| 1. |
Dai Shimizu, Kenzui Taniue, Yusuke Matsui, Hiroshi Haeno, Hiromitsu Araki, Fumihito Miura, Mitsuko Fukunaga, Kenji Shiraishi, Yuji Miyamoto, Seiichi Tsukamoto, Aya Komine, Yuta Kobayashi, Akihiro Kitagawa, Yukihiro Yoshikawa, Kuniaki Sato, Tomoko Saito, Shuhei Ito, Takaaki Masuda, Atsushi Niida, Makoto Suzuki, Hideo Baba, Takashi Ito, Nobuyoshi Akimitsu, Yasuhiro Kodera, Koshi Mimori, Pan-cancer methylome analysis for cancer diagnosis and classification of cancer cell of origin., Cancer gene therapy, 10.1038/s41417-021-00401-w, 2021.11, The accurate and early diagnosis and classification of cancer origin from either tissue or liquid biopsy is crucial for selecting the appropriate treatment and reducing cancer-related mortality. Here, we established the CAncer Cell-of-Origin (CACO) methylation panel using the methylation data of the 28 types of cancer in The Cancer Genome Atlas (7950 patients and 707 normal controls) as well as healthy whole blood samples (95 subjects). We showed that the CACO methylation panel had high diagnostic potential with high sensitivity and specificity in the discovery (maximum AUC = 0.998) and validation (maximum AUC = 1.000) cohorts. Moreover, we confirmed that the CACO methylation panel could identify the cancer cell type of origin using the methylation profile from liquid as well as tissue biopsy, including primary, metastatic, and multiregional cancer samples and cancer of unknown primary, independent of the methylation analysis platform and specimen preparation method. Together, the CACO methylation panel can be a powerful tool for the classification and diagnosis of cancer.. |
| 2. |
Zhaonan Zou, Tazro Ohta, Fumihito Miura, Shinya Oki, ChIP-Atlas 2021 update: a data-mining suite for exploring epigenomic landscapes by fully integrating ChIP-seq, ATAC-seq and Bisulfite-seq data., Nucleic acids research, 10.1093/nar/gkac199, 2022.03, ChIP-Atlas (https://chip-atlas.org) is a web service providing both GUI- and API-based data-mining tools to reveal the architecture of the transcription regulatory landscape. ChIP-Atlas is powered by comprehensively integrating all data sets from high-throughput ChIP-seq and DNase-seq, a method for profiling chromatin regions accessible to DNase. In this update, we further collected all the ATAC-seq and whole-genome bisulfite-seq data for six model organisms (human, mouse, rat, fruit fly, nematode, and budding yeast) with the latest genome assemblies. These together with ChIP-seq data can be visualized with the Peak Browser tool and a genome browser to explore the epigenomic landscape of a query genomic locus, such as its chromatin accessibility, DNA methylation status, and protein-genome interactions. This epigenomic landscape can also be characterized for multiple genes and genomic loci by querying with the Enrichment Analysis tool, which, for example, revealed that inflammatory bowel disease-associated SNPs are the most significantly hypo-methylated in neutrophils. Therefore, ChIP-Atlas provides a panoramic view of the whole epigenomic landscape. All datasets are free to download via either a simple button on the web page or an API.. |
| 3. |
Kentaro Inoue, Hiromitsu Araki, Fumihito Miura, Takashi Ito, Transcriptome dynamics during cholesterol-induced transdifferentiation of human coronary artery smooth muscle cells: A Gene Ontology-centric clustering approach., Biochemistry and biophysics reports, 10.1016/j.bbrep.2021.101061, 27, 101061-101061, 2021.09, Macrophage-like cells derived from vascular smooth muscle cells (SMCs) play critical roles in atherogenesis, and DNA hydroxymethylation was implicated in transdifferentiation. We examined transcriptomes and (hydroxy)methylomes of human coronary artery SMCs during cholesterol-induced transdifferentiation. A unique approach of exhaustive identification of differentially expressed genes followed by Gene Ontology-centric clustering facilitated deeper understanding of multifaceted modulations of genes involved in extracellular matrix organization, angiogenesis, cell migration, hypoxia response, and cholesterol biosynthesis. Intriguingly, type I interferon response was transiently activated, presumably forming an immuno-metabolic circuit with cholesterol metabolism. Neither global nor DEG-proximal changes were evident in (hydroxy)methylation. These results would not only provide a unique data resource for atherosclerosis research but present a potentially useful approach in transcriptome data interpretation.. |
| 4. |
Hidenari Hirata, Atsushi Niida, Nobuyuki Kakiuchi, Ryutaro Uchi, Keishi Sugimachi, Takaaki Masuda, Tomoko Saito, Shun-Ichiro Kageyama, Yushi Motomura, Shuhei Ito, Tadamasa Yoshitake, Daisuke Tsurumaru, Yusuke Nishimuta, Akira Yokoyama, Takanori Hasegawa, Kenichi Chiba, Yuichi Shiraishi, Junyan Du, Fumihito Miura, Masaru Morita, Yasushi Toh, Masakazu Hirakawa, Yoshiyuki Shioyama, Takashi Ito, Tetsuo Akimoto, Satoru Miyano, Tatsuhiro Shibata, Masaki Mori, Yutaka Suzuki, Seishi Ogawa, Kousei Ishigami, Koshi Mimori, The Evolving Genomic Landscape of Esophageal Squamous Cell Carcinoma Under Chemoradiotherapy., Cancer research, 10.1158/0008-5472.CAN-21-0653, 81, 19, 4926-4938, 2021.10, Esophageal squamous cell carcinoma (ESCC) often recurs after chemoradiotherapy, and the prognosis of ESCC after chemoradiotherapy has not improved over the past few decades. The mutation process in chemoradiotherapy-resistant clones and the functional relevance of genetic alterations remain unclear. To address these problems, we performed whole-exome sequencing of 52 tumor samples from 33 patients with ESCC who received radiotherapy combined with 5-fluorouracil/platinum. In multiregion analyses of pretreatment and locally recurrent lesions from five cases, most driver gene-altered clones remained under chemoradiotherapy selection pressure, while few driver gene alterations were acquired at recurrence. The mutation signatures of recurrent ESCC, including increased deletion frequency and platinum dose-dependent base substitution signatures, were substantially different from those of primary ESCC and reflected the iatrogenic impacts of chemoradiotherapy. Single-region analysis of 28 pretreatment tumors indicated that focal copy-number gain at the MYC locus was significantly associated with poor progression-free survival and overall survival after chemoradiotherapy. MYC gain remained throughout the chemoradiotherapy course and potentially contributes to intrinsic resistance to chemoradiotherapy. Consistent with these findings, MYC copy number and mRNA and protein levels in ESCC cell lines correlated positively with resistance to radiotherapy, and MYC knockdown improved sensitivity to radiotherapy. Overall, these data characterize the clonal evolution process induced by chemoradiotherapy and clinically relevant associations for genetic alterations in ESCC. These findings increase our understanding of therapeutic resistance and support the rationale for precision chemoradiotherapy. SIGNIFICANCE: Whole-exome sequencing reveals the genetic evolution of ESCC during chemoradiotherapy, highlighting MYC gain in pretreatment tumors as a potential marker of therapy resistance.. |
| 5. |
Yuki Takada, Ruken Yaman-Deveci, Takayuki Shirakawa, Jafar Sharif, Shin-Ichi Tomizawa, Fumihito Miura, Takashi Ito, Michio Ono, Kuniko Nakajima, Yoko Koseki, Fuyuko Shiotani, Kei-Ichiro Ishiguro, Kazuyuki Ohbo, Haruhiko Koseki, Maintenance DNA methylation in pre-meiotic germ cells regulates meiotic prophase by facilitating homologous chromosome pairing., Development (Cambridge, England), 10.1242/dev.194605, 148, 10, 2021.05, Heterochromatin-related epigenetic mechanisms, such as DNA methylation, facilitate pairing of homologous chromosomes during the meiotic prophase of mammalian spermatogenesis. In pro-spermatogonia, de novo DNA methylation plays a key role in completing meiotic prophase and initiating meiotic division. However, the role of maintenance DNA methylation in the regulation of meiosis, especially in the adult, is not well understood. Here, we reveal that NP95 (also known as UHRF1) and DNMT1 - two essential proteins for maintenance DNA methylation - are co-expressed in spermatogonia and are necessary for meiosis in male germ cells. We find that Np95- or Dnmt1-deficient spermatocytes exhibit spermatogenic defects characterized by synaptic failure during meiotic prophase. In addition, assembly of pericentric heterochromatin clusters in early meiotic prophase, a phenomenon that is required for subsequent pairing of homologous chromosomes, is disrupted in both mutants. Based on these observations, we propose that DNA methylation, established in pre-meiotic spermatogonia, regulates synapsis of homologous chromosomes and, in turn, quality control of male germ cells. Maintenance DNA methylation, therefore, plays a role in ensuring faithful transmission of both genetic and epigenetic information to offspring.. |
