Combining our full-length cDNA method and the massively parallel sequencing technology, we developed a simple method to collect precise positional information of transcriptional start sites (TSSs) together with digital information of the gene-expression levels in a high throughput manner. We applied this method to observe gene-expression changes in a colon cancer cell line cultured in normoxic and hypoxic conditions. We generated more than 100 million 36-base TSS-tag sequences and revealed comprehensive features of hypoxia responsive alterations in the transcriptional landscape of the human genome. The features include presence of inducible 'hot regions' in 54 genomic regions, 220 novel hypoxia inducible promoters that may drive non-protein-coding transcripts, 191 hypoxia responsive alternative promoters and detailed views of 120 novel as well as known hypoxia responsive genes. We further analyzed hypoxic response of different cells using additional 60 million TSS-tags and found that the degree of the gene-expression changes were different among cell lines, possibly reflecting cellular robustness against hypoxia. The novel dynamic figure of the human gene transcriptome will deepen our understanding of the transcriptional program of the human genome as well as bringing new insights into the biology of cancer cells in hypoxia.

Using full-length cDNA sequences, we compared alternative splicing (AS) in humans and mice. The alignment of the human and mouse genomes showed that 86% of 199 426 total exons in human AS variants were conserved in the mouse genome. Of the 20 392 total human AS variants, however, 59% consisted of all conserved exons. Comparing AS patterns between human and mouse transcripts revealed that only 431 transcripts from 189 loci were perfectly conserved AS variants. To exclude the possibility that the full-length human cDNAs used in the present study, especially those with retained introns, were cloning artefacts or prematurely spliced transcripts, we experimentally validated 34 such cases. Our results indicate that even retained-intron type transcripts are typically expressed in a highly controlled manner and interact with translating ribosomes. We found non-conserved AS exons to be predominantly outside the coding sequences (CDSs). This suggests that non-conserved exons in the CDSs of transcripts cause functional constraint. These findings should enhance our understanding of the relationship between AS and species specificity of human genes.

Full-Parasites (http://fullmal.hgc.jp/) is a transcriptome database of apicomplexa parasites, which include Plasmodium and Toxoplasma species. The latest version of Full-Parasites contains a total of 105,786 EST sequences from 12 parasites, of which 5925 full-length cDNAs have been completely sequenced. Full-Parasites also contain more than 30 million transcription start sites (TSS) for Plasmodium falciparum (Pf) and Toxoplasma gondii (Tg), which were identified using our novel oligo-capping-based protocol. Various types of cDNA data resources were interconnected with our original database functionalities. Specifically, in this update, we have included two unique RNA-Seq data sets consisting of 730 million mapped RNA-Seq tags. One is a dataset of 16 time-lapse experiments of cultured bradyzoite differentiation for Tg. The other dataset includes 31 clinical samples of Pf. Parasite RNA was extracted together with host human RNA, and the extracted mixed RNA was used for RNA sequencing, with the expectation that gene expression information from the host and parasite would be simultaneously represented. By providing the largest unique full-length cDNA and dynamic transcriptome data, Full-Parasites is useful for understanding host-parasite interactions and will help to eventually elucidate how monophyletic organisms have evolved to become parasites by adopting complex life cycles.

Massively parallel, tag-based sequencing systems, such as the SOLiD system, hold the promise of revolutionizing the study of whole genome gene expression due to the number of data points that can be generated in a simple and cost-effective manner. We describe the development of a 5'-end transcriptome workflow for the SOLiD system and demonstrate the advantages in sensitivity and dynamic range offered by this tag-based application over traditional approaches for the study of whole genome gene expression. 5'-end transcriptome analysis was used to study whole genome gene expression within a colon cancer cell line, HT-29, treated with the DNA methyltransferase inhibitor, 5-aza-2'-deoxycytidine (5Aza). More than 20 million 25-base 5'-end tags were obtained from untreated and 5Aza-treated cells and matched to sequences within the human genome. Seventy three percent of the mapped unique tags were associated with RefSeq cDNA sequences, corresponding to approximately 14,000 different protein-coding genes in this single cell type. The level of expression of these genes ranged from 0.02 to 4,704 transcripts per cell. The sensitivity of a single sequence run of the SOLiD platform was 100-1,000 fold greater than that observed from 5'end SAGE data generated from the analysis of 70,000 tags obtained by Sanger sequencing. The high-resolution 5'end gene expression profiling presented in this study will not only provide novel insight into the transcriptional machinery but should also serve as a basis for a better understanding of cell biology.

