Biology subjects

Tashiro, S.

Publications and source records attributed to Tashiro, S..

9 recordsLinked to original sources

bioRxiv2024

Spatial organization of supercoil dynamics during DNA replication

Progression of DNA replication inevitably generates helical tension and resulting super-helical structures, or supercoils, that originate from the double helix of DNA. As DNA polymerases progress, negative supercoils accumulate in the wake of the replication fork, thereby impeding fork progression unless the supercoils are resolved topologically. Using super-resolution microscopy combined with spatial distribution analysis, here we describe that SMC5/6-mediated confinement of negative supercoils lies at the basis of releasing them by topoisomerase Top2A. Along with DNA replication, SMC5/6 progressively associates with chromatin and locally increases the density of supercoil clusters behind the fork, allowing Top2A to efficiently target and release the accumulated supercoils. These processes are essential to complete DNA replication prior to mitosis and therefore to ensure genome stability. Remarkably, we found that HeLa cells over-accumulate poorly confined negative supercoils beyond the processing capacity of cellular SMC5/6, which may exemplify the condition associated with genome instability of cancer cells.

cell biology↗

bioRxiv2024

Local chromatin decompaction shapes mitotic chromosome landscape

Unregulated activation of transcription on mitotic chromosomal arms can be detrimental to faithful chromosome segregation1-3, although a subset of genes are up-regulated during mitosis4, raising the question as to how gene activation is coordinated in the context of mitotic chromosome reorganization. Here we investigate the fine structure of mitotic chromosomes in fission yeast Schizosaccharomyces pombe and reveal that intense decompaction of local chromatin upon mitotic gene activation helps shape the mitotic chromosome landscape. More specifically, we show that binding of mitosis-specific transcription factors, Ace2 and MluI binding factor (MBF)5,6, to gene promoters followed by transcriptional elongation can locally induce chromatin decompaction during mitosis. Interestingly, local decompaction-competent genes can establish chromatin boundaries that demarcate long-range contact domains, indicating tight coupling between local and global genome organizations. Furthermore, efficient local decompaction requires enough transcriptional elongation distance and histone removal, providing a mechanistic insight into the decompaction-associated domain boundary formation. Given that the demarcation of large, self-associating domains is critical for faithful chromosome segregation7, we propose that decompaction-competent genes can be a key determinant of mitotic chromosome configuration, thereby underpinning the maintenance of genome integrity over generations, and thus their distribution along chromosomes can be subject to evolutionary selection.

molecular biology↗

bioRxiv2024

A role for condensin-mediator interaction in mitotic chromosomal organization

Eukaryotic genomes are organized by condensin into 3D chromosomal architectures suitable for chromosomal segregation during mitosis. However, molecular mechanisms underlying the condensin-mediated chromosomal organization remain largely unclear. Here, we investigate the role of newly identified interaction between the Cnd1 condensin and Pmc4 mediator subunits in fission yeast, Schizosaccharomyces pombe. We develop a condensin mutation, cnd1-K658E, that impairs the condensin-mediator interaction and find that this mutation diminishes condensin-mediated chromatin domains during mitosis and causes chromosomal segregation defects. The condensin-mediator interaction is involved in recruiting condensin to highly transcribed genes and mitotically activated genes, the latter of which demarcate condensin-mediated domains. Furthermore, this study predicts that mediator-driven transcription of mitotically activated genes contributes to forming domain boundaries via phase separation. This study provides a novel insight into how genome-wide gene expression during mitosis is transformed into the functional chromosomal architecture suitable for chromosomal segregation. O_FIG O_LINKSMALLFIG WIDTH=189 HEIGHT=200 SRC="FIGDIR/small/600663v1_ufig1.gif" ALT="Figure 1"> View larger version (46K): org.highwire.dtl.DTLVardef@488ab5org.highwire.dtl.DTLVardef@8a7a73org.highwire.dtl.DTLVardef@16d416eorg.highwire.dtl.DTLVardef@1e2d3db_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract C_FIG In BriefMitotic chromosomes are organized by condensin into an architecture suitable for their segregation, although molecular mechanisms underlying how condensin assembles the functional chromosomal architecture remain unclear. This study with the fission yeast model organism shows that the protein interaction between condensin and mediator is involved in chromosomal segregation by forming a mitotic chromosomal architecture. A similar mechanism involving the condensin-mediator interaction is likely conserved in human cells, implying the evolutionary conservation of this mechanism. HighlightsO_LIThe Cnd1 condensin subunit interacts with the Pmc4 mediator component in fission yeast. C_LIO_LIThe condensin-mediator interaction is required to recruit condensin to highly transcribed and mitotically activated genes. C_LIO_LIThe condensin-mediator interaction is required to form chromatin domains during mitosis. C_LIO_LIMediator participates in the boundary formation of mitotic chromatin domains. C_LI