| 6. |
Kiyoshi Yoshioka, Hiroshi Nagahisa, Fumihito Miura, Hiromitsu Araki, Yasutomi Kamei, Yasuo Kitajima, Daiki Seko, Jumpei Nogami, Yoshifumi Tsuchiya, Narihiro Okazaki, Akihiko Yonekura, Seigo Ohba, Yoshinori Sumita, Ko Chiba, Kosei Ito, Izumi Asahina, Yoshihiro Ogawa, Takashi Ito, Yasuyuki Ohkawa, Yusuke Ono, Hoxa10 mediates positional memory to govern stem cell function in adult skeletal muscle, SCIENCE ADVANCES, 10.1126/sciadv.abd7924, 7, 24, 2021.06, Muscle stem cells (satellite cells) are distributed throughout the body and have heterogeneous properties among muscles. However, functional topographical genes in satellite cells of adult muscle remain unidentified. Here, we show that expression of Homeobox-A (Hox-A) cluster genes accompanied with DNA hypermethylation of the Hox-A locus was robustly maintained in both somite-derived muscles and their associated satellite cells in adult mice, which recapitulates their embryonic origin. Somite-derived satellite cells were clearly separated from cells derived from cranial mesoderm in Hoxa10 expression. Hoxa10 inactivation led to genomic instability and mitotic catastrophe in somite-derived satellite cells in mice and human. Satellite cell-specific Hoxa10 ablation in mice resulted in a decline in the regenerative ability of somite-derived muscles, which were unobserved in cranial mesoderm-derived muscles. Thus, our results show that Hox gene expression profiles instill the embryonic history in satellite cells as positional memory, potentially modulating region-specific pathophysiology in adult muscles.. |
| 7. |
Wakako Kuribayashi, Motohiko Oshima, Naoki Itokawa, Shuhei Koide, Yaeko Nakajima-Takagi, Masayuki Yamashita, Satoshi Yamazaki, Bahityar Rahmutulla, Fumihito Miura, Takashi Ito, Atsushi Kaneda, Atsushi Iwama, Limited rejuvenation of aged hematopoietic stem cells in young bone marrow niche., The Journal of experimental medicine, 10.1084/jem.20192283, 218, 3, 2021.03, Hematopoietic stem cells (HSCs) exhibit functional alterations, such as reduced regenerative capacity and myeloid-biased differentiation, with age. The HSC niche, which is essential for the maintenance of HSCs, also undergoes marked changes with aging. However, it has been technically challenging to directly evaluate the contribution of niche aging to age-associated HSC alterations without niche-damaging myeloablation in HSC transplantation assays. We herein transplanted an excess of aged HSCs into young mice without preconditioning. Although aged HSCs successfully engrafted in the intact young bone marrow niche, they poorly regenerated downstream progenitors and exhibited persistent myeloid-biased differentiation, resulting in no significant functional rejuvenation. Transcriptome and methylome analyses revealed that the young niche largely restored the transcriptional profile of aged HSCs, but not their DNA methylation profiles. Therefore, the restoration of the young niche is insufficient for rejuvenating HSC functions, highlighting a key role for age-associated cell-intrinsic defects in HSC aging.. |
| 8. |
Nobuhiko Hamazaki, Hirohisa Kyogoku, Hiromitsu Araki, Fumihito Miura, Chisako Horikawa, Norio Hamada, So Shimamoto, Orie Hikabe, Kinichi Nakashima, Tomoya S Kitajima, Takashi Ito, Harry G Leitch, Katsuhiko Hayashi, Reconstitution of the oocyte transcriptional network with transcription factors., Nature, 10.1038/s41586-020-3027-9, 589, 7841, 264-269, 2021.01, During female germline development, oocytes become a highly specialized cell type and form a maternal cytoplasmic store of crucial factors. Oocyte growth is triggered at the transition from primordial to primary follicle and is accompanied by dynamic changes in gene expression1, but the gene regulatory network that controls oocyte growth remains unknown. Here we identify a set of transcription factors that are sufficient to trigger oocyte growth. By investigation of the changes in gene expression and functional screening using an in vitro mouse oocyte development system, we identified eight transcription factors, each of which was essential for the transition from primordial to primary follicle. Notably, enforced expression of these transcription factors swiftly converted pluripotent stem cells into oocyte-like cells that were competent for fertilization and subsequent cleavage. These transcription-factor-induced oocyte-like cells were formed without specification of primordial germ cells, epigenetic reprogramming or meiosis, and demonstrate that oocyte growth and lineage-specific de novo DNA methylation are separable from the preceding epigenetic reprogramming in primordial germ cells. This study identifies a core set of transcription factors for orchestrating oocyte growth, and provides an alternative source of ooplasm, which is a unique material for reproductive biology and medicine.. |
| 9. |
Yuhei Sangatsuda, Fumihito Miura, Hiromitsu Araki, Masahiro Mizuguchi, Nobuhiro Hata, Daisuke Kuga, Ryusuke Hatae, Yojiro Akagi, Takeo Amemiya, Yutaka Fujioka, Yasuhito Arai, Tatsuhiro Shibata, Koji Yoshimoto, Takashi Ito, Koji Iihara, BASE-RESOLUTION METHYLOMES OF GLIOMAS BEARING HISTONE H3.3 MUTATIONS REVEAL A G34 MUTANT-SPECIFIC SIGNATURE SHARED WITH BONE TUMORS, NEURO-ONCOLOGY, 22, 468-468, 2020.12. |
| 10. |
Yuhei Sangatsuda, Fumihito Miura, Hiromitsu Araki, Masahiro Mizoguchi, Nobuhiro Hata, Daisuke Kuga, Ryusuke Hatae, Yojiro Akagi, Takeo Amemiya, Yutaka Fujioka, Yasuhito Arai, Akihiko Yoshida, Tatsuhiro Shibata, Koji Yoshimoto, Koji Iihara, Takashi Ito, Base-resolution methylomes of gliomas bearing histone H3.3 mutations reveal a G34 mutant-specific signature shared with bone tumors, Scientific Reports, 10.1038/s41598-020-73116-x, 10, 1, 16162-16162, 2020.12, © 2020, The Author(s). Two recurrent mutations, K27M and G34R/V, in H3F3A, encoding non-canonical histone H3.3, are reported in pediatric and young adult gliomas, whereas G34W mutation is prevalent in bone tumors. In contrast to K27M mutation, it remains elusive how G34 mutations affect the epigenome. Here we performed whole-genome bisulfite sequencing of four G34R-mutated gliomas and the G34V-mutated glioma cell line KNS-42 for comparison with gliomas harboring K27M and no mutations in H3F3A and with G34W-mutated bone tumors. G34R-mutated gliomas exhibited lower global methylation levels, similar CpG island (CGI) methylation levels, and compromised hypermethylation of telomere-proximal CGIs, compared to the other two glioma subgroups. Hypermethylated regions specific to G34R-mutated gliomas were enriched for CGIs, including those of OLIG1, OLIG2, and canonical histone genes in the HIST1 cluster. They were notably hypermethylated in osteosarcomas with, but not without, G34W mutation. Independent component analysis revealed that G34 mutation-specific components shared a significant similarity between glioma and osteosarcoma, suggesting that G34 mutations exert characteristic methylomic effects regardless of the tumor tissue-of-origin. CRISPR/Cas9-mediated disruption of G34V-allele in KNS-42 cells led to demethylation of a subset of CGIs hypermethylated in G34R-mutated gliomas. These findings will provide a basis for elucidating epigenomic roles of G34 oncohistone in tumorigenesis.. |
| 11. |
Asuka Higo, Noriko Saihara, Fumihito Miura, Yoko Higashi, Megumi Yamada, Shojiro Tamaki, Tasuku Ito, Yoshiaki Tarutani, Tomoaki Sakamoto, Masayuki Fujiwara, Tetsuya Kurata, Yoichiro Fukao, Satoru Moritoh, Rie Terada, Toshinori Kinoshita, Takashi Ito, Tetsuji Kakutani, Ko Shimamoto, Hiroyuki Tsuji, DNA methylation is reconfigured at the onset of reproduction in rice shoot apical meristem, Nature Communications, 10.1038/s41467-020-17963-2, 11, 1, 4079-4079, 2020.12, © 2020, The Author(s). DNA methylation is an epigenetic modification that specifies the basic state of pluripotent stem cells and regulates the developmental transition from stem cells to various cell types. In flowering plants, the shoot apical meristem (SAM) contains a pluripotent stem cell population which generates the aerial part of plants including the germ cells. Under appropriate conditions, the SAM undergoes a developmental transition from a leaf-forming vegetative SAM to an inflorescence- and flower-forming reproductive SAM. While SAM characteristics are largely altered in this transition, the complete picture of DNA methylation remains elusive. Here, by analyzing whole-genome DNA methylation of isolated rice SAMs in the vegetative and reproductive stages, we show that methylation at CHH sites is kept high, particularly at transposable elements (TEs), in the vegetative SAM relative to the differentiated leaf, and increases in the reproductive SAM via the RNA-dependent DNA methylation pathway. We also show that half of the TEs that were highly methylated in gametes had already undergone CHH hypermethylation in the SAM. Our results indicate that changes in DNA methylation begin in the SAM long before germ cell differentiation to protect the genome from harmful TEs.. |
