Biology subjects

Ishii, T.

Publications and source records attributed to Ishii, T..

28 records · Page 2Linked to original sources

bioRxiv2022

Plasticity in centromere organization: Holocentromeres can consist of merely a few megabase-sized satellite arrays

The centromere is the chromosome region where the microtubules attach during cell division. In contrast to monocentric chromosomes with one centromere location, holocentric species usually distribute hundreds of centromere units along the entire chromatid. We assembled the chromosome-scale reference genome and analyzed the holocentromere and (epi)genome organization of the lilioid Chionographis japonica. Remarkably, each of its holocentric chromatids consists of only 7 to 11 evenly-spaced megabase-sized centromere-specific histone H3-positive units. These units contain satellite arrays of 23 and 28 bp-long monomers capable of forming palindromic structures. Like monocentric species, C. japonica forms distinctly clustered centromeres in chromocenters at interphase. Additionally, the large-scale eu- and heterochromatin arrangement differs between C. japonica and other known holocentric species. Using polymer simulations, we modeled the formation of prometaphase line-like holocentromeres from interphase centromere clusters. Our findings broaden the knowledge about the diversity of centromere organization, showing that holocentricity is not restricted to species with numerous and small centromere units.

plant biology↗

bioRxiv2022

Hepatocyte Differentiation From Mouse Liver Ductal Organoids By Transducing Four Liver-Specific Transcription Factors

Background & AimsHepatocyte sources that are expandable in vitro are required for liver regenerative medicine and to elucidate the mechanisms underlying the physiological functions of the liver. Liver ductal organoids (LDOs) comprise liver tissue stem cells with a bipotential capacity to differentiate into hepatocyte and cholangiocyte lineages, and can thus serve as a hepatocyte source. However, using current differentiation methods LDOs differentiate into immature hepatocytes while retaining strong cholangiocyte characteristics. We thus investigated an alternative differentiation method for LDOs to achieve hepatocyte maturation. MethodsWe extracted 12 candidate transcription factors to induce hepatocyte differentiation by comparing their gene expression in LDOs and liver tissues. After evaluating the effects of these transcription factors on LDOs, we analyzed the comprehensive gene expression profile, protein expression, and hepatic function in the transduced organoids. ResultsWe identified a combination of four transcription factors, Hnf4a, Foxa1, Prox1, and Hlf, which upregulated hepatic lineage markers and downregulated cholangiocyte markers. Differentiation-induced LDOs showed more hepatocyte-specific characteristics than those with the conventional method, enhancing the transition from cholangiocyte to hepatocyte lineage and hepatic functions, such as liver-specific protein synthesis, lipid droplet deposition, and ammonia detoxification. ConclusionTransduction of the four transcription factors (Hnf4a, Foxa1, Prox1, Hlf) is a promising strategy to promote the differentiation of LDOs to obtain mature hepatocyte-like cells with better functionality.

cell biology↗

bioRxiv2021

A stepwise route to domesticate rice by controlling seed shattering and panicle shape

Asian rice (Oryza sativa L.) is consumed by more than half of the worlds population. Despite its global importance, the process of early rice domestication remains unclear. During domestication, wild rice (O. rufipogon Griff.) acquired non-seed-shattering behaviour, allowing humans to increase grain yield. Previous studies argued that the sh4 mutation triggered a reduction in seed shattering during rice domestication; but our experiments using wild introgression lines of O. rufipogon show that the domesticated sh4 allele alone is insufficient for shattering loss. Here, we identified the interaction between three key mutations associated with the interruption of abscission layer formation and panicle architecture that were causal in early rice domestication. An interruption of abscission layer formation requires both sh4 and qSH3 mutations, presenting an apparent barrier to the selection of shattering loss. We identified a causal single-nucleotide polymorphism at qSH3 within the seed-shattering gene OsSh1, which is conserved in indica and japonica subspecies but absent in the circum-aus group of rice. Through harvest experiments, we demonstrated that seed shattering alone did not significantly impact yield; rather yield increases were observed with closed panicle formation controlled by SPR3, which is further augmented by the integration of sh4 and qSH3 alleles. Complementary manipulation of seed shattering and panicle shape result in a mechanically stable panicle structure. We propose a stepwise route for the earliest phase of rice domestication, wherein selection of visible SPR3-controlled closed panicle morphology was instrumental in the sequential recruitment of sh4 and qSH3, which led to the loss of shattering. Significance StatementRice is one of the most important crops worldwide. Loss of seed shattering in domesticated rice, previously attributed to single mutations such as those in sh4, is considered the principal genetic change which resulted in yield increases. However, we show that sh4 alone is insufficient and other genes, such as qSH3, are required to cause abscission layer disruption. The evolution of non-seed-shattering therefore required multiple mutations. Furthermore, shattering loss in genetic backgrounds of wild rice does not correspondingly increase yields. We have identified an interaction in which a second trait, closed panicle formation controlled by SPR3, that both increases the yield and facilitates recruitment of sh4 and qSH3, which synergistically augment yield, leading to a stepwise model for rice domestication.

plant biology↗

bioRxiv2021

Liver ductal organoids reconstruct intrahepatic biliary trees in decellularized liver grafts.

