Biology subjects

Takahashi, M.

Publications and source records attributed to Takahashi, M..

At least 73 records · Page 4Linked to original sources

bioRxiv2023

Cotranscriptional demethylation induces global loss of H3K4me2 from active genes in Arabidopsis

Based on studies of animals and yeasts, methylation of histone H3 lysine 4 (H3K4me1/2/3, for mono-, di-, and tri-methylation, respectively) is regarded as the key epigenetic modification of transcriptionally active genes. In plants, however, H3K4me2 correlates negatively with transcription, and the regulatory mechanisms of this counterintuitive H3K4me2 distribution in plants remain largely unexplored. A previous genetic screen for factors regulating plant regeneration identified Arabidopsis LYSINE-SPECIFIC DEMETHYLASE 1-LIKE 3 (LDL3), which is a major H3K4me2 demethylase. Here, we show that LDL3-mediated H3K4me2 demethylation depends on the transcription elongation factor Paf1C and phosphorylation of the C-terminal domain (CTD) of RNA polymerase II (RNAPII). In addition, LDL3 binds to phosphorylated RNAPII. These results suggest that LDL3 is recruited to transcribed genes by binding to elongating RNAPII and demethylates H3K4me2 cotranscriptionally. Importantly, the negative correlation between H3K4me2 and transcription is disrupted in the ldl3 mutant, demonstrating the genome-wide impacts of the transcription-driven LDL3 pathway to control H3K4me2 in plants. Our findings implicate H3K4me2 in plants as chromatin memory for transcriptionally repressive states, which ensures robust gene control.

plant biology↗

bioRxiv2022

Haplotype-resolved de novo genome assemblies of four coniferous tree species

Coniferous trees in gymnosperm are an important source of wood production. Because of their long lifecycle, the breeding programs of coniferous tree are time- and labor-consuming. Genomics could accelerate the selection of superior trees or clones in the breeding programs; however, the genomes of coniferous trees are generally giant in size and exhibit high heterozygosity. Therefore, the generation of long contiguous genome assemblies of coniferous species has been difficult. In this study, we optimized the DNA library preparation protocols and employed high-fidelity (HiFi) long-read sequencing technology to sequence and assemble the genomes of four coniferous tree species, Larix kaempferi, Chamaecyparis obtusa, Cryptomeria japonica, and Cunninghamia lanceolata. Genome assemblies of the four species totaled 13.5 Gb (L. kaempferi), 8.5 Gb (C. obtusa), 9.2 Gb (C. japonica), and 11.7 Gb (C. lanceolata), which covered 99.6% of the estimated genome sizes on average. The contig N50 value, which indicates assembly contiguity, ranged from 1.2 Mb in C. obtusa to 16.0 Mb in L. kaempferi, and the assembled sequences contained, on average, 89.2% of the single-copy orthologs conserved in embryophytes. Assembled sequences representing alternative haplotypes covered 70.3-95.1% of the genomes, suggesting that the four coniferous tree genomes exhibit high heterozygosity levels. The genome sequence information obtained in this study represents a milestone in tree genetics and genomics, and will facilitate gene discovery, allele mining, phylogenetics, and evolutionary studies in coniferous trees, and accelerate forest tree breeding programs.

genomics↗

bioRxiv2022

PNPO-PLP Axis Senses Prolonged Hypoxia by Regulating Lysosomal Activity

Oxygen is critical for all metazoan organisms on the earth and impacts various biological processes in physiological and pathological conditions. While oxygen sensing systems inducing acute hypoxic response, including HIF pathway, have been identified, those operating in prolonged hypoxia remain to be elucidated. Here, we show that pyridoxine 5-phosphate oxidase (PNPO) that catalyzes bioactivation of vitamin B6 serves as an oxygen sensor and regulates lysosomal activity in macrophages. Decline of PNPO activity under prolonged hypoxia reduced an active form of vitamin B6, pyridoxal 5-phosphate (PLP), and inhibited lysosomal activity, leading to the augmentation of inflammatory response of macrophages. The PNPO-PLP axis creates a distinct layer of oxygen sensing, which gradually turns down and up the PLP-dependent metabolism according to prolonged changes in oxygen availability.

biochemistry↗

bioRxiv2022

Nicotinamide-N-methyltransferase is essential for SAM and 1-methylnicotinamide homeostasis in the AML12 hepatocyte cell line

