Biology subjects

Suzuki, T.

Publications and source records attributed to Suzuki, T..

At least 235 records · Page 13Linked to original sources

bioRxiv2020

Removal of repressive histone marks creates epigenetic memory of recurring heat in Arabidopsis

Acclimation to high temperature increases plants tolerance of subsequent lethal high temperatures1-3. Although epigenetic regulation of plant gene expression is well studied, how plants maintain a memory of environmental changes over time remains unclear. Here, we show that JUMONJI (JMJ) proteins4-8, demethylases involved in histone H3 lysine 27 trimethylation (H3K27me3), are necessary for Arabidopsis thaliana heat acclimation. Acclimation induces sustained H3K27me3 demethylation at key HEAT SHOCK PROTEIN (HSP) loci by JMJs, poising the HSP genes for subsequent activation. Upon sensing heat after a 3-day interval, JMJs directly reactivate HSP genes. Finally, jmj mutants fail to maintain heat memory under fluctuating field temperature conditions. Our findings of an epigenetic memory mechanism involving histone demethylases may have implications for environmental adaptation of field plants.

plant biology↗

bioRxiv2020

Environmental RNA interference in two-spotted spider mite, Tetranychus urticae, reveals dsRNA processing requirements for efficient RNAi response

Comprehensive understanding of pleiotropic roles of RNAi machinery highlighted the conserved chromosomal functions of RNA interference. The consequences of the evolutionary variation in the core RNAi pathway genes are mostly unknown, but may lead to the species-specific functions associated with gene silencing. The two-spotted spider mite, Tetranychus urticae, is a major polyphagous chelicerate pest capable of feeding on over 1,100 plant species and developing resistance to pesticides used for its control. A well annotated genome, susceptibility to RNAi and economic importance, make T. urticae an excellent candidate for development of an RNAi protocol that enables high-throughput genetic screens and RNAi-based pest control. Here, we show that the length of the exogenous dsRNA critically determines its processivity and ability to induce RNAi in vivo. A combination of the long dsRNAs and the use of dye to trace the ingestion of dsRNA enabled the identification of genes involved in membrane transport and 26S proteasome degradation as sensitive RNAi targets. Our data demonstrate that environmental RNAi can be an efficient reverse genetics and pest control tool in T. urticae. In addition, the species-specific properties together with the variation in the components of the RNAi machinery make T. urticae a potent experimental system to study the evolution of RNAi pathways.

molecular biology↗

bioRxiv2020

Enhancer Remodeling Promotes Tumor-Initiating Activity in NRF2-Activated Non-Small Cell Lung Cancers

Transcriptional dysregulation, which can be caused by genetic and epigenetic alterations, is a fundamental feature of many cancers. A key cytoprotective transcriptional activator, NRF2, is often aberrantly activated in non-small cell lung cancers (NSCLCs) and supports both aggressive tumorigenesis and therapeutic resistance. Herein, we found that persistently activated NRF2 in NSCLCs generates enhancers at gene loci that are not normally regulated by transiently activated NRF2 under physiological conditions. Elevated accumulation of CEBPB in NRF2-activated NSCLCs was found to be one of the prerequisites for establishment of the unique NRF2-dependent enhancers, among which NOTCH3 enhancer was shown to be critical for the promotion of tumor-initiating activity. Enhancer remodeling mediated by NRF2-CEBPB cooperativity promotes tumor-initiating activity and drives malignancy of NRF2-activated NSCLCs via establishment of the NRF2-NOTCH3 regulatory axis.

cancer biology↗

bioRxiv2020

Multidrug treatment with nelfinavir and cepharanthine against COVID-19

Antiviral treatments targeting the emerging coronavirus disease 2019 (COVID-19) are urgently required. We screened a panel of already-approved drugs in a cell culture model of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and identified two new antiviral agents: the HIV protease inhibitor Nelfinavir and the anti-inflammatory drug Cepharanthine. In silico modeling shows Nelfinavir binds the SARS-CoV-2 main protease consistent with its inhibition of viral replication, whilst Cepharanthine inhibits viral attachment and entry into cells. Consistent with their different modes of action, in vitro assays highlight a synergistic effect of this combined treatment to limit SARS-CoV-2 proliferation. Mathematical modeling in vitro antiviral activity coupled with the known pharmacokinetics for these drugs predicts that Nelfinavir will facilitate viral clearance. Combining Nelfinavir/Cepharanthine enhanced their predicted efficacy to control viral proliferation, to ameliorate both the progression of disease and risk of transmission. In summary, this study identifies a new multidrug combination treatment for COVID-19.

