Biology subjects

Tanaka, K.

Publications and source records attributed to Tanaka, K..

99 records · Page 6Linked to original sources

bioRxiv2019

Multifaceted Hi-C benchmarking: what makes a difference in chromosome-scale genome scaffolding?

BackgroundHi-C, a derivative of chromosome conformation capture (3C) targeting the whole genome, was originally developed as a means for characterizing chromatin conformation. More recently, this method has also been frequently employed in elongating nucleotide sequences obtained by de novo genome sequencing and assembly, in which the number of resultant sequences rarely converge into the chromosome number. Despite the prevailing and irreplaceable use, sample preparation methods for Hi-C have not been intensively discussed, especially from the standpoint of genome scaffolding.\n\nResultsTo gain insights into the best practice of Hi-C scaffolding, we performed a multifaceted methodological comparison using vertebrate samples and optimized various factors during sample preparation, sequencing, and computation. As a result, we have identified some key factors that help improve Hi-C scaffolding including the choice and preparation of tissues, library preparation conditions, and restriction enzyme(s), as well as the choice of scaffolding program and its usage.\n\nConclusionsThis study provides the first comparison of multiple sample preparation kits/protocols and computational programs for Hi-C scaffolding, by an academic third party. We introduce a customized protocol designated the inexpensive and controllable Hi-C (iconHi-C) protocol, in which the optimal conditions revealed by this study have been incorporated, and release the resultant chromosome-scale genome assembly of the Chinese softshell turtle Pelodiscus sinensis.

genomics↗

bioRxiv2019

Benchmarking Single-Cell RNA Sequencing Protocols for Cell Atlas Projects

Single-cell RNA sequencing (scRNA-seq) is the leading technique for charting the molecular properties of individual cells. The latest methods are scalable to thousands of cells, enabling in-depth characterization of sample composition without prior knowledge. However, there are important differences between scRNA-seq techniques, and it remains unclear which are the most suitable protocols for drawing cell atlases of tissues, organs and organisms. We have generated benchmark datasets to systematically evaluate techniques in terms of their power to comprehensively describe cell types and states. We performed a multi-center study comparing 13 commonly used single-cell and single-nucleus RNA-seq protocols using a highly heterogeneous reference sample resource. Comparative and integrative analysis at cell type and state level revealed marked differences in protocol performance, highlighting a series of key features for cell atlas projects. These should be considered when defining guidelines and standards for international consortia, such as the Human Cell Atlas project.

genomics↗

bioRxiv2019

GUN1 regulates tetrapyrrole biosynthesis

The biogenesis of the photosynthetic apparatus in developing chloroplasts requires the assembly of proteins encoded on both nuclear and chloroplast genomes1. To co-ordinate this process there needs to be communication between these organelles, and while we have a good understanding of how the nucleus controls chloroplast development, how the chloroplast communicates with the nucleus at this time is still essentially unknown2. What we do know comes from pioneering work in which a series of genomes uncoupled (gun) mutants were identified that show elevated nuclear gene expression after chloroplast damage3. Of the six reported gun mutations, five are in tetrapyrrole biosynthesis proteins4-6 and this has led to the development of a model for chloroplast-to-nucleus retrograde signaling in which ferrochelatase 1 (FC1)-dependent heme synthesis generates a positive signal promoting expression of photosynthesis-related genes6. However, the molecular consequences of the strongest of the gun mutants, gun17, is unknown, preventing the development of a unifying hypothesis for chloroplast-to-nucleus signaling. Here, we show that GUN1 directly binds to heme and other metal-porphyrins, affects flux through the tetrapyrrole biosynthesis pathway and can increase the chelatase activity of FC1. These results raise the possibility that the signaling role of GUN1 may be manifested through changes in tetrapyrrole metabolism and supports a role for tetrapyrroles as mediators of a single biogenic chloroplast-to-nucleus retrograde signaling pathway.