Nicotinamide-N-methyltransferase (NNMT) is an enzyme that consumes S-adenosyl-methionine (SAM) and nicotinamide (NAM) to produce S-adenosyl-homocysteine (SAH) and 1-methylnicotinamide (MNAM). How much NNMT contributes to the quantity regulation of these four metabolites depends on whether NNMT is a major consumer or producer of these metabolites, which varies among various cellular contexts. Yet, whether NNMT critically regulates these metabolites in the AML12 hepatocyte cell line has been unexplored. To address this, we knock down Nnmt in AML12 cells and investigate the effects of Nnmt RNAi on metabolism and gene expression. We find that Nnmt RNAi accumulates SAM and SAH, whereas it reduces MNAM with NAM being unaltered. These results indicate that NNMT is a significant consumer of SAM and critical for MNAM production in this cell line. Moreover, transcriptome analyses reveal that altered SAM and MNAM homeostasis is accompanied by various detrimental molecular phenotypes, as exemplified by the down-regulations of lipogenic genes such as Srebf1. Consistent with this, oil-red O-staining experiments demonstrate the decrease of total lipids upon Nnmt RNAi. These results suggest that NNMT maintains proper SAM and MNAM homeostasis, providing an additional example where NNMT plays a critical role in regulating SAM and MNAM metabolism.

biochemistry↗

bioRxiv2022

NRF2 activation induces NADH-reductive stress providing a metabolic vulnerability in lung cancer

Multiple cancers regulate oxidative stress by activating the transcription factor NRF2 through mutation of its negative regulator KEAP1. NRF2 has been studied extensively in KEAP1-mutant cancers, however the role of this pathway in cancers with wildtype KEAP1 remains poorly understood. To answer this question, we induced NRF2 via pharmacological inactivation of KEAP1 in a panel of 50+ non-small lung cancer cell lines. Unexpectedly, marked decreases in viability were observed in >13% of the cell lines--an effect that was completely rescued by NRF2 ablation. Genome-wide and targeted CRISPR screens revealed that NRF2 induces NADH-reductive stress, through the upregulation of the NAD+-consuming enzyme ALDH3A1. Leveraging these findings, we show that cells treated with KEAP1 inhibitors or those with endogenous KEAP1 mutations are selectively vulnerable to Complex I inhibition, which impairs NADH oxidation capacity and potentiates reductive stress. Thus, we identify reductive stress as a metabolic vulnerability in NRF2-activated lung cancers.

molecular biology↗

bioRxiv2022

FT-GO: a multiplex fluorescent tyramide signal amplification system for histochemical analysis

Tyramide signal amplification (TSA) is a highly sensitive method for histochemical analysis. Previously, we reported a TSA system, BT-GO, for bright-filed imaging. Here, we develop FT-GO (Fluorochromized Tyramide-Glucose Oxidase) as a multiplex fluorescent TSA system. FT-GO involves peroxidase-catalyzed deposition of FT with hydrogen peroxide produced in enzymatic reaction between glucose and glucose oxidase. We showed that FT-GO enhanced immunofluorescence signals while maintaining low background signals. Compared with indirect immunofluorescence detections, FT-GO demonstrated a more widespread distribution of monoaminergic projection systems in mouse and marmoset brains. For multiplex labeling with FT-GO, we quenched Ab-conjugated peroxidase using sodium azide. We applied FT-GO to multiplex fluorescent in situ hybridization, and succeeded in labeling neocortical interneuron subtypes by coupling with immunofluorescence. FT-GO immunofluorescence further increased the detectability of an adeno-associated virus tracer. Given its simplicity and a staining with a high signal-to-noise ratio, FT-GO would provide a versatile platform for histochemical analysis.