DBTSS (http://dbtss.hgc.jp/) was originally constructed as a collection of uniquely determined transcriptional start sites (TSSs) in humans and some other species in 2002. Since then, it has been regularly updated and in recent updates epigenetic information has also been incorporated because such information is useful for characterizing the biological relevance of these TSSs/downstream genes. In the newest release, Release 9, we further integrated public and original single nucleotide variation (SNV) data into our database. For our original data, we generated SNV data from genomic analyses of various cancer types, including 97 lung adenocarcinomas and 57 lung small cell carcinomas from Japanese patients as well as 26 cell lines of lung cancer origin. In addition, we obtained publically available SNV data from other cancer types and germline variations in total of 11,322 individuals. With these updates, users can examine the association between sequence variation pattern in clinical lung cancers with its corresponding TSS-seq, RNA-seq, ChIP-seq and BS-seq data. Consequently, DBTSS is no longer a mere storage site for TSS information but has evolved into an integrative platform of a variety of genome activity data.

We detected and characterized the binding sites of the representative Rest complex components Rest, Sin3A, and Lsd1. We compared their binding patterns in mouse embryonic stem (ES) cells and epiblast stem (EpiS) cells. We found few Rest sites unique to the EpiS cells. The ES-unique site features were distinct from those of the common sites, namely, the signal intensities were weaker, and the characteristic gene function categories differed. Our analyses showed that the Rest binding sites do not always overlap with the Sin3A and Lsd1 binding sites. The Sin3A binding pattern differed remarkably between the ES and EpiS cells and was accompanied by significant changes in acetylated-histone patterns in the surrounding regions. A series of transcriptome analyses in the same cell types unexpectedly showed that the putative target gene transcript levels were not dramatically different despite dynamic changes in the Rest complex binding patterns and chromatin statuses, which suggests that Rest is not the sole determinant of repression at its targets. Nevertheless, we identified putative Rest targets with explicitly enhanced transcription upon Rest knock-down in 143 and 60 common and ES-unique Rest target genes, respectively. Among such sites, several genes are involved in ES cell proliferation. In addition, we also found that long, intergenic non-coding RNAs were apparent Rest targets and shared similar features with the protein-coding target genes. Interestingly, such non-coding target genes showed less conservation through evolution than protein-coding targets. As a result of differences in the components and targets of the Rest complex, its functional roles may differ in ES and EpiS cells.

Whole exome sequencing (WES) has become a common tool for identifying genetic causes of human inherited disorders, and it has also recently been applied to canine genome research. We conducted WES analysis of neuroaxonal dystrophy (NAD), a neurodegenerative disease that sporadically occurs worldwide in Papillon dogs. The disease is considered an autosomal recessive monogenic disease, which is histopathologically characterized by severe axonal swelling, known as "spheroids," throughout the nervous system. By sequencing all eleven DNA samples from one NAD-affected Papillon dog and her parents, two unrelated NAD-affected Papillon dogs, and six unaffected control Papillon dogs, we identified 10 candidate mutations. Among them, three candidates were determined to be "deleterious" by in silico pathogenesis evaluation. By subsequent massive screening by TaqMan genotyping analysis, only the PLA2G6 c.1579G>A mutation had an association with the presence or absence of the disease, suggesting that it may be a causal mutation of canine NAD. As a human homologue of this gene is a causative gene for infantile neuroaxonal dystrophy, this canine phenotype may serve as a good animal model for human disease. The results of this study also indicate that WES analysis is a powerful tool for exploring canine hereditary diseases, especially in rare monogenic hereditary diseases.

