Biology subjects

Endo, T.

Publications and source records attributed to Endo, T..

31 records · Page 2Linked to original sources

bioRxiv2023

Haploid-resolved and chromosome-scale genome assembly in Citrus unshiu and its parental species, C. nobilis and C. kinokuni

Citrus, a member of the Rutaceae family, is a widely cultivated crop with numerous cultivars. In Japan, citrus fruits account for a significant portion of agricultural production. Although several new citrus varieties have been developed through conventional breeding programs, satsuma mandarin remains the dominant cultivar. In this study, chromosome-scale and haploid-resolved reference genome sequences of satsuma mandarin (Citrus unshiu Marc) and its parental varaieties, kishu mandarin (C. kinokuni hort. ex Tanaka) and kunenbo mandarin (C. nobilis Lour. var. kunip Tanaka) were generated using long-read sequencing and Hi-C technologies. The comparison of haploid and unphased genomes revealed structural differences between them, indicating distinct regions in each haploid. In addition, genetic linkage maps were constructed, and genetic and physical distances were compared. The results showed variations in polymorphism density across different regions of the chromosomes. Together, the obtained results provide valuable insights into the genomic characteristics and structural variations of satsuma mandarin and related citrus varieties. These insights will lead to the further elucidation and improvement of citrus cultivars through genome breeding strategies.

genomics↗

bioRxiv2023

Targeting retrovirus-derived Rtl8a and 8b causes late onset obesity and neurodevelopmental defects

Retrotransposon Gag-like (RTL) 8A, 8B and 8C are triplet genes of uncertain function that form a cluster on the X chromosome. They are eutherian-specific genes presumably derived from a certain retrovirus. Here, we demonstrate that Rtl8a and Rtl8b play an important role in growth and behavior via brain functions in the hypothalamus and prefrontal cortex. Rtl8a and Rtl8b double knockout (DKO) mice exhibited overgrowth due to hyperphagia from young adulthood and reduced social responses, increased apathy-like behavior. RTL8A and RTL8B proteins are localized to both the nucleus and cytoplasm of neurons presumably due to an N-terminal nuclear localization signal-like sequence. An increment in nucleus size was also detected in the neurons in the prefrontal cortex, suggesting neuronal dysfunction. These data give another strong evidence that retrovirus-derived acquired genes contributed to the establishment of the current eutherian developmental system in a wide variety of ways. Summary statementRtl8a and Rtl8b double knockout mice exhibited late onset obesity and neurodevelopmental defects, demonstrating that these eutherian specific retrovirus-derived acquired genes encoding proteins with only 113 amino acids play important roles in the brain presumably via their functions in the hypothalamus and prefrontal cortex.

molecular biology↗

bioRxiv2023

Fgf10 mutant newts can regenerate normal limbs despite severe developmental hindlimb defects

In the amniote limb, FGF10 is essential for limb bud initiation and outgrowth. However, whether this function is broadly conserved in tetrapods and/or involved in adult limb regeneration remains unknown. To tackle this question, we established an Fgf10 null mutant line in the newt Pleurodeles waltl which have amazing regenerative ability. While Fgf10 mutant forelimbs develop normally, the hindlimbs exhibit severe digit reduction, fail to ossify the zeugopod, and downregulate FGF target genes like Sall1, Runx1 and Hoxa11/d11. Despite these developmental defects, Fgf10 mutants were able to regenerate near-normal hindlimbs. Together, our results suggest an important role for Fgf10 in hindlimb digit formation and zeugopod ossification during development, but little or no function in regeneration, suggesting that different mechanisms operate during limb regeneration versus development.

developmental biology↗

bioRxiv2023

Identification of a novel enhancer essential for Satb1 expression in TH2 cells and activated ILC2s.

