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Yasushi Hiraoka

Publications and source records attributed to Yasushi Hiraoka.

At least 19 recordsLinked to original sources
PubMed2026

Integrative modeling of the genome structure and dynamics in fission yeast.

Genome organization in the nucleus is highly structured and dynamic. Recent advances in genomic technology have enabled the measurement of genome-wide architecture and locus-specific motion, yielding contact maps and live-cell trajectories. However, these outcomes are derived from different modalities and are not directly comparable, with their quantitative integration being a key challenge. Here we establish a genome-wide live-cell imaging platform in fission yeast Schizosaccharomyces pombe, tracking 131 chromosomal loci, along with the spindle pole body (SPB) and nucleolus, to construct a quantitative map of locus dynamics. By integrating these dynamics with contact data through polymer modeling of Hi-C data, we build a physics-based "digital twin" of the S. pombe genome consistent with the spatiotemporal dynamics of interphase chromatin. We validate it against genome-wide mobility patterns and known architectural features, including centromere and telomere clustering. The model also identifies distinct dynamical regimes: centromere- and telomere-proximal loci relax within [Formula: see text]150 s, whereas the remaining loci relax within [Formula: see text]70 s. We measure semiperiodic dynamics of SPB motion, including a characteristic peak near 225 s and [Formula: see text] fluctuations. We use the model with SPB-directed forcing to show how these low-frequency components propagate through the genome to drive genome-wide chromatin displacements. Together, this predictive physics-based modeling framework integrates genome structure and dynamics to reveal how nuclear mechanical driving forces shape chromosome motion, linking mechanically driven chromatin responses to genome maintenance and regulation.

Schizosaccharomyces↗
PubMed2006

Reconstruction of the kinetochore during meiosis in fission yeast Schizosaccharomyces pombe.

During the transition from mitosis to meiosis, the kinetochore undergoes significant reorganization, switching from a bipolar to a monopolar orientation. To examine the centromere proteins that are involved in fundamental reorganization in meiosis, we observed the localization of 22 mitotic and 2 meiotic protein components of the kinetochore during meiosis in living cells of the fission yeast. We found that the 22 mitotic proteins can be classified into three groups: the Mis6-like group, the NMS (Ndc80-Mis12-Spc7) group, and the DASH group, based on their meiotic behavior. Mis6-like group proteins remain at the centromere throughout meiosis. NMS group proteins disappear from the centromere at the onset of meiosis and reappear at the centromere in two steps in late prophase. DASH group proteins appear shortly before metaphase of meiosis I. These observations suggest that Mis6-like group proteins constitute the structural basis of the centromere and that the NMS and DASH group proteins reassemble to establish the functional metaphase kinetochore. On the other hand, the meiosis-specific protein Moa1, which plays an important role in forming the meiotic monopolar kinetochore, is loaded onto the centromere significantly earlier than the NMS group, whereas another meiosis-specific protein, Sgo1, is loaded at times similar to the NMS group.

Amino Acid Sequence↗
PubMed2006

Meiotic cohesins modulate chromosome compaction during meiotic prophase in fission yeast.

The meiotic cohesin Rec8 is required for the stepwise segregation of chromosomes during the two rounds of meiotic division. By directly measuring chromosome compaction in living cells of the fission yeast Schizosaccharomyces pombe, we found an additional role for the meiotic cohesin in the compaction of chromosomes during meiotic prophase. In the absence of Rec8, chromosomes were decompacted relative to those of wild-type cells. Conversely, loss of the cohesin-associated protein Pds5 resulted in hypercompaction. Although this hypercompaction requires Rec8, binding of Rec8 to chromatin was reduced in the absence of Pds5, indicating that Pds5 promotes chromosome association of Rec8. To explain these observations, we propose that meiotic prophase chromosomes are organized as chromatin loops emanating from a Rec8-containing axis: the absence of Rec8 disrupts the axis, resulting in disorganized chromosomes, whereas reduced Rec8 loading results in a longitudinally compacted axis with fewer attachment points and longer chromatin loops.

Cell Cycle Proteins↗
PubMed2006

Selective elimination of messenger RNA prevents an incidence of untimely meiosis.

