Showing papers by "Yutaka Suzuki published in 2014"

A comprehensive assessment of RNA-seq accuracy, reproducibility and information content by the Sequencing Quality Control Consortium

Abstract: We present primary results from the Sequencing Quality Control (SEQC) project, coordinated by the US Food and Drug Administration. Examining Illumina HiSeq, Life Technologies SOLiD and Roche 454 platforms at multiple laboratory sites using reference RNA samples with built-in controls, we assess RNA sequencing (RNA-seq) performance for junction discovery and differential expression profiling and compare it to microarray and quantitative PCR (qPCR) data using complementary metrics. At all sequencing depths, we discover unannotated exon-exon junctions, with >80% validated by qPCR. We find that measurements of relative expression are accurate and reproducible across sites and platforms if specific filters are used. In contrast, RNA-seq and microarrays do not provide accurate absolute measurements, and gene-specific biases are observed for all examined platforms, including qPCR. Measurement performance depends on the platform and data analysis pipeline, and variation is large for transcript-level profiling. The complete SEQC data sets, comprising >100 billion reads (10Tb), provide unique resources for evaluating RNA-seq analyses for clinical and regulatory settings.

A single female-specific piRNA is the primary determiner of sex in the silkworm

Abstract: The silkworm Bombyx mori uses a WZ sex determination system that is analogous to the one found in birds and some reptiles. In this system, males have two Z sex chromosomes, whereas females have Z and W sex chromosomes. The silkworm W chromosome has a dominant role in female determination, suggesting the existence of a dominant feminizing gene in this chromosome. However, the W chromosome is almost fully occupied by transposable element sequences, and no functional protein-coding gene has been identified so far. Female-enriched PIWI-interacting RNAs (piRNAs) are the only known transcripts that are produced from the sex-determining region of the W chromosome, but the function(s) of these piRNAs are unknown. Here we show that a W-chromosome-derived, female-specific piRNA is the feminizing factor of B. mori. This piRNA is produced from a piRNA precursor which we named Fem. Fem sequences were arranged in tandem in the sex-determining region of the W chromosome. Inhibition of Fem-derived piRNA-mediated signalling in female embryos led to the production of the male-specific splice variants of B. mori doublesex (Bmdsx), a gene which acts at the downstream end of the sex differentiation cascade. A target gene of Fem-derived piRNA was identified on the Z chromosome of B. mori. This gene, which we named Masc, encoded a CCCH-type zinc finger protein. We show that the silencing of Masc messenger RNA by Fem piRNA is required for the production of female-specific isoforms of Bmdsx in female embryos, and that Masc protein controls both dosage compensation and masculinization in male embryos. Our study characterizes a single small RNA that is responsible for primary sex determination in the WZ sex determination system.

Genome sequence of the tsetse fly (Glossina morsitans ): Vector of African trypanosomiasis

Abstract: Tsetse flies are the sole vectors of human African trypanosomiasis throughout sub-Saharan Africa. Both sexes of adult tsetse feed exclusively on blood and contribute to disease transmission. Notable differences between tsetse and other disease vectors include obligate microbial symbioses, viviparous reproduction, and lactation. Here, we describe the sequence and annotation of the 366-megabase Glossina morsitans morsitans genome. Analysis of the genome and the 12,308 predicted protein–encoding genes led to multiple discoveries, including chromosomal integrations of bacterial (Wolbachia) genome sequences, a family of lactation-specific proteins, reduced complement of host pathogen recognition proteins, and reduced olfaction/chemosensory associated genes. These genome data provide a foundation for research into trypanosomiasis prevention and yield important insights with broad implications for multiple aspects of tsetse biology.

Phytochrome controls alternative splicing to mediate light responses in Arabidopsis

Abstract: Plants monitor the ambient light conditions using several informational photoreceptors, including red/far-red light absorbing phytochrome Phytochrome is widely believed to regulate the transcription of light-responsive genes by modulating the activity of several transcription factors Here we provide evidence that phytochrome significantly changes alternative splicing (AS) profiles at the genomic level in Arabidopsis, to approximately the same degree as it affects steady-state transcript levels mRNA sequencing analysis revealed that 1,505 and 1,678 genes underwent changes in their AS and steady-state transcript level profiles, respectively, within 1 h of red light exposure in a phytochrome-dependent manner Furthermore, we show that splicing factor genes were the main early targets of AS control by phytochrome, whereas transcription factor genes were the primary direct targets of phytochrome-mediated transcriptional regulation We experimentally validated phytochrome-induced changes in the AS of genes that are involved in RNA splicing, phytochrome signaling, the circadian clock, and photosynthesis Moreover, we show that phytochrome-induced AS changes of SPA1-RELATED 3, the negative regulator of light signaling, physiologically contributed to promoting photomorphogenesis Finally, photophysiological experiments demonstrated that phytochrome transduces the signal from its photosensory domain to induce light-dependent AS alterations in the nucleus Taking these data together, we show that phytochrome directly induces AS cascades in parallel with transcriptional cascades to mediate light responses in Arabidopsis

