism
7.
PLoS Genet ; 15(6): e1008061, 2019 06.
Article in English | MEDLINE | ID: mdl-31170156

ABSTRACT

The nuclear pore complex (NPC) forms a gateway for nucleocytoplasmic transport. The outer ring protein complex of the NPC (the Nup107-160 subcomplex in humans) is a key component for building the NPC. Nup107-160 subcomplexes are believed to be symmetrically localized on the nuclear and cytoplasmic sides of the NPC. However, in S. pombe immunoelectron and fluorescence microscopic analyses revealed that the homologous components of the human Nup107-160 subcomplex had an asymmetrical localization: constituent proteins spNup132 and spNup107 were present only on the nuclear side (designated the spNup132 subcomplex), while spNup131, spNup120, spNup85, spNup96, spNup37, spEly5 and spSeh1 were localized only on the cytoplasmic side (designated the spNup120 subcomplex), suggesting the complex was split into two pieces at the interface between spNup96 and spNup107. This contrasts with the symmetrical localization reported in other organisms. Fusion of spNup96 (cytoplasmic localization) with spNup107 (nuclear localization) caused cytoplasmic relocalization of spNup107. In this strain, half of the spNup132 proteins, which interact with spNup107, changed their localization to the cytoplasmic side of the NPC, leading to defects in mitotic and meiotic progression similar to an spNup132 deletion strain. These observations suggest the asymmetrical localization of the outer ring spNup132 and spNup120 subcomplexes of the NPC is necessary for normal cell cycle progression in fission yeast.


Subject(s)
Nuclear Pore Complex Proteins/genetics , Nuclear Pore/genetics , Schizosaccharomyces pombe Proteins/genetics , Active Transport, Cell Nucleus/genetics , Cell Cycle/genetics , Cell Division/genetics , Cell Nucleus/genetics , Cell Nucleus/ultrastructure , Cytoplasm/genetics , Cytoplasm/ultrastructure , Humans , Meiosis/genetics , Microscopy, Fluorescence , Nuclear Envelope/genetics , Nuclear Pore/ultrastructure , Protein Binding/genetics , Saccharomyces cerevisiae/genetics , Schizosaccharomyces/genetics
8.
PLoS Genet ; 15(6): e1008129, 2019 06.
Article in English | MEDLINE | ID: mdl-31206516

ABSTRACT

H3K9 methylation (H3K9me) is a conserved marker of heterochromatin, a transcriptionally silent chromatin structure. Knowledge of the mechanisms for regulating heterochromatin distribution is limited. The fission yeast JmjC domain-containing protein Epe1 localizes to heterochromatin mainly through its interaction with Swi6, a homologue of heterochromatin protein 1 (HP1), and directs JmjC-mediated H3K9me demethylation in vivo. Here, we found that loss of epe1 (epe1Δ) induced a red-white variegated phenotype in a red-pigment accumulation background that generated uniform red colonies. Analysis of isolated red and white colonies revealed that silencing of genes involved in pigment accumulation by stochastic ectopic heterochromatin formation led to white colony formation. In addition, genome-wide analysis of red- and white-isolated clones revealed that epe1Δ resulted in a heterogeneous heterochromatin distribution among clones. We found that Epe1 had an N-terminal domain distinct from its JmjC domain, which activated transcription in both fission and budding yeasts. The N-terminal transcriptional activation (NTA) domain was involved in suppression of ectopic heterochromatin-mediated red-white variegation. We introduced a single copy of Epe1 into epe1Δ clones harboring ectopic heterochromatin, and found that Epe1 could reduce H3K9me from ectopic heterochromatin but some of the heterochromatin persisted. This persistence was due to a latent H3K9me source embedded in ectopic heterochromatin. Epe1H297A, a canonical JmjC mutant, suppressed red-white variegation, but entirely failed to remove already-established ectopic heterochromatin, suggesting that Epe1 prevented stochastic de novo deposition of ectopic H3K9me in an NTA-dependent but JmjC-independent manner, while its JmjC domain mediated removal of H3K9me from established ectopic heterochromatin. Our results suggest that Epe1 not only limits the distribution of heterochromatin but also controls the balance between suppression and retention of heterochromatin-mediated epigenetic diversification.