microbiology↗

bioRxiv2020

Dynamics of the cell fate specifications during female gametophyte development in Arabidopsis

The female gametophytes of angiosperms contain cells with distinct functions, such as those that enable reproduction via pollen tube attraction and fertilization. Although the female gametophyte undergoes unique developmental processes, such as several rounds of nuclear division without cell plate formation, and the final cellularization, it remains unknown when and how the cell fate is determined during their development. Here, we visualized the living dynamics of female gametophyte development and performed transcriptome analysis of its individual cell types, to assess the cell fate specifications in Arabidopsis thaliana. We recorded time lapses of the nuclear dynamics and cell plate formation from the one-nucleate stage to the seven-cell stage after cellularization, using the in vitro ovule culture system. The movies showed that the nuclear division occurred along the micropylar-chalazal axis. During cellularization, the polar nuclei migrated while associating with forming edge of the cell plate. Then, each polar nucleus migrated to fuse linearly towards each other. We also tracked the gene expression dynamics and identified that the expression of the MYB98pro::GFP, a synergid-specific marker, was initiated before cellularization, and then restricted to the synergid cells after cellularization. This indicated that cell fates are determined immediately after cellularization. Transcriptome analysis of the female gametophyte cells of the wild type and myb98 mutant, revealed that the myb98 synergid cells had the egg cell-like gene expression profile. Although in the myb98, the egg cell-specific gene expressions were properly initiated only in the egg cells after cellularization, but subsequently expressed ectopically in one of the two synergid cells. These results, together with the various initiation timings of the egg cell-specific genes suggest the complex regulation of the individual gametophyte cells, such as cellularization-triggered fate initiation, MYB98-dependent fate maintenance, cell morphogenesis, and organelle positioning. Our system of live-cell imaging and cell-type-specific gene expression analysis provides insights into the dynamics and mechanisms of cell fate specifications in the development of female gametophytes in plants.

plant biology↗

bioRxiv2020

Host-parasite tissue adhesion by a secreted type of β-1,4-glucanase in the parasitic plant Phtheirospermum japonicum

Tissue adhesion between plant species occurs both naturally and artificially. Parasitic plants establish symbiotic relationship with host plants by adhering tissues at roots or stems. Plant grafting, on the other hand, is a widely used technique in agriculture to adhere tissues of two stems. While compatibility of tissue adhesion in plant grafting is often limited within close relatives, parasitic plants exhibit much wider compatibilities. For example, the Orobanchaceae parasitic plant Striga hermonthica is able to infect Poaceae crop plants, causing a serious agricultural loss. Here we found that the model Orobanchaceae parasite plant Phtheirospermum japonicum can be grafted on to interfamily species, such as Arabidopsis, a Brassicaceae plant. To understand molecular basis of tissue adhesion between distant plant species, we conducted comparative transcriptome analyses on both infection and grafting by P. japonicum on Arabidopsis. Through gene clustering, we identified genes upregulated during these tissue adhesion processes, which include cell proliferation- and cell wall modification-related genes. By comparing with a transcriptome dataset of interfamily grafting between Nicotiana and Arabidopsis, we identified 9 genes commonly induced in tissue adhesion between distant species. Among them, we showed a gene encoding secreted type of {beta}-1,4-glucanase plays an important role for plant parasitism. Our data provide insights into the molecular commonality between parasitism and grafting in plants. Significance StatementComprehensive sequential RNA-Seq datasets for parasitic infection of the root and grafting of the stem between P. japonicum and Arabidopsis revealed that molecular events of parasitism and grafting are substantially different and only share a part of events such as cell proliferation and cell wall modification. This study demonstrated that a secreted type of {beta}-1,4-glucanase gene expressed in cells located at the parasite-host interface as an important factor for parasitism in the Orobanchaceae.

