Biology subjects

Nagasaka, K.

Publications and source records attributed to Nagasaka, K..

13 recordsLinked to original sources

bioRxiv2026

Selective activation of LH-dependent transcriptional pathways determines ovulatory follicles in the hierarchical ovary of cloudy catshark

The number of offspring produced per reproductive cycle is generally explained by a luteinizing hormone receptor (LHR)-threshold model, in which only follicles expressing sufficient levels of LHR respond to the LH surge and proceed to ovulation. Using the cloudy catshark as a model, we reveal a distinct mechanism that determines the fate of ovulatory (F1) and non-ovulatory (F2) follicles independently of LHR abundance. The cloudy catshark produces only two eggs per reproductive cycle by ovulating a pair of F1 follicles from its single ovary. Both F1 and F2 follicles express comparable levels of lhr, indicating that both retain the capacity to receive the LH signal. Nevertheless, LH stimulation selectively activated ovulation-associated responses in F1 follicles. These included progesterone production via star2 upregulation, as well as transcriptional programs including RUNX transcription factors, prostaglandin signaling (ptgs2, ptger1), and matrix metalloproteinases. Moreover, direct activation of cAMP pathway, bypassing LHR activation, induced progesterone production in F1 but not F2 follicles, indicating that the critical difference in ovulatory responsiveness lies downstream of cAMP. These results demonstrate that follicular ovulatory fate is determined, at least in part, by follicle-specific transcriptional responses to cAMP, rather than by LHR abundance alone, challenging the prevailing LHR-threshold model of ovulatory regulation in vertebrates.

physiology↗

bioRxiv2025

PDS5 proteins control genome architecture by limiting the lifetime of cohesin-NIPBL complexes

Cohesin-NIPBL complexes extrude genomic DNA into loops that are constrained by CTCF boundaries. This process has important regulatory functions and weakens the separation between euchromatic and heterochromatic compartments. Cohesin can also bind PDS5A or PDS5B, which do not support loop extrusion but are required for the formation of CTCF boundaries. How PDS5 proteins perform this function is unknown. Here we show by in vitro single-molecule imaging that PDS5 proteins stop loop extrusion by facilitating the dissociation of NIPBL from cohesin. Hi-C experiments suggest that this function is required for the establishment of CTCF boundaries in cells. In silico modelling indicates that PDS5 proteins enable the separation between compartments by limiting cohesins velocity and chromatin-residence time. The degree of this compartmentalization depends on the frequency with which chromatin is extruded relative to the time it takes for compartments to form. These results identify PDS5 proteins as key regulators of genome organization. HighlightsO_LIPDS5 proteins stop loop extrusion by facilitating dissociation of NIPBL from cohesin. C_LIO_LIPDS5 proteins strengthen CTCF boundaries by limiting the lifetime of cohesin-NIPBL. C_LIO_LIPDS5 proteins regulate compartmentalization. C_LIO_LICompartmentalization is governed by polymer relaxation and loop extrusion dynamics. C_LI