| 12. |
Kenjiro Shirane, Fumihito Miura, Takashi Ito, Matthew C. Lorincz, NSD1-deposited H3K36me2 directs de novo methylation in the mouse male germline and counteracts Polycomb-associated silencing, Nature Genetics, 10.1038/s41588-020-0689-z, 52, 10, 1088-1098, 2020.10, © 2020, The Author(s), under exclusive licence to Springer Nature America, Inc. De novo DNA methylation (DNAme) in mammalian germ cells is dependent on DNMT3A and DNMT3L. However, oocytes and spermatozoa show distinct patterns of DNAme. In mouse oocytes, de novo DNAme requires the lysine methyltransferase (KMTase) SETD2, which deposits H3K36me3. We show here that SETD2 is dispensable for de novo DNAme in the male germline. Instead, the lysine methyltransferase NSD1, which broadly deposits H3K36me2 in euchromatic regions, plays a critical role in de novo DNAme in prospermatogonia, including at imprinted genes. However, males deficient in germline NSD1 show a more severe defect in spermatogenesis than Dnmt3l−/− males. Notably, unlike DNMT3L, NSD1 safeguards a subset of genes against H3K27me3-associated transcriptional silencing. In contrast, H3K36me2 in oocytes is predominantly dependent on SETD2 and coincides with H3K36me3. Furthermore, females with NSD1-deficient oocytes are fertile. Thus, the sexually dimorphic pattern of DNAme in mature mouse gametes is orchestrated by distinct profiles of H3K36 methylation.. |
| 13. |
Shinya Tanaka, Wataru Ise, Takeshi Inoue, Ayako Ito, Chisato Ono, Yoshihito Shima, Shuhei Sakakibara, Manabu Nakayama, Kentaro Fujii, Ikuo Miura, Jafar Sharif, Haruhiko Koseki, Pandelakis A. Koni, Indu Raman, Quan Zhen Li, Masato Kubo, Katsunori Fujiki, Ryuichiro Nakato, Katsuhiko Shirahige, Hiromitsu Araki, Fumihito Miura, Takashi Ito, Eiryo Kawakami, Yoshihiro Baba, Tomohiro Kurosaki, Tet2 and Tet3 in B cells are required to repress CD86 and prevent autoimmunity, Nature Immunology, 10.1038/s41590-020-0700-y, 21, 8, 950-961, 2020.08, © 2020, The Author(s), under exclusive licence to Springer Nature America, Inc. A contribution of epigenetic modifications to B cell tolerance has been proposed but not directly tested. Here we report that deficiency of ten–eleven translocation (Tet) DNA demethylase family members Tet2 and Tet3 in B cells led to hyperactivation of B and T cells, autoantibody production and lupus-like disease in mice. Mechanistically, in the absence of Tet2 and Tet3, downregulation of CD86, which normally occurs following chronic exposure of self-reactive B cells to self-antigen, did not take place. The importance of dysregulated CD86 expression in Tet2- and Tet3-deficient B cells was further demonstrated by the restriction, albeit not complete, on aberrant T and B cell activation following anti-CD86 blockade. Tet2- and Tet3-deficient B cells had decreased accumulation of histone deacetylase 1 (HDAC1) and HDAC2 at the Cd86 locus. Thus, our findings suggest that Tet2- and Tet3-mediated chromatin modification participates in repression of CD86 on chronically stimulated self-reactive B cells, which contributes, at least in part, to preventing autoimmunity.. |
| 14. |
Keisuke Yoshida, Toshio Maekawa, Nhung Hong Ly, Shin-ichiro Fujita, Masafumi Muratani, Minami Ando, Yuki Katou, Hiromitsu Araki, Fumihito Miura, Katsuhiko Shirahige, Mariko Okada, Takashi Ito, Bruno Chatton, Shunsuke Ishii, ATF7-Dependent Epigenetic Changes Are Required for the Intergenerational Effect of a Paternal Low-Protein Diet, MOLECULAR CELL, 10.1016/j.molcel.2020.02.028, 78, 3, 445-+, 2020.05, Paternal dietary conditions may contribute to metabolic disorders in offspring. We have analyzed the role of the stress-dependent epigenetic regulator cyclic AMP-dependent transcription factor 7 (ATF7) in paternal low-protein diet (pLPD)-induced gene expression changes in mouse liver. Atf7(+/- )mutations cause an offspring phenotype similar to that caused by pLPD, and the effect of pLPD almost vanished when paternal Atf7(+/-) mice were used. ATF7 binds to the promoter regions of similar to 2,300 genes, including cholesterol biosynthesis-related and tRNA genes in testicular germ cells (TGCs). LPD induces ATF7 phosphorylation by p38 via reactive oxygen species (ROS) in TGCs. This leads to the release of ATF7 and a decrease in histone H3K9 dimethylation (H3K9me2) on its target genes. These epigenetic changes are maintained and induce expression of some tRNA fragments in spermatozoa. These results indicate that LPD-induced and ATF7-dependent epigenetic changes in TGCs play an important role in paternal diet-induced metabolic reprograming in offspring.. |
| 15. |
Keisuke Yoshida, Toshio Maekawa, Nhung Hong Ly, Shin ichiro Fujita, Masafumi Muratani, Minami Ando, Yuki Katou, Hiromitsu Araki, Fumihito Miura, Katsuhiko Shirahige, Mariko Okada, Takashi Ito, Bruno Chatton, Shunsuke Ishii, ATF7-Dependent Epigenetic Changes Are Required for the Intergenerational Effect of a Paternal Low-Protein Diet, Molecular Cell, 10.1016/j.molcel.2020.02.028, 2020.01, Paternal dietary conditions may contribute to metabolic disorders in offspring. We have analyzed the role of the stress-dependent epigenetic regulator cyclic AMP-dependent transcription factor 7 (ATF7) in paternal low-protein diet (pLPD)-induced gene expression changes in mouse liver. Atf7+/– mutations cause an offspring phenotype similar to that caused by pLPD, and the effect of pLPD almost vanished when paternal Atf7+/– mice were used. ATF7 binds to the promoter regions of ∼2,300 genes, including cholesterol biosynthesis-related and tRNA genes in testicular germ cells (TGCs). LPD induces ATF7 phosphorylation by p38 via reactive oxygen species (ROS) in TGCs. This leads to the release of ATF7 and a decrease in histone H3K9 dimethylation (H3K9me2) on its target genes. These epigenetic changes are maintained and induce expression of some tRNA fragments in spermatozoa. These results indicate that LPD-induced and ATF7-dependent epigenetic changes in TGCs play an important role in paternal diet-induced metabolic reprograming in offspring.. |
| 16. |
Hiromitsu Araki, Fumihito Miura, Akira Watanabe, Chikako Morinaga, Fumiyo Kitaoka, Yuko Kitano, Noriko Sakai, Yumiko Shibata, Motoki Terada, So Goto, Shinya Yamanaka, Masayo Takahashi, Takashi Ito, Base-Resolution Methylome of Retinal Pigment Epithelial Cells Used in the First Trial of Human Induced Pluripotent Stem Cell-Based Autologous Transplantation, Stem Cell Reports, 10.1016/j.stemcr.2019.08.014, 13, 4, 761-774, 2019.10, In this article, Ito and colleagues delineated the base-resolution methylome of iPSC-derived retinal pigment epithelial (RPE) cell used in the first-in-human trial of autologous transplantation. The methylome closely resembled that of native RPE cell. Intriguingly, it became even closer to its natural counterpart upon xenotransplantation to mouse, demonstrating its plasticity to respond appropriately to the in vivo environment.. |
| 17. |
Hiromitsu Araki, Fumihito Miura, Akira Watanabe, Chikako Morinaga, Fumiyo Kitaoka, Yuko Kitano, Noriko Sakai, Yumiko Shibata, Motoki Terada, So Goto, Shinya Yamanaka, Masayo Takahashi, Takashi Ito, Base-Resolution Methylome of Retinal Pigment Epithelial Cells Used in the First Trial of Human Induced Pluripotent Stem Cell-Based Autologous Transplantation, Stem Cell Reports, 10.1016/j.stemcr.2019.08.014, 13, 4, 761-774, 2019.10, © 2019 The Author(s) In this article, Ito and colleagues delineated the base-resolution methylome of iPSC-derived retinal pigment epithelial (RPE) cell used in the first-in-human trial of autologous transplantation. The methylome closely resembled that of native RPE cell. Intriguingly, it became even closer to its natural counterpart upon xenotransplantation to mouse, demonstrating its plasticity to respond appropriately to the in vivo environment.. |
| 18. |