plant biology↗

bioRxiv2020

Cell-cell adhesion in plant grafting is facilitated by β-1,4-glucanases

Plant grafting is conducted for vegetative propagation in plants, whereby a piece of living tissue is attached to another tissue through establishment of cell-cell adhesion. Plant grafting has a long history in agriculture and has been applied to improve crop traits for thousands of years1. Plant grafting has mostly relied on the natural ability of a plant for wound healing. However, the compatibility of cell-cell adhesion typically limits graft combinations to closely related species2-4, and the mechanism by which cell-cell adhesion of injured tissues is established is largely unknown. Here, we show that a subclade of {beta}-1,4-glucanases secreted into the extracellular region facilitates cell-cell adhesion near the graft interface. Nicotiana shows a propensity for cell-cell adhesion with a diverse range of angiosperms, including vegetables, fruit trees, and monocots, in which cell wall reconstruction was promoted in a similar manner to conventional intrafamily grafting5-7. Using transcriptomic approaches, we identified a specific clade of {beta}-1,4-glucanases that is upregulated during grafting in successful graft combinations but not in incompatible grafts and precedes graft adhesion in inter- and intrafamily grafts. Grafting was facilitated with an overexpressor of the {beta}-1,4-glucanase and, using Nicotiana stem as an interscion, we produced tomato fruits on rootstocks from other plant families. Our results demonstrate that the mechanism of cell-cell adhesion is partly conserved in plants and is a potential target to enhance plant grafting techniques.

plant biology↗

bioRxiv2020

Mitochondrial dysfunction underlying sporadic inclusion body myositis is ameliorated by the mitochondrial homing drug MA-5

Sporadic inclusion body myositis (sIBM) is the most common idiopathic inflammatory myopathy, and several reports have suggested that mitochondrial abnormalities are involved in its etiology. We recruited 9 sIBM patients and found significant histological changes and an elevation of growth differential factor 15 (GDF15), a marker of mitochondrial disease, strongly suggesting the involvement of mitochondrial dysfunction. Bioenergetic analysis of sIBM patient myoblasts revealed impaired mitochondrial function. Decreased ATP production, reduced mitochondrial size and reduced mitochondrial dynamics were also observed in sIBM myoblasts. Cell vulnerability to oxidative stress also suggested the existence of mitochondrial dysfunction. Mitochonic acid-5 (MA-5) increased the cellular ATP level, reduced mitochondrial ROS, and provided protection against sIBM myoblast death. MA-5 also improved the survival of sIBM skin fibroblasts as well as mitochondrial morphology and dynamics in these cells. The reduction in the gene expression levels of Opa1 and Drp1 was also reversed by MA-5, suggesting the modification of the fusion/fission process. These data suggest that MA-5 may provide an alternative therapeutic strategy for treating not only mitochondrial diseases but also sIBM.

neuroscience↗

bioRxiv2020

PLZF is a new substrate of CRBN with thalidomide and 5-hydroxythalidomide

Thalidomide induces cereblon (CRBN)-dependent degradation of proteins. Human cytochrome P450s are thought to provide two monohydroxylated metabolites from thalidomide, and the metabolites are also considered to be involved in thalidomide effects. However, it remains unclear. We report that human PLZF/ZBTB16 is a target protein of CRBN with thalidomide and its derivatives, and that 5-hydroxythalidomide has high potential for degrading PLZF. Using a human transcription factor protein array produced by a wheat cell-free protein synthesis system, PLZF was found to bind to CRBN with thalidomide. PLZF is degraded by the CRL4CRBN complex with thalidomide and its derivatives. Mutagenesis analysis revealed that both 1st and 3rd zinc finger domains conserved in vertebrates are recognized for thalidomide-dependent binding and degradation by CRBN. In chicken limbs, knockdown of Plzf induced skeletal abnormalities, and Plzf was degraded after thalidomide or 5-hydroxythalidomide treatment. Our findings suggest that PLZF is a pivotal substrate involving thalidomide-induced teratogenesis.