neuroscience↗

bioRxiv2022

DHCR7 as a novel regulator of ferroptosis in hepatocytes

Recent evidence indicates that ferroptosis is implicated in the pathophysiology of various liver diseases; however, the mechanism of ferroptosis regulation in the liver is poorly understood. Here, using the whole-genome screening approach, we identified 7-dehydrocholesterol reductase (DHCR7), the terminal enzyme of cholesterol biosynthesis, as a novel regulator of ferroptosis in hepatocytes. Genetic and pharmacological inhibition (with AY9944) of DHCR7 suppressed lipid peroxidation and ferroptosis in human hepatocellular carcinoma Huh-7 cells. DHCR7 inhibition increased its substrate, 7-dehydrocholesterol (7-DHC), and extrinsic 7-DHC supplementation in turn suppressed ferroptosis. On the other hand, cholesterol deprivation had no effect on ferroptosis. A 7-DHC-derived oxysterol metabolite, 3{beta},5-dihydroxycholest-7-en-6-one (DHCEO), was increased by a ferroptosis inducer RSL-3 in DHCR7-deficient cells, suggesting that the ferroptosis-suppressive effect of DHCR7 inhibition was driven by intracellular 7-DHC as a radical scavenger. While extrinsic 7-DHC supplementation suppressed ferroptosis in various cancer cells, pharmacological DHCR7 inhibition by AY9944 showed cell-type specific effects, which could be explained by high DHCR7 expression in Huh-7 cells. We further showed that AY9944 suppressed ferroptosis in murine primary hepatocytes in vitro and systemic administration of AY9944 inhibited hepatic ischemia-reperfusion injury in vivo. These findings provide new insights into the regulatory mechanism of liver ferroptosis and suggest that DHCR7 inhibition is a potential therapeutic option for ferroptosis-related liver diseases.

molecular biology↗

bioRxiv2022

Comparative single-cell transcriptomes of dose and time dependent epithelial-mesenchymal spectrums

Epithelial-mesenchymal transition (EMT) is a cellular process involved in development and disease progression. Intermediate EMT states were observed in tumors and fibrotic tissues, but previous in vitro studies focused on time-dependent responses with single doses of signals; it was unclear whether single-cell transcriptomes support stable intermediates observed in diseases. Here, we performed single-cell RNA-sequencing with human mammary epithelial cells treated with multiple doses of TGF-{beta}. We found that dose-dependent EMT harbors multiple intermediate states at nearly steady state. Comparisons of dose- and time-dependent EMT transcriptomes revealed that the dose-dependent data enable higher sensitivity to detect genes associated EMT. We identified cell clusters unique to time-dependent EMT, reflecting cells en route to stable states. Combining dose- and time-dependent cell clusters gave rise to accurate prognosis for cancer patients. Our transcriptomic data and analyses uncover a stable EMT continuum at the single-cell resolution, and complementary information of two types of single-cell experiments.

genomics↗

bioRxiv2022

Genetically encoded sensors for analysing neurotransmission among synaptically-connected neurons

Anatomical connectome mapping in small areas of the nervous system as well as large-scale detection of neuronal activity patterns have been respectively achieved; however, it is still challenging to evaluate the functional connections among anatomically-connected neurons in a large-scale nervous system. We have developed a novel method to visualize neurotransmission named Split Protein HEmispheres for REconstitution (Sphere). By splitting a sensor into two fragments and expressing them in pre- and postsynaptic neurons separately, functional neurotransmitter sensors can be reconstituted only at the synapses between those neurons. We developed a Sphere-SF-iGluSnFR to measure glutamate levels, and further demonstrated that this system is functional in cultured cells, worms, and mouse brains. Moreover, this system is applicable to sensors other than glutamate, and colour variants have also been developed. This could allow for brain-wide imaging of functional synaptic transmission among particular neurons and identification of important neuronal circuits in the nervous system.

neuroscience↗

bioRxiv2022

Influenza A virus agnostic receptor tropism revealed using a novel biological system with terminal sialic acid-knockout cells