Identification of the carotenoid modifying gene PALE YELLOW PETAL 1 as an essential factor in xanthophyll esterification and yellow flower pigmentation in tomato (Solanum lycopersicum)

Abstract: Summary Xanthophylls, the pigments responsible for yellow to red coloration, are naturally occurring carotenoid compounds in many colored tissues of plants. These pigments are esterified within the chromoplast; however, little is known about the mechanisms underlying their accumulation in flower organs. In this study, we characterized two allelic tomato (Solanum lycopersicum L.) mutants, pale yellow petal (pyp) 1-1 and pyp1-2, that have reduced yellow color intensity in the petals and anthers due to loss-of-function mutations. Carotenoid analyses showed that the yellow flower organs of wild-type tomato contained high levels of xanthophylls that largely consisted of neoxanthin and violaxanthin esterified with myristic and/or palmitic acids. Functional disruption of PYP1 resulted in loss of xanthophyll esters, which was associated with a reduction in the total carotenoid content and disruption of normal chromoplast development. These findings suggest that xanthophyll esterification promotes the sequestration of carotenoids in the chromoplast and that accumulation of these esters is important for normal chromoplast development. Next-generation sequencing coupled with map-based positional cloning identified the mutant alleles responsible for the pyp1 phenotype. PYP1 most likely encodes a carotenoid modifying protein that plays a vital role in the production of xanthophyll esters in tomato anthers and petals. Our results provide insight into the molecular mechanism underlying the production of xanthophyll esters in higher plants, thereby shedding light on a longstanding mystery.

Aberrant transcriptional regulations in cancers: genome, transcriptome and epigenome analysis of lung adenocarcinoma cell lines

Abstract: Here we conducted an integrative multi-omics analysis to understand how cancers harbor various types of aberrations at the genomic, epigenomic and transcriptional levels. In order to elucidate biological relevance of the aberrations and their mutual relations, we performed whole-genome sequencing, RNA-Seq, bisulfite sequencing and ChIP-Seq of 26 lung adenocarcinoma cell lines. The collected multi-omics data allowed us to associate an average of 536 coding mutations and 13,573 mutations in promoter or enhancer regions with aberrant transcriptional regulations. We detected the 385 splice site mutations and 552 chromosomal rearrangements, representative cases of which were validated to cause aberrant transcripts. Averages of 61, 217, 3687 and 3112 mutations are located in the regulatory regions which showed differential DNA methylation, H3K4me3, H3K4me1 and H3K27ac marks, respectively. We detected distinct patterns of aberrations in transcriptional regulations depending on genes. We found that the irregular histone marks were characteristic to EGFR and CDKN1A, while a large genomic deletion and hyper-DNA methylation were most frequent for CDKN2A. We also used the multi-omics data to classify the cell lines regarding their hallmarks of carcinogenesis. Our datasets should provide a valuable foundation for biological interpretations of interlaced genomic and epigenomic aberrations.

CLOCK-Controlled Polyphonic Regulation of Circadian Rhythms through Canonical and Noncanonical E-Boxes

Abstract: In mammalian circadian clockwork, the CLOCK-BMAL1 complex binds to DNA enhancers of target genes and drives circadian oscillation of transcription. Here we identified 7,978 CLOCK-binding sites in mouse liver by chromatin immunoprecipitation-sequencing (ChIP-Seq), and a newly developed bioinformatics method, motif centrality analysis of ChIP-Seq (MOCCS), revealed a genome-wide distribution of previously unappreciated noncanonical E-boxes targeted by CLOCK. In vitro promoter assays showed that CACGNG, CACGTT, and CATG(T/C)G are functional CLOCK-binding motifs. Furthermore, we extensively revealed rhythmically expressed genes by poly(A)-tailed RNA-Seq and identified 1,629 CLOCK target genes within 11,926 genes expressed in the liver. Our analysis also revealed rhythmically expressed genes that have no apparent CLOCK-binding site, indicating the importance of indirect transcriptional and posttranscriptional regulations. Indirect transcriptional regulation is represented by rhythmic expression of CLOCK-regulated transcription factors, such as Kruppel-like factors (KLFs). Indirect posttranscriptional regulation involves rhythmic microRNAs that were identified by small-RNA-Seq. Collectively, CLOCK-dependent direct transactivation through multiple E-boxes and indirect regulations polyphonically orchestrate dynamic circadian outputs.