molecular biology↗

bioRxiv2025

Giant viruses specific to deep oceans show persistent presence and activity

Giant viruses (GVs) of the phyla Nucleocytoviricota and Mirusviricota are large double-stranded DNA viruses that infect diverse eukaryotic hosts and impact biogeochemical cycles. Their diversity and ecological roles have been well studied in the photic layer of the ocean, but less is known about their activity, population dynamics, and adaptive strategies in the aphotic layers. Here, we conducted eight seasonal time-series samplings of the surface and mesopelagic layers at a coastal site in Muroto, Japan, and integrated 18S metabarcoding, metagenomic, and metatranscriptomic data to investigate deep-sea GVs and their potential hosts. The analysis identified 48 GV genomes including six that were exclusively detected in the mesopelagic layer. Notably, these mesopelagic-specific GVs showed persistent activity across seasons. To investigate the global deep-sea-specific GV distribution, we compiled GV reference genomic data from the OceanDNA MAG project and other resources, and analyzed 1,890 marine metagenomes. This revealed 101 deep-sea-specific GVs, distributed across the GV phylogenetic tree, indicating that adaptation to deep-sea environments has occurred in multiple lineages. One clade enriched with deep-sea-specific GVs included one GV identified in our Muroto sampling, which displayed a wide geographic distribution. Seventy-six KEGG orthologs and 74 Pfam domains were specifically enriched in deep-sea-specific GVs, encompassing functions related to the ubiquitin system, energy metabolism, and nitrogen acquisition. These findings support the scenario that distinct GV lineages have adapted to hosts in aphotic marine environments by altering their gene repertoire to thrive in this unique habitat. IMPORTANCEGiant viruses are widespread in the ocean surface and are key in shaping marine ecosystems by infecting phytoplankton and other protists. However, little is known about their activity and adaptive strategies in deep-sea environments. In this study, we performed metagenomic and metatranscriptomic analyses of seawater samples collected from a coastal site in Japan and discovered giant virus genomes showing persistent transcriptional activity across seasons in the deep-sea water. Using a global marine dataset, we further uncovered the widespread existence of deep-sea-specific giant viruses and characterized their unique gene repertoire, which likely facilitates adaptation to the limited availability of light and organic compounds in the aphotic zone. These findings expand our understanding of giant virus ecology in the dark ocean.

genomics↗

bioRxiv2024

Cohesin supercoils DNA during loop extrusion

Cohesin extrudes genomic DNA into loops that promote chromatin assembly, gene regulation and recombination. Here we show that cohesin introduces negative supercoils into extruded DNA. Supercoiling requires engagement of cohesins ATPase heads, DNA clamping by these heads, and a DNA binding site on cohesins hinge, indicating that cohesin supercoils DNA when constraining it between the hinge and the clamp. Our results suggest that DNA extrusion stops once cohesin reaches its stall torque during supercoiling, and a cohesin mutant predicted to stall at lower torque forms shorter loops in cells. These results indicate that supercoiling is an integral part of the loop extrusion mechanism and that cohesin controls genome architecture not only by looping DNA but also by supercoiling it.

biochemistry↗

bioRxiv2023

Injectable microcapillary network hydrogels engineered by liquid-liquid phase separation for stem cell transplantation

Injectable hydrogels are promising carriers for cell delivery in regenerative medicine. However, injectable hydrogels composed of crosslinked polymer networks are often non porous and prevent biological communication with host tissues through signals, nutrients, oxygen, and cells, thereby limiting graft survival and tissue integration. Here we report injectable hydrogels with liquid-liquid phase separation-induced microcapillary networks ({micro}CN) as stem cell-delivering scaffolds. The molecular modification of gelatin with hydrogen bonding moieties induced liquid-liquid phase separation when mixed with unmodified gelatin to form {micro}CN structures in the hydrogels. Through spatiotemporally controlled covalent crosslinking and dissolution processes, porous {micro}CN structures were formed in the hydrogels, which can enhance mass transport and cellular activity. The encapsulation of cells with injectable {micro}CN hydrogels improved cellular adhesion, spreading, migration, and proliferation. Transplantation of mesenchymal stem cells with injectable {micro}CN hydrogels enhanced graft survival and recovered hindlimb ischemia by enhancing material-tissue communication with biological signals and cells through {micro}CN. This facile approach may serve as an advanced scaffold for improving stem cell transplantation therapies in regenerative medicine.

bioengineering↗

bioRxiv2022

Cohesin mediates DNA loop extrusion and sister chromatid cohesion by distinct mechanisms

Cohesin connects CTCF binding sites and other genomic loci in cis to form chromatin loops, and replicated DNA molecules in trans to mediate sister chromatid cohesion. Whether cohesin uses distinct or related mechanisms to perform these functions is unknown. Here we describe a cohesin hinge mutant, which can extrude DNA into loops but is unable to mediate cohesion. Our results suggest that the latter defect arises during cohesion establishment. The observation that cohesins cohesion and loop extrusion activities can be separated indicates that cohesin uses distinct mechanisms to perform these two functions. Unexpectedly, the same hinge mutant can also not be stopped by CTCF boundaries as well as wildtype cohesin. This suggests that cohesion establishment and cohesins interaction with CTCF boundaries depend on related mechanisms and raises the possibility that both require transient hinge opening to entrap DNA inside the cohesin ring.