Much remains unknown about the molecular regulation of meiosis. Here we show that meiosis-specific transcripts are selectively removed if expressed during vegetative growth in fission yeast. These messenger RNAs contain a cis-acting region--which we call the DSR--that confers this removal via binding to a YTH-family protein Mmi1. Loss of Mmi1 function severely impairs cell growth owing to the untimely expression of meiotic transcripts. Microarray analysis reveals that at least a dozen such meiosis-specific transcripts are eliminated by the DSR-Mmi1 system. Mmi1 remains in the form of multiple nuclear foci during vegetative growth. At meiotic prophase these foci precipitate to a single focus, which coincides with the dot formed by the master meiosis-regulator Mei2. A meiotic arrest due to the loss of the Mei2 dot is released by a reduction in Mmi1 activity. We propose that Mei2 turns off the DSR-Mmi1 system by sequestering Mmi1 to the dot and thereby secures stable expression of meiosis-specific transcripts.

Meiosis↗
PubMed2006

ORFeome cloning and global analysis of protein localization in the fission yeast Schizosaccharomyces pombe.

Cloning of the entire set of an organism's protein-coding open reading frames (ORFs), or 'ORFeome', is a means of connecting the genome to downstream 'omics' applications. Here we report a proteome-scale study of the fission yeast Schizosaccharomyces pombe based on cloning of the ORFeome. Taking advantage of a recombination-based cloning system, we obtained 4,910 ORFs in a form that is readily usable in various analyses. First, we evaluated ORF prediction in the fission yeast genome project by expressing each ORF tagged at the 3' terminus. Next, we determined the localization of 4,431 proteins, corresponding to approximately 90% of the fission yeast proteome, by tagging each ORF with the yellow fluorescent protein. Furthermore, using leptomycin B, an inhibitor of the nuclear export protein Crm1, we identified 285 proteins whose localization is regulated by Crm1.

Antifungal Agents↗
PubMed2006

A defect in protein farnesylation suppresses a loss of Schizosaccharomyces pombe tsc2+, a homolog of the human gene predisposing to tuberous sclerosis complex.

Mutations in the human Tsc1 and Tsc2 genes predispose to tuberous sclerosis complex (TSC), a disorder characterized by the wide spread of benign tumors. Tsc1 and Tsc2 proteins form a complex and serve as a GTPase-activating protein (GAP) for Rheb, a GTPase regulating a downstream kinase, mTOR. The genome of Schizosaccharomyces pombe contains tsc1(+) and tsc2(+), homologs of human Tsc1 and Tsc2, respectively. In this study we analyzed the gene expression profile on a genomewide scale and found that deletion of either tsc1(+) or tsc2(+) affects gene induction upon nitrogen starvation. Three hours after nitrogen depletion genes encoding permeases and genes required for meiosis are less induced. Under the same condition, retrotransposons, G1-cyclin (pas1(+)), and inv1(+) are more induced. We also demonstrate that a mutation (cpp1-1) in a gene encoding a beta-subunit of a farnesyltransferase can suppress most of the phenotypes associated with deletion of tsc1(+) or tsc2(+). When a mutant of rhb1(+) (homolog of human Rheb), which bypasses the requirement of protein farnesylation, was expressed, the cpp1-1 mutation could no longer suppress, indicating that deficient farnesylation of Rhb1 contributes to the suppression. On the basis of these results, we discuss TSC pathology and possible improvement in chemotherapy for TSC.

Base Sequence↗
PubMed2006

Meiotic proteins bqt1 and bqt2 tether telomeres to form the bouquet arrangement of chromosomes.

In many organisms, meiotic chromosomes are bundled at their telomeres to form a "bouquet" arrangement. The bouquet formation plays an important role in homologous chromosome pairing and therefore progression of meiosis. As meiotic telomere clustering occurs in response to mating pheromone signaling in fission yeast, we looked for factors essential for bouquet formation among genes induced under mating pheromone signaling. This genome-wide search identified two proteins, Bqt1 and Bqt2, that connect telomeres to the spindle-pole body (SPB; the centrosome equivalent in fungi). Neither Bqt1 nor Bqt2 alone functions as a connector, but together the two proteins form a bridge between Rap1 (a telomere protein) and Sad1 (an SPB protein). Significantly, when both Bqt1 and Bqt2 are ectopically expressed in mitotic cells, they also form a bridge between Rap1 and Sad1. Thus, a complex including Bqt1 and Bqt2 is essential for connecting telomeres to the SPB.