Fumihito Miura, Yukiko Shibata, Miki Miura, Yuhei Sangatsuda, Osamu Hisano, Hiromitsu Araki, Takashi Ito, Highly efficient single-stranded DNA ligation technique improves low-input whole-genome bisulfite sequencing by post-bisulfite adaptor tagging, Nucleic acids research, 10.1093/nar/gkz435, 47, 15, e85, 2019.09, Whole-genome bisulfite sequencing (WGBS) is the current gold standard of methylome analysis. Post-bisulfite adaptor tagging (PBAT) is an increasingly popular WGBS protocol because of high sensitivity and low bias. PBAT originally relied on two rounds of random priming for adaptor-tagging of single-stranded DNA (ssDNA) to attain high efficiency but at a cost of library insert length. To overcome this limitation, we developed terminal deoxyribonucleotidyl transferase (TdT)-assisted adenylate connector-mediated ssDNA (TACS) ligation as an alternative to random priming. In this method, TdT attaches adenylates to the 3'-end of input ssDNA, which are then utilized by RNA ligase as an efficient connector to the ssDNA adaptor. A protocol that uses TACS ligation instead of the second random priming step substantially increased the lengths of PBAT library fragments. Moreover, we devised a dual-library strategy that splits the input DNA to prepare two libraries with reciprocal adaptor polarity, combining them prior to sequencing. This strategy ensured an ideal base-color balance to eliminate the need for DNA spike-in for color compensation, further improving the throughput and quality of WGBS. Adopting the above strategies to the HiSeq X Ten and NovaSeq 6000 platforms, we established a cost-effective, high-quality WGBS, which should accelerate various methylome analyses.. |
| 19. |
Fumihito Miura, Yukiko Shibata, Miki Miura, Yuhei Sangatsuda, Osamu Hisano, Hiromitsu Araki, Takashi Ito, Highly efficient single-stranded DNA ligation technique improves low-input whole-genome bisulfite sequencing by post-bisulfite adaptor tagging, Nucleic Acids Research, 10.1093/nar/gkz435, 47, 15, e85-e85, 2019.09, © 2019 The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research. Whole-genome bisulfite sequencing (WGBS) is the current gold standard of methylome analysis. Post-bisulfite adaptor tagging (PBAT) is an increasingly popular WGBS protocol because of high sensitivity and low bias. PBAT originally relied on two rounds of random priming for adaptor-tagging of single-stranded DNA (ssDNA) to attain high efficiency but at a cost of library insert length. To overcome this limitation, we developed terminal deoxyribonucleotidyl transferase (TdT)-assisted adenylate connector-mediated ssDNA (TACS) ligation as an alternative to random priming. In this method, TdT attaches adenylates to the 3′-end of input ssDNA, which are then utilized by RNA ligase as an efficient connector to the ssDNA adaptor. A protocol that uses TACS ligation instead of the second random priming step substantially increased the lengths of PBAT library fragments. Moreover, we devised a dual-library strategy that splits the input DNA to prepare two libraries with reciprocal adaptor polarity, combining them prior to sequencing. This strategy ensured an ideal base-color balance to eliminate the need for DNA spike-in for color compensation, further improving the throughput and quality of WGBS. Adopting the above strategies to the HiSeq X Ten and NovaSeq 6000 platforms, we established a cost-effective, high-quality WGBS, which should accelerate various methylome analyses.. |
| 20. |
Taito Matsuda, Takashi Irie, Shutaro Katsurabayashi, Yoshinori Hayashi, Tatsuya Nagai, Nobuhiko Hamazaki, Aliya Mari D. Adefuin, Fumihito Miura, Takashi Ito, Hiroshi Kimura, Katsuhiko Shirahige, Tadayuki Takeda, Katsunori Iwasaki, Takuya Imamura, Kinichi Nakashima, Pioneer Factor NeuroD1 Rearranges Transcriptional and Epigenetic Profiles to Execute Microglia-Neuron Conversion, Neuron, 10.1016/j.neuron.2018.12.010, 101, 3, 472-485.e7, 2019.02, Matsuda et al. report direct neuronal conversion of microglia induced by the expression of a single transcription factor, NeuroD1, which occupies bivalent epigenetic domains for neuronal gene induction. NeuroD1 can also converts microglia into neurons in the adult mouse striatum.. |
| 21. |
Taito Matsuda, Takashi Irie, Shutaro Katsurabayashi, Yoshinori Hayashi, Tatsuya Nagai, Nobuhiko Hamazaki, Aliya Mari D. Adefuin, Fumihito Miura, Takashi Ito, Hiroshi Kimura, Katsuhiko Shirahige, Tadayuki Takeda, Katsunori Iwasaki, Takuya Imamura, Kinichi Nakashima, Pioneer Factor NeuroD1 Rearranges Transcriptional and Epigenetic Profiles to Execute Microglia-Neuron Conversion, Neuron, 10.1016/j.neuron.2018.12.010, 101, 3, 472-485.e7, 2019.02, © 2018 Elsevier Inc. Matsuda et al. report direct neuronal conversion of microglia induced by the expression of a single transcription factor, NeuroD1, which occupies bivalent epigenetic domains for neuronal gene induction. NeuroD1 can also converts microglia into neurons in the adult mouse striatum.. |
| 22. |
Natsuko Hama, Yasushi Totoki, Fumihito Miura, Kenji Tatsuno, Mihoko Saito-Adachi, Hiromi Nakamura, Yasuhito Arai, Fumie Hosoda, Tomoko Urushidate, Shoko Ohashi, Wakako Mukai, Nobuyoshi Hiraoka, Hiroyuki Aburatani, Takashi Ito, Tatsuhiro Shibata, Epigenetic landscape influences the liver cancer genome architecture, Nature Communications, 10.1038/s41467-018-03999-y, 9, 1, 2018.12, The accumulations of different types of genetic alterations such as nucleotide substitutions, structural rearrangements and viral genome integrations and epigenetic alterations contribute to carcinogenesis. Here, we report correlation between the occurrence of epigenetic features and genetic aberrations by whole-genome bisulfite, whole-genome shotgun, long-read, and virus capture sequencing of 373 liver cancers. Somatic substitutions and rearrangement breakpoints are enriched in tumor-specific hypo-methylated regions with inactive chromatin marks and actively transcribed highly methylated regions in the cancer genome. Individual mutation signatures depend on chromatin status, especially, signatures with a higher transcriptional strand bias occur within active chromatic areas. Hepatitis B virus (HBV) integration sites are frequently detected within inactive chromatin regions in cancer cells, as a consequence of negative selection for integrations in active chromatin regions. Ultra-high structural instability and preserved unmethylation of integrated HBV genomes are observed. We conclude that both precancerous and somatic epigenetic features contribute to the cancer genome architecture.. |
| 23. |
Keisuke Yoshida, Masafumi Muratani, Hiromitsu Araki, Fumihito Miura, Takehiro Suzuki, Naoshi Dohmae, Yuki Katou, Katsuhiko Shirahige, Takashi Ito, Shunsuke Ishii, Mapping of histone-binding sites in histone replacement-completed spermatozoa, Nature Communications, 10.1038/s41467-018-06243-9, 9, 1, 2018.12, The majority of histones are replaced by protamines during spermatogenesis, but small amounts are retained in mammalian spermatozoa. Since nucleosomes in spermatozoa influence epigenetic inheritance, it is important to know how histones are distributed in the sperm genome. Conflicting data, which may result from different conditions used for micrococcal nuclease (MNase) digestion, have been reported: retention of nucleosomes at either gene promoter regions or within distal gene-poor regions. Here, we find that the swim-up sperm used in many studies contain about 10% population of sperm which have not yet completed the histone-to-protamine replacement. We develop a method to purify histone replacement-completed sperm (HRCS) and to completely solubilize histones from cross-linked HRCS without MNase digestion. Our results indicate that histones are retained at specific promoter regions in HRCS. This method allows the study of epigenetic status in mature sperm.. |
| 24. |