genomics↗

bioRxiv2023

Dose-dependent effects of histone methyltransferase NSD2 on site-specific double-strand break repair

Histone modifications are catalyzed and recognized by specific proteins to regulate dynamic DNA metabolism processes. NSD2 is a histone H3 lysine 36 (H3K36)-specific methyltransferase that associates both with various transcription regulators and DNA repair factors. Specifically, it has been implicated in the repair of DNA double-strand breaks (DSBs); however, the role of NSD2 during DSB repair remains enigmatic. Here, we show that NSD2 does not accumulate at DSB sites and that the localization of NSD2 at chromatin is maintained even after DSB formation. Using three different DSB repair reporter systems, which contained the endonuclease site in the active thymidine kinase gene (TK) locus, we demonstrated separate dose-dependent effects of NSD2 on HR, canonical-NHEJ (c-NHEJ), and non-canonical-NHEJ (non-c-NHEJ). Endogenous NSD2 has a role in repressing non-c-NHEJ, without affecting DSB repair efficiency by HR or total NHEJ. Furthermore, overexpression of NSD2 promotes c-NHEJ repair suppressing HR repair. Therefore, we propose that NSD2 has functions in chromatin integrity at the active regions during DSB repair.

cell biology↗

bioRxiv2021

Rec8 cohesin-mediated loop-axis chromatin architecture is required for meiotic recombination

During meiotic prophase, cohesin-dependent axial structures are formed in the synaptonemal complex (SC). However, the functional correlation between these structures and cohesion remains elusive. Here, we examined the formation of cohesin-dependent axial structures in the fission yeast Schizosaccharomyces pombe. This organism forms atypical SCs composed of linear elements (LinEs) resembling the lateral elements of SC but lacking the transverse filaments. Hi-C analysis using a highly synchronous population of meiotic S. pombe cells revealed that the axis-loop chromatin structure formed in meiotic prophase was dependent on the Rec8 cohesin complex. In contrast, the Rec8-mediated formation of the axis-loop structure occurred in cells lacking components of LinEs. To dissect the functions of Rec8, we identified a rec8-F204S mutant that lost the ability to assemble the axis-loop structure without losing cohesion of sister chromatids. This mutant showed defects in the formation of the axis-loop structure and LinE assembly and thus exhibited reduced meiotic recombination. Collectively, our results demonstrate that the Rec8-dependent axis-loop structure provides a structural platform essential for LinE assembly, facilitating meiotic recombination of homologous chromosomes, independently of its role in sister chromatid cohesion.

cell biology↗

bioRxiv2021

Distinctive nuclear zone for RAD51-mediated homologous recombinational DNA repair

Genome-based functions are inseparable from the dynamic higher-order architecture of the cell nucleus. In this context, the repair of DNA damage is coordinated by precise spatiotemporal controls that target and regulate the repair machinery required to maintain genome integrity. However, the mechanisms that pair damaged DNA with intact template for repair by homologous recombination (HR) without illegitimate recombination remain unclear. This report highlights the intimate relationship between nuclear architecture and HR in mammalian cells. RAD51, the key recombinase of HR, forms spherical foci in S/G2 phases spontaneously. Using super-resolution microscopy, we show that following induction of DNA double-strand breaks RAD51 foci at damaged sites elongate to bridge between intact and damaged sister chromatids; this assembly occurs within bundle-shaped distinctive nuclear zones, requires interactions of RAD51 with various factors, and precedes ATP-dependent events involved the recombination of intact and damaged DNA. We observed a time-dependent transfer of single-stranded DNA overhangs, generated during HR, into such zones. Our observations suggest that RAD51-mediated homologous pairing during HR takes place within the distinctive nuclear zones to execute appropriate recombination.

