Possibilities of skin coat color-dependent risks and risk factors of squamous cell carcinoma and deafness of domestic cats inferred via RNA-seq data.

The transcriptome data of skin cells from domestic cats with brown, orange, and white coats were analyzed using a public database to investigate the possible relationship between coat color-related gene expression and squamous cell carcinoma risk, as well as the mechanism of deafness in white cats. We found that the ratio of the expression level of genes suppressing squamous cell carcinoma to that of genes promoting squamous cell carcinoma might be considerably lower than the theoretical estimation in skin cells with orange and white coats in white-spotted cat. We also found the possibility of the frequent production of KIT lacking the first exon (d1KIT) in skin cells with white coats, and d1KIT production exhibited a substantial negative correlation with the expression of SOX10, which is essential for melanocyte formation and adjustment of hearing function. Additionally, the production of d1KIT was expected to be due to the insulating activity of the feline endogenous retrovirus 1 (FERV1) LTR in the first intron of KIT by its CTCF binding sequence repeat. These results contribute to basic veterinary research to understand the relationship between cat skin coat and disease risk, as well as the underlying mechanism.

Assembly of continuous high-resolution draft genome sequence of Hemicentrotus pulcherrimus using long-read sequencing.

The update of the draft genome assembly of sea urchin, Hemicentrotus pulcherrimus, which is widely studied in East Asia as a model organism of early development, was performed using Oxford nanopore long-read sequencing. The updated assembly provided ~600-Mb genome sequences divided into 2,163 contigs with N50 = 516 kb. BUSCO completeness score and transcriptome model mapping ratio (TMMR) of the present assembly were obtained as 96.5% and 77.8%, respectively. These results were more continuous with higher resolution than those by the previous version of H. pulcherrimus draft genome, HpulGenome_v1, where the number of scaffolds = 16,251 with a total of ~100 Mb, N50 = 143 kb, BUSCO completeness score = 86.1%, and TMMR = 55.4%. The obtained genome contained 36,055 gene models that were consistent with those in other echinoderms. Additionally, two tandem repeat sequences of early histone gene locus containing 47 copies and 34 copies of all histone genes, and 185 of the homologous sequences of the interspecifically conserved region of the Ars insulator, ArsInsC, were obtained. These results provide further advance for genome-wide research of development, gene regulation, and intranuclear structural dynamics of multicellular organisms using H. pulcherrimus.

CRISPR-Cas9-Mediated Gene Knockout in a Non-Model Sea Urchin, Heliocidaris crassispina.

Sea urchins have been used as model organisms in developmental biology research and the genomes of several sea urchin species have been sequenced. Recently, genome editing technologies have become available for sea urchins, and methods for gene knockout using the CRISPRCas9 system have been established. Heliocidaris crassispina is an important marine fishery resource with edible gonads. Although H. crassispina has been used as a biological research material, its genome has not yet been published, and it is a non-model sea urchin for molecular biology research. However, as recent advances in genome editing technology have facilitated genome modification in non-model organisms, we applied genome editing using the CRISPR-Cas9 system to H. crassispina. In this study, we targeted genes encoding ETS transcription factor (HcEts) and pigmentation-related polyketide synthase (HcPks1). Gene fragments were isolated using primers designed by inter-specific sequence comparisons within Echinoidea. When Ets gene was targeted using two sgRNAs, one successfully introduced mutations and impaired skeletogenesis. In the Pks1 gene knockout, when two sgRNAs targeting the close vicinity of the site corresponding to the target site that showed 100% mutagenesis efficiency of the Pks1 gene in Hemicentrotus pulcherrimus, mutagenesis was not observed. However, two other sgRNAs targeting distant sites efficiently introduced mutations. In addition, Pks1 knockout H. crassispina exhibited an albino phenotype in the pluteus larvae and adult sea urchins after metamorphosis. This indicates that the CRISPRCas9 system can be used to modify the genome of the non-model sea urchin H. crassispina.

Partial exogastrulation due to apical-basal polarity of F-actin distribution disruption in sea urchin embryo by omeprazole.