Subject(s)
Epigenomics , Heterochromatin/genetics , Nuclear Proteins/genetics , Schizosaccharomyces pombe Proteins/genetics , Schizosaccharomyces/genetics , Chromatin Assembly and Disassembly/genetics , Chromosomal Proteins, Non-Histone/genetics , Gene Silencing , Histones/genetics , Jumonji Domain-Containing Histone Demethylases/genetics , Methylation , Mutation
9.
Sci Rep ; 9(1): 7159, 2019 05 09.
Article in English | MEDLINE | ID: mdl-31073221

ABSTRACT

The nucleosome, composed of DNA and a histone core, is the basic structural unit of chromatin. The fission yeast Schizosaccharomyces pombe has two genes of histone H2A, hta1+ and hta2+; these genes encode two protein species of histone H2A (H2Aα and H2Aß, respectively), which differ in three amino acid residues, and only hta2+ is upregulated during meiosis. However, it is unknown whether S. pombe H2Aα and H2Aß have functional differences. Therefore, in this study, we examined the possible functional differences between H2Aα and H2Aß during meiosis in S. pombe. We found that deletion of hta2+, but not hta1+, causes defects in chromosome segregation and spore formation during meiosis. Meiotic defects in hta2+ deletion cells were rescued by expressing additional copies of hta1+ or by expressing hta1+ from the hta2 promoter. This indicated that the defects were caused by insufficient amounts of histone H2A, and not by the amino acid residue differences between H2Aα and H2Aß. Microscopic observation attributed the chromosome segregation defects to anaphase bridge formation in a chromosomal region at the repeats of ribosomal RNA genes (rDNA repeats). These results suggest that histone H2A insufficiency affects the chromatin structures of rDNA repeats, leading to chromosome missegregation in S. pombe.


Subject(s)
Chromosome Segregation/physiology , Chromosomes, Fungal/metabolism , DNA, Ribosomal/genetics , Histones/genetics , Schizosaccharomyces pombe Proteins/genetics , Schizosaccharomyces/metabolism , Anaphase , Chromatin/metabolism , Histones/deficiency , Histones/metabolism , Promoter Regions, Genetic , Protein Subunits/deficiency , Protein Subunits/genetics , Protein Subunits/metabolism , Schizosaccharomyces pombe Proteins/metabolism , Spores, Fungal/metabolism , Up-Regulation
10.
J Cell Sci ; 132(10)2019 05 15.
Article in English | MEDLINE | ID: mdl-30975915

ABSTRACT

In eukaryotic cells, chromosomes are confined to the nucleus, which is compartmentalized by the nuclear membranes; these are continuous with the endoplasmic reticulum membranes. Maintaining the homeostasis of these membranes is an important cellular activity performed by lipid metabolic enzymes. However, how lipid metabolic enzymes affect nuclear membrane functions remains to be elucidated. We found that the very-long-chain fatty acid elongase Elo2 is located in the nuclear membrane and prevents lethal defects associated with nuclear membrane ruptures in mutants of the nuclear membrane proteins Lem2 and Bqt4 in the fission yeast Schizosaccharomyces pombe. Lipid composition analysis shows that t20:0/24:0 phytoceramide (a conjugate of C20:0 phytosphingosine and C24:0 fatty acid) is a major ceramide species in S. pombe The quantity of this ceramide is reduced in the absence of Lem2, and restored by increased expression of Elo2. Furthermore, loss of S. pombe Elo2 can be rescued by its human orthologs. These results suggest that the conserved very-long-chain fatty acid elongase producing the ceramide component is essential for nuclear membrane integrity and cell viability in eukaryotes.This article has an associated First Person interview with the first author of the paper.


