Kosuke Hashimoto

Deep transcriptome profiling of mammalian stem cells supports a regulatory role for retrotransposons in pluripotency maintenance

The importance of microRNAs and long noncoding RNAs in the regulation of pluripotency has been documented; however, the noncoding components of stem cell gene networks remain largely unknown. Here we investigate the role of noncoding RNAs in the pluripotent state, with particular emphasis on nuclear and retrotransposon-derived transcripts. We have performed deep profiling of the nuclear and cytoplasmic transcriptomes of human and mouse stem cells, identifying a class of previously undetected stem cell–specific transcripts. We show that long terminal repeat (LTR)-derived transcripts contribute extensively to the complexity of the stem cell nuclear transcriptome. Some LTR-derived transcripts are associated with enhancer regions and are likely to be involved in the maintenance of pluripotency.

An integrated expression atlas of miRNAs and their promoters in human and mouse

MicroRNAs (miRNAs) are short non-coding RNAs with key roles in cellular regulation. As part of the fifth edition of the Functional Annotation of Mammalian Genome (FANTOM5) project, we created an integrated expression atlas of miRNAs and their promoters by deep-sequencing 492 short RNA (sRNA) libraries, with matching Cap Analysis Gene Expression (CAGE) data, from 396 human and 47 mouse RNA samples. Promoters were identified for 1,357 human and 804 mouse miRNAs and showed strong sequence conservation between species. We also found that primary and mature miRNA expression levels were correlated, allowing us to use the primary miRNA measurements as a proxy for mature miRNA levels in a total of 1,829 human and 1,029 mouse CAGE libraries. We thus provide a broad atlas of miRNA expression and promoters in primary mammalian cells, establishing a foundation for detailed analysis of miRNA expression patterns and transcriptional control regions.

CAGE profiling of ncRNAs in hepatocellular carcinoma reveals widespread activation of retroviral LTR promoters in virus-induced tumors

An increasing number of noncoding RNAs (ncRNAs) have been implicated in various human diseases including cancer; however, the ncRNA transcriptome of hepatocellular carcinoma (HCC) is largely unexplored. We used CAGE to map transcription start sites across various types of human and mouse HCCs with emphasis on ncRNAs distant from protein-coding genes. Here, we report that retroviral LTR promoters, expressed in healthy tissues such as testis and placenta but not liver, are widely activated in liver tumors. Despite HCC heterogeneity, a subset of LTR-derived ncRNAs were more than 10-fold up-regulated in the vast majority of samples. HCCs with a high LTR activity mostly had a viral etiology, were less differentiated, and showed higher risk of recurrence. ChIP-seq data show that MYC and MAX are associated with ncRNA deregulation. Globally, CAGE enabled us to build a mammalian promoter map for HCC, which uncovers a new layer of complexity in HCC genomics.

Nuclear transcriptome profiling of induced pluripotent stem cells and embryonic stem cells identify non-coding loci resistant to reprogramming

Identification of functionally relevant differences between induced pluripotent stem cells (iPSC) and reference embryonic stem cells (ESC) remains a central question for therapeutic applications. Differences in gene expression between iPSC and ESC have been examined by microarray and more recently with RNA-SEQ technologies. We here report an in depth analyses of nuclear and cytoplasmic transcriptomes, using the CAGE (cap analysis of gene expression) technology, for 5 iPSC clones derived from mouse lymphocytes B and 3 ESC lines. This approach reveals nuclear transcriptomes significantly more complex in ESC than in iPSC. Hundreds of yet not annotated putative non-coding RNAs and enhancer-associated transcripts specifically transcribed in ESC have been detected and supported with epigenetic and chromatin-chromatin interactions data. We identified super-enhancers transcriptionally active specifically in ESC and associated with genes implicated in the maintenance of pluripotency. Similarly, we detected non-coding transcripts of yet unknown function being regulated by ESC specific super-enhancers. Taken together, these results demonstrate that current protocols of iPSC reprogramming do not trigger activation of numerous cis-regulatory regions. It thus reinforces the need for already suggested deeper monitoring of the non-coding transcriptome when characterizing iPSC clones. Such differences in regulatory transcript expression may indeed impact their potential for clinical applications.