Avian or human influenza A viruses bind preferentially to avian- or human-type sialic acid receptors, respectively, indicating that receptor tropism is an important factor for determining the viral host range. However, there are currently no reliable methods for analyzing receptor tropism biologically under physiological conditions. Here, we established a novel system using MDCK cells with avian- or human-type sialic acid receptors and with both sialic acid receptors knocked out (KO). When we examined the replication of human and avian influenza viruses in these KO cells, we observed unique viral receptor tropism that could not be detected using a conventional solid-phase sialylglycan binding assay, which directly assesses physical binding between the virus and sialic acids. Furthermore, we serially passaged an engineered avian-derived H4N5 influenza virus, whose PB2 gene was deleted, in avian-type receptor-KO cells stably expressing PB2 to select a mutant with enhanced replication in KO cells; however, its binding to human-type sialylglycan was undetectable using the solid-phase binding assay. These data indicate that a panel of sialic acid receptor-KO cells could be a useful tool for determining the biological receptor tropism of influenza A viruses. Moreover, the PB2-KO virus experimental system could help to safely and efficiently identify the mutations required for avian influenza viruses to adapt to human cells that could trigger a new influenza pandemic. Author summaryInfluenza A virus initiates infection via hemagglutinin by binding to avian- or human-type receptors. The acquisition of mutations that allow avian virus hemagglutinins (HAs) to recognize human-type receptors is mandatory for the transmission of avian influenza viruses to humans, which could lead to a pandemic. Therefore, it is important to detect such mutation(s) in animal influenza viruses for pandemic surveillance and risk assessment. In this study, we established a novel system using a set of genetically engineered MDCK cells with knocked out sialic acid receptors to biologically evaluate the receptor tropism for influenza A viruses. Using this system, we observed unique receptor tropism in several virus strains that was undetectable using conventional solid-phase binding assays that measure physical binding between the virus and artificially synthesized sialylglycans. This study makes a significant contribution to the literature because our findings suggest the pitfall of conventional receptor binding assay and the existence of a sialic acid-independent pathway for viral infection. In addition, our system could be safely used to identify mutations that could acquire human-type receptor tropism. Thus, this system could contribute not only toward basic analyses, such as elucidating the mechanism of influenza virus host range determination, but also the surveillance of viruses of animal origin that could be capable of infecting via human-type receptors, triggering a new influenza pandemic.

microbiology↗

bioRxiv2022

Self-organization, quality control, and preclinical studies of human iPSC-derived retinal sheets for tissue-transplantation therapy

Three-dimensional retinal organoids (3D-retinas) are a promising graft source for transplantation therapy. We previously developed self-organizing culture for 3D-retina generation from human pluripotent stem cells (hPSCs). Here we present a quality control method and preclinical studies for tissue-sheet transplantation. Self-organizing hPSCs differentiated into both retinal and off-target tissues. Gene expression analyses identified the major off-target tissues as eye-related, cortex-like, and spinal cord-like tissues. For quality control, we developed a qPCR-based test in which each hPSC-derived neuroepithelium was dissected into two tissue-sheets: inner-central sheet for transplantation and outer-peripheral sheet for qPCR to ensure retinal tissue selection. During qPCR, tissue-sheets were stored for 3-4 days using a newly developed preservation method. In a rat tumorigenicity study, no transplant-related adverse events were observed. In retinal degeneration model rats, retinal transplants differentiated into mature photoreceptors and exhibited light responses in electrophysiology assays. These results demonstrate our rationale toward self-organizing retinal sheet transplantation therapy.

neuroscience↗

bioRxiv2022

Apoplastic CBM1-interacting proteins bind conserved carbohydrate binding module 1 motifs in fungal hydrolases to counter pathogen invasion

When infecting plants, fungal pathogens secrete cell wall degrading enzymes (CWDEs) that break down cellulose and hemicellulose, the primary components of plant cell walls. Some fungal CWDEs contain a unique domain, named the carbohydrate binding module (CBM), that facilitates their access to polysaccharides. However, little is known about how plants counteract pathogen degradation of their cell walls. Here, we show that the rice cysteine-rich repeat secretion protein OsCBMIP binds to and inhibits xylanase MoCel10A of the blast fungus pathogen Magnaporthe oryzae, interfering with its access to the rice cell wall and degradation of rice xylan. We found binding of OsCBMIP to various CBM1-containing enzymes, suggesting it has a general role in inhibiting the catalytic activities of fungal enzymes. OsCBMIP is localized to the apoplast, and its expression is strongly induced in leaves infected with M. oryzae. Remarkably, knockdown of OsCBMIP reduced rice defense against M. oryzae, demonstrating that inhibition of CBM1-containing fungal enzymes by OsCBMIP is crucial for rice defense. We also identified additional CBMIP-related proteins from Arabidopsis thaliana and Setaria italica, indicating that a wide range of plants counteract pathogens through this mechanism. SummaryPlants have evolved various activity-inhibiting proteins as a defense against fungal cell wall degrading enzymes (CWDEs), but how plants counteract the function of fungal enzymes containing carbohydrate binding modules (CBMs) remains unknown. Here, we demonstrate that OsCBMIP, a member of the cysteine-rich repeat secretion protein family, interacts with fungal CBM1. OsCBMIP binding to CBM1 of a blast fungal xylanase blocks access to cellulose, resulting in the inhibition of xylanase enzymatic activity. Our study provides significant insights into plant countermeasure against CWDEs in the apoplastic space during plant-fungal pathogen interactions. It also reveals a molecular function of the DUF26 domain widely distributed in plant proteins.