Subject(s)
Acetyltransferases/metabolism , Fatty Acid Elongases/metabolism , Membrane Proteins/metabolism , Nuclear Envelope/metabolism , Schizosaccharomyces pombe Proteins/metabolism , Schizosaccharomyces/metabolism , Humans , Saccharomyces cerevisiae Proteins/metabolism
11.
Genes Cells ; 23(3): 122-135, 2018 Mar.
Article in English | MEDLINE | ID: mdl-29292846

ABSTRACT

Inner nuclear membrane (INM) proteins are thought to play important roles in modulating nuclear organization and function through their interactions with chromatin. However, these INM proteins share redundant functions in metazoans that pose difficulties for functional studies. The fission yeast Schizosaccharomyces pombe exhibits a relatively small number of INM proteins, and molecular genetic tools are available to separate their redundant functions. In S. pombe, it has been reported that among potentially redundant INM proteins, Lem2 displays a unique genetic interaction with another INM protein, Bqt4, which is involved in anchoring telomeres to the nuclear envelope. Double mutations in the lem2 and bqt4 genes confer synthetic lethality during vegetative growth. Here, we show that Lem2 is retained at the nuclear envelope through its interaction with Bqt4, as the loss of Bqt4 results in the exclusive accumulation of Lem2 to the spindle pole body (SPB). An N-terminal nucleoplasmic region of Lem2 bears affinity to both Bqt4 and the SPB in a competitive manner. In contrast, the synthetic lethality of the lem2 bqt4 double mutant is suppressed by the C-terminal region of Lem2. These results indicate that the N-terminal and C-terminal domains of Lem2 show independent functions with respect to Bqt4.


Subject(s)
DNA-Binding Proteins/metabolism , Membrane Proteins/metabolism , Nuclear Envelope/metabolism , Nuclear Proteins/metabolism , Schizosaccharomyces pombe Proteins/metabolism , Schizosaccharomyces/metabolism , Telomere/metabolism , DNA, Fungal/genetics , DNA, Fungal/metabolism , Nuclear Envelope/genetics , Protein Interaction Domains and Motifs , Schizosaccharomyces/growth & development , Telomere/genetics
12.
Proc Natl Acad Sci U S A ; 114(52): E11208-E11217, 2017 12 26.
Article in English | MEDLINE | ID: mdl-29237752

ABSTRACT

Some long noncoding RNAs (ncRNAs) transcribed by RNA polymerase II (RNAPII) are retained on chromatin, where they regulate RNAi and chromatin structure. The molecular basis of this retention remains unknown. We show that in fission yeast serine 7 (Ser7) of the C-terminal domain (CTD) of RNAPII is required for efficient siRNA generation for RNAi-dependent heterochromatin formation. Surprisingly, Ser7 facilitates chromatin retention of nascent heterochromatic RNAs (hRNAs). Chromatin retention of hRNAs and siRNA generation requires both Ser7 and an RNA-binding activity of the chromodomain of Chp1, a subunit of the RNA-induced transcriptional silencing (RITS) complex. Furthermore, RITS associates with RNAPII in a Ser7-dependent manner. We propose that Ser7 promotes cotranscriptional chromatin retention of hRNA by recruiting the RNA-chromatin connector protein Chp1, which facilitates RNAi-dependent heterochromatin formation. Our findings reveal a function of the CTD code: linking ncRNA transcription to RNAi for heterochromatin formation.


Subject(s)
Cell Cycle Proteins/metabolism , Heterochromatin/metabolism , RNA Polymerase II/metabolism , RNA, Fungal/metabolism , RNA, Long Noncoding/metabolism , RNA, Small Interfering/metabolism , Schizosaccharomyces pombe Proteins/metabolism , Schizosaccharomyces/metabolism , Cell Cycle Proteins/genetics , Heterochromatin/genetics , Protein Domains , RNA Polymerase II/genetics , RNA, Fungal/genetics , RNA, Long Noncoding/genetics , RNA, Small Interfering/genetics , Schizosaccharomyces/genetics , Schizosaccharomyces pombe Proteins/genetics , Serine/genetics , Serine/metabolism
13.
FEBS Lett ; 591(7): 1029-1040, 2017 04.
Article in English | MEDLINE | ID: mdl-28245054

ABSTRACT

In meiosis, two rounds of nuclear division occur consecutively without DNA replication between the divisions. We isolated a fission yeast mutant in which the nucleus divides only once to generate two spores, as opposed to four, in meiosis. In this mutant, we found that the initiation codon of the slp1+ gene is converted to ATA, producing a reduced amount of Slp1. As a member of the Fizzy family of anaphase-promoting complex/cyclosome (APC/C) activators, Slp1 is essential for vegetative growth; however, the mutant allele shows a phenotype only in meiosis. Slp1 insufficiency delays degradation of maturation-promoting factor at the first meiotic division, and another APC/C activator, Fzr1, which acts late in meiosis, terminates meiosis immediately after the delayed first division to produce two viable spores.