Gastrulation is a universal process in the morphogenesis of many animal embryos. Although morphological and molecular events in gastrulation have been well studied, the mechanical driving forces and underlying regulatory mechanisms are not fully understood. Here, we investigated the gastrulation of embryos of a sea urchin, Hemicentrotus pulcherrimus, which involves the invagination of a single-layered vegetal plate into the blastocoel. We observed that omeprazole, a proton pump inhibitor capable of perturbing the left-right asymmetry of sea urchin embryo, induced "partial exogastrulation" where the secondary invagination proceeds outward. During early gastrulation, intracellular apical-basal polarity of F-actin distribution in vegetal half was higher than those in animal half, while omeprazole treatment disturbed the apical-basal polarity of F-actin distribution in vegetal half. Furthermore, gastrulation stopped and even partial exogastrulation did not occur when F-actin polymerization or degradation in whole embryo was partially inhibited via RhoA or YAP1 knockout. A mathematical model of the early gastrulation reproduced the shapes of both normal and exogastrulating embryos using cell-dependent cytoskeletal features based on F-actin. Additionally, such cell position-dependent intracellular F-actin distributions might be regulated by intracellular pH distributions. Therefore, apical-basal polarity of F-actin distribution disrupted by omeprazole may induce the partial exogastrulation via anomalous secondary invagination.

The crucial role of CTCF in mitotic progression during early development of sea urchin.

CCCTC-binding factor (CTCF), an insulator protein with 11 zinc fingers, is enriched at the boundaries of topologically associated domains (TADs) in eukaryotic genomes. In this study, we isolated and analyzed the cDNAs encoding HpCTCF, the CTCF homolog in the sea urchin Hemicentrotus pulcherrimus, to investigate its expression patterns and functions during the early development of sea urchin. HpCTCF contains nine zinc fingers corresponding to fingers 2-10 of the vertebrate CTCF. Expression pattern analysis revealed that HpCTCF mRNA was detected at all developmental stages and in the entire embryo. Upon expressing the HpCTCF-GFP fusion protein in early embryos, we observed its uniform distribution within interphase nuclei. However, during mitosis, it disappeared from the chromosomes and subsequently reassembled on the chromosome during telophase. Moreover, the morpholino-mediated knockdown of HpCTCF resulted in mitotic arrest during the morula to blastula stage. Most of the arrested chromosomes were not phospholylated at serine 10 of histone H3, indicating that mitosis was arrested at the telophase by HpCTCF depletion. Furthermore, impaired sister chromatid segregation was observed using time-lapse imaging of HpCTCF-knockdown embryos. Thus, HpCTCF is essential for mitotic progression during the early development of sea urchins, especially during the telophase-to-interphase transition. However, the normal development of pluteus larvae in CRISPR-mediated HpCTCF-knockout embryos suggests that disruption of zygotic HpCTCF expression has little effect on embryonic and larval development.

Dynamic changes in the interchromosomal interaction of early histone gene loci during development of sea urchin.

The nuclear positioning and chromatin dynamics of eukaryotic genes are closely related to the regulation of gene expression, but they have not been well examined during early development, which is accompanied by rapid cell cycle progression and dynamic changes in nuclear organization, such as nuclear size and chromatin constitution. In this study, we focused on the early development of the sea urchin Hemicentrotus pulcherrimus and performed three-dimensional fluorescence in situ hybridization of gene loci encoding early histones (one of the types of histone in sea urchin). There are two non-allelic early histone gene loci per sea urchin genome. We found that during the morula stage, when the early histone gene expression levels are at their maximum, interchromosomal interactions were often formed between the early histone gene loci on separate chromosomes and that the gene loci were directed to locate to more interior positions. Furthermore, these interactions were associated with the active transcription of the early histone genes. Thus, such dynamic interchromosomal interactions may contribute to the efficient synthesis of early histone mRNA during the morula stage of sea urchin development.

Establishment of knockout adult sea urchins by using a CRISPR-Cas9 system.

Sea urchins are used as a model organism for research on developmental biology and gene regulatory networks during early development. Gene knockdown by microinjection of morpholino antisense oligonucleotide (MASO) has been used to analyze gene function in early sea urchin embryos. However, as the effect of MASO is not long lasting, it is impossible to perturb genes expressed during late development by MASO. Recent advances in genome editing technologies have enabled gene modification in various organisms. We previously reported genome editing in the sea urchin Hemicentrotus pulcherrimus using zinc-finger nuclease (ZFN) and transcription activator-like effector nuclease (TALEN); however, the efficiencies of these technologies were not satisfactory. Here, we applied clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated nuclease 9 (Cas9) technology to knock out the Pks1 gene in H. pulcherrimus. When sgRNAs targeting Pks1, which is required for the biosynthesis of larval pigment, were microinjected into fertilized eggs with SpCas9 mRNA, high-efficiency mutagenesis was achieved within 24 hr post fertilization and SpCas9/sgRNA-injected pluteus larvae had an albino phenotype. One of the sgRNAs yielded 100% mutagenesis efficiency, and no off-target effect was detected. In addition, the albino phenotype was maintained in juvenile sea urchins after metamorphosis, and the knockout sea urchins survived for at least one year and grew to albino adult sea urchins. These findings suggest that knockout adult sea urchins were successfully established and the CRISPR-Cas9 system is a feasible method for analyzing gene functions from late developmental to adult stage.