cell biology↗

bioRxiv2020

Complete sequences of Schizosaccharomyces pombe subtelomeres reveal multiple patterns of genome variation

Genome sequences have been determined for many model organisms; however, repetitive regions such as centromeres, telomeres, and subtelomeres have not yet been sequenced completely. Here, we report the complete sequences of subtelomeric homologous (SH) regions of the fission yeast Schizosaccharomyces pombe. We overcame technical difficulties to obtain subtelomeric repetitive sequences by constructing strains that possess single SH regions. Whole sequences of SH regions revealed that each SH region consists of two distinct parts: the telomere-proximal part with mosaics of multiple common segments showing high variation among subtelomeres and strains, and the telomere-distal part showing high sequence similarity among subtelomeres with some insertions and deletions. The newly sequenced SH regions showed differences in nucleotide sequences and common segment composition compared to those in the S. pombe genome database (PomBase), which is in striking contrast to the regions outside of SH, where mutations are rarely detected. Furthermore, we identified new subtelomeric RecQ-type helicase genes, tlh3 and tlh4, which add to the already known tlh1 and tlh2, and found that the tlh1-4 genes show high sequence variation. Our results indicate that SH sequences are highly polymorphic and hot spots for genome variation. These features of subtelomeres may have contributed to genome diversity and, conversely, various diseases.

genomics↗

bioRxiv2019

A Single Defined Sister Chromatid Fusion Destabilizes Cell Cycle through Micronuclei Formation

Chromosome fusion is deleterious among oncogenic chromosome rearrangements, and has been proposed to cause multiple tumor-driving abnormalities. Conventional methodologies, however, lack the strictness of the experimental controls on the fusion such as the exact timing, the number and the types of fusion in a given cell. Here, we developed a human cell-based sister chromatid fusion visualization system (FuVis), in which a single defined sister chromatid fusion is induced by CRISPR/Cas9 concomitantly with mCitrine expression. Fused chromosome developed numerical and structural abnormalities, including chromosome fragmentation, an indicative of eventual chromothripsis. Live cell imaging and hierarchical Bayesian modeling indicated that micronucleus (MN) is generated in the first few cell cycle, and that cells with MN tend to possess cell cycle abnormalities. These results demonstrate that, although most cells can tolerate a single fusion, even a single sister chromatid fusion destabilizes cell cycle through MN formation.

cell biology↗

bioRxiv2018

Visualizing multiple inter-organelle contact sites using the organelle-targeted split-GFP system

Functional integrity of eukaryotic organelles relies on direct physical contacts between distinct organelles. However, the entity of organelle-tethering factors is not well understood due to lack of means to analyze inter-organelle interactions in living cells. Here we evaluate the split-GFP system for visualizing organelle contact sites in vivo and show its advantages and disadvantages. We observed punctate GFP signals from the split-GFP fragments targeted to any pairs of organelles among the ER, mitochondria, peroxisomes, vacuole and lipid droplets in yeast cells, which suggests that these organelles form contact sites with multiple organelles simultaneously although it is difficult to rule out the possibilities that these organelle contacts sites are artificially formed by the irreversible associations of the split-GFP probes. Importantly, split-GFP signals in the overlapped regions of the ER and mitochondria were mainly co-localized with ERMES, an authentic ER-mitochondria tethering structure, suggesting that split-GFP assembly depends on the preexisting inter-organelle contact sites. We also confirmed that the split-GFP system can be applied to detection of the ER-mitochondria contact sites in HeLa cells. We thus propose that the split-GFP system is a potential tool to observe and analyze inter-organelle contact sites in living yeast and mammalian cells.

cell biology↗