Subject(s)
Cdc20 Proteins/metabolism , Cdh1 Proteins/metabolism , Meiosis , Schizosaccharomyces pombe Proteins/metabolism , Schizosaccharomyces/metabolism , Anaphase-Promoting Complex-Cyclosome/metabolism , Blotting, Western , Cdc20 Proteins/genetics , Cdh1 Proteins/genetics , Cell Nucleus Division/genetics , Microscopy, Fluorescence , Mutation , Schizosaccharomyces/genetics , Schizosaccharomyces pombe Proteins/genetics , Spores, Fungal/genetics , Spores, Fungal/metabolism , Time Factors
14.
Biochem Biophys Res Commun ; 482(4): 896-901, 2017 Jan 22.
Article in English | MEDLINE | ID: mdl-27890612

ABSTRACT

Schizosaccharomyces pombe, which has a small genome but shares many physiological functions with higher eukaryotes, is a useful single-cell, model eukaryotic organism. In particular, many features concerning chromatin structure and dynamics, including heterochromatin, centromeres, telomeres, and DNA replication origins, are well conserved between S. pombe and higher eukaryotes. However, the S. pombe nucleosome, the fundamental structural unit of chromatin, has not been reconstituted in vitro. In the present study, we established the method to purify S. pombe histones H2A, H2B, H3, and H4, and successfully reconstituted the S. pombe nucleosome in vitro. Our thermal stability assay and micrococcal nuclease treatment assay revealed that the S. pombe nucleosome is markedly unstable and its DNA ends are quite accessible, as compared to the canonical human nucleosome. These findings are important to understand the mechanisms of epigenetic genomic DNA regulation in fission yeast.


Subject(s)
Histones/chemistry , Nucleosomes/chemistry , Schizosaccharomyces pombe Proteins/chemistry , Schizosaccharomyces/chemistry , Amino Acid Sequence , DNA, Fungal/chemistry , Histones/isolation & purification , Humans , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Schizosaccharomyces/cytology , Schizosaccharomyces pombe Proteins/isolation & purification , Sequence Alignment
15.
Genes Cells ; 21(8): 812-32, 2016 Aug.
Article in English | MEDLINE | ID: mdl-27334362

ABSTRACT

Inner nuclear membrane proteins interact with chromosomes in the nucleus and are important for chromosome activity. Lem2 and Man1 are conserved members of the LEM-domain nuclear membrane protein family. Mutations of LEM-domain proteins are associated with laminopathy, but their cellular functions remain unclear. Here, we report that Lem2 maintains genome stability in the fission yeast Schizosaccharomyces pombe. S. pombe cells disrupted for the lem2(+) gene (lem2∆) showed slow growth and increased rate of the minichromosome loss. These phenotypes were prominent in the rich culture medium, but not in the minimum medium. Centromeric heterochromatin formation was augmented upon transfer to the rich medium in wild-type cells. This augmentation of heterochromatin formation was impaired in lem2∆ cells. Notably, lem2∆ cells occasionally exhibited spontaneous duplication of genome sequences flanked by the long-terminal repeats of retrotransposons. The resulting duplication of the lnp1(+) gene, which encodes an endoplasmic reticulum membrane protein, suppressed lem2∆ phenotypes, whereas the lem2∆ lnp1∆ double mutant showed a severe growth defect. A combination of mutations in Lem2 and Bqt4, which encodes a nuclear membrane protein that anchors telomeres to the nuclear membrane, caused synthetic lethality. These genetic interactions imply that Lem2 cooperates with the nuclear membrane protein network to regulate genome stability.