Natsuko Hama, Yasushi Totoki, Fumihito Miura, Kenji Tatsuno, Mihoko Saito-Adachi, Hiromi Nakamura, Yasuhito Arai, Fumie Hosoda, Tomoko Urushidate, Shoko Ohashi, Wakako Mukai, Nobuyoshi Hiraoka, Hiroyuki Aburatani, Takashi Ito, Tatsuhiro Shibata, Epigenetic landscape influences the liver cancer genome architecture, Nature Communications, 10.1038/s41467-018-03999-y, 9, 1, 1643-1643, 2018.12, © 2018 The Author(s). The accumulations of different types of genetic alterations such as nucleotide substitutions, structural rearrangements and viral genome integrations and epigenetic alterations contribute to carcinogenesis. Here, we report correlation between the occurrence of epigenetic features and genetic aberrations by whole-genome bisulfite, whole-genome shotgun, long-read, and virus capture sequencing of 373 liver cancers. Somatic substitutions and rearrangement breakpoints are enriched in tumor-specific hypo-methylated regions with inactive chromatin marks and actively transcribed highly methylated regions in the cancer genome. Individual mutation signatures depend on chromatin status, especially, signatures with a higher transcriptional strand bias occur within active chromatic areas. Hepatitis B virus (HBV) integration sites are frequently detected within inactive chromatin regions in cancer cells, as a consequence of negative selection for integrations in active chromatin regions. Ultra-high structural instability and preserved unmethylation of integrated HBV genomes are observed. We conclude that both precancerous and somatic epigenetic features contribute to the cancer genome architecture.. |
| 25. |
Keisuke Yoshida, Masafumi Muratani, Hiromitsu Araki, Fumihito Miura, Takehiro Suzuki, Naoshi Dohmae, Yuki Katou, Katsuhiko Shirahige, Takashi Ito, Shunsuke Ishii, Mapping of histone-binding sites in histone replacement-completed spermatozoa, Nature Communications, 10.1038/s41467-018-06243-9, 9, 1, 3885-3885, 2018.12, © 2018, The Author(s). The majority of histones are replaced by protamines during spermatogenesis, but small amounts are retained in mammalian spermatozoa. Since nucleosomes in spermatozoa influence epigenetic inheritance, it is important to know how histones are distributed in the sperm genome. Conflicting data, which may result from different conditions used for micrococcal nuclease (MNase) digestion, have been reported: retention of nucleosomes at either gene promoter regions or within distal gene-poor regions. Here, we find that the swim-up sperm used in many studies contain about 10% population of sperm which have not yet completed the histone-to-protamine replacement. We develop a method to purify histone replacement-completed sperm (HRCS) and to completely solubilize histones from cross-linked HRCS without MNase digestion. Our results indicate that histones are retained at specific promoter regions in HRCS. This method allows the study of epigenetic status in mature sperm.. |
| 26. |
Moe Miyoshi, Masayuki Sato, Kenji Saito, Lila Otani, Katsuhiko Shirahige, Fumihito Miura, Takashi Ito, Huijuan Jia, Hisanori Kato, Maternal protein restriction alters the renal ptger1 DNA methylation state in SHRSP offspring, Nutrients, 10.3390/nu10101436, 10, 10, 2018.10, We previously reported that maternal protein restriction (LP) during pregnancy increases salt sensitivity in offspring using the Stroke-Prone Spontaneously Hypertensive Rat (SHRSP). In the present study, we focus on DNA methylation profiles of prostaglandin E receptor 1 gene (ptger1), which is known to be associated with hypertension. We evaluated the ptger1 DNA methylation status via bisulfite sequencing, and analyzed the expression of ptger1-related genes. The results of these analyses showed that, compared to controls, the LP-S offspring exhibited both marked ptger1 hypermethylation, and significantly increased ptger1 expression. Moreover, they also exhibited significantly decreased expression of the downstream gene epithelial Na+ channel alpha (enacα). Interestingly, LP offspring that were provided with a standard water drinking supply (W) also exhibited increased ptger1 methylation and expression. Together, these results suggest that maternal protein restriction during pregnancy modulates the renal ptger1 DNA methylation state in SHRSP offspring, and thereby likely mediates ptger1 and enacα gene expression to induce salt sensitivity.. |
| 27. |
Moe Miyoshi, Masayuki Sato, Kenji Saito, Lila Otani, Katsuhiko Shirahige, Fumihito Miura, Takashi Ito, Huijuan Jia, Hisanori Kato, Maternal protein restriction alters the renal ptger1 DNA methylation state in SHRSP offspring, Nutrients, 10.3390/nu10101436, 10, 10, 2018.10, © 2018 by the authors. Licensee MDPI, Basel, Switzerland. We previously reported that maternal protein restriction (LP) during pregnancy increases salt sensitivity in offspring using the Stroke-Prone Spontaneously Hypertensive Rat (SHRSP). In the present study, we focus on DNA methylation profiles of prostaglandin E receptor 1 gene (ptger1), which is known to be associated with hypertension. We evaluated the ptger1 DNA methylation status via bisulfite sequencing, and analyzed the expression of ptger1-related genes. The results of these analyses showed that, compared to controls, the LP-S offspring exhibited both marked ptger1 hypermethylation, and significantly increased ptger1 expression. Moreover, they also exhibited significantly decreased expression of the downstream gene epithelial Na+ channel alpha (enacα). Interestingly, LP offspring that were provided with a standard water drinking supply (W) also exhibited increased ptger1 methylation and expression. Together, these results suggest that maternal protein restriction during pregnancy modulates the renal ptger1 DNA methylation state in SHRSP offspring, and thereby likely mediates ptger1 and enacα gene expression to induce salt sensitivity.. |
| 28. |
Fumihito Miura, Tomoko Fujino, Kanako Kogashi, Yukiko Shibata, Miki Miura, Hiroyuki Isobe, Takashi Ito, Triazole linking for preparation of a next-generation sequencing library from single-stranded DNA, Nucleic acids research, 10.1093/nar/gky452, 46, 16, e95, 2018.09, Next-generation sequencing of single-stranded DNA (ssDNA) is attracting increased attention from a wide variety of research fields. Accordingly, various methods are actively being tested for the efficient adaptor-tagging of ssDNA. We conceived a novel chemo-enzymatic method termed terminal deoxynucleotidyl transferase (TdT)-assisted, copper-catalyzed azide-alkyne cycloaddition (CuAAC)-mediated ssDNA ligation (TCS ligation). In this method, TdT is used to incorporate a single 3'-azide-modified dideoxyribonucleotide onto the 3'-end of target ssDNA, followed by CuAAC-mediated click ligation of the azide-incorporated 3'-end to a 5'-ethynylated synthetic adaptor. This report presents the first proof-of-principle application of TCS ligation with its use in the preparation of a next-generation sequencing library.. |
| 29. |
Fumihito Miura, Tomoko Fujino, Kanako Kogashi, Yukiko Shibata, Miki Miura, Hiroyuki Isobe, Takashi Ito, Triazole linking for preparation of a next-generation sequencing library from single-stranded DNA, Nucleic acids research, 10.1093/nar/gky452, 46, 16, e95-e95, 2018.09, Next-generation sequencing of single-stranded DNA (ssDNA) is attracting increased attention from a wide variety of research fields. Accordingly, various methods are actively being tested for the efficient adaptor-tagging of ssDNA. We conceived a novel chemo-enzymatic method termed terminal deoxynucleotidyl transferase (TdT)-assisted, copper-catalyzed azide-alkyne cycloaddition (CuAAC)-mediated ssDNA ligation (TCS ligation). In this method, TdT is used to incorporate a single 3'-azide-modified dideoxyribonucleotide onto the 3'-end of target ssDNA, followed by CuAAC-mediated click ligation of the azide-incorporated 3'-end to a 5'-ethynylated synthetic adaptor. This report presents the first proof-of-principle application of TCS ligation with its use in the preparation of a next-generation sequencing library.. |
| 30. |
Eri Arai, Fumihito Miura, Yasushi Totoki, Satoshi Yamashita, Ying Tian, Masahiro Gotoh, Hidenori Ojima, Hiroyuki Nakagawa, Yoriko Takahashi, Hiromi Nakamura, Natsuko Hama, Mamoru Kato, Hiroshi Kimura, Yutaka Suzuki, Takashi Ito, Tatsuhiro Shibata, Yae Kanai, Epigenome mapping of human normal purified hepatocytes
Personal epigenome variation and genome-epigenome correlation, Epigenomics, 10.2217/epi-2017-0111, 10, 7, 955-979, 2018.07, Aim: The aim of this study was to reveal the epigenome landscape of human normal hepatocytes. Materials & methods: Cells purified from partial hepatectomy specimens of Japanese patients were subjected to whole-genome bisulfite sequencing using postbisulfite adaptor tagging, chromatin immunoprecipitation sequencing, RNA sequencing and whole-genome sequencing. Results: CHG and CHH methylations were inversely associated with gene expression. Histone modification profiles of personal differentially methylated regions (pDMRs) differed considerably among samples. pDMRs were observed around the transcription start sites of genes whose expression is reportedly regulated by CpG methylation. pDMRs were frequently observed in the vicinity of single-nucleotide variations and insertions/deletions. Conclusion: Genetic variations may induce epigenetic variations, generating individual differences in the phenotypes of normal hepatocytes through variations in expression.. |