Cell Nucleus↗
PubMed2006

Modulation of Alp4 function in Schizosaccharomyces pombe induces novel phenotypes that imply distinct functions for nuclear and cytoplasmic gamma-tubulin complexes.

The gamma-tubulin complex acts as a nucleation unit for microtubule assembly. It remains unknown, however, how spatial and temporal regulation of the complex activity affects microtubule-mediated cellular processes. Alp4 is one of the essential components of the S. pombe gamma-tubulin complex. We show here that overproduction of a carboxy-terminal form of Alp4 (Alp4C) and its derivatives tagged to a nuclear localization signal or to a nuclear export signal affect localization of gamma-tubulin complexes and induces novel phenotypes that reflect distinct functions of nuclear and cytoplasmic gamma-tubulin complexes. Nuclear Alp4C induces a Wee1-dependent G2 delay, reduces the levels of the gamma-tubulin complex at the spindle pole body, and results in defects in mitotic progression including spindle assembly, cytoplasmic microtubule disassembly, and chromosome segregation. In contrast, cytoplasmic Alp4C induces oscillatory nuclear movement and affects levels of cell polarity markers, Bud6 and Tip1, at the cell ends. These results demonstrate that regulation of nuclear gamma-tubulin complex activity is essential for cell cycle progression through the G2/M boundary and M phase, whereas regulation of cytoplasmic gamma-tubulin complex activity is important for nuclear positioning and cell polarity control during interphase.

Cell Cycle Proteins↗
PubMed2006

The carboxy-terminus of Alp4 alters microtubule dynamics to induce oscillatory nuclear movement led by the spindle pole body in Schizosaccharomyces pombe.

Alp4 is an essential component of the S. pombe gamma-tubulin complex. Overproduction of the carboxy-terminus of Alp4 induces oscillatory nuclear movement led by the spindle pole body (SPB). The movement is not dependent on cytoplasmic dynein dhc1, or kinesin-related proteins pkl1 and klp2. Rates of SPB movement correlate with elongation rates of microtubules (MTs) extending backwards from the moving SPB (backward-extending MTs), showing that pushing forces exerted by backward-extending MTs move the nucleus via the SPB. These backward-extending MTs are more stable than those of control cells and, thus, are able to push the SPB further towards the cell end, inducing nuclear oscillation with larger amplitudes than in control cells. SPB movement is biased towards the new end of the cell where levels of the CLIP170 homolog Tip1 increase, suggesting that the movement is related to MT-mediated cell polarity control. These results demonstrate that the carboxy-terminus of Alp4 alters MT dynamics and induces nuclear oscillation by modulating a nuclear positioning mechanism based on the balance of MT pushing forces, and suggest that regulation of gamma-tubulin complex activity is important for controlling MT dynamics and nuclear positioning.

Biological Clocks↗
PubMed2005

Assembly of additional heterochromatin distinct from centromere-kinetochore chromatin is required for de novo formation of human artificial chromosome.

Alpha-satellite (alphoid) DNA is necessary for de novo formation of human artificial chromosomes (HACs) in human cultured cells. To investigate the relationship among centromeric, transcriptionally permissive and non-permissive chromatin assemblies on de novo HAC formation, we constructed bacterial artificial chromosome (BAC)-based linear HAC vectors whose left vector arms are occupied by beta geo coding genes with or without a functional promoter in addition to a common marker gene on the right arm. Although HACs were successfully generated from the vectors with promoter-less constructs on the left arm in HT1080 cells, we failed to generate a stable HAC from the vectors with a functional promoter on the left arm. Despite this failure in HAC formation, centromere components (CENP-A, CENP-B and CENP-C) assembled at the integration sites correlating with a transcriptionally active state of both marker genes on the vector arms. However, on the stable HAC, chromatin immunoprecipitation analysis showed that HP1alpha and trimethyl histone H3-K9 were enriched at the non-transcribing left vector arm. A transcriptionally active state on both vector arms is not compatible with heterochromatin formation on the introduced BAC DNA, suggesting that epigenetic assembly of heterochromatin is distinct from centromere chromatin assembly and is required for the establishment of a stable artificial chromosome.