Methylome diversification through changes in DNA methyltransferase sequence specificity.

Abstract: Epigenetic modifications such as DNA methylation have large effects on gene expression and genome maintenance. Helicobacter pylori, a human gastric pathogen, has a large number of DNA methyltransferase genes, with different strains having unique repertoires. Previous genome comparisons suggested that these methyltransferases often change DNA sequence specificity through domain movement—the movement between and within genes of coding sequences of target recognition domains. Using single-molecule real-time sequencing technology, which detects N6-methyladenines and N4-methylcytosines with single-base resolution, we studied methylated DNA sites throughout the H. pylori genome for several closely related strains. Overall, the methylome was highly variable among closely related strains. Hypermethylated regions were found, for example, in rpoB gene for RNA polymerase. We identified DNA sequence motifs for methylation and then assigned each of them to a specific homology group of the target recognition domains in the specificity-determining genes for Type I and other restriction-modification systems. These results supported proposed mechanisms for sequence-specificity changes in DNA methyltransferases. Knocking out one of the Type I specificity genes led to transcriptome changes, which suggested its role in gene expression. These results are consistent with the concept of evolution driven by DNA methylation, in which changes in the methylome lead to changes in the transcriptome and potentially to changes in phenotype, providing targets for natural or artificial selection.

A new lncRNA, APTR, associates with and represses the CDKN1A/p21 promoter by recruiting polycomb proteins.

Abstract: Long noncoding RNAs (lncRNAs) have emerged as a major regulator of cell physiology, but many of which have no known function. CDKN1A/p21 is an important inhibitor of the cell-cycle, regulator of the DNA damage response and effector of the tumor suppressor p53, playing a crucial role in tumor development and prevention. In order to identify a regulator for tumor progression, we performed an siRNA screen of human lncRNAs required for cell proliferation, and identified a novel lncRNA, APTR, that acts in trans to repress the CDKN1A/p21 promoter independent of p53 to promote cell proliferation. APTR associates with the promoter of CDKN1A/p21 and this association requires a complementary-Alu sequence encoded in APTR. A different module of APTR associates with and recruits the Polycomb repressive complex 2 (PRC2) to epigenetically repress the p21 promoter. A decrease in APTR is necessary for the induction of p21 after heat stress and DNA damage by doxorubicin, and the levels of APTR and p21 are anti-correlated in human glioblastomas. Our data identify a new regulator of the cell-cycle inhibitor CDKN1A/p21 that acts as a proliferative factor in cancer cell lines and in glioblastomas and demonstrate that Alu elements present in lncRNAs can contribute to targeting regulatory lncRNAs to promoters.

Interactive transcriptome analysis of malaria patients and infecting Plasmodium falciparum

Abstract: To understand the molecular mechanisms of parasitism in vivo, it is essential to elucidate how the transcriptomes of the human hosts and the infecting parasites affect one another. Here we report the RNA-seq analysis of 116 Indonesian patients infected with the malaria parasite Plasmodium falciparum (Pf). We extracted RNAs from their peripheral blood as a mixture of host and parasite transcripts and mapped the RNA-seq tags to the human and Pf reference genomes to separate the respective tags. We were thus able to simultaneously analyze expression patterns in both humans and parasites. We identified human and parasite genes and pathways that correlated with various clinical data, which may serve as primary targets for drug developments. Of particular importance, we revealed characteristic expression changes in the human innate immune response pathway genes including TLR2 and TICAM2 that correlated with the severity of the malaria infection. We also found a group of transcription regulatory factors, JUND, for example, and signaling molecules, TNFAIP3, for example, that were strongly correlated in the expression patterns of humans and parasites. We also identified several genetic variations in important anti-malaria drug resistance-related genes. Furthermore, we identified the genetic variations which are potentially associated with severe malaria symptoms both in humans and parasites. The newly generated data should collectively lay a unique foundation for understanding variable behaviors of the field malaria parasites, which are far more complex than those observed under laboratory conditions.