| 31. |
Eri Arai, Fumihito Miura, Yasushi Totoki, Satoshi Yamashita, Ying Tian, Masahiro Gotoh, Hidenori Ojima, Hiroyuki Nakagawa, Yoriko Takahashi, Hiromi Nakamura, Natsuko Hama, Mamoru Kato, Hiroshi Kimura, Yutaka Suzuki, Takashi Ito, Tatsuhiro Shibata, Yae Kanai, Epigenome mapping of human normal purified hepatocytes: Personal epigenome variation and genome-epigenome correlation, Epigenomics, 10.2217/epi-2017-0111, 10, 7, 955-979, 2018.07, © 2018 Yae Kanai. Aim: The aim of this study was to reveal the epigenome landscape of human normal hepatocytes. Materials & methods: Cells purified from partial hepatectomy specimens of Japanese patients were subjected to whole-genome bisulfite sequencing using postbisulfite adaptor tagging, chromatin immunoprecipitation sequencing, RNA sequencing and whole-genome sequencing. Results: CHG and CHH methylations were inversely associated with gene expression. Histone modification profiles of personal differentially methylated regions (pDMRs) differed considerably among samples. pDMRs were observed around the transcription start sites of genes whose expression is reportedly regulated by CpG methylation. pDMRs were frequently observed in the vicinity of single-nucleotide variations and insertions/deletions. Conclusion: Genetic variations may induce epigenetic variations, generating individual differences in the phenotypes of normal hepatocytes through variations in expression.. |
| 32. |
Fumihito Miura, Takashi Ito, Post-bisulfite adaptor tagging for PCR-free whole-genome bisulfite sequencing, Methods in Molecular Biology, 10.1007/978-1-4939-7481-8_7, 1708, 123-136, 2018.01, Post-bisulfite adaptor tagging (PBAT) is a highly efficient procedure to construct libraries for whole-genome bisulfite sequencing (WGBS). PBAT attaches adaptors to bisulfite-converted genomic DNA to circumvent bisulfite-induced degradation of library DNA inherent to conventional WGBS protocols. Consequently, it enables PCR-free WGBS from nanogram quantities of mammalian DNA, thereby serving as an invaluable tool for methylomics.. |
| 33. |
Soh Yamazaki, Yoshihiko Tanaka, Hiromitsu Araki, Akira Kohda, Fumiyuki Sanematsu, Tomoko Arasaki, Xuefeng Duan, Fumihito Miura, Takaharu Katagiri, Ryodai Shindo, Hiroyasu Nakano, Takashi Ito, Yoshinori Fukui, Shogo Endo, Hideki Sumimoto, The AP-1 transcription factor JunB is required for Th17 cell differentiation, Scientific Reports, 10.1038/s41598-017-17597-3, 7, 1, 2017.12, Interleukin (IL)-17-producing T helper (Th17) cells are crucial for host defense against extracellular microbes and pathogenesis of autoimmune diseases. Here we show that the AP-1 transcription factor JunB is required for Th17 cell development. Junb-deficient CD4+ T cells are able to develop in vitro into various helper T subsets except Th17. The RNA-seq transcriptome analysis reveals that JunB is crucial for the Th17-specific gene expression program. Junb-deficient mice are completely resistant to experimental autoimmune encephalomyelitis, a Th17-mediated inflammatory disease, and naive T helper cells from such mice fail to differentiate into Th17 cells. JunB appears to activate Th17 signature genes by forming a heterodimer with BATF, another AP-1 factor essential for Th17 differentiation. The mechanism whereby JunB controls Th17 cell development likely involves activation of the genes for the Th17 lineage-specifying orphan receptors RORγt and RORα and reduced expression of Foxp3, a transcription factor known to antagonize RORγt function.. |
| 34. |
Soh Yamazaki, Yoshihiko Tanaka, Hiromitsu Araki, Akira Kohda, Fumiyuki Sanematsu, Tomoko Arasaki, Xuefeng Duan, Fumihito Miura, Takaharu Katagiri, Ryodai Shindo, Hiroyasu Nakano, Takashi Ito, Yoshinori Fukui, Shogo Endo, Hideki Sumimoto, The AP-1 transcription factor JunB is required for Th17 cell differentiation, Scientific Reports, 10.1038/s41598-017-17597-3, 7, 1, 17402-17402, 2017.12, © 2017 The Author(s). Interleukin (IL)-17-producing T helper (Th17) cells are crucial for host defense against extracellular microbes and pathogenesis of autoimmune diseases. Here we show that the AP-1 transcription factor JunB is required for Th17 cell development. Junb-deficient CD4+ T cells are able to develop in vitro into various helper T subsets except Th17. The RNA-seq transcriptome analysis reveals that JunB is crucial for the Th17-specific gene expression program. Junb-deficient mice are completely resistant to experimental autoimmune encephalomyelitis, a Th17-mediated inflammatory disease, and naive T helper cells from such mice fail to differentiate into Th17 cells. JunB appears to activate Th17 signature genes by forming a heterodimer with BATF, another AP-1 factor essential for Th17 differentiation. The mechanism whereby JunB controls Th17 cell development likely involves activation of the genes for the Th17 lineage-specifying orphan receptors RORγt and RORα and reduced expression of Foxp3, a transcription factor known to antagonize RORγt function.. |
| 35. |
Tsukasa Sanosaka, Takuya Imamura, Nobuhiko Hamazaki, Muh Chyi Chai, Katsuhide Igarashi, Maky Ideta-Otsuka, Fumihito Miura, Takashi Ito, Nobuyuki Fujii, Kazuho Ikeo, Kinichi Nakashima, DNA Methylome Analysis Identifies Transcription Factor-Based Epigenomic Signatures of Multilineage Competence in Neural Stem/Progenitor Cells, Cell Reports, 10.1016/j.celrep.2017.08.086, 20, 12, 2992-3003, 2017.09, Regulation of the epigenome during in vivo specification of brain stem cells is still poorly understood. Here, we report DNA methylome analyses of directly sampled cortical neural stem and progenitor cells (NS/PCs) at different development stages, as well as those of terminally differentiated cortical neurons, astrocytes, and oligodendrocytes. We found that sequential specification of cortical NS/PCs is regulated by two successive waves of demethylation at early and late development stages, which are responsible for the establishment of neuron- and glia-specific low-methylated regions (LMRs), respectively. The regulatory role of demethylation of the gliogenic genes was substantiated by the enrichment of nuclear factor I (NFI)-binding sites. We provide evidence that de novo DNA methylation of neuron-specific LMRs establishes glia-specific epigenotypes, essentially by silencing neuronal genes. Our data highlight the in vivo implications of DNA methylation dynamics in shaping epigenomic features that confer the differentiation potential of NS/PCs sequentially during development.. |
| 36. |
Tsukasa Sanosaka, Takuya Imamura, Nobuhiko Hamazaki, Muh Chyi Chai, Katsuhide Igarashi, Maky Ideta-Otsuka, Fumihito Miura, Takashi Ito, Nobuyuki Fujii, Kazuho Ikeo, Kinichi Nakashima, DNA Methylome Analysis Identifies Transcription Factor-Based Epigenomic Signatures of Multilineage Competence in Neural Stem/Progenitor Cells, Cell Reports, 10.1016/j.celrep.2017.08.086, 20, 12, 2992-3003, 2017.09, © 2017 The Author(s) Regulation of the epigenome during in vivo specification of brain stem cells is still poorly understood. Here, we report DNA methylome analyses of directly sampled cortical neural stem and progenitor cells (NS/PCs) at different development stages, as well as those of terminally differentiated cortical neurons, astrocytes, and oligodendrocytes. We found that sequential specification of cortical NS/PCs is regulated by two successive waves of demethylation at early and late development stages, which are responsible for the establishment of neuron- and glia-specific low-methylated regions (LMRs), respectively. The regulatory role of demethylation of the gliogenic genes was substantiated by the enrichment of nuclear factor I (NFI)-binding sites. We provide evidence that de novo DNA methylation of neuron-specific LMRs establishes glia-specific epigenotypes, essentially by silencing neuronal genes. Our data highlight the in vivo implications of DNA methylation dynamics in shaping epigenomic features that confer the differentiation potential of NS/PCs sequentially during development.. |
| 37. |