Coelacanths are known as "living fossils," as they show remarkable morphological resemblance to the fossil record and belong to the most primitive lineage of living Sarcopterygii (lobe-finned fishes and tetrapods). Coelacanths may be key to elucidating the tempo and mode of evolution from fish to tetrapods. Here, we report the genome sequences of five coelacanths, including four Latimeria chalumnae individuals (three specimens from Tanzania and one from Comoros) and one L. menadoensis individual from Indonesia. These sequences cover two African breeding populations and two known extant coelacanth species. The genome is ∼2.74 Gbp and contains a high proportion (∼60%) of repetitive elements. The genetic diversity among the individuals was extremely low, suggesting a small population size and/or a slow rate of evolution. We found a substantial number of genes that encode olfactory and pheromone receptors with features characteristic of tetrapod receptors for the detection of airborne ligands. We also found that limb enhancers of bmp7 and gli3, both of which are essential for limb formation, are conserved between coelacanth and tetrapods, but not ray-finned fishes. We expect that some tetrapod-like genes may have existed early in the evolution of primitive Sarcopterygii and were later co-opted to adapt to terrestrial environments. These coelacanth genomes will provide a cornerstone for studies to elucidate how ancestral aquatic vertebrates evolved into terrestrial animals.

Transcription start sites (TSSs) with pronounced and phased nucleosome arrays downstream and nucleosome-depleted regions upstream of TSSs are observed in various species.

About 1 million expressed sequence tag (EST) sequences comprising 125.3 Mb nucleotides were accreted from 51 cDNA libraries constructed from a variety of tissues and organs under a range of conditions, including abiotic stresses and pathogen challenges in common wheat (Triticum aestivum). Expressed sequence tags were assembled with stringent parameters after processing with inbuild scripts, resulting in 37,138 contigs and 215,199 singlets. In the assembled sequences, 10.6% presented no matches with existing sequences in public databases. Functional characterization of wheat unigenes by gene ontology annotation, mining transcription factors, full-length cDNA, and miRNA targeting sites were carried out. A bioinformatics strategy was developed to discover single-nucleotide polymorphisms (SNPs) within our large EST resource and reported the SNPs between and within (homoeologous) cultivars. Digital gene expression was performed to find the tissue-specific gene expression, and correspondence analysis was executed to identify common and specific gene expression by selecting four biotic stress-related libraries. The assembly and associated information cater a framework for future investigation in functional genomics.

The mouse embryonal carcinoma cell line ATDC5 provides an excellent model system for chondrogenesis in vitro. To understand better the molecular mechanisms of endochondral bone formation, we investigated gene expression profiles during the differentiation course of ATDC5 cells, using an in-house microarray harboring full-length-enriched cDNAs. For 28 days following chondrogenic induction, 507 genes were up- or down-regulated at least 1.5-fold. These genes were classified into five clusters based on their expression patterns. Genes for growth factor and cytokine pathways were significantly enriched in the cluster characterized by increases in expression during late stages of chondrocyte differentiation. mRNAs for decorin and osteoglycin, which have been shown to bind to transforming growth factors-beta and bone morphogenetic proteins, respectively, were found in this cluster and were detected in hypertrophic chondrocytes of developing mouse bones by in situ hybridization analysis. Taken together with assigned functions of individual genes in the cluster, interdigitated interaction between a number of intercellular signaling molecules is likely to take place in the late chondrogenic stage for autocrine and paracrine regulation among chondrocytes, as well as for chemoattraction and stimulation of progenitor cells of other lineages.

The current RNA-Seq method analyses fragments of mRNAs, from which it is occasionally difficult to reconstruct the entire transcript structure. Here, we performed and evaluated the recent procedure for full-length cDNA sequencing using the Nanopore sequencer MinION. We applied MinION RNA-Seq for various applications, which would not always be easy using the usual RNA-Seq by Illumina. First, we examined and found that even though the sequencing accuracy was still limited to 92.3%, practically useful RNA-Seq analysis is possible. Particularly, taking advantage of the long-read nature of MinION, we demonstrate the identification of splicing patterns and their combinations as a form of full-length cDNAs without losing precise information concerning their expression levels. Transcripts of fusion genes in cancer cells can also be identified and characterized. Furthermore, the full-length cDNA information can be used for phasing of the SNPs detected by WES on the transcripts, providing essential information to identify allele-specific transcriptional events. We constructed a catalogue of full-length cDNAs in seven major organs for two particular individuals and identified allele-specific transcription and splicing. Finally, we demonstrate that single-cell sequencing is also possible. RNA-Seq on the MinION platform should provide a novel approach that is complementary to the current RNA-Seq.