Diverse stochasticity leads a colony of ants to optimal foraging.

A mathematical model of garden ants (Lasius japonicus) is introduced herein to investigate the relationship between the distribution of the degree of stochasticity in following pheromone trails and the group foraging efficiency. Numerical simulations of the model indicate that depending on the systematic change of the feeding environment, the optimal distribution of stochasticity shifts from a mixture of almost deterministic and mildly stochastic ants to a contrasted mixture of almost deterministic ants and highly stochastic ants. In addition, the interaction between the stochasticity and the pheromone path regulates the dynamics of the foraging efficiency optimization. Stochasticity could strengthen the collective efficiency when the variance in the sensitivity to pheromone for ants is introduced in the model.

Prediction of nucleosome positioning by the incorporation of frequencies and distributions of three different nucleotide segment lengths into a general pseudo k-tuple nucleotide composition.

MOTIVATION: Nucleosome positioning plays important roles in many eukaryotic intranuclear processes, such as transcriptional regulation and chromatin structure formation. The investigations of nucleosome positioning rules provide a deeper understanding of these intracellular processes. RESULTS: Nucleosome positioning prediction was performed using a model consisting of three types of variables characterizing a DNA sequence-the number of five-nucleotide sequences, the number of three-nucleotide combinations in one period of a helix, and mono- and di-nucleotide distributions in DNA fragments. Using recently proposed stringent benchmark datasets with low biases for Saccharomyces cerevisiae, Homo sapiens, Caenorhabditis elegans and Drosophila melanogaster, the present model was shown to have a better prediction performance than the recently proposed predictors. This model was able to display the common and organism-dependent factors that affect nucleosome forming and inhibiting sequences as well. Therefore, the predictors developed here can accurately predict nucleosome positioning and help determine the key factors influencing this process. CONTACT: awa@hiroshima-u.ac.jpSupplementary information: Supplementary data are available at Bioinformatics online.

Cilia play a role in breaking left-right symmetry of the sea urchin embryo.

Left-right asymmetry of bilaterian animals is established during early development. In mice, frogs and fishes, the ciliated left-right organizer plays an essential role in establishing bilateral asymmetry, and leftward flow of extracellular fluid generated by ciliary motion results in Nodal activity on the left side. However, H(+) /K(+) -ATPase activity is also involved in the determination of left-right asymmetry in a variety of animals, and it has been thought to be an ancestral mechanism in deuterostomes. In sea urchin, the determination of the left-right asymmetry based on H(+) /K(+) -ATPase activity was already clarified, but it remains to be uncovered whether ciliary motion is involved in the left-right asymmetry of the embryo. Here, we show evidence that ciliary motion is involved in the establishment of left-right asymmetry of sea urchin embryo. Furthermore, we show that the initial cilia generated on small micromeres during the early stage of embryogenesis may be involved in this process. These results suggest that the cilia-mediated mechanism for the determination of left-right asymmetry may be acquired at the base of the deuterostomes.

Noise-plasticity correlations of gene expression in the multicellular organism Arabidopsis thaliana.

Gene expression levels exhibit stochastic variations among genetically identical organisms under the same environmental conditions (called gene expression "noise" or phenotype "fluctuation"). In yeast and Escherichia coli, positive correlations have been found between such gene expression noise and "plasticity" with environmental variations. To determine the universality of such correlations in both unicellular and multicellular organisms, we focused on the relationships between gene expression "noise" and "plasticity" in Arabidopsis thaliana, a multicellular model organism. In recent studies on yeast and E. coli, only some gene groups with specific properties of promoter architecture, average expression levels, and functions exhibited strong noise-plasticity correlations. However, we found strong noise-plasticity correlations for most gene groups in Arabidopsis; additionally, promoter architecture, functional essentiality of genes, and circadian rhythm appeared to have only a weak influence on the correlation strength. The differences in the characteristics of noise-plasticity correlations may result from three-dimensional chromosomal structures and/or circadian rhythm.

Mathematical model of structural changes in nuclear speckle.