cell biology↗

bioRxiv2022

CTCF is a DNA-tension-dependent barrier to cohesin-mediated DNA loop extrusion

In eukaryotes, genomic DNA is extruded into loops by cohesin1. By restraining this process, the DNA-binding protein CTCF generates topologically associating domains (TADs)2-4 that play key roles in gene regulation and recombination during development and disease1,5-8. How CTCF establishes TAD boundaries and to what extent these are permeable to cohesin is unknown9. To address these questions, we visualize interactions of single CTCF and cohesin molecules on DNA in vitro. We show that CTCF is sufficient to block diffusing cohesin, possibly reflecting how cohesive cohesin accumulates at TAD boundaries, as well as to block loop-extruding cohesin, reflecting how CTCF establishes TAD boundaries. CTCF functions asymmetrically, as predicted, but unexpectedly is dependent on DNA tension. Moreover, CTCF regulates cohesins loop extrusion activity by changing its direction and by inducing loop shrinkage. Our data indicate that CTCF is not, as previously assumed, simply a barrier to cohesin-mediated loop extrusion but is an active regulator of this process, where the permeability of TAD boundaries can be modulated by DNA tension. These results reveal mechanistic principles of how CTCF controls loop extrusion and genome architecture.

biophysics↗

bioRxiv2022

Degenerate oligonucleotide primer MIG-seq: an effective PCR-based method for high-throughput genotyping

Multiplexed inter-simple sequence repeats genotyping by sequencing (MIG-seq) is an next-generation sequencing library construction method developed for the analysis of DNA in ecology. Although MIG-seq can generate libraries from low-quality DNA, few polymorphisms can be obtained in species with small genomes. In this study, we developed degenerate oligonucleotide primer MIG-seq (dpMIG-seq) as an effective polymorphism discovery method that allows for variation in the number of polymorphisms while retaining the advantages of MIG-seq, including independence from DNA quality. In dpMIG-seq, a proportion of the simple sequence repeats in the primer sequence of the first PCR in MIG-seq was changed to degenerate oligonucleotides to enable annealing to a wider range of sequences. In tests of several crop species other than wheat, the number of loci that could be sequenced using dpMIG-seq with a data volume of 0.3 gigabases (Gb) was increased compared with that sequenced using MIG-seq. In wheat, the number of polymorphisms obtained via dpMIG-seq was higher than that obtained via MIG-seq when a data volume of about [≥]2 Gb was obtained. In dpMIG-seq, different loci could be sequenced by changing the positions of the degenerate oligonucleotides. By applying dpMIG-seq, we constructed a linkage map consisting of 5,142 markers for the rice inter-subspecies F2 population, and we detected quantitative trait loci for heading date in the regions where known heading-related genes were located. Overall, our results show that dpMIG-seq is a useful tool for the genetic analysis of crop species.

genetics↗

bioRxiv2021

Hotmelt tissue adhesive with supramolecularly-controlled sol-gel transition for preventing postoperative abdominal adhesion

Postoperative adhesion is a serious and frequent complication, but there is currently no reliable anti-adhesive barrier available due to low tissue adhesiveness, undesirable chemical reactions, and poor operability. Here, we report a single-syringe hotmelt tissue adhesive to prevent postoperative abdominal adhesions. Through the augmentation of intermolecular hydrogen bonding by conjugation of the ureidopyrimidinone unit to tendon-derived gelatin, the sol-gel transition behavior of gelatin was supramolecularly-controlled, which provided a hotmelt tissue adhesive that dissolves upon warming over 40 {degrees}C and glues at 37 {degrees}C. This functionalization improved the key features necessary for an anti-adhesive barrier, including bulk mechanical strength, tissue adhesive properties, underwater stability, and anti-adhesive property. This hotmelt tissue adhesive with excellent tissue adhesiveness, biocompatibility, and operability has enormous potential to prevent postoperative complications.

bioengineering↗

bioRxiv2019

ESCO1 and CTCF enable formation of long chromatin loops by protecting cohesinSTAG1 from WAPL