plant biology↗

bioRxiv2021

Cryo-sensitive aggregation triggers NLRP3 inflammasome assemblyin cryopyrin-associated periodic syndrome

Cryopyrin-associated periodic syndrome (CAPS) is an autoinflammatory syndrome caused by mutations of NLRP3, which was originally identified as cryopyrin. Familial cold autoinflammatory syndrome (FCAS), the mildest form of CAPS, is characterized by cold-induced inflammation induced by the overproduction of IL-1{beta}. However, the molecular mechanism of how mutated NLRP3 causes inflammasome activation in CAPS remains unclear. Here, we found that CAPS-associated NLRP3 mutants form cryo-sensitive aggregates that function as a scaffold for inflammasome activation. Cold exposure promoted inflammasome assembly and subsequent IL-1{beta} release triggered by mutated NLRP3. While K+ efflux was dispensable, Ca2+ was indispensable for mutated NLRP3-mediated inflammasome assembly. Notably, Ca2+ influx was induced during mutated NLRP3-mediated inflammasome assembly. Furthermore, caspase-1 inhibition prevented Ca2+ influx and inflammasome assembly induced by the mutated NLRP3, suggesting a feed-forward Ca2+ influx loop triggered by mutated NLRP3. Thus, the mutated NLRP3 forms cryo-sensitive aggregates to promote inflammasome assembly distinct from canonical NLRP3 inflammasome activation.

immunology↗

bioRxiv2021

A specific eIF4A paralog facilitates LARP1-mediated translation repression during mTORC1 inhibition

Eukaryotic translation initiation factor (eIF) 4A -- a DEAD-box RNA-binding protein -- plays an essential role in translation initiation. Two mammalian eIF4A paralogs, eIF4A1 and eIF4A2, have been assumed to be redundant because of their high homology, and the difference in their functions has been poorly understood. Here, we show that eIF4A1, but not eIF4A2, enhances translational repression during the inhibition of mechanistic target of rapamycin complex 1 (mTORC1), an essential kinase complex controlling cell proliferation. RNA-immunoprecipitation sequencing (RIP-Seq) of the two eIF4A paralogs revealed that eIF4A1 preferentially binds to mRNAs containing terminal oligopyrimidine (TOP) motifs, whose translation is rapidly repressed upon mTOR inhibition. This biased interaction depends on a La-related RNA-binding protein, LARP1. Ribosome profiling revealed that the deletion of EIF4A1, but not EIF4A2, rendered the translation of TOP mRNAs resistant to mTOR inactivation. Moreover, eIF4A1 enhances the affinity between TOP mRNAs and LARP1 and thus ensures stronger translation repression upon mTORC1 inhibition. Our data show that the distinct protein interactions of these highly homologous translation factor paralogs shape protein synthesis during mTORC1 inhibition and provide a unique example of the repressive role of a universal translation activator.

molecular biology↗

bioRxiv2021

Uterus-specific transcriptional regulation underlies eggshell pigment production in Japanese quail

The precursor of heme, protoporphyrin IX (PPIX), accumulates abundantly in the uterus of birds, such as Japanese quail, Coturnix japonica, resulting in brown-speckled eggshells. The molecular basis of PPIX production in the uterus remains largely unknown. Here, we investigated the cause of low PPIX production in a classical Japanese quail mutant exhibiting white eggshells by comparing its gene expression in the uterus with that of the wild type using transcriptome analysis and performed genetic linkage mapping to identify the causative genomic region of the white eggshell phenotype. We showed that 11 genes, including the 5-aminolevulinic acid synthase 1 (ALAS1) and ferroxidase hephaestin-like 1 (HEPHL1) genes, were specifically upregulated in the wild-type uterus and downregulated in the mutant. We mapped the 172 kb candidate genomic region on chromosome 6, which contains several genes, including a part of the paired-like homeodomain 3 (PITX3), which encodes a transcription factor. ALAS1, HEPHL1, and PITX3 were expressed in the apical cells of the luminal epithelium and lamina propria cells of the uterine mucosa of the wild-type quail, and their expression was downregulated in these cells of the mutant quail. Biochemical analysis using uterine homogenates indicated that the restricted availability of 5-aminolevulinic acid is the main cause of low PPIX production. These results suggest that uterus-specific transcriptional regulation of heme-biosynthesis-related genes is an evolutionarily acquired mechanism of eggshell pigment production in Japanese quail.