Nuclear speckles are nuclear bodies consisting of populations of small and irregularly shaped droplet-like molecular condensates that contain various splicing factors. Recent experiments have revealed the following structural features of nuclear speckles: (I) Each molecular condensate contains SON and SRRM2 proteins, and MALAT1 non-coding RNA surrounds these condensates; (II) During normal interphase of the cell cycle in multicellular organisms, these condensates are broadly distributed throughout the nucleus. In contrast, when cell transcription is suppressed, the condensates fuse and form strongly condensed spherical droplets; (III) SON is dispersed spatially in MALAT1 knocked-down cells and MALAT1 is dispersed in SON knocked-down cells because of the collapse of the nuclear speckles. However, the detailed interactions among the molecules that are mechanistically responsible for the structural variation remain unknown. In this study, a coarse-grained molecular dynamics model of the nuclear speckle was developed by considering the dynamics of SON, SRRM2, MALAT1, and pre-mRNA as representative components of the condensates. The simulations reproduced the structural changes, which were used to predict the interaction network among the representative components of the condensates.

Bifurcation analysis of the transition of dune shapes under a unidirectional wind.

A bifurcation analysis of dune shape transition is made. By use of a reduced model of dune morphodynamics, the Dune Skeleton model, we elucidate the transition mechanism between different shapes of dunes under unidirectional wind. It was found that the decrease in the total amount of sand in the system and/or the lateral sand flow shifts the stable state from a straight transverse dune to a wavy transverse dune through a pitchfork bifurcation. A further decrease causes wavy transverse dunes to shift into barchans through a Hopf bifurcation. These bifurcation structures reveal the transition mechanism of dune shapes under unidirectional wind.

Mathematical model of chromosomal dynamics during DNA double strand break repair in budding yeast.

During the repair of double-strand breaks (DSBs) in DNA, active mobilizations for conformational changes in chromosomes have been widely observed in eukaryotes, from yeast to animal and plant cells. DSB-damaged loci in the yeast genome showed increased mobility and relocation to the nuclear periphery. However, the driving forces behind DSB-induced chromatin dynamics remain unclear. In this study, mathematical models of normal and DSB-damaged yeast chromosomes were developed to simulate their structural dynamics. The effects of histone degradation in the whole nucleus and the change in the physical properties of damaged loci due to the binding of SUMOylated repair proteins were considered in the model of DSB-induced chromosomes based on recent experimental results. The simulation results reproduced DSB-induced changes to structural and dynamical features by which the combination of whole nuclear histone degradation and the rigid structure formation of repair protein accumulations on damaged loci were suggested to be primary contributors to the process by which damaged loci are relocated to the nuclear periphery.

Epigenetic-structural changes in X chromosomes promote Xic pairing during early differentiation of mouse embryonic stem cells.

X chromosome inactivation center (Xic) pairing occurs during the differentiation of embryonic stem (ES) cells from female mouse embryos, and is related to X chromosome inactivation, the circadian clock, intra-nucleus architecture, and metabolism. However, the mechanisms underlying the identification and approach of X chromosome pairs in the crowded nucleus are unclear. To elucidate the driving force of Xic pairing, we developed a coarse-grained molecular dynamics model of intranuclear chromosomes in ES cells and in cells 2 days after the onset of differentiation (2-day cells) by considering intrachromosomal epigenetic-structural feature-dependent mechanics. The analysis of the experimental data showed that X-chromosomes exhibit the rearrangement of their distributions of open/closed chromatin regions on their surfaces during cell differentiation. By simulating models where the excluded volume effects of closed chromatin regions are stronger than those of open chromatin regions, such rearrangement of open/closed chromatin regions on X-chromosome surfaces promoted the mutual approach of the Xic pair. These findings suggested that local intrachromosomal epigenetic features may contribute to the regulation of cell species-dependent differences in intranuclear architecture.

Molecular dynamics analysis of biomolecular systems including nucleic acids.

With the recent progress in structural biology and genome biology, structural dynamics of molecular systems that include nucleic acids has attracted attention in the context of gene regulation. The structure-function relationship is an important topic that highlights the importance of the physicochemical properties of nucleotides, as well as that of amino acids in proteins. Simulations are a useful tool for the detailed analysis of molecular dynamics that complement experiments in molecular biology; however, molecular simulation of nucleic acids is less well developed than that of proteins partly due to the physical nature of nucleic acids. In this review, we briefly describe the current status and future directions of the field as a guide to promote collaboration between experimentalists and computational biologists.

Methylome data derived from maternal-zygotic DNA methyltransferase 3aa-/- zebrafish.