pharmacology and toxicology↗

bioRxiv2020

The tRNA pseudouridine synthase TruB1 regulates the maturation and function of let-7 miRNA

Let-7 is an evolutionary conserved microRNA that mediates post-transcriptional gene silencing to regulate a wide range of biological processes, including development, differentiation, and tumor suppression. Let-7 biogenesis is tightly regulated by several RNA-binding proteins, including Lin28A/B, which represses let-7 maturation. To identify new regulators of let-7, we devised a cell-based functional screen of RNA-binding proteins using a let-7 sensor luciferase reporter, and identified the tRNA pseudouridine synthase, TruB1. TruB1 enhanced maturation specifically of let-7 family members. Rather than inducing pseudouridylation of the miRNAs, HITS-CLIP (High throughput sequencing crosslinking immunoprecipitation) and biochemical analyses revealed direct binding between endogenous TruB1 and the stem-loop structure of pri-let-7, which also binds Lin28A/B. TruB1 selectively enhanced the interaction between pri-let-7 and the microprocessor DGCR-8, which mediates miRNA maturation. Finally, TruB1 suppressed cell proliferation, which was mediated in part by let-7. Altogether, we reveal an unexpected function for TruB1 in promoting let-7 maturation and function.

molecular biology↗

bioRxiv2020

Helicobacter small RNA regulates host adaptation and carcinogenesis

Type-1 carcinogenic Helicobacter pylori that is known to evolve during long-term infection, enters the stomach orally and causes gastric cancer using the carcinogenic protein CagA1. However, little is known about the adaptation mechanisms of H. pylori when the environment changes from the outside to the inside of the living body. Here we show that small non-coding RNA HPnc4160 is a crucial novel RNA molecule of H. pylori that negatively regulates bacterial-host adaptation and gastric cancer. H. pylori isolated from gerbils stomachs eight weeks post-infection acquired mutations in the increased number of T-repeats within the upstream region of the HPnc4160 coding region, which leads to reduced HPnc4160 expression levels that also seen in cancer patients-derived H. pylori. By comparing RNA-seq and iTRAQ analysis between wild-type and hpnc4160 deficient mutant strains, we identified eight targets of HPnc4160 including cagA and unknown factors. Mice infection experiment revealed that the hpnc4160 deficient mutant had a higher number of colonized bacteria in the mice stomach than the wild-type strain, indicating that reduced expression levels of HPnc4160 was important for bacterial host adaptation. The expression level of HPnc4160 was lower in the clinical isolates derived from gastric cancer patients compared with non-cancer-derived strains, while the mRNA expression levels of target factors were higher. Our findings highlight the first discovery that HPnc4160 is an important small RNA for bacteria to adapt to the host environment leading to gastric carcinogenesis.

microbiology↗

bioRxiv2020

Glial insulin regulates cooperative or antagonistic Golden goal/Flamingo interactions during photoreceptor axon navigation

Transmembrane protein Golden goal (Gogo) interacts with the atypical cadherin Flamingo to direct R8 photoreceptor axons in the Drosophila visual system. However, the precise mechanisms underlying Gogo regulation during columnar- and layer-specific R8 axon targeting are unknown. Our studies demonstrated that the insulin secreted from surface and cortex glia switches the phosphorylation status of Gogo, thereby regulating its two distinct functions in this process. Nonphosphorylated Gogo mediates the initial recognition of the glial protrusion in the center of the medulla column, whereas phosphorylated Gogo suppresses horizontal filopodia extension by counteracting Flamingo to maintain one axon to one column ratio. Later, Gogo expression ceases during the midpupal developmental stage, thus allowing R8 filopodia to extend vertically into the M3 layer. These results demonstrate that the long- and short-range signaling between the glia and R8 axon growth cones regulates growth cone dynamics in a stepwise manner, and thus shape the entire organization of the visual systems functional neuronal circuit.

neuroscience↗

bioRxiv2019

Detection of genome-wide low-frequency mutations with Paired-End and Complementary Consensus Sequencing (PECC-Seq) revealed end-repair derived artifacts as residual errors