Lung cancer is a major cause of death worldwide, with lung adenocarcinoma being the most frequently diagnosed subtype in Japan. Finding the target of an anticancer drug can improve lung adenocarcinoma treatments. Polo-like kinase 1 (PLK1) is an essential mitotic kinase in mitotic progression, and PLK1 inhibition induces cell cycle arrest and apoptosis in tumor cells. In addition, a variety of PLK1 inhibitors have been identified for cancer treatments. In this study, we looked for the target gene of the anticancer drug that has synergy with PLK1 inhibitors. We identified karyopherin beta 1 (KPNB1) as a possible target for lung adenocarcinoma treatment. We found that PLK1 inhibition decreased KPNB1 expression in lung adenocarcinoma cells and KPNB1 depletion inhibited cell proliferation via apoptosis. The same apoptosis signaling pathway may be activated because the expression of common apoptosis-related genes was decreased by PLK1 and KPNB1 silencing; however, the time course of cell growth inhibition was somewhat different. Cell cycle analysis showed that KPNB1 depletion increased the proportion of cells at the G0/G1 phase, although cells also accumulated at the G2/M phase in PLK1-depleted cells. Our findings suggest that decreased KPNB1 expression may be associated with the apoptosis induced by PLK1 inhibition.

The evolution of sex chromosomes and mating loci in organisms with UV systems of sex/mating type determination in haploid phases via genes on UV chromosomes is not well understood. We report the structure of the mating type (MT) locus and its evolutionary history in the green seaweed Ulva partita, which is a multicellular organism with an isomorphic haploid-diploid life cycle and mating type determination in the haploid phase. Comprehensive comparison of a total of 12.0 and 16.6 Gb of genomic next-generation sequencing data for mt- and mt+ strains identified highly rearranged MT loci of 1.0 and 1.5 Mb in size and containing 46 and 67 genes, respectively, including 23 gametologs. Molecular evolutionary analyses suggested that the MT loci diverged over a prolonged period in the individual mating types after their establishment in an ancestor. A gene encoding an RWP-RK domain-containing protein was found in the mt- MT locus but was not an ortholog of the chlorophycean mating type determination gene MID. Taken together, our results suggest that the genomic structure and its evolutionary history in the U. partita MT locus are similar to those on other UV chromosomes and that the MT locus genes are quite different from those of Chlorophyceae.

Chromosomal interactions regulate genome functions, such as transcription, via dynamic chromosomal organization in the nucleus. In this study, we attempted to identify genomic regions that physically bind to the promoter region of the Pax5 gene, which encodes a master regulator for B cell lineage commitment, in a chicken B cell line, DT40, with the goal of obtaining mechanistic insight into transcriptional regulation through chromosomal interaction. We found that the Pax5 promoter bound to multiple genomic regions using locus-specific chromatin immunoprecipitation (locus-specific ChIP), a method for locus-specific isolation of target genomic regions, in combination with next-generation sequencing (NGS). Comparing chromosomal interactions in wild-type DT40 with those in a macrophage-like counterpart, we found that some of the identified chromosomal interactions were organized in a B cell-specific manner. In addition, deletion of a B cell-specific interacting genomic region in chromosome 11, which was marked by active enhancer histone modifications, resulted in moderate but significant down-regulation of Pax5 transcription. Together, these results suggested that Pax5 transcription in DT40 is regulated by B cell-specific inter-chromosomal interactions. Moreover, these analyses showed that locus-specific ChIP combined with NGS analysis is useful for non-biased identification of functional genomic regions that physically interact with a locus of interest.