genetics↗

bioRxiv2021

An isogenic panel of single App knock-in mouse models of Alzheimer's disease confers differential profiles of β-secretase inhibition and endosomal abnormalities

We previously developed single App knock-in mouse models of Alzheimers disease (AD) that harbor the Swedish and Beyreuther/Iberian mutations with or without the Arctic mutation (AppNL- G-F and AppNL-F mice). These models showed the development of amyloid {beta} peptide (A{beta}) pathology, neuroinflammation and cognitive impairment with aging. We have now generated App knock-in mice devoid of the Swedish mutations (AppG-F mice) and some additional mutants to address the following two questions: [1] Do the Swedish mutations influence the mode of {beta}-secretase inhibitor action in vivo? [2] Does the quantity of C-terminal fragment of amyloid precursor protein (APP) generated by {beta}-secretase (CTF-{beta}) affect endosomal properties as previously reported as well as other pathological events? A{beta} pathology was exhibited by AppG-F mice from 6 to 8 months of age, and was accompanied by microglial and astrocyte activation. We found that a {beta}-secretase inhibitor, verubecestat, inhibited A{beta} production in AppG-F mice, but not in AppNL-G-F mice, indicating that the AppG-F mice are more suitable for preclinical studies of {beta}-secretase inhibition given that most AD patients do not carry Swedish mutations. We also found that the quantity of CTF-{beta} generated by various App knock-in mutants failed to correlate with endosomal alterations or enlargement, implying that CTF-{beta}, endosomal abnormalities, or both are unlikely to play a major role in AD pathogenesis. This is the first AD mouse model ever described that recapitulates amyloid pathology in the brain without the presence of Swedish mutations and without relying on the overexpression paradigm. Thus, experimental comparisons between different App knock-in mouse lines will potentially provide new insights into our understanding of the etiology of AD.

neuroscience↗

bioRxiv2021

Genes for tRNA recycling are upregulated in response to infection with Theiler's mouse encephalitis virus

The concept of tRNA recycling has recently emerged from the studies of ribosome-associated quality control. Therein tRNase ZS removes the 2', 3'>p from the ANKZF1-cleaved tRNA and the subsequent TRNT1 action re-generates the intact tRNA. To know the roles of the tRNA recycling in vivo, we investigated how viral infection affects the tRNA recycling system by analyzing the mRNA levels of tRNase ZS and TRNT1. We found that both genes in HeLa cells are upregulated in response to infection of Theilers mouse encephalitis virus but not to that of an influenza A virus. Upregulation was also observed in cells infected with encephalomyocarditis virus with reduced efficiency. The levels of the IFN-{beta} mRNA appeared to positively correlate with those of the tRNase ZS and TRNT1 mRNAs. The tRNase ZS gene may be regulated post-transcriptionally in the cells infected with Theilers mouse encephalitis virus.

immunology↗

bioRxiv2021

A small-molecule fluorescent probe for live-cell imaging of endocytosis

Endocytosis involves plasma membrane-derived vesicles for the recycling of intra- and extracellular components. Increasing evidence suggests that endocytosis is related to maintaining intracellular homeostasis and defense against disease. Consequently, investigation of the endocytic pathway attracts considerable scientific interest. This study reports live-cell imaging of endocytosis using the newly-developed fluorescent probe ECGreen. We demonstrate that ECGreen is not membrane permeable and its fluorescence signal increases in acidic conditions. Because of these characteristics, ECGreen remains on the plasma membrane, and then shows increased fluorescence when it is internalized into the acidic vesicles formed in the endocytic process. ECGreen allows direct observation of the internalized vesicle; it is a valuable new probe for endocytic imaging.

biochemistry↗