Hidehiro Toh, Kenjiro Shirane, Fumihito Miura, Naoki Kubo, Kenji Ichiyanagi, Katsuhiko Hayashi, Mitinori Saitou, Mikita Suyama, Takashi Ito, Hiroyuki Sasaki, Software updates in the illumina hiseq platform affect whole-genome bisulfite sequencing, BMC Genomics, 10.1186/s12864-016-3392-9, 18, 1, 2017.01, Background: Methylation of cytosine in genomic DNA is a well-characterized epigenetic modification involved in many cellular processes and diseases. Whole-genome bisulfite sequencing (WGBS), such as MethylC-seq and post-bisulfite adaptor tagging sequencing (PBAT-seq), uses the power of high-throughput DNA sequencers and provides genome-wide DNA methylation profiles at single-base resolution. However, the accuracy and consistency of WGBS outputs in relation to the operating conditions of high-throughput sequencers have not been explored. Results: We have used the Illumina HiSeq platform for our PBAT-based WGBS, and found that different versions of HiSeq Control Software (HCS) and Real-Time Analysis (RTA) installed on the system provided different global CpG methylation levels (approximately 5% overall difference) for the same libraries. This problem was reproduced multiple times with different WGBS libraries and likely to be associated with the low sequence diversity of bisulfite-converted DNA. We found that HCS was the major determinant in the observed differences. To determine which version of HCS is most suitable for WGBS, we used substrates with predetermined CpG methylation levels, and found that HCS v2.0.5 is the best among the examined versions. HCS v2.0.12 showed the poorest performance and provided artificially lower CpG methylation levels when 5-methylcytosine is read as guanine (first read of PBAT-seq and second read of MethylC-seq). In addition, paired-end sequencing of low diversity libraries using HCS v2.2.38 or the latest HCS v2.2.58 was greatly affected by cluster densities. Conclusions: Software updates in the Illumina HiSeq platform can affect the outputs from low-diversity sequencing libraries such as WGBS libraries. More recent versions are not necessarily the better, and HCS v2.0.5 is currently the best for WGBS among the examined HCS versions. Thus, together with other experimental conditions, special care has to be taken on this point when CpG methylation levels are to be compared between different samples by WGBS.. |
| 38. |
Hidehiro Toh, Kenjiro Shirane, Fumihito Miura, Naoki Kubo, Kenji Ichiyanagi, Katsuhiko Hayashi, Mitinori Saitou, Mikita Suyama, Takashi Ito, Hiroyuki Sasaki, Software updates in the illumina hiseq platform affect whole-genome bisulfite sequencing, BMC Genomics, 10.1186/s12864-016-3392-9, 18, 1, 31-31, 2017.01, © 2017 The Author(s). Background: Methylation of cytosine in genomic DNA is a well-characterized epigenetic modification involved in many cellular processes and diseases. Whole-genome bisulfite sequencing (WGBS), such as MethylC-seq and post-bisulfite adaptor tagging sequencing (PBAT-seq), uses the power of high-throughput DNA sequencers and provides genome-wide DNA methylation profiles at single-base resolution. However, the accuracy and consistency of WGBS outputs in relation to the operating conditions of high-throughput sequencers have not been explored. Results: We have used the Illumina HiSeq platform for our PBAT-based WGBS, and found that different versions of HiSeq Control Software (HCS) and Real-Time Analysis (RTA) installed on the system provided different global CpG methylation levels (approximately 5% overall difference) for the same libraries. This problem was reproduced multiple times with different WGBS libraries and likely to be associated with the low sequence diversity of bisulfite-converted DNA. We found that HCS was the major determinant in the observed differences. To determine which version of HCS is most suitable for WGBS, we used substrates with predetermined CpG methylation levels, and found that HCS v2.0.5 is the best among the examined versions. HCS v2.0.12 showed the poorest performance and provided artificially lower CpG methylation levels when 5-methylcytosine is read as guanine (first read of PBAT-seq and second read of MethylC-seq). In addition, paired-end sequencing of low diversity libraries using HCS v2.2.38 or the latest HCS v2.2.58 was greatly affected by cluster densities. Conclusions: Software updates in the Illumina HiSeq platform can affect the outputs from low-diversity sequencing libraries such as WGBS libraries. More recent versions are not necessarily the better, and HCS v2.0.5 is currently the best for WGBS among the examined HCS versions. Thus, together with other experimental conditions, special care has to be taken on this point when CpG methylation levels are to be compared between different samples by WGBS.. |
| 39. |
Eri Arai, Fumihito Miura, Yasushi Totoki, Satoshi Yamashita, Ying Tian, Masahiro Gotoh, Hidenori Ojima, Hiroyuki Nakagawa, Yoriko Takahashi, Hiromi Nakamura, Natsuko Hama, Mamoru Kato, Hiroshi Kimura, Yutaka Suzuki, Takashi Ito, Tatsuhiro Shibata, Yae Kanai, Epigenome landscape of human normal purified hepatocytes: analysis by the International Human Epigenome Consortium (IHEC), CANCER RESEARCH, 10.1158/1538-7445.AM2016-4517, 76, 2016.07. |
| 40. |
Tasuku Koike, Takuya Wakai, Yuko Jincho, Akihiko Sakashita, Hisato Kobayashi, Eiji Mizutani, Sayaka Wakayama, Fumihito Miura, Takashi Ito, Tomohiro Kono, DNA methylation errors in cloned mouse sperm by germ line barrier evasion, Biology of Reproduction, 10.1095/biolreprod.116.138677, 94, 6, 2016.06, The germ line reprogramming barrier resets parental epigenetic modifications according to sex, conferring totipotency to mammalian embryos upon fertilization. However, it is not known whether epigenetic errors are committed during germ line reprogramming that are then transmitted to germ cells, and consequently to offspring. We addressed this question in the present study by performing a genome-wide DNA methylation analysis using a target postbisulfite sequencing method in order to identify DNA methylation errors in cloned mouse sperm. The sperm genomes of two somatic cell-cloned mice (CL1 and CL7) contained significantly higher numbers of differentially methylated CpG sites (P1/40.0045 and P1/40.0116). As a result, they had higher numbers of differentially methylated CpG islands. However, there was no evidence that these sites were transmitted to the sperm genome of offspring. These results suggest that DNA methylation errors resulting from embryo cloning are transmitted to the sperm genome by evading the germ line reprogramming barrier.. |
| 41. |
Tasuku Koike, Takuya Wakai, Yuko Jincho, Akihiko Sakashita, Hisato Kobayashi, Eiji Mizutani, Sayaka Wakayama, Fumihito Miura, Takashi Ito, Tomohiro Kono, DNA methylation errors in cloned mouse sperm by germ line barrier evasion, Biology of Reproduction, 10.1095/biolreprod.116.138677, 94, 6, 128, 2016.06, © 2016 by the Society for the Study of Reproduction, Inc. The germ line reprogramming barrier resets parental epigenetic modifications according to sex, conferring totipotency to mammalian embryos upon fertilization. However, it is not known whether epigenetic errors are committed during germ line reprogramming that are then transmitted to germ cells, and consequently to offspring. We addressed this question in the present study by performing a genome-wide DNA methylation analysis using a target postbisulfite sequencing method in order to identify DNA methylation errors in cloned mouse sperm. The sperm genomes of two somatic cell-cloned mice (CL1 and CL7) contained significantly higher numbers of differentially methylated CpG sites (P1/40.0045 and P1/40.0116). As a result, they had higher numbers of differentially methylated CpG islands. However, there was no evidence that these sites were transmitted to the sperm genome of offspring. These results suggest that DNA methylation errors resulting from embryo cloning are transmitted to the sperm genome by evading the germ line reprogramming barrier.. |
| 42. |