Influenza A viruses cause seasonal epidemics and occasional pandemics. The emergence of viruses resistant to neuraminidase (NA) inhibitors and M2 ion channel inhibitors underlines the need for alternate anti-influenza drugs with novel mechanisms of action. Here, we report the discovery of a host factor as a potential target of anti-influenza drugs. By using cell-based virus replication screening of a chemical library and several additional assays, we identified clonidine as a new anti-influenza agent in vitro. We found that clonidine, which is an agonist of the alpha2-adrenergic receptor (α2-AR), has an inhibitory effect on the replication of various influenza virus strains. α2-AR is a Gi-type G protein-coupled receptor that reduces intracellular cyclic AMP (cAMP) levels. In-depth analysis showed that stimulation of α2-ARs leads to impairment of influenza virus replication and that α2-AR agonists inhibit the virus assembly step, likely via a cAMP-mediated pathway. Although clonidine administration did not reduce lung virus titers or prevent body weight loss, it did suppress lung edema and improve survival in a murine lethal infection model. Clonidine may thus protect against lung damage caused by influenza virus infection. Our results identify α2-AR-mediated signaling as a key pathway to exploit in the development of anti-influenza agents.

Image-based cell/colony analyses offer promising solutions to compensate for the lack of quality control (QC) tools for noninvasive monitoring of cultured cells, a regulatory challenge in regenerative medicine. Here, the feasibility of two image analysis algorithms, optical flow and normalised cross-correlation, to noninvasively measure cell/colony motion in human primary oral keratinocytes for screening the proliferative capacity of cells in the early phases of cell culture were examined. We applied our software to movies converted from 96 consecutive time-lapse phase-contrast images of an oral keratinocyte culture. After segmenting the growing colonies, two indices were calculated based on each algorithm. The correlation between each index of the colonies and their proliferative capacity was evaluated. The software was able to assess cell/colony motion noninvasively, and each index reflected the observed cell kinetics. A positive linear correlation was found between cell/colony motion and proliferative capacity, indicating that both algorithms are potential tools for QC.

The spindle assembly checkpoint (SAC) is a surveillance mechanism that prevents unequal segregation of chromosomes during mitosis. Abnormalities in the SAC are associated with chromosome instability and resultant aneuploidy. This study was performed to evaluate the SAC competence in canine malignant melanoma (CMM) using four aneuploid cell lines (CMeC1, CMeC2, KMeC, and LMeC). After treatment with nocodazole, a microtubule disrupting agent, CMeC1, KMeC, and LMeC cells were arrested in M phase, whereas CMeC2 cells were not arrested, and progressed into the next cell cycle phase without cytokinesis. Chromosome spread analysis revealed a significantly increased rate of premature sister chromatid separation in CMeC2 cells. Expression of the phosphorylated form of the SAC regulator, monopolar spindle 1 (Mps1), was lower in CMeC2 cells than in the other CMM cell lines. These results indicate that the SAC is defective in CMeC2 cells, which may partially explain aneuploidy in CMM. Thus, CMeC2 cells may be useful for further studies of the SAC mechanism in CMM and in determining the relationship between SAC incompetence and aneuploidy.

Nonalcoholic steatohepatitis (NASH) occurs in the context of aberrant metabolism. Glutaminolysis is required for metabolic reprograming of hepatic stellate cells (HSCs) and liver fibrogenesis in mice. However, it is unclear how changes in HSC glutamine metabolism contribute to net changes in hepatic glutaminolytic activity during fibrosis progression, or whether this could be used to track fibrogenic activity in NASH. We postulated that increased HSC glutaminolysis marks active scarring in NASH.

To better understand the disruptions of transcriptional regulations and gene expression in lung cancers, we constructed a multi-omics catalogue of the responses of lung cancer cells to a series of chemical compounds. We generated and analyzed 3,240 RNA-seq and 3,393 ATAC-seq libraries obtained from 23 cell lines treated with 95 well-annotated compounds. To demonstrate the power of the created multi-omics resource, we attempted to identify drugs that could induce the designated changes alone or in combination. The basal multi-omics information was first integrated into co-expression modules. Among these modules, we identified a stress response module that may be a promising drug intervention target, as new combinations of compounds that could be used to regulate this module and the consequent phenotypic appearance of cancer cells have been identified. We believe that the multi-omics profiles generated in this study and the strategy used to stratify them will lead to more rational and efficient development of anticancer drugs.