plant biology↗

bioRxiv2018

Microhomology-mediated CRISPR/Cas9-based method for genome editing in fission yeast

The CRISPR/Cas9 system enables the editing of genomes of numerous organisms through the induction of the double-strand breaks (DSB) at specific chromosomal targets. We improved the CRISPR/Cas9 system to ease the direct introduction of a point mutation or a tagging sequence into the chromosome by combining it with the microhomology mediated end joining (MMEJ)-based genome editing in fission yeast. We constructed convenient cloning vectors, which possessed a guide RNA (gRNA) expression module, or the humanized Streptococcus pyogenes Cas9 gene that is expressed under the control of an inducible promoter to avoid the needless expression, or both a gRNA and Cas9 gene. Using this system, we attempted the MMEJ-mediated genome editing and found that the MMEJ-mediated method provides high-frequency genome editing at target loci without the need of a long donor DNA. Using short oligonucleotides, we successfully introduced point mutations into two target genes at high frequency. We also precisely integrated the sequences for epitope and GFP tagging using donor DNA possessing microhomology into the target loci, which enabled us to obtain cells expressing N-terminally tagged fusion proteins. This system could expedite genome editing in fission yeast, and could be applicable to other organisms.

genetics↗

bioRxiv2018

Constitutively active RAS in S. pombe causes persistent Cdc42 signalling but only transient MAPK activation

The small GTPase RAS is a signalling hub for many pathways and oncogenic human RAS mutations are assumed to over-activate all of its downstream pathways. We tested this assumption in fission yeast, where, RAS-mediated pheromone signalling (PS) activates the MAPKSpk1 and Cdc42 pathways. Unexpectedly, we found that constitutively active Ras1.G17V induced immediate but only transient MAPKSpk1 activation, whilst Cdc42 activation persisted. Immediate but transient MAPKSpk1 activation was also seen in the deletion mutant of Cdc42-GEFScd1, a Cdc42 activator. We built a mathematical model using PS negative-feedback circuits and competition between the two Ras1 effectors, MAPKKKByr2 and Cdc42-GEFScd1. The model robustly predicted the MAPKSpk1 activation dynamics of an additional 21 PS mutants. Supporting the model, we showed that a recombinant Cdc42-GEFScd1 fragment competes with MAPKKKByr2 for Ras1 binding. Our study has established a concept that the constitutively active RAS propagates differently to downstream pathways where the system prevents MAPK overactivation. HighlightsO_LIConstitutively active Ras1.GV prolongs Cdc42 activation in S. pombe pheromone signalling C_LIO_LIRas1.GV results in an immediate but only transient MAPKSpk1 activation C_LIO_LIThe RAS effector pathways MAPKSpk1 and Cdc42 compete with each other for active Ras1 C_LIO_LIPredictive modelling explains MAPKSpk1 activation dynamics in 24 signaling-mutants C_LI eTOC BlurbS. pombe Ras1 activates the MAPKSpk1 and Cdc42 pathways. Kelsall et al. report that the constitutively active Ras1.G17V mutation, which causes morphological anomalies, induces prolonged Cdc42 activation but only a transient MAPKSpk1 activation followed by attenuation. Mathematical modelling and biochemical data suggest a competition between the MAPKSpk1 and Cdc42 pathways for active Ras1.

molecular biology↗

bioRxiv2018

HERC2 promotes BLM and WRN to suppress G-quadruplex DNA

BLM and WRN are RecQ DNA helicases essential for genomic stability. Here we demonstrate that HERC2, a HECT E3 ligase, is critical for their functions to suppress G-quadruplex (G4) DNA. HERC2 interacts with BLM, WRN, and replication protein A (RPA) complexes during S-phase of the cell cycle. Depletion of HERC2 dissociates RPA from BLM and WRN complexes and significantly increases G4 formation. Triple depletion revealed that HERC2 has an epistatic relationship with BLM and WRN in their G4- suppressing function. In vitro, HERC2 releases RPA onto single-stranded DNA (ssDNA), rather than anchoring onto RPA-coated ssDNA. CRISPR/Cas9-mediated deletion of the catalytic ubiquitin-binding site of HERC2 causes RPA accumulation in the helicase complexes and increases G4, indicating an essential role for E3 activity in G4 suppression. Both HERC2 depletion and E3 inactivation sensitize cells to the G4-interacting compounds, telomestatin and pyridostatin. Overall, HERC2 is a master regulator of G4 suppression and affects the sensitivity of cells to G4 stabilizers.