To improve the accuracy and the cost-efficiency of next-generation sequencing in ultralow-frequency mutation detection, we developed the Paired-End and Complementary Consensus Sequencing (PECC-Seq), a PCR-free duplex consensus sequencing approach. PECC-Seq employed shear points as endogenous barcodes to identify consensus sequences from the overlap in the shortened, complementary DNA strands-derived paired-end reads for sequencing error correction. With the high accuracy of PECC-Seq, we identified the characteristic base substitution errors introduced by the end-repair process of mechanical fragmentation-based library preparations, which were prominent at the terminal 6 bp of the library fragments in the 5-NpCpA-3 or 5-NpCpT-3 trinucleotide context. As demonstrated at the human genome scale (TK6 cells), after removing these potential end-repair artifacts from the terminal 6 bp, PECC-Seq could reduce the sequencing error frequency to mid-10-7 with a relatively low sequencing depth. For TA base pairs, the background error rate could be suppressed to mid-10-8. In mutagen-treated TK6, slight increases in mutagen treatment-related mutant frequencies could be detected, indicating the potential of PECC-Seq in detecting genome-wide ultra-rare mutations. In addition, our finding on the patterns of end-repair artifacts may provide new insights in further reducing technical errors not only for PECC-Seq, but also for other next-generation sequencing techniques.

genomics↗

bioRxiv2019

ppGpp functions as an alarmone in metazoa

Guanosine 3,5-bis(pyrophosphate) (ppGpp) functions as a second messenger in bacteria to adjust their physiology in response to environmental changes. In recent years, the ppGpp-specific hydrolase, metazoan SpoT homolog-1 (Mesh1), was shown to have important roles for growth under nutrient deficiency in Drosophila melanogaster. Curiously, however, ppGpp has never been detected in animal cells, and therefore the physiological relevance of this molecule, if any, in metazoans has not been established. Here, we report the detection of ppGpp in Drosophila and human cells and demonstrate that ppGpp accumulation induces metabolic changes, cell death, and eventually lethality in Drosophila. Our results provide the first evidence of the existence and function of the ppGpp-dependent stringent response in animals.

molecular biology↗

bioRxiv2019

Transcriptome Analysis of Desquamative Interstitial Pneumonia (DIP)

Desquamative interstitial pneumonia (DIP) is a rare diffuse parenchymal lung disease of unclear etiology. A recent study showed that mice overexpressing granulocyte macrophage colony-stimulating factor (GM-CSF) in lungs develop DIP-like disease, suggesting that pulmonary GM-CSF may be involved in the pathogenesis of DIP. To determine if GM-CSF is involved in human DIP lungs, we performed transcriptome analysis on human DIP lung tissue. We also extended transcriptome analysis to respiratory bronchiolitis-associated interstitial lung disease (RB-ILD), which has been thought to be in the same spectrum of disease. The analysis revealed that DIP has a distinct transcriptome profile compared to both RB-ILD and non-diseased lung controls. It also suggested that GM-CSF was a key upstream regulator in the DIP transcriptome and that the GM-CSF signaling pathway was highly activated in DIP tissue. Further bioinformatics analysis using xCell, a novel computational method that assesses enrichments of individual cell types based on gene expression, suggested that DIP is enriched for gene signatures of macrophages and other immune cells such as dendritic cells and B cells. In conclusion, our analysis shows that DIP is characterized by a GM-CSF signature, and thus GM-CSF is likely to be involved in the pathogenesis of DIP. Our analysis also suggests that immune cells other than alveolar macrophages, such as B cells, may also be involved in the pathogenesis of DIP.

molecular biology↗

bioRxiv2019

Large scale genome-wide association study in a Japanese population identified 45 novel susceptibility loci for 22 diseases