Subject(s)
Chromatin Assembly and Disassembly/genetics , Heterochromatin/genetics , Schizosaccharomyces pombe Proteins/genetics , Schizosaccharomyces/genetics , Endoplasmic Reticulum/genetics , Lamin Type A/genetics , Mutation , Nuclear Envelope/genetics , Nuclear Proteins/genetics , Telomere/genetics
16.
Nucleic Acids Res ; 44(9): 4147-62, 2016 05 19.
Article in English | MEDLINE | ID: mdl-26792892

ABSTRACT

In budding yeast, Set2 catalyzes di- and trimethylation of H3K36 (H3K36me2 and H3K36me3) via an interaction between its Set2-Rpb1 interaction (SRI) domain and C-terminal repeats of RNA polymerase II (Pol2) phosphorylated at Ser2 and Ser5 (CTD-S2,5-P). H3K36me2 is sufficient for recruitment of the Rpd3S histone deacetylase complex to repress cryptic transcription from transcribed regions. In fission yeast, Set2 is also responsible for H3K36 methylation, which represses a subset of RNAs including heterochromatic and subtelomeric RNAs, at least in part via recruitment of Clr6 complex II, a homolog of Rpd3S. Here, we show that CTD-S2P-dependent interaction of fission yeast Set2 with Pol2 via the SRI domain is required for formation of H3K36me3, but not H3K36me2. H3K36me3 silenced heterochromatic and subtelomeric transcripts mainly through post-transcriptional and transcriptional mechanisms, respectively, whereas H3K36me2 was not enough for silencing. Clr6 complex II appeared not to be responsible for heterochromatic silencing by H3K36me3. Our results demonstrate that H3K36 methylation has multiple outputs in fission yeast; these findings provide insights into the distinct roles of H3K36 methylation in metazoans, which have different enzymes for synthesis of H3K36me1/2 and H3K36me3.


Subject(s)
Gene Expression Regulation, Fungal , Gene Silencing , Histones/metabolism , Protein Processing, Post-Translational , Schizosaccharomyces/genetics , Chromosomes, Fungal/genetics , Chromosomes, Fungal/ultrastructure , Genes, Fungal , Heterochromatin/genetics , Heterochromatin/ultrastructure , Histone-Lysine N-Methyltransferase/chemistry , Histone-Lysine N-Methyltransferase/physiology , Methylation , Protein Interaction Domains and Motifs , RNA Polymerase II/chemistry , RNA Polymerase II/metabolism , RNA Stability , Schizosaccharomyces/metabolism , Schizosaccharomyces pombe Proteins/chemistry , Schizosaccharomyces pombe Proteins/physiology , Telomere/genetics , Transcription, Genetic
17.
Sci Rep ; 5: 15617, 2015 Oct 21.
Article in English | MEDLINE | ID: mdl-26486373

ABSTRACT

In ribosome biogenesis, a large fraction of ribosomes is used for producing ribosomal proteins themselves. Here, we applied simulation and experimentation to determine what fraction of ribosomes should be allocated for the synthesis of ribosomal proteins to optimize cellular economy for growth. We define the "r-fraction" as the fraction of mRNA of the ribosomal protein genes out of the total mRNA, and we simulated the effect of the r-fraction on the number of ribosomes. We then empirically measured the amount of protein and RNA in fission yeast cells cultured with high and low nitrogen sources. In the cells cultured with a low nitrogen source, the r-fraction decreased from 0.46 to 0.42 with a 40% reduction of rRNA, but the reduction of the total protein was smaller at 30%. These results indicate that the r-fraction is internally controlled to optimize the efficiency of protein synthesis at a limited cellular cost.


Subject(s)
Nitrogen/metabolism , Ribosomal Proteins/biosynthesis , Ribosomes/genetics , Gene Expression Regulation, Fungal , RNA, Messenger/biosynthesis , Ribosomal Proteins/metabolism , Ribosomes/metabolism , Schizosaccharomyces/genetics , Schizosaccharomyces/metabolism
18.
Sci Rep ; 5: 12720, 2015 Jul 30.
Article in English | MEDLINE | ID: mdl-26223950