Pigment dispersing factor regulates ecdysone biosynthesis via bombyx neuropeptide G protein coupled receptor-B2 in the prothoracic glands of Bombyx mori.

Abstract: Ecdysone is the key hormone regulating insect growth and development. Ecdysone synthesis occurs in the prothoracic glands (PGs) and is regulated by several neuropeptides. Four prothoracicotropic and three prothoracicostatic factors have been identified to date, suggesting that ecdysone biosynthesis is intricately regulated. Here, we demonstrate that the neuropeptide pigment dispersing factor (PDF) stimulates ecdysone biosynthesis and that this novel signaling pathway partially overlaps with the prothoracicotropic hormone (PTTH) signaling pathway. We performed transcriptome analysis and focused on receptors predominantly expressed in the PGs. From this screen, we identified a candidate orphan G protein coupled receptor (GPCR), Bombyx neuropeptide GPCR-B2 (BNGR-B2). BNGR-B2 was predominantly expressed in ecdysteroidogenic tissues, and the expression pattern in the PGs corresponded to the ecdysteroid titer in the hemolymph. Furthermore, we identified PDF as a ligand for BNGR-B2. PDF stimulated ecdysone biosynthesis in the PGs, but the stimulation was only observed in the PGs during a specific larval stage. PDF did not affect the transcript level of known ecdysone biosynthetic enzymes, and inhibiting transcription did not suppress ecdysone biosynthesis, suggesting that the effects of PDF might be mediated by translational regulation and/or post-translational modification. In addition, the participation of protein kinase A (PKA), phosphatidylinositol 3-kinase (PI3K), target of rapamycin (TOR) and eukaryotic translation initiation factor 4E (eIF4E)-binding protein (4E-BP) in the PDF signaling pathway was discovered.

Sall1 Maintains Nephron Progenitors and Nascent Nephrons by Acting as Both an Activator and a Repressor

Abstract: The balanced self-renewal and differentiation of nephron progenitors are critical for kidney development and controlled, in part, by the transcription factor Six2, which antagonizes canonical Wnt signaling-mediated differentiation. A nuclear factor, Sall1, is expressed in Six2-positive progenitors as well as differentiating nascent nephrons, and it is essential for kidney formation. However, the molecular functions and targets of Sall1, especially the functions and targets in the nephron progenitors, remain unknown. Here, we report that Sall1 deletion in Six2-positive nephron progenitors results in severe progenitor depletion and apoptosis of the differentiating nephrons in mice. Analysis of mice with an inducible Sall1 deletion revealed that Sall1 activates genes expressed in progenitors while repressing genes expressed in differentiating nephrons. Sall1 and Six2 co-occupied many progenitor-related gene loci, and Sall1 bound to Six2 biochemically. In contrast, Sall1 did not bind to the Wnt4 locus suppressed by Six2. Sall1-mediated repression was also independent of its binding to DNA. Thus, Sall1 maintains nephron progenitors and their derivatives by a unique mechanism, which partly overlaps but is distinct from that of Six2: Sall1 activates progenitor-related genes in Six2-positive nephron progenitors and represses gene expression in Six2-negative differentiating nascent nephrons.

Development and validation of a new high-throughput method to investigate the clonality of HTLV-1-infected cells based on provirus integration sites

Abstract: Transformation and clonal proliferation of T-cells infected with human T-cell leukemia virus type-I (HTLV-1) cause adult T-cell leukemia. We took advantage of next-generation sequencing technology to develop and internally validate a new methodology for isolating integration sites and estimating the number of cells in each HTLV-1-infected clone (clone size). Initial analysis was performed with DNA samples from infected individuals. We then used appropriate controls with known integration sites and clonality status to confirm the accuracy of our system, which indeed had the least errors among the currently available techniques. Results suggest potential clinical and biological applications of the new method.

Whole exome sequencing in Thai patients with retinitis pigmentosa reveals novel mutations in six genes.