STAP cells are derived from ES cells

Two reports claiming a novel cellular reprogramming phenomenon, stimulus-triggered acquisition of pluripotency (STAP), were published in Nature last year, but then subsequently retracted. The identity of STAP cells and STAP-derived stem cells, however, has remained undetermined. Here we report the results of a whole-genome sequencing (WGS) investigation of STAP-related samples kept mainly at the RIKEN Center for Developmental Biology. We show that all purported STAP stem-cell lines were contaminated with embryonic stem (ES) cells, and that chimaeric mice and teratomas supposedly derived from STAP cells instead show ES cell contribution. The original article reported that exposure to low pH can reprogram differentiated cells into unique pluripotent cells (STAP cells), from which two secondary cell lines were established; ES-like STAP stem cells and trophoblast stem-like Fgf4-induced stem cells capable of generating placental cells. Because STAP cells were not maintained as frozen stocks, we first performed WGS of 15 genomic DNA samples in total, including three representative STAP stem-cell lines with different genetic backgrounds, an Fgf4-induced stem-cell line, and seven ES cell lines established at the Wakayama laboratory before or during the STAP study (Extended Data Table 1). We determined genome-wide patterns of single-nucleotide polymorphisms (SNPs) that distinguish mouse strains 129/Sv (129) and C57BL/6 (B6), as well as green fluorescent protein (GFP) transgene types (Supplementary Methods and Extended Data Fig. 1a). No samples from the Oct4-GFP Fgf4-induced stem cells described in the original letter were found (Oct4 is also known as Pou5f1). The STAP stem-cell line FLS and the Fgf4-induced stem-cell line CTS were reported to carry a homozygous insertion of a single cag-gfp transgene with the genetic background of 129 female 3 B6 male (Extended Data Table 1). However, these cell lines had co-insertions of two GFP transgenes, sperm-specific acrosin-promoter-gfp and ubiquitously expressed cag-gfp (hereafter designated Acr/cag-gfp) at chromosome 3, which originated from an Acr/cag-GFP B6 mouse strain not described in the STAP papers. These STAP cell lines were then compared with four ES cell lines—FES1, FES2, and two nuclear transfer ES lines (ntESG1 and ntESG2) (ref. 9)—established from crossing the Acr/cag-GFP mouse strain with 129 mice in the Wakayama laboratory in 2005 (Extended Data Fig. 1a and Extended Data Table 1). FES1 and FES2 cells shared homologous SNP patterns with these STAP cell lines over the entire genome, including the 129 X chromosome, while ntESG1 and ntESG2 cells bearing B6 X chromosome were excluded from the comparison. Furthermore, these STAP cell lines shared two genomic characteristics with FES1, but not FES2; first, two chromosomal deletions (Fig. 1a) are present only in FES1 and all Acr/cag-GFP STAP stem-cell sublines, but not in the other cell lines examined, in the paternal Acr/cag-GFP mice (frozen stock in 2010), or in potential maternal 129 substrains available in Japan. Second, FES1 and the STAP cell lines with Acr/cag-GFP share

Deficiency of multidrug resistance 2 contributes to cell transformation through oxidative stress

Summary The transporter of phosphatidylcholine Mdr2/MDR3 not only plays an essential role for bile formation but also is involved in the maintenance of lipid homeostasis. Deficiency of Mdr2 leads to accumulation of ROS, cell transformation and susceptibility to intestinal carcinogenesis.

Characterization of Novel Transcripts of Human Papillomavirus Type 16 Using Cap Analysis Gene Expression Technology

ABSTRACT We have performed cap-analysis gene expression (CAGE) sequencing to identify the regulatory networks that orchestrate genome-wide transcription in human papillomavirus type 16 (HPV16)-positive cervical cell lines of different grades: W12E, SiHa, and CaSki. Additionally, a cervical intraepithelial neoplasia grade 1 (CIN1) lesion was assessed for identifying the transcriptome expression profile. Here we have precisely identified a novel antisense noncoding viral transcript in HPV16. In conclusion, CAGE sequencing should pave the way for understanding a diversity of viral transcript expression.