Cell Line, Tumor↗
PubMed2005

Characterization of rec15, an early meiotic recombination gene in Schizosaccharomyces pombe.

In S. pombe strains mutant for rec15 aberrant ascus morphology, reduced spore viability and severe reduction of meiotic recombination was detected. Genetic and cytological analysis identified frequent interruption of meiosis after the first division, and nondisjunction I, as the main segregation errors in the mutant. Chromosome segregation at meiosis I was not random in rec15, suggesting the presence of a backup system for correct segregation of achiasmate chromosomes. The analysis of meiotic progression in time-course experiments revealed that the major meiotic events, such as the onset of premeiotic DNA synthesis, of horse-tail nuclear movement, and of the first meiotic division occurred earlier in rec15 than in wild-type. The early onset of meiotic events is a novel observation for an early recombination mutant and implies a function of rec15 protein already at or before DNA synthesis.

Aneuploidy↗
PubMed2005

Dissociation of the Nuf2-Ndc80 complex releases centromeres from the spindle-pole body during meiotic prophase in fission yeast.

In the fission yeast Schizosaccharomyces pombe, centromeres remain clustered at the spindle-pole body (SPB) during mitotic interphase. In contrast, during meiotic prophase centromeres dissociate from the SPB. Here we examined the behavior of centromere proteins in living meiotic cells of S. pombe. We show that the Nuf2-Ndc80 complex proteins (Nuf2, Ndc80, Spc24, and Spc25) disappear from the centromere in meiotic prophase when the centromeres are separated from the SPB. The centromere protein Mis12 also dissociates during meiotic prophase; however, Mis6 remains throughout meiosis. When cells are induced to meiosis by inactivation of Pat1 kinase (a key negative regulator of meiosis), centromeres remain associated with the SPB during meiotic prophase. However, inactivation of Nuf2 by a mutation causes the release of centromeres from the SPB in pat1 mutant cells, suggesting that the Nuf2-Ndc80 complex connects centromeres to the SPB. We further found that removal of the Nuf2-Ndc80 complex from the centromere and centromere-SPB dissociation are caused by mating pheromone signaling. Because pat1 mutant cells also show aberrant chromosome segregation in the first meiotic division and this aberration is compensated by mating pheromone signaling, dissociation of the Nuf2-Ndc80 complex may be associated with remodeling of the kinetochore for meiotic chromosome segregation.

Base Sequence↗
PubMed2005

Ect2 and MgcRacGAP regulate the activation and function of Cdc42 in mitosis.

Although Rho regulates cytokinesis, little was known about the functions in mitosis of Cdc42 and Rac. We recently suggested that Cdc42 works in metaphase by regulating bi-orient attachment of spindle microtubules to kinetochores. We now confirm the role of Cdc42 by RNA interference and identify the mechanisms for activation and down-regulation of Cdc42. Using a pull-down assay, we found that the level of GTP-Cdc42 elevates in metaphase, whereas the level of GTP-Rac does not change significantly in mitosis. Overexpression of dominant-negative mutants of Ect2 and MgcRacGAP, a Rho GTPase guanine nucleotide exchange factor and GTPase activating protein, respectively, or depletion of Ect2 by RNA interference suppresses this change of GTP-Cdc42 in mitosis. Depletion of Ect2 also impairs microtubule attachment to kinetochores and causes prometaphase delay and abnormal chromosomal segregation, as does depletion of Cdc42 or expression of the Ect2 and MgcRacGAP mutants. These results suggest that Ect2 and MgcRacGAP regulate the activation and function of Cdc42 in mitosis.

Autoantigens↗
PubMed2005

The constitutive centromere component CENP-50 is required for recovery from spindle damage.