The genome organizer, special AT-rich binding protein-1 (SATB1) functions to globally regulate gene networks during primary T cells development and plays a pivotal role in lineage-specification in CD4+ helper-, CD8+ cytotoxic- and FOXP3+ regulatory-T cell subsets. However, it remains unclear how Satb1 gene expression is controlled, particularly in effector T cell function. Here, by using a novel reporter mouse strain expressing SATB1-Venus and genome editing, we have identified a cis-regulatory enhancer, essential for maintaining Satb1 expression specifically in TH2 cells. This enhancer is occupied by STAT6 and interacts with Satb1 promoters through chromatin looping in TH2 cells. Reduction of Satb1 expression, by the lack of this enhancer, resulted in elevated IL-5 expression in TH2 cells. In addition, we found that Satb1 is induced in activated group 2 innate lymphoid cells (ILC2s) through this enhancer. Collectively, these results provide novel insights into how Satb1 expression is regulated in TH2 cells and ILC2s during type 2 immune responses.

immunology↗

bioRxiv2022

Development of a fluorescence reporter system to quantify transcriptional activity of endogenous p53 in living cells

The tumor suppressor p53 plays a central role in cellular stress responses by regulating transcription of multiple target genes. The temporal dynamics of p53 are thought to be important for its function: it encode input information and are decoded to induce distinct cellular phenotypes. However it remains unclear to what extent the temporal dynamics of p53 reflects the activity of p53-induced gene expression. In this study, we report a multiplexed reporter system that allows us to visualize the transcriptional activity of p53 at the single cell level. Our reporter system features simple and sensitive observation of the transcriptional activity of endogenous p53 to the response elements of various target genes. Using this system, we show that the transcriptional activation of p53 exhibits strong cell-to-cell heterogeneity. The transcriptional activation of p53 by etoposide is highly dependent on the cell cycle but not by UV-C. Finally, we show that our reporter system allows simultaneous visualization of the transcriptional activity of p53 and cell cycle. Our reporter system can thus be a useful tool for studying biological processes involving the p53 signaling pathway. Summary statementTumor suppressor protein p53 is one of the hub factor of the cellular stress responses. Our novel reporter system allows us to visualize the transcriptional activity of endogenous p53 easily and precisely.

cell biology↗

bioRxiv2022

Non-classical glycosylation determines intracellular trafficking of APP and Aβ production

A primary pathology of Alzheimers disease (AD) is A{beta} deposition in brain parenchyma and blood vessels, the latter being called cerebral amyloid angiopathy (CAA). Parenchymal amyloid plaques presumably originate from neuronal A{beta} precursor protein (APP), but vascular amyloid deposits origins remain unclear. Endothelial APP expression in APP-knock-in mice was recently shown to expand CAA pathology, highlighting endothelial APPs importance. Furthermore, two types of endothelial APP--with and without O-glycans--have been biochemically identified, but only the former is cleaved for A{beta} production, indicating the critical relationship between APP O-glycosylation and processing. Here, we analyzed APP glycosylation and its intracellular trafficking in neurons and endothelial cells. Although protein glycosylation is generally believed to precede cell surface trafficking, which was true for neuronal APP, we unexpectedly observed that APP lacking O-glycans is externalized to the endothelial cell surface and transported back to the Golgi apparatus, where it then acquires O-glycans. Knockdown of genes encoding enzymes initiating APP O-glycosylation significantly reduced A{beta} production, suggesting this non-classical glycosylation pathway contributes to CAA pathology and is a novel therapeutic target.

neuroscience↗

bioRxiv2021

Whole-genome sequencing of Vero E6 (C1008) and comparative analysis of four Vero cell sublines