INTRODUCTORY PARAGRAPHThe overwhelming majority of participants in current genetic studies are of European ancestry1-3, limiting our genetic understanding of complex disease in non-European populations. To address this, we aimed to elucidate polygenic disease biology in the East Asian population by conducting a genome-wide association study (GWAS) with 212,453 Japanese individuals across 42 diseases. We detected 383 independent signals in 331 loci for 30 diseases, among which 45 loci were novel (P < 5 x 10-8). Compared with known variants, novel variants have lower frequency in European populations but comparable frequency in East Asian populations, suggesting the advantage of this study in discovering these novel variants. Three novel signals were in linkage disequilibrium (r2 > 0.6) with missense variants which are monomorphic in European populations (1000 Genomes Project) including rs11235604(p.R220W of ATG16L2, a autophagy-related gene) associated with coronary artery disease. We further investigated enrichment of heritability within 2,868 annotations of genome-wide transcription factor occupancy, andidentified 378 significant enrichments across nine diseases (FDR < 0.05) (e.g. NF-{kappa}B for immune-related diseases). This large-scale GWAS in a Japanese population provides insights into the etiology of common complex diseases and highlights the importance of performing GWAS in non-European populations.

genetics↗

bioRxiv2019

eIF5a reduction via decreased Klf5 leads to cell senescence by mitochondrial fission in VSMCs

Though dysregulation of mitochondrial dynamics has been linked to cellular senescence, which contributes to advanced age-related disorders, it is unclear how Klf5, an essential transcriptional factor of cardiovascular remodeling, mediates the link between mitochondrial dynamics and vascular smooth muscle cell (VSMC) senescence. Here we show that Klf5 downregulation in VSMCs is correlated with rupture of abdominal aortic aneurysm (AAA), an age-related vascular disease. Mice lacking Klf5 in VSMCs exacerbate vascular senescence and progression of Ang II-induced AAA by facilitating ROS formation. Klf5 knockdown enhances, while Klf5 overexpression suppresses mitochondrial fission. Mechanistically, Klf5 activates eIF5a transcription through binding to the promoter of eIF5a, which in turn preserves mitochondrial integrity by interacting with Mfn1. Accordingly, decreased expression of eIF5a elicited by Klf5 downregulation leads to mitochondrial fission and excessive ROS production. Inhibition of mitochondrial fission decreases ROS production and VSMC senescence. Our studies provide a potential therapeutic target for age-related vascular disorders.

biochemistry↗

bioRxiv2019

Assessment of a western blot signal for the Bcnt/Cfdp1, a tentative component of Srcap chromatin remodeling complex; trial to overcome off-target problems

The BCNT (Bucentaur) protein family is characterized by a conserved amino acid sequence at the C-terminus (BCNT-C domain) and plays an essential role in gene expression and chromosomal maintenance in fungi, fly, and chicken. The mammalian Bucentaur/Craniofacial developmental protein 1 (Bcnt/Cfdp1) is also a tentative component of the Srcap (SNF2-Related CBP Activator Protein) chromatin remodeling complex, but little is known about its properties, partly because there are few suitable antibodies to detect the endogenous protein. We used multiple anti-Bcnt/Cfdp1 antibodies against unrelated immunogens derived from BCNT-C domain and mouse-specific N-terminal peptide. To assign western blot signals and evaluate these antibodies, we utilized a stem cell line from mutant embryos of mouse Bcnt/Cfdp1, whose mRNA expression levels were reduced to 75% of the parental cells. In western blotting of these mutant and parental cell extracts with the anti-Bcnt/Cfdp1 antibodies, mouse Bcnt/Cfdp1 was detected as a doublet of approximately 45 kDa. LC-MS/MS analysis of the corresponding doublet for the Flag-tagged mouse Bcnt/Cfdp1 constitutively expressed in T-REx 293 cell (a HEK293 derivative) exhibited that the upper band was much more phosphorylated than the lower band and that there was preferential Ser phosphorylation in the WESF motif in the BCNT-C domain. Western blot with these validated antibodies indicated a preferential expression of Bcnt/Cfdp1 in the early stages of brain development in mouse and rat, which is consistent with the expression of Bcnt/Cfdp1 mRNA. This article describes the evaluation of anti-Bcnt/Cfdp1 antibodies, including a scheme to prepare a potential negative control for western blot, and discusses immune-cross reactions with off-target proteins, particularly immunoreaction probabilities.

biochemistry↗