Three-dimensional scaffolds decellularized from native organs are a promising technique to establish engineered liver grafts and overcome the current shortage of donor organs. However, limited sources of bile duct cells and inappropriate cell distribution in bioengineered liver grafts have hindered their practical application. Organoid technology is anticipated to be an excellent tool for the advancement of regenerative medicine. In the present study, we reconstructed intrahepatic bile ducts in a rat decellularized liver graft by recellularization with liver ductal organoids. Using an ex vivo perfusion culture system, we demonstrated the biliary characteristics of repopulated mouse liver organoids, which maintained bile duct markers and reconstructed biliary tree-like networks with luminal structures. We also established a method for the co-recellularization with engineered bile ducts and primary hepatocytes, revealing the appropriate cell distribution to mimic the native liver. We then utilized this model in human organoids to demonstrate the reconstructed bile ducts. Our results show that liver ductal organoids are a potential cell source for bile ducts from bioengineered liver grafts using three-dimensional scaffolds.

bioengineering↗

bioRxiv2020

Interspecific introgression and natural selection in the evolution of Japanese apricot (Prunus mume)

O_LIDomestication and population differentiation in crops involve considerable phenotypic changes. The logs of these evolutionary paths, including natural/artificial selection, can be found in the genomes of the current populations. However, these profiles have been little studied in tree crops, which have specific characters, such as long generation time and clonal propagation, maintaining high levels of heterozygosity. C_LIO_LIWe conducted exon-targeted resequencing of 129 genomes in the genus Prunus, mainly Japanese apricot (Prunus mume), and apricot (P. armeniaca), plum (P. salicina), and peach (P. persica). Based on their genome-wide single nucleotide polymorphisms merged with published resequencing data of 79 Chinese P. mume cultivars, we inferred complete and ongoing population differentiation in P. mume. C_LIO_LISliding window characterization of the indexes for genetic differentiation identified interspecific fragment introgressions between P. mume and related species (plum and apricot). These regions often exhibited strong selective sweeps formed in the paths of establishment or formation of substructures of P. mume, suggesting that P. mume has frequently imported advantageous genes from other species in the subgenus Prunus as adaptive evolution. C_LIO_LIThese findings shed light on the complicated nature of adaptive evolution in a tree crop that has undergone interspecific exchange of genome fragments with natural/artificial selection. C_LI

evolutionary biology↗

bioRxiv2020

Interspike intervals within retinal spike bursts combinatorially encode multiple stimulus features

Neurons in various regions of the brain generate spike bursts. While the number of spikes within a burst has been shown to carry information, information coding by interspike intervals (ISIs) is less well understood. In particular, a burst with k spikes has k-1 intraburst ISIs, and these k-1 ISIs could theoretically encode k-1 independent values. In this study, we demonstrate that such combinatorial coding occurs for retinal bursts. By recording ganglion cell spikes from isolated salamander retinae, we found that intraburst ISIs encode oscillatory light sequences that are much faster than the light intensity modulation encoded by the number of spikes. When a burst has three spikes, the two intraburst ISIs combinatorially encode the amplitude and phase of the oscillatory sequence. Analysis of trial-to-trial variability suggested that intraburst ISIs are regulated by two independent mechanisms responding to orthogonal oscillatory components, one of which is common to bursts with different number of spikes. Therefore, the retina encodes multiple stimulus features by exploiting all degrees of freedom of burst spike patterns, i.e., the spike number and multiple intraburst ISIs. Author SummaryNeurons in various regions of the brain generate spike bursts. Bursts are typically composed of a few spikes generated within dozens of milliseconds, and individual bursts are separated by much longer periods of silence ([~]hundreds of milliseconds). Recent evidence indicates that the number of spikes in a burst, the interspike intervals (ISIs), and the overall duration of a burst, as well as the timing of burst onset, encode information. However, it remains unknown whether multiple ISIs within a single burst encode multiple independent information contents. Here we demonstrate that such combinatorial ISI coding occurs for spike bursts in the retina. We recorded ganglion cell spikes from isolated salamander retinae stimulated with computer-generated movies. Visual response analyses indicated that multiple ISIs within a single burst combinatorially encode the phase and amplitude of oscillatory light sequences, which are different from the stimulus feature encoded by the spike number. The result demonstrates that the retina encodes multiple stimulus features by exploiting all degrees of freedom of burst spike patterns, i.e., the spike number and multiple intraburst ISIs. Because synaptic transmission in the visual system is highly sensitive to ISIs, the combinatorial ISI coding must have a major impact on visual information processing.