Genomic DNA methylation is an epigenetic marker mediated by DNA methyltransferases (Dnmts); in vertebrates, it comprises of a maintenance DNA methyltransferase, Dnmt1, and two de novo DNA methyltransferases (Dnmt3a and Dnmt3b). In zebrafish, there are two homologs of the mammalian Dnmt3a: Dnmt3aa and Dnmt3ab. A knockout (KO) mutant of zebrafish dnmt3aa was generated using the CRISPR/Cas9 genome-editing system as a new model for DNA methylation research. Since zebrafish dnmt3aa KO mutants were viable and fertile, a maternal-zygotic dnmt3aa deficient mutant (MZdnmt3aa) was generated. We performed whole-genome bisulfite sequencing (WGBS) to reveal the DNA methylation profile using this mutant and identified genomic regions with altered CpG methylation as differentially methylated regions (DMRs) in this mutant compared to those in the wild-type fish. We provided novel raw and processed datasets using the MZdnmt3aa KO mutant, and the raw data of WGBS are available through the Gene Expression Omnibus (GEO), accession number GSE178690.

Identification of aberrant transcription termination at specific gene loci with DNA hypomethylated transcription termination sites caused by DNA methyltransferase deficiency.

CpG methylation of genomic DNA is a well-known repressive epigenetic marker in eukaryotic transcription, and DNA methylation of promoter regions is correlated with gene silencing. In contrast to the promoter regions, the function of DNA methylation during transcription termination remains to be elucidated. A recent study revealed that mouse DNA methyltransferase 3a (Dnmt3a) mainly functions in de novo methylation in the promoter and gene body regions, including transcription termination sites (TTSs), during development. To investigate the relationship between DNA methylation overlapping the TTSs and transcription termination, we performed bioinformatics analysis using six pre-existing Dnmt-/- mouse cell datasets: four types of neurons (three Dnmt3a-/- and one Dnmt1-/- mutants) and two types of embryonic fibroblasts (MEFs) (Dnmt3a-/- and Dnmt3b-/- mutants). Combined analyses using methylome and transcriptome data revealed that read counts downstream of hypomethylated TTSs were increased in three types of neurons (two Dnmt3a-/- and one Dnmt1-/- mutants). Among these, an increase in chimeric transcripts downstream of the TTSs was observed in Dnmt3a-/- mature olfactory sensory neurons and Dnmt3a-/- agouti-related peptide (protein)-producing neurons, thereby indicating that read-through occurs in hypomethylated TTSs at specific gene loci in these two mutants. Conversely, in Dnmt3a-/- MEFs, we detected reductions in read counts downstream of hypomethylated TTSs. These results indicate that the hypomethylation of TTSs can both positively and negatively regulate transcription termination, dependent on Dnmt and cell types. This study is the first to identify the aberrant termination of transcription at specific gene loci with DNA hypomethylated TTSs attributable to Dnmt deficiency.

Ribosomal protein L5 facilitates rDNA-bundled condensate and nucleolar assembly.

The nucleolus is the site of ribosome assembly and formed through liquid-liquid phase separation. Multiple ribosomal DNA (rDNA) arrays are bundled in the nucleolus, but the underlying mechanism and significance are unknown. In the present study, we performed high-content screening followed by image profiling with the wndchrm machine learning algorithm. We revealed that cells lacking a specific 60S ribosomal protein set exhibited common nucleolar disintegration. The depletion of RPL5 (also known as uL18), the liquid-liquid phase separation facilitator, was most effective, and resulted in an enlarged and un-separated sub-nucleolar compartment. Single-molecule tracking analysis revealed less-constrained mobility of its components. rDNA arrays were also unbundled. These results were recapitulated by a coarse-grained molecular dynamics model. Transcription and processing of ribosomal RNA were repressed in these aberrant nucleoli. Consistently, the nucleoli were disordered in peripheral blood cells from a Diamond-Blackfan anemia patient harboring a heterozygous, large deletion in RPL5 Our combinatorial analyses newly define the role of RPL5 in rDNA array bundling and the biophysical properties of the nucleolus, which may contribute to the etiology of ribosomopathy.

Structural dynamics of DNA depending on methylation pattern.

DNA methylation is associated with a number of biological phenomena, and plays crucial roles in epigenetic regulation of eukaryotic gene expression. It is also suggested that DNA methylation alters the mechanical properties of DNA molecules, which is likely to affect epigenetic regulation. However, it has not been systematically investigated how methylation changes the structural and dynamic features of DNA. In this research, to elucidate the effects of methylation on DNA mechanics, a fully atomic molecular dynamics simulation of double-stranded DNA with several methylation patterns was performed. Through the analysis of the relative positioning of the nucleotides (base-step variables), characteristic changes in terms of local flexibility were observed, which further affected the overall DNA geometry and stiffness. These findings may serve as a basis for a discussion on methylation-dependent DNA dynamics in physiological conditions.