We identified CENP-50 as a novel kinetochore component. We found that CENP-50 is a constitutive component of the centromere that colocalizes with CENP-A and CENP-H throughout the cell cycle in vertebrate cells. To determine the precise role of CENP-50, we examined its role in centromere function by generating a loss-of-function mutant in the chicken DT40 cell line. The CENP-50 knockout was not lethal; however, the growth rate of cells with this mutation was slower than that of wild-type cells. We observed that the time for CENP-50-deficient cells to complete mitosis was longer than that for wild-type cells. Centromeric localization of CENP-50 was abolished in both CENP-H- and CENP-I-deficient cells. Coimmunoprecipitation experiments revealed that CENP-50 interacted with the CENP-H/CENP-I complex in chicken DT40 cells. We also observed severe mitotic defects in CENP-50-deficient cells with apparent premature sister chromatid separation when the mitotic checkpoint was activated, indicating that CENP-50 is required for recovery from spindle damage.

Amino Acid Sequence↗
PubMed2004

Cadherin activity is required for activity-induced spine remodeling.

Neural activity induces the remodeling of pre- and postsynaptic membranes, which maintain their apposition through cell adhesion molecules. Among them, N-cadherin is redistributed, undergoes activity-dependent conformational changes, and is required for synaptic plasticity. Here, we show that depolarization induces the enlargement of the width of spine head, and that cadherin activity is essential for this synaptic rearrangement. Dendritic spines visualized with green fluorescent protein in hippocampal neurons showed an expansion by the activation of AMPA receptor, so that the synaptic apposition zone may be expanded. N-cadherin-venus fusion protein laterally dispersed along the expanding spine head. Overexpression of dominant-negative forms of N-cadherin resulted in the abrogation of the spine expansion. Inhibition of actin polymerization with cytochalasin D abolished the spine expansion. Together, our data suggest that cadherin-based adhesion machinery coupled with the actin-cytoskeleton is critical for the remodeling of synaptic apposition zone.

Actins↗
PubMed2004

Activation of the pheromone-responsive MAP kinase drives haploid cells to undergo ectopic meiosis with normal telomere clustering and sister chromatid segregation in fission yeast.

Meiosis is a process of importance for sexually reproducing eukaryotic organisms. In the fission yeast Schizosaccharomyces pombe, meiosis normally proceeds in a diploid zygote which is produced by conjugation of haploid cells of opposite mating types. We demonstrate that activation of the pheromone-responsive MAPK, Spk1, by the ectopic expression of a constitutively active form of Byr1 (MAPKK for Spk1) induced the cells to undergo meiosis while in the haploid state. Moreover, the induction of meiosis required Mei2 (a key positive regulator of meiosis), but did not require Mei3; Mei3 is normally required to inactivate the Pat1 kinase (a negative regulator of Mei2) thereby allowing Mei2 to drive meiosis. Therefore, expression of a constitutively active form of Byr1 activates Mei2 without the need of Mei3. In cells induced to undergo meiosis by activating the Spk1 MAPK signaling pathway, telomeres clustered at the spindle pole body (SPB) and centromeres detached normally from the SPB during meiotic prophase, and the cells showed the correct segregation of sister chromatids during meiotic divisions. In contrast, in meiosis induced by inactivation of Pat1, sister chromatids segregate precociously during the first meiotic division. Thus, these results suggest that activation of Spk1 drives meiosis in S. pombe.

Binding Sites↗
PubMed2004

Nuclear structure-associated TIF2 recruits glucocorticoid receptor and its target DNA.

Assembly of multi-protein complexes on promoter and enhancer elements is a prerequisite for onset of gene transcription. At the beginning of this process, transcription factors are thought to act as nucleating centers for complex formation through the binding of their target DNA sequences, and thereafter recruit coactivators. Here, we investigated this process of assembly by determining the distribution of the glucocorticoid receptor (GR) and its coactivator, TIF2. Both endogenously and ectopically expressed TIF2 were shown to form foci in the nucleus, and GR could be recruited to the TIF2 foci upon GR agonist but not antagonist treatment. Moreover, we show that the coactivators, p300 and PCAF, are also recruited to the TIF2 foci. The TIF2 foci could recruit GR carrying a microinjected GR responsive element. We propose that TIF2 provides a nuclear compartment that allows the assembly of multi-protein complexes required for GR-mediated gene activation.

Animals↗