neuroscience↗

bioRxiv2020

Unequal contribution of two paralogous centromeric histones to function the cowpea centromere

The legume cowpea (Vigna unguiculata, 2n=2x=22) has significant tolerance to drought and heat stress. Here we analysed and manipulated cowpea centromere-specific histone H3 (CENH3) genes, aiming to establish a centromere-based doubled-haploid method for use in genetic improvement of this dryland crop in future. Cowpea encodes two functional CENH3 variants (CENH3.1 and CENH3.2) and two CENH3 pseudogenes. Phylogenetic analysis suggests that gene duplication of CENH3 occurred independently during the speciation of V. unguiculata and the related V. mungo without a genome duplication event. Both functional cowpea CENH3 variants are transcribed, and the corresponding proteins are intermingled in subdomains of different types of centromere sequences in a tissue-specific manner together with the outer kinetochore protein CENPC. CENH3.2 is removed from the generative cell of mature pollen, while CENH3.1 persists. Differences between both CENH3 paralogs are restricted to the N-terminus. The complete CRISPR/Cas9-based inactivation of CENH3.1 resulted in delayed vegetative growth and sterility, indicating that this variant is needed for plant development and reproduction. By contrast, CENH3.2 knockout individuals did not show obvious defects during vegetative and reproductive development, suggesting that the gene is an early stage of subfunctionalization or pseudogenization. One-sentence summaryThe two paralogous centromeric histones (CENH3) of cowpea contribute unequal to the function of the centromere.

genetics↗

bioRxiv2019

High-throughput screening of high protein producer budding yeast using gel microdrop technology

The need for protein production has been growing over the years in various industries. We here present a high-throughput screening strategy to isolate high producer budding yeast clones from a mutagenized cell population using gel microdrop (GMD) technology. We use a microfluidic water-in-oil (W/O) emulsion method to produce monodisperse GMDs and a microfluidic cell sorter for damage-free sorting of GMDs by fluorescently quantifying secreted proteins. As a result, this high-throughput GMD screening method effectively selects high producer clones and improves protein production up to five-fold. We speculate that this screening strategy can be applied, in principle, to select any types of high producer cells (bacterial, fungal, mammalian, etc.) which produce arbitrary target protein as it does not depend on enzymes to be produced.

bioengineering↗

bioRxiv2019

Estrogen induces mammary ductal dysplasia.

Mammary ductal dysplasia is a phenotype observed in precancerous lesions and early-stage breast cancer. However, the mechanism of dysplasia formation remains elusive. Here we show, by establishing a novel dysplasia model system, that estrogen, a female hormone, has the potential to cause mammary ductal dysplasia. We injected estradiol (E2), the most active form of estrogen, daily into scid mice with a defect in nonhomologous end joining repair and observed dysplasia formation with cell proliferation at day 30. Moreover, we found that isoflavones inhibited E2-induced dysplasia formation. Our dysplasia model system provides insight into the mechanistic understanding of breast tumorigenesis, and the development of breast cancer prevention.

cancer biology↗

bioRxiv2018

Direct binding of Cdt2 to PCNA is important for targeting the CRL4Cdt2 E3 ligase activity to Cdt1

The CRL4Cdt2 ubiquitin ligase complex is an essential regulator of cell-cycle progression and genome stability, ubiquitinating substrates such as p21, Set8 and Cdt1, via a display of substrate degrons on PCNA. Here, we examine the hierarchy of the ligase and substrate recruitment kinetics onto PCNA at sites of DNA replication. We demonstrate that the C-terminal end of Cdt2 bears a PCNA interaction protein motif (PIP box, Cdt2PIP), which is necessary and sufficient for binding of Cdt2 to PCNA. Cdt2PIP binds PCNA directly with high affinity, two orders of magnitude tighter than the PIP box of Cdt1. X-ray crystallographic structures of PCNA bound to Cdt2PIP and Cdt1PIP show that the peptides occupy all three binding sites of the trimeric PCNA ring. Mutating Cdt2PIP weakens the interaction with PCNA, rendering CRL4Cdt2 less effective in Cdt1 ubiquitination and leading to defects in Cdt1 degradation. The molecular mechanism we present suggests a new paradigm for bringing substrates to the CRL4-type ligase, where the substrate receptor and substrates bind to a common multivalent docking platform to enable subsequent ubiquitination.\n\nSummary blurbThe C-terminal end of Cdt2 contains a PIP-box for binding to PCNA to promote CRL4Cdt2 function, creating a new paradigm, where the substrate receptor and substrates bind to a common multivalent docking platform for ubiquitination.

molecular biology↗