molecular biology↗

bioRxiv2018

Soil microbial habitats in an extreme desert Mars-analogue environment

Sediments in the hyper-arid core of the Atacama Desert are a terrestrial analogue to Mars regolith. Understanding the distribution and drivers of microbial life in the sediment may give critical clues on how to search for biosignatures on Mars. Here, we identify the spatial distribution of highly specialised bacterial communities in previously unexplored depth horizons of subsurface sediments. We deployed an autonomous rover in a mission-relevant Martian drilling scenario with manual sample validation. Subsurface communities were delineated by depth related to sediment moisture. Geochemical analysis indicated soluble salts and minerology that influenced water bio-availability, particularly in deeper sediments. Colonization was also patchy and uncolonized sediment was associated with indicators of extreme osmotic challenge. The study identifies linkage between biocomplexity, moisture and geochemistry in Mars-like sediments at the limit of habitability and demonstrates feasibility of the rover-mounted drill for future Mars sample recovery.

microbiology↗

bioRxiv2018

Patterns of polymorphism, selection and linkage disequilibrium in the subgenomes of the allopolyploid Arabidopsis kamchatica

Although genome duplication is widespread in wild and crop plants, little is known about genome-wide selection due to the complexity of polyploid genomes. In allopolyploid species, the patterns of purifying selection and adaptive substitutions would be affected by masking owing to duplicated genes or homeologs as well as by effective population size. We resequenced 25 distribution-wide accessions of the allotetraploid Arabidopsis kamchatica, which has a relatively small genome size (450 Mb) derived from the diploid species A. halleri and A. lyrata. The level of nucleotide polymorphism and linkage disequilibrium decay were comparable to A. thaliana, indicating the feasibility of association studies. A reduction in purifying selection compared with parental species was observed. Interestingly, the proportion of adaptive substitutions () was significantly positive in contrast to the majority of plant species. A recurrent pattern observed in both frequency and divergence-based neutrality tests is that the genome-wide distributions of both subgenomes were similar, but the correlation between homeologous pairs was low. This may increase the opportunity of different evolutionary trajectories such as in the HMA4 gene involved in heavy metal hyperaccumulation.

evolutionary biology↗

bioRxiv2017

Cell Type Specific Survey of Epigenetic Modifications by Tandem Chromatin Immunoprecipitation Sequencing

BackgroundThe nervous system of higher eukaryotes is composed of numerous types of neurons and glia that together orchestrate complex neuronal responses. However, this complex pool of cells typically poses analytical challenges in investigating gene expression profiles and their epigenetic basis for specific cell types. Here, we developed a novel method that enables cell type-specific analyses of epigenetic modifications using tandem chromatin immunoprecipitation sequencing (tChIP-Seq).\n\nResultsFLAG-tagged histone H2B, a constitutive chromatin component, was first expressed in Camk2a-positive pyramidal cortical neurons and used to purify chromatin in a cell type-specific manner. Subsequent chromatin immunoprecipitation using antibodies against H3K4me3--an active promoter mark--allowed us to survey neuron-specific coding and non-coding transcripts. Indeed, tChIP-Seq identified hundreds of genes associated with neuronal functions and genes with unknown functions expressed in cortical neurons.\n\nConclusionstChIP-Seq thus provides a versatile approach to investigating the epigenetic modifications of particular cell types in vivo.

molecular biology↗