Eukaryotic genomes are folded into loops. It is thought that these are formed by cohesin complexes via extrusion, either until loop expansion is arrested by CTCF or until cohesin is removed from DNA by WAPL. Although WAPL limits cohesins chromatin residence time to minutes, it has been reported that some loops exist for hours. How these loops can persist is unknown. We show that during G1-phase, mammalian cells contain acetylated cohesinSTAG1 which binds chromatin for hours, whereas cohesinSTAG2 binds chromatin for minutes. Our results indicate that CTCF and the acetyltransferase ESCO1 protect a subset of cohesinSTAG1 complexes from WAPL, thereby enable formation of long and presumably long-lived loops, and that ESCO1, like CTCF, contributes to boundary formation in chromatin looping. Our data are consistent with a model of nested loop extrusion, in which acetylated cohesinSTAG1 forms stable loops between CTCF sites, demarcating the boundaries of more transient cohesinSTAG2 extrusion activity.

cell biology↗

bioRxiv2019

Absolute quantification of cohesin, CTCF and their regulators in human cells

The organisation of mammalian genomes into loops and topologically associating domains (TADs) contributes to chromatin structure, gene expression and recombination. Loops and TADs are formed by cohesin and positioned by CTCF. In proliferating cells, cohesin also mediates sister chromatid cohesion, which is essential for chromosome segregation. Current models of chromatin folding and cohesion are based on assumptions of how many cohesin and CTCF molecules organise the genome. Here we have measured absolute copy numbers and dynamics of cohesin, CTCF, NIPBL, WAPL and sororin by mass spectrometry, fluorescence-correlation spectroscopy and fluorescence recovery after photobleaching in HeLa cells. In G1-phase there are ~245,000 cohesin complexes, of which ~139,000 are on chromatin. Comparison with chromatin immunoprecipitation-sequencing data implies that some genomic cohesin and CTCF enrichment sites are unoccupied in single cells at any one time. We discuss the implications of these findings for how cohesin can contribute to genome organisation and cohesion.

cell biology↗

bioRxiv2019

Werner syndrome helicase is a selective vulnerability of microsatellite instability high tumor cells

Targeted cancer therapy is based on exploiting selective dependencies of tumor cells. By leveraging recent large-scale genomic profiling and functional screening of cancer cell lines we identified Werner syndrome helicase (WRN) as a novel specific vulnerability of microsatellite instability-high (MSI-H) cancer cells. MSI, caused by defective mismatch repair is frequently detected in human malignancies, in particular in colorectal, endometrial and gastric cancers. We demonstrate that WRN inactivation selectively impairs the viability of MSI-H but not microsatellite stable (MSS) colorectal and endometrial cancer cell lines. In MSI-H cells, WRN loss results in the emergence of chromosome breaks, chromatin bridges and micronuclei highlighting defective genome integrity. WRN variants harboring mutations abrogating the ATPase function of WRN helicase fail to rescue the viability phenotype of WRN-depleted MSI-H colorectal cells. Our study suggests that pharmacological inhibition of WRN helicase function might represent a novel opportunity to develop a targeted therapy for MSI-H cancers.

cancer biology↗

bioRxiv2017

CTCF, WAPL and PDS5 proteins control the formation of TADs and loops by cohesin

Mammalian genomes are organized into compartments, topologically-associating domains (TADs) and loops to facilitate gene regulation and other chromosomal functions. Compartments are formed by nucleosomal interactions, but how TADs and loops are generated is unknown. It has been proposed that cohesin forms these structures by extruding loops until it encounters CTCF, but direct evidence for this hypothesis is missing. Here we show that cohesin suppresses compartments but is essential for TADs and loops, that CTCF defines their boundaries, and that WAPL and its PDS5 binding partners control the length of chromatin loops. In the absence of WAPL and PDS5 proteins, cohesin passes CTCF sites with increased frequency, forms extended chromatin loops, accumulates in axial chromosomal positions (vermicelli) and condenses chromosomes to an extent normally only seen in mitosis. These results show that cohesin has an essential genome-wide function in mediating long-range chromatin interactions and support the hypothesis that cohesin creates these by loop extrusion, until it is delayed by CTCF in a manner dependent on PDS5 proteins, or until it is released from DNA by WAPL.

genomics↗