Epi-drivers and cancer-testis genes

Wang et al . (1) reported new candidate cancer drivers by studying cancer-testis expression patterns in large cohorts of normal and cancer samples. The authors assumed that few cancer can be fully explained by the mutation drivers (mut-drivers) and thus they focused on studying epigenetic drivers (epi-drivers); genes that are altered by epigenetic mechanisms and confer selective growth advantage (2). The authors reasoned that since epi-drivers are aberrantly expressed in cancer due to underlying epigenetic alterations, it is possible to search for epi-drivers by using transcriptomics data. This is a very tempting idea since little is known about epi-drivers and their role in cancer is relatively poorly understood as compared to mut-drivers. However, epigenetics mechanisms alone do not fully explain altered gene expression, which can be caused by multiple factors such as mutations in promoter regions, DNA copy number alterations, mutations of the up-stream transcription factor or changes in signaling. For example in our recent transcriptome study of genes de-regulated and activated across multiple cancer types (3) we opted for not calling the broadly de-regulated genes as “epi-drivers”. Interestingly, Wang et al . regarded MEIOB (meiosis specific with OB domains) to be an epi-driver, while also noting that its expression is correlated with (and probably driven by) arm level and focal DNA copy number alterations (CNAs). While MEIOB is a reported cancer driver (4,5) that is differentially expressed in cancer, there is no epigenetic data to regard MEIOB as an epi-driver.

Genomics dissection of the zebrafish heart

Development of the embryonic head is determined by the activity of gene regulatory networks (GRN) driven by transcription factors such as LHX1 and OTX2. Analysis of genetic mutants has revealed that loss of Lhx1 function at sequential steps of embryonic head formation leads to the truncation of head structures. We have implemented Lhx1-expressing embryonic stem cells (ESCs) to generate embryos for the identification of the in vivo ensemble of LHX1 downstream target genes. We have generated embryos from ESCs harbouring a conditional Lhx1 transgene and have confirmed inducible activation of Lhx1 in the embryos that are rendered amenable for RNA-seq and ChIP-seq analyses. Meta-analysis of gene expression profiles of differentiating Lhx1-expressing ESCs and the anterior germ layers of mouse gastrula-staged embryos, and ChIPseq data on the transcriptional targets of the LHX1 orthologue in Xenopus embryos has revealed a Head GRN, consisting of 92 genes as putative targets of LHX1 in the mouse. Among the putative targets, the functional attribute of nine that have no known phenotype are being studied by analysis of the chimeras derived from genome-edited ESCs. Preliminary findings of these studies will be presented.

Single-nucleotide-resolution mapping of HBV promoters in infected human livers and hepatocellular carcinoma

ABSTRACT Hepatitis B virus (HBV) is a major cause of liver diseases, including hepatocellular carcinoma (HCC), and more than 650,000 people die annually due to HBV-associated liver failure. Extensive studies of individual promoters have revealed that heterogeneous RNA 5′ ends contribute to the complexity of HBV transcriptome and proteome. Here, we provide a comprehensive map of HBV transcription start sites (TSSs) in human liver, HCC, and blood, as well as several experimental replication systems, at a single-nucleotide resolution. Using CAGE (cap analysis of gene expression) analysis of 16 HCC/nontumor liver pairs, we identify 17 robust TSSs, including a novel promoter for the X gene located in the middle of the gene body, which potentially produces a shorter X protein translated from the conserved second start codon, and two minor antisense transcripts that might represent viral noncoding RNAs. Interestingly, transcription profiles were similar in HCC and nontumor livers, although quantitative analysis revealed highly variable patterns of TSS usage among clinical samples, reflecting precise regulation of HBV transcription initiation at each promoter. Unlike the variety of TSSs found in liver and HCC, the vast majority of transcripts detected in HBV-positive blood samples are pregenomic RNA, most likely generated and released from liver. Our quantitative TSS mapping using the CAGE technology will allow better understanding of HBV transcriptional responses in further studies aimed at eradicating HBV in chronic carriers. IMPORTANCE Despite the availability of a safe and effective vaccine, HBV infection remains a global health problem, and current antiviral protocols are not able to eliminate the virus in chronic carriers. Previous studies of the regulation of HBV transcription have described four major promoters and two enhancers, but little is known about their activity in human livers and HCC. We deeply sequenced the HBV RNA 5′ ends in clinical human samples and experimental models by using a new, sensitive and quantitative method termed cap analysis of gene expression (CAGE). Our data provide the first comprehensive map of global TSS distribution over the entire HBV genome in the human liver, validating already known promoters and identifying novel locations. Better knowledge of HBV transcriptional activity in the clinical setting has critical implications in the evaluation of therapeutic approaches that target HBV replication.