Abstract: PURPOSE To identify disease-causing mutations and describe genotype-phenotype correlations in Thai patients with nonsyndromic retinitis pigmentosa (RP). METHODS Whole exome sequencing was performed in 20 unrelated patients. Eighty-six genes associated with RP, Leber congenital amaurosis, and cone-rod dystrophy were analyzed for variant detection. RESULTS Seventeen variants (13 novel and 4 known) in 13 genes were identified in 11 patients. These variants include 10 missense substitutions, 2 nonsense mutations, 3 deletions, 1 insertion, and 1 splice site change. Nine patients with identified inheritance patterns carried a total of 10 potentially pathogenic mutations located in genes CRB1, C8orf37, EYS, PROM1, RP2, and USH2A. Three of the nine patients also demonstrated additional heterozygous variants in genes ABCA4, GUCY2D, RD3, ROM1, and TULP1. In addition, two patients carried variants of uncertain significance in genes FSCN2 and NR2E3. The RP phenotypes of our patients were consistent with previous reports. CONCLUSIONS This is the first report of mutations in Thai RP patients. These findings are useful for genotype-phenotype comparisons among different ethnic groups.

Occupancy of tissue-specific cis -regulatory modules by Otx2 and TLE/Groucho for embryonic head specification

Abstract: Head specification by the head-selector gene, orthodenticle (otx), is highly conserved among bilaterian lineages. However, the molecular mechanisms by which Otx and other transcription factors (TFs) interact with the genome to direct head formation are largely unknown. Here we employ ChIP-seq and RNA-seq approaches in Xenopus tropicalis gastrulae and find that occupancy of the corepressor, TLE/Groucho, is a better indicator of tissue-specific cis-regulatory modules (CRMs) than the coactivator p300, during early embryonic stages. On the basis of TLE binding and comprehensive CRM profiling, we define two distinct types of Otx2- and TLE-occupied CRMs. Using these devices, Otx2 and other head organizer TFs (for example, Lim1/Lhx1 (activator) or Goosecoid (repressor)) are able to upregulate or downregulate a large battery of target genes in the head organizer. An underlying principle is that Otx marks target genes for head specification to be regulated positively or negatively by partner TFs through specific types of CRMs.

Large hypomethylated domains serve as strong repressive machinery for key developmental genes in vertebrates

Abstract: DNA methylation is a fundamental epigenetic modification in vertebrate genomes and a small fraction of genomic regions is hypomethylated. Previous studies have implicated hypomethylated regions in gene regulation, but their functions in vertebrate development remain elusive. To address this issue, we generated epigenomic profiles that include base-resolution DNA methylomes and histone modification maps from both pluripotent cells and mature organs of medaka fish and compared the profiles with those of human ES cells. We found that a subset of hypomethylated domains harbor H3K27me3 (K27HMDs) and their size positively correlates with the accumulation of H3K27me3. Large K27HMDs are conserved between medaka and human pluripotent cells and predominantly contain promoters of developmental transcription factor genes. These key genes were found to be under strong transcriptional repression, when compared with other developmental genes with smaller K27HMDs. Furthermore, human-specific K27HMDs show an enrichment of neuronal activity-related genes, which suggests a distinct regulation of these genes in medaka and human. In mature organs, some of the large HMDs become shortened by elevated DNA methylation and associate with sustained gene expression. This study highlights the significance of domain size in epigenetic gene regulation. We propose that large K27HMDs play a crucial role in pluripotent cells by strictly repressing key developmental genes, whereas their shortening consolidates long-term gene expression in adult differentiated cells.

Discovery of osmotic sensitive transcription factors in fish intestine via a transcriptomic approach

Abstract: Teleost intestine is crucial for seawater acclimation by sensing osmolality of imbibed seawater and regulating drinking and water/ion absorption. Regulatory genes for transforming intestinal function have not been identified. A transcriptomic approach was used to search for such genes in the intestine of euryhaline medaka. Quantitative RNA-seq by Illumina Hi-Seq Sequencing method was performed to analyze intestinal gene expression 0 h, 1 h, 3 h, 1 d, and 7 d after seawater transfer. Gene ontology (GO) enrichment results showed that cell adhesion, signal transduction, and protein phosphorylation gene categories were augmented soon after transfer, indicating a rapid reorganization of cellular components and functions. Among >50 transiently up-regulated transcription factors selected via co-expression correlation and GO selection, five transcription factors, including CEBPB and CEBPD, were confirmed by quantitative PCR to be specific to hyperosmotic stress, while others were also up-regulated after freshwater control transfer, including some well-known osmotic-stress transcription factors such as SGK1 and TSC22D3/Ostf1. Protein interaction networks suggest a high degree of overlapping among the signaling of transcription factors that respond to osmotic and general stresses, which sheds light on the interpretation of their roles during hyperosmotic stress and emergency. Since cortisol is an important hormone for seawater acclimation as well as for general stress in teleosts, emergency and osmotic challenges could have been evolved in parallel and resulted in the overlapped signaling networks. Our results revealed important interactions among transcription factors and offer a multifactorial perspective of genes involved in seawater acclimation.