The Vero cell line is an immortalized cell line established from kidney epithelial cells of the African green monkey. A variety of sublines have been established from the original cell line, which display different characteristics. In this study, we determined the whole-genome sequence of Vero E6 (C1008) and performed comparative analysis among Vero JCRB 0111, Vero CCL-81, Vero 76 and Vero E6. Analysis of the copy number changes and loss of heterozygosity revealed that all sublines share a large deletion and loss of heterozygosity on chromosome 12, which harbors type I interferon and CDKN2 gene clusters. We identified a substantial number of genetic differences among the sublines including single nucleotide variants, indels, and copy number variations. The spectrum of single nucleotide variants indicated a close genetic relationship between Vero JCRB0111 and Vero CCL-81, and between Vero 76 and Vero E6, and a considerable genetic gap between the former two and the latter two lines. In contrast, we confirmed the pattern of genomic integration sites of simian endogenous retroviral sequences, which was consistent among the sublines. We identified subline-specific/enriched loss of function and missense variants, which potentially contribute to the differences in response to viral infection among the Vero sublines. In particular, we focused on Vero E6-specific/enriched variants and identified four genes (IL1RAP, TRIM25, RB1CC1, and ATG2A) that contained missense variants specific or enriched in Vero E6. In addition, we found that V739I variants of ACE2, which functions as the receptor for SARS-CoV-2, were heterozygous in Vero JCRB0111, Vero CCL-81, and Vero 76; however, Vero E6 contained the allele with isoleucine, resulting from the loss of one of the X chromosomes.

genomics↗

bioRxiv2020

DAZL mediates a broad translational program regulating expansion and differentiation of spermatogonial progenitors

Fertility across metazoa requires the germline-specific DAZ family of RNA-binding proteins. Here we examine whether DAZL directly regulates progenitor spermatogonia using a conditional genetic mouse model and in vivo biochemical approaches combined with chemical synchronization of spermatogenesis. We find that the absence of Dazl impairs both expansion and differentiation of the spermatogonial progenitor population. In undifferentiated spermatogonia, DAZL binds the 3 UTRs of [~]2,500 protein-coding genes. Some targets are known regulators of spermatogonial proliferation and differentiation while others are broadly expressed, dosage-sensitive factors that control transcription and RNA metabolism. DAZL binds 3 UTR sites conserved across vertebrates at a UGUU(U/A) motif. By assessing ribosome occupancy in undifferentiated spermatogonia, we find that DAZL increases translation of its targets. In total, DAZL orchestrates a broad translational program that amplifies protein levels of key spermatogonial and gene regulatory factors to promote the expansion and differentiation of progenitor spermatogonia.

developmental biology↗

bioRxiv2020

Identification of characteristic genomic markers in human hepatoma Huh7 and Huh7.5.1-8 cell lines

The human hepatoma-derived Huh7 cell line and its derivatives (Huh7.5 and Huh7.5.1) have been widely used as a convenient experimental substitute for primary hepatocytes. In particular, these cell lines represent host cells suitable for propagating the hepatitis C virus (HCV) in vitro. The Huh7.5.1-8 cell line, a subline of Huh7.5.1, can propagate HCV more efficiently than its parental cells. To provide genomic information for cells quality control, we performed whole-genome sequencing of Huh7 and Huh7.5.1-8 and identified their characteristic genomic deletions, some of which are applicable to an in-house test for cell authentication. Among the genes related to HCV infection and replication, 53 genes were found to carry missense or loss-of-function mutations likely specific to the Huh7 and/or Huh7.5.1-8. Eight genes, including DDX58 (RIG-I), BAX, EP300, and SPP1 (osteopontin), contained mutations observed only in Huh7.5.1-8 or mutations with higher frequency in Huh7.5.1-8. These mutations might be relevant to phenotypic differences between the two cell lines and may also serve as genetic markers to distinguish Huh7.5.1-8 cells from the ancestral Huh7 cells.

genomics↗

bioRxiv2019

A non-canonical unfolded protein response pathway and mitochondrial dynamics control the number of ER-mitochondria contact sites

Mitochondria maintain their morphology and functions through the optimized balance between the mitochondrial fusion and division. Here we report a novel role of mitochondrial dynamics in controlling the number of ER-mitochondria encounter structure (ERMES) clusters in a yeast cell. Loss of mitochondrial fusion or division caused the increased or decreased number, respectively, of ERMES foci observed in cells. ERMES complexes, therefore, appear to cluster with each other and mitochondrial division may inhibit undesired ERMES hyper-clustering. Furthermore, our microscopic analyses suggest that ER stress induces dissociation of ERMES clusters, increasing the number of ERMES foci even in the absence of Ire1 and Hac1, which are essential factors for the UPR response. Interestingly, we found that ER stress leads to expansion of both the ER and mitochondrial membranes in an ERMES function-dependent manner. These findings imply that a cell is equipped with two independent regulatory mechanisms controlling the number of ER-mitochondria contact sites to meet the cellular as well as environmental demands.