ABSTRACT

Faithful DNA replication is a prerequisite for cell proliferation. Several cytological studies have shown that chromosome structures alter in the S-phase of the cell cycle. However, the molecular mechanisms behind the alteration of chromosome structures associated with DNA replication have not been elucidated. Here, we investigated chromatin structures and acetylation of specific histone residues during DNA replication using the meiotic nucleus of the fission yeast Schizosaccharomyces pombe. The S. pombe meiotic nucleus provides a unique opportunity for measuring the levels of compaction of chromatin along the chromosome in a defined orientation. By direct measurement of chromatin compaction in living cells, we demonstrated that decompaction of chromatin occurs during meiotic DNA replication. This chromatin decompaction was suppressed by depletion of histone acetyltransferase Mst1 or by arginine substitution of specific lysine residues (K8 and K12) of histone H4. These results suggest that acetylation of histone H4 residues K8 and K12 plays a critical role in loosening chromatin structures during DNA replication.


Subject(s)
Chromatin Assembly and Disassembly/physiology , DNA Replication/physiology , DNA, Fungal/biosynthesis , Histones/metabolism , Schizosaccharomyces pombe Proteins/metabolism , Schizosaccharomyces/metabolism , Acetylation , DNA, Fungal/genetics , Histones/genetics , S Phase/physiology , Schizosaccharomyces/genetics , Schizosaccharomyces pombe Proteins/genetics
19.
Nat Commun ; 6: 7753, 2015 Jul 24.
Article in English | MEDLINE | ID: mdl-26205977

ABSTRACT

It is generally believed that silent chromatin is condensed and transcriptionally active chromatin is decondensed. However, little is known about the relationship between the condensation levels and gene expression. Here we report the condensation levels of interphase chromatin in the fission yeast Schizosaccharomyces pombe examined by super-resolution fluorescence microscopy. Unexpectedly, silent chromatin is less condensed than the euchromatin. Furthermore, the telomeric silent regions are flanked by highly condensed chromatin bodies, or 'knobs'. Knob regions span ∼50 kb of sequence devoid of methylated histones. Knob condensation is independent of HP1 homologue Swi6 and other gene silencing factors. Disruption of methylation at lysine 36 of histone H3 (H3K36) eliminates knob formation and gene repression at the subtelomeric and adjacent knob regions. Thus, epigenetic marks at H3K36 play crucial roles in the formation of a unique chromatin structure and in gene regulation at those regions in S. pombe.


Subject(s)
Chromatin/metabolism , Schizosaccharomyces/metabolism , Gene Silencing , Microscopy, Fluorescence
20.
PLoS One ; 10(3): e0120109, 2015.
Article in English | MEDLINE | ID: mdl-25767875

ABSTRACT

Microtubules play important roles in organelle transport, the maintenance of cell polarity and chromosome segregation and generally form bundles during these processes. The fission yeast gene scp3+ was identified as a multicopy suppressor of the cps3-81 mutant, which is hypersensitive to isopropyl N-3-chlorophenylcarbamate (CIPC), a poison that induces abnormal multipolar spindle formation in higher eukaryotes. In this study, we investigated the function of Scp3 along with the effect of CIPC in the fission yeast Schizosaccharomyces pombe. Microscopic observation revealed that treatment with CIPC, cps3-81 mutation and scp3+ gene deletion disturbed the orientation of microtubules in interphase cells. Overexpression of scp3+ suppressed the abnormal orientation of microtubules by promoting bundling. Functional analysis suggested that Scp3 functions independently from Ase1, a protein largely required for the bundling of the mitotic spindle. A strain lacking the ase1+ gene was more sensitive to CIPC, with the drug affecting the integrity of the mitotic spindle, indicating that CIPC has a mitotic target that has a role redundant with Ase1. These results suggested that multiple systems are independently involved to ensure microtubule orientation by bundling in fission yeast.


Subject(s)
Microtubules/physiology , Schizosaccharomyces pombe Proteins/physiology , Chlorpropham/toxicity , Green Fluorescent Proteins/metabolism , Microscopy, Fluorescence , Microtubule-Associated Proteins/genetics , Microtubule-Associated Proteins/metabolism , Microtubules/drug effects , Plasmids/genetics , Schizosaccharomyces , Schizosaccharomyces pombe Proteins/genetics , Schizosaccharomyces pombe Proteins/metabolism
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