Showing papers by "Sumio Sugano published in 2014"
TL;DR: It is shown 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.
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.
407 citations
TL;DR: An integrative multi-omics analysis to understand how cancers harbor various types of aberrations at the genomic, epigenomic and transcriptional levels 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.
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.
98 citations
TL;DR: 7,978 CLOCK-binding sites in mouse liver are identified 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.
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.
97 citations
TL;DR: DNA sequence motifs for methylation were identified and assigned to a specific homology group of the target recognition domains in the specificity-determining genes for Type I and other restriction-modification systems, which supported proposed mechanisms for sequence-specificity changes in DNA methyltransferases.
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.
83 citations
TL;DR: The 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 lnc RNAs to promoters.
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.
79 citations
TL;DR: It is reported that Sall1 deletion in Six2-positive nephron progenitors results in severe progenitor depletion and apoptosis of the differentiating nephrons in mice, and a unique mechanism, which partly overlaps but is distinct from that of Six2, is maintained.
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.
56 citations
TL;DR: Next-generation sequencing technology was developed and internally validated a new methodology for isolating integration sites and estimating the number of cells in each HTLV-1-infected clone (clone size), which indeed had the least errors among the currently available techniques.
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.
54 citations
TL;DR: ChIP-seq and RNA-seq approaches in Xenopus tropicalis gastrulae are employed and it is found 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.
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.
45 citations
TL;DR: It is proposed 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.
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.
43 citations
TL;DR: Analysis of putative associations between 34, 45, and 103 miRNAs and 164, 391, and 81 mRNAs via Argonaute1 or Ago2 immunoprecipitation experiments in a colon cancer cell line identified potential hypoxia-induced miRNA-mRNA associations, which included several well-characterized cancer-related genes as novel putative miRNA targets.
30 citations
TL;DR: The draft to date has been developed under the auspices of the Regulatory and Ethics Working Group of the Global Alliance for Genomics and Health, in collaboration with many individuals, organizations and committees around the world.
Abstract: The draft to date has been developed under the auspices of the Regulatory and Ethics Working Group of the Global Alliance for Genomics and Health, in collaboration with many individuals, organizations and committees around the world.
TL;DR: Perturbation of copper homeostasis induced profound and early developmental arrest of P. falciparum, and may be applied in the development of effective antimalarial strategies.
Abstract: Background: Malaria continues to be a devastating disease. The elucidation of factors inducing asexual growth versus arrest of Plasmodium falciparum can provide information about the development of the parasite, and may help in the search for novel malaria medication. Based on information from genome-wide transcriptome profiling of different developmental stages of P. falciparum, we investigated the critical importance of copper homeostasis in the developmental succession of P. falciparum with regard to three aspects of copper function. These were:1) inhibition of copper-binding proteins, 2) copper-ion chelation, and 3) down-regulated expression of genes encoding copper-binding proteins associated with a specific growth-promoting factor. Results: Inhibition of copper-binding proteins with tetrathiomolybdate (TTM) caused cessation of growth of the parasite. TTM arrested the parasite irreversibly during the trophozoite to schizont stage progression. Target molecules for TTM may be present in P. falciparum. The involvement of copper ions in developmental arrest was also investigated by copper-ion chelating methods, which indicated a critical function of reduced copper ions (Cu 1+ ) in the parasite during the early developmental stage. Copper ions, not only in the parasite but also in host cells, were targets of the chelators. Chelation of Cu 1+ caused blockage of trophozoite progression from the ring stage. Profound growth arrest was detected in parasites cultured in a chemically defined medium containing hexadecanoic acid alone as a growth-promoting factor. This developmental arrest was associated with down-regulated expression of genes encoding copper-binding proteins. Cis-9-octadecenoic acid completely prevented the down-regulation of gene expression and developmental arrest that were observed with the use of hexadecanoic acid. Conclusions: The critical importance of copper homeostasis in early developmental stages of P. falciparum was confirmed. Perturbation of copper homeostasis induced profound and early developmental arrest of P. falciparum. These findings should help to elucidate the mechanisms behind the development of P. falciparum, and may be applied in the development of effective antimalarial strategies.
TL;DR: These libraries were useful for assembling the internal structure of previously uncharacterized alternative promoter products, as well as intervening transcripts, and were also useful for identifying fusion gene transcripts in cancerous cells.
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.
TL;DR: 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.
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.
TL;DR: 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 are provided.
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.
TL;DR: It is demonstrated that BmHP1a binds near TSSs of highly expressed euchromatic genes and positively regulates their expression, revealing a novel mode of transcriptional regulation mediated by HP1 proteins.
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.
TL;DR: A complete sequence of mitochondrial genome of echolocating megabat Rousettus leschenaulti is presented and phylogenetic analysis based on mitochondrial genomes of eighteen bat species, including R. lescenaulti, supports division of Chiroptera into Yinpterochiroptera and Yangochioptera.
Abstract: We present a complete sequence of mitochondrial genome of echolocating megabat Rousettus leschenaulti (16,704 nt, GenBank record KC702803) and provide its annotation. We also show that phylogenetic analysis based on mitochondrial genomes of eighteen bat species, including R. leschenaulti, supports division of Chiroptera into Yinpterochiroptera and Yangochiroptera.
01 Nov 2014
TL;DR: In this paper, the authors proposed an analysis framework based on the minimum description length principle for extracting useful information from eQTL data, which is achieved by minimizing the stochastic complexity of the data by using the universal normalized maximum likelihood code as the global code length optimization criterion.
Abstract: Rapid development of genome sequencing technologies enables novel insights into the mechanisms of complex disease through Big Data analysis Physicians can nowadays assay a patient's gene variants and gene expression patterns in a timely manner and use the obtained data to study an individual's susceptibility to complex disease and unravel the underlying mechanisms of disease pathogenesis Massive amounts of correlated genotype, gene expression, and clinical data are collected in eQTL datasets In this work, we propose an analysis framework based on the minimum description length principle for extracting useful information from eQTL data This is achieved by minimizing the stochastic complexity of the data by using the universal normalized maximum likelihood code as the global code length optimization criterion The algorithm simultaneously identifies disease associated features, extracts the optimal model of the complex disease, and estimates its parameters Applied to a simulated eQTL dataset, our framework successfully reveals the underlying mechanisms of a hypothetical complex disease interaction network