Induction of virulence gene expression in Staphylococcus aureus by pulmonary surfactant.

Abstract: We performed a genomewide analysis using a next-generation sequencer to investigate the effect of pulmonary surfactant on gene expression in Staphylococcus aureus, a clinically important opportunistic pathogen. RNA sequence (RNA-seq) analysis of bacterial transcripts at late log phase revealed 142 genes that were upregulated >2-fold following the addition of pulmonary surfactant to the culture medium. Among these genes, we confirmed by quantitative reverse transcription-PCR analysis that mRNA amounts for genes encoding ESAT-6 secretion system C (EssC), an unknown hypothetical protein (NWMN_0246; also called pulmonary surfactant-inducible factor A [PsiA] in this study), and hemolysin gamma subunit B (HlgB) were increased 3- to 10-fold by the surfactant treatment. Among the major constituents of pulmonary surfactant, i.e., phospholipids and palmitate, only palmitate, which is the most abundant fatty acid in the pulmonary surfactant and a known antibacterial substance, stimulated the expression of these three genes. Moreover, these genes were also induced by supplementing the culture with detergents. The induction of gene expression by surfactant or palmitate was not observed in a disruption mutant of the sigB gene, which encodes an alternative sigma factor involved in bacterial stress responses. Furthermore, each disruption mutant of the essC, psiA, and hlgB genes showed attenuation of both survival in the lung and host-killing ability in a murine pneumonia model. These findings suggest that S. aureus resists membrane stress caused by free fatty acids present in the pulmonary surfactant through the regulation of virulence gene expression, which contributes to its pathogenesis within the lungs of the host animal.

Natural history of diminutive colorectal polyps: Long-term prospective observation by colonoscopy

Abstract: Background and Aim Endoscopic removal of colorectal adenomatous polyps effectively prevents cancer. However, the treatment strategy for diminutive polyps (diameter ≤5 mm) remains controversial. Understanding the natural history of diminutive polyps is a prerequisite to their effective management. We prospectively examined the natural history of diminutive polyps by long-term surveillance colonoscopy. Methods A total of 207 polyps detected in 112 patients from December 1991 through March 2002 were studied. To avoid potential effects on size and morphological characteristics, all polyps were selected randomly and were followed without biopsy. Polyp size was estimated by comparing the lesion with the diameter of a biopsy forceps. Results Mean follow up was 7.8 years (SD, 4.8; range, 1.0–18.6; median, 7.5; interquartile range 3.4–11.2). Twenty-four polyps were resected endoscopically, and the histopathological diagnosis was mucosal high-grade neoplasia (Category 4) for one polyp, and mucosal low-grade neoplasia (Category 3) for 23 polyps. Mean linear size of the polyps was 3.2 mm (SD, 1.0; range, 1.3–5.0) at initial colonoscopy and 3.8 mm (SD 1.6; range 1.3–10.0) at final colonoscopy (P < 0.01). Left-sided polyps showed a higher growth rate than right-sided polyps, and a type IIIL2 pit pattern was associated with a lower growth rate than a type IIIL1 pattern. Conclusion We clarified the natural history of diminutive polyps by long-term follow-up colonoscopy. The benign course of diminutive polyps should be considered in the design of treatment strategies.

Genome-wide analysis of the chromatin composition of histone H2A and H3 variants in mouse embryonic stem cells

Abstract: Genome-wide distribution of the majority of H2A and H3 variants (H2A, H2AX, H2AZ, macroH2A, H3.1, H3.2 and H3.3) was simultaneously investigated in mouse embryonic stem cells by chromatin immunoprecipitation sequencing. Around the transcription start site, histone variant distribution differed between genes possessing promoters of high and low CpG density, regardless of their expression levels. In the intergenic regions, regulatory elements were enriched in H2A.Z and H3.3, whereas repeat elements were abundant in H2A and macroH2A, and H3.1, respectively. Analysis of H2A and H3 variant combinations composing nucleosomes revealed that the H2A.Z and H3.3 combinations were present at a higher frequency throughout the genome than the other combinations, suggesting that H2A.Z and H3.3 associate preferentially with each other to comprise the nucleosomes independently of genome region. Finally, we found that chromatin was unstable only in regions where it was enriched in both H2A.Z and H3.3, but strongly quantified stable in regions in which only H3.3 was abundant. Therefore, histone variant composition is an important determinant of chromatin structure, which is associated with specific genomic functions.