cell biology↗

bioRxiv2019

Exhaustive identification of conserved upstream open reading frames with potential translational regulatory functions from animal genomes

Upstream open reading frames (uORFs) are present in the 5-untranslated regions of many eukaryotic mRNAs, and some peptides encoded by these regions play important regulatory roles in controlling main ORF (mORF) translation. We previously developed a novel pipeline, ESUCA, to comprehensively identify plant uORFs encoding functional peptides, based on genome-wide identification of uORFs with conserved peptide sequences (CPuORFs). Here, we applied ESUCA to diverse animal genomes, because animal CPuORFs have been identified only by comparing uORF sequences between a limited number of species, and how many previously identified CPuORFs encode regulatory peptides is unclear. By using ESUCA, 1,517 (1,373 novel and 144 known) CPuORFs were extracted from four evolutionarily divergent animal genomes. We examined the effects of 17 human CPuORFs on mORF translation using transient expression assays. Through these analyses, we identified seven novel regulatory CPuORFs that repressed mORF translation in a sequence-dependent manner, including one conserved only among Eutheria. We discovered a much higher number of animal CPuORFs than previously identified. Since most human CPuORFs identified in this study are conserved across a wide range of Eutheria or a wider taxonomic range, many CPuORFs encoding regulatory peptides are expected to be found in the identified CPuORFs.

bioinformatics↗

bioRxiv2019

ESUCA: a pipeline for genome-wide identification of upstream open reading frames with evolutionarily conserved sequences and determination of the taxonomic range of their conservation

BackgroundUpstream open reading frames (uORFs) in the 5'-untranslated regions (5'-UTRs) of certain eukaryotic mRNAs encode evolutionarily conserved functional peptides, such as cis-acting regulatory peptides that control translation of downstream main ORFs (mORFs). For genome-wide searches for uORFs with conserved peptide sequences (CPuORFs), comparative genomic studies have been conducted, in which uORF sequences were compared between selected species. To increase chances of identifying CPuORFs, we previously developed an approach in which uORF sequences were compared using BLAST between Arabidopsis and any other plant species with available transcript sequence databases. If this approach is applied to multiple plant species belonging to phylogenetically distant clades, it is expected to further comprehensively identify CPuORFs conserved in various plant lineages, including those conserved among relatively small taxonomic groups.\n\nResultsTo efficiently compare uORF sequences among many species and efficiently identify CPuORFs conserved in various taxonomic lineages, we developed a novel pipeline, ESUCA. We applied ESUCA to the genomes of five angiosperm species, which belong to phylogenetically distant clades, and selected CPuORFs conserved among at least three different orders. Through these analyses, we identified 88 novel CPuORF families. As expected, ESUCA analysis of each of the five angiosperm genomes identified many CPuORFs that were not identified from ESUCA analyses of the other four species. However, unexpectedly, these CPuORFs include those conserved in wide taxonomic ranges, indicating that the approach used here is useful not only for comprehensive identification of narrowly conserved CPuORFs but also for that of widely conserved CPuORFs. Examination of the effects of 11 selected CPuORFs on mORF translation revealed that CPuORFs conserved only in relatively narrow taxonomic ranges can have sequence-dependent regulatory effects, suggesting that most of the identified CPuORFs are conserved because of functional constraints of their encoded peptides.\n\nConclusionsThis study demonstrates that ESUCA is capable of efficiently identifying CPuORFs likely to be conserved because of the functional importance of their encoded peptides. Furthermore, our data show that the approach in which uORF sequences from multiple species are compared with those of many other species, using ESUCA, is highly effective in comprehensively identifying CPuORFs conserved in various taxonomic ranges.

bioinformatics↗

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↗