Construction of mate pair full-length cDNAs libraries and characterization of transcriptional start sites and termination sites

Abstract: To identify and characterize transcript structures ranging from transcriptional start sites (TSSs) to poly(A)-addition sites (PASs), we constructed and analyzed human TSS/PAS mate pair full-length cDNA libraries from 14 tissue types and four cell lines. The collected information enabled us to define TSS cluster (TSC) and PAS cluster (PAC) relationships for a total of 8530/9400 RefSeq genes, as well as 4251/5618 of their putative alternative promoters/terminators and 4619/4605 intervening transcripts, respectively. Analyses of the putative alternative TSCs and alternative PACs revealed that their selection appeared to be mostly independent, with rare exceptions. In those exceptional cases, pairs of transcript units rarely overlapped one another and were occasionally separated by Rad21/CTCF. We also identified a total of 172 similar cases in which TSCs and PACs spanned adjacent but distinct genes. In these cases, different transcripts may utilize different functional units of a particular gene or of adjacent genes. This approach was also useful for identifying fusion gene transcripts in cancerous cells. Furthermore, we could construct cDNA libraries in which 3′-end mate pairs were distributed randomly over the transcripts. These libraries were useful for assembling the internal structure of previously uncharacterized alternative promoter products, as well as intervening transcripts.

A comparison of the rest complex binding patterns in embryonic stem cells and epiblast stem cells.

Abstract: 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.

Comprehensive analysis of mutually exclusive alternative splicing in C. elegans

Abstract: Mutually exclusive selection of one exon in a cluster of exons is a rare form of alternative pre-mRNA splicing, yet suggests strict regulation. However, the repertoires of regulation mechanisms for the mutually exclusive (ME) splicing in vivo are still unknown. Here, we experimentally explore putative ME exons in C. elegans to demonstrate that 29 ME exon clusters in 27 genes are actually selected in a mutually exclusive manner. Twenty-two of the clusters consist of homologous ME exons. Five clusters have too short intervening introns to be excised between the ME exons. Fidelity of ME splicing relies at least in part on nonsense-mediated mRNA decay for 14 clusters. These results thus characterize all the repertoires of ME splicing in this organism.

RNA sequencing: from sample preparation to analysis.

Abstract: The introduction of RNA sequencing (RNA-seq), as a direct result of rapid progression of next-generation sequencing technologies, has revolutionized the world of transcriptomics. It enables quantification of the complete set of RNA with all its isoforms in a given cell, in far greater accuracy than before. Here, we provide a step-by-step guide to the sample preparation in RNA-seq and an overview of the bioinformatic analysis that is required following the cDNA sequencing.

Silkworm HP1a transcriptionally enhances highly expressed euchromatic genes via association with their transcription start sites

Abstract: Heterochromatin protein 1 (HP1) is an evolutionarily conserved protein across different eukaryotic species and is crucial for heterochromatin establishment and maintenance. The silkworm, Bombyx mori, encodes two HP1 proteins, BmHP1a and BmHP1b. In order to investigate the role of BmHP1a in transcriptional regulation, we performed genome-wide analyses of the transcriptome, transcription start sites (TSSs), chromatin modification states and BmHP1a-binding sites of the silkworm ovary-derived BmN4 cell line. We identified a number of BmHP1a-binding loci throughout the silkworm genome and found that these loci included TSSs and frequently co-occurred with neighboring euchromatic histone modifications. In addition, we observed that genes with BmHP1a-associated TSSs were relatively highly expressed in BmN4 cells. RNA interference-mediated BmHP1a depletion resulted in the transcriptional repression of highly expressed genes with BmHP1a-associated TSSs, whereas genes not coupled with BmHP1a-binding regions were less affected by the treatment. These results demonstrate that BmHP1a binds near TSSs of highly expressed euchromatic genes and positively regulates their expression. Our study revealed a novel mode of transcriptional regulation mediated by HP1 proteins.