Takao Yokoyama, Fumihito Miura, Hiromitsu Araki, Kohji Okamura, Takashi Ito, Changepoint detection in base-resolution methylome data reveals a robust signature of methylated domain landscape, BMC Genomics, 10.1186/s12864-015-1809-5, 16, 1, 2015.08, Background: Base-resolution methylome data generated by whole-genome bisulfite sequencing (WGBS) is often used to segment the genome into domains with distinct methylation levels. However, most segmentation methods include many parameters to be carefully tuned and/or fail to exploit the unsurpassed resolution of the data. Furthermore, there is no simple method that displays the composition of the domains to grasp global trends in each methylome. Results: We propose to use changepoint detection for domain demarcation based on base-resolution methylome data. While the proposed method segments the methylome in a largely comparable manner to conventional approaches, it has only a single parameter to be tuned. Furthermore, it fully exploits the base-resolution of the data to enable simultaneous detection of methylation changes in even contrasting size ranges, such as focal hypermethylation and global hypomethylation in cancer methylomes. We also propose a simple plot termed methylated domain landscape (MDL) that globally displays the size, the methylation level and the number of the domains thus defined, thereby enabling one to intuitively grasp trends in each methylome. Since the pattern of MDL often reflects cell lineages and is largely unaffected by data size, it can serve as a novel signature of methylome. Conclusions: Changepoint detection in base-resolution methylome data followed by MDL plotting provides a novel method for methylome characterization and will facilitate global comparison among various WGBS data differing in size and even species origin.. |
| 43. |
Takao Yokoyama, Fumihito Miura, Hiromitsu Araki, Kohji Okamura, Takashi Ito, Changepoint detection in base-resolution methylome data reveals a robust signature of methylated domain landscape, BMC Genomics, 10.1186/s12864-015-1809-5, 16, 1, 594, 2015.08, © 2015 Yokoyama et al. Background: Base-resolution methylome data generated by whole-genome bisulfite sequencing (WGBS) is often used to segment the genome into domains with distinct methylation levels. However, most segmentation methods include many parameters to be carefully tuned and/or fail to exploit the unsurpassed resolution of the data. Furthermore, there is no simple method that displays the composition of the domains to grasp global trends in each methylome. Results: We propose to use changepoint detection for domain demarcation based on base-resolution methylome data. While the proposed method segments the methylome in a largely comparable manner to conventional approaches, it has only a single parameter to be tuned. Furthermore, it fully exploits the base-resolution of the data to enable simultaneous detection of methylation changes in even contrasting size ranges, such as focal hypermethylation and global hypomethylation in cancer methylomes. We also propose a simple plot termed methylated domain landscape (MDL) that globally displays the size, the methylation level and the number of the domains thus defined, thereby enabling one to intuitively grasp trends in each methylome. Since the pattern of MDL often reflects cell lineages and is largely unaffected by data size, it can serve as a novel signature of methylome. Conclusions: Changepoint detection in base-resolution methylome data followed by MDL plotting provides a novel method for methylome characterization and will facilitate global comparison among various WGBS data differing in size and even species origin.. |
| 44. |
Jack M. Colicchio, Fumihito Miura, John K. Kelly, Takashi Ito, Lena C. Hileman, DNA methylation and gene expression in Mimulus guttatus, BMC Genomics, 10.1186/s12864-015-1668-0, 16, 1, 2015.07, Background: The presence of methyl groups on cytosine nucleotides across an organism's genome (methylation) is a major regulator of genome stability, crossing over, and gene regulation. The capacity for DNA methylation to be altered by environmental conditions, and potentially passed between generations, makes it a prime candidate for transgenerational epigenetic inheritance. Here we conduct the first analysis of the Mimulus guttatus methylome, with a focus on the relationship between DNA methylation and gene expression. Results: We present a whole genome methylome for the inbred line Iron Mountain 62 (IM62). DNA methylation varies across chromosomes, genomic regions, and genes. We develop a model that predicts gene expression based on DNA methylation (R2 = 0.2). Post hoc analysis of this model confirms prior relationships, and identifies novel relationships between methylation and gene expression. Additionally, we find that DNA methylation is significantly depleted near gene transcriptional start sites, which may explain the recently discovered elevated rate of recombination in these same regions. Conclusions: The establishment here of a reference methylome will be a useful resource for the continued advancement of M. guttatus as a model system. Using a model-based approach, we demonstrate that methylation patterns are an important predictor of variation in gene expression. This model provides a novel approach for differential methylation analysis that generates distinct and testable hypotheses regarding gene expression.. |
| 45. |
Jack M. Colicchio, Fumihito Miura, John K. Kelly, Takashi Ito, Lena C. Hileman, DNA methylation and gene expression in Mimulus guttatus, BMC Genomics, 10.1186/s12864-015-1668-0, 16, 1, 507, 2015.07, © 2015 Colicchio et al. Background: The presence of methyl groups on cytosine nucleotides across an organism's genome (methylation) is a major regulator of genome stability, crossing over, and gene regulation. The capacity for DNA methylation to be altered by environmental conditions, and potentially passed between generations, makes it a prime candidate for transgenerational epigenetic inheritance. Here we conduct the first analysis of the Mimulus guttatus methylome, with a focus on the relationship between DNA methylation and gene expression. Results: We present a whole genome methylome for the inbred line Iron Mountain 62 (IM62). DNA methylation varies across chromosomes, genomic regions, and genes. We develop a model that predicts gene expression based on DNA methylation (R2 = 0.2). Post hoc analysis of this model confirms prior relationships, and identifies novel relationships between methylation and gene expression. Additionally, we find that DNA methylation is significantly depleted near gene transcriptional start sites, which may explain the recently discovered elevated rate of recombination in these same regions. Conclusions: The establishment here of a reference methylome will be a useful resource for the continued advancement of M. guttatus as a model system. Using a model-based approach, we demonstrate that methylation patterns are an important predictor of variation in gene expression. This model provides a novel approach for differential methylation analysis that generates distinct and testable hypotheses regarding gene expression.. |
| 46. |
Fumihito Miura, Takashi Ito, Highly sensitive targeted methylome sequencing by post-bisulfite adaptor tagging, DNA Research, 10.1093/dnares/dsu034, 22, 1, 13-18, 2015.02, © The Author 2014. Published by Oxford University Press on behalf of Kazusa DNA Research Institute. The current gold standard method for methylome analysis is whole-genome bisulfite sequencing (WGBS), but its cost is substantial, especially for the purpose of multi-sample comparison of large methylomes. Shotgun bisulfite sequencing of target-enriched DNA, or targeted methylome sequencing (TMS), can be a flexible, cost-effective alternative to WGBS. However, the current TMS protocol requires a considerable amount of input DNA and hence is hardly applicable to samples of limited quantity. Here we report a method to overcome this limitation by using post-bisulfite adaptor tagging (PBAT), in which adaptor tagging is conducted after bisulfite treatment to circumvent bisulfite-induced loss of intact sequencing templates, thereby enabling TMS of a 100-fold smaller amount of input DNA with far fewer cycles of polymerase chain reaction than in the current protocol. We thus expect that the PBAT-mediated TMS will serve as an invaluable method in epigenomics.. |
| 47. |
Fumihito Miura, Takashi Ito, Highly sensitive targeted methylome sequencing by post-bisulfite adaptor tagging, DNA Research, 10.1093/dnares/dsu034, 22, 1, 13-18, 2015.01, The current gold standard method for methylome analysis is whole-genome bisulfite sequencing (WGBS), but its cost is substantial, especially for the purpose of multi-sample comparison of large methylomes. Shotgun bisulfite sequencing of target-enriched DNA, or targeted methylome sequencing (TMS), can be a flexible, cost-effective alternative to WGBS. However, the current TMS protocol requires a considerable amount of input DNA and hence is hardly applicable to samples of limited quantity. Here we report a method to overcome this limitation by using post-bisulfite adaptor tagging (PBAT), in which adaptor tagging is conducted after bisulfite treatment to circumvent bisulfite-induced loss of intact sequencing templates, thereby enabling TMS of a 100-fold smaller amount of input DNA with far fewer cycles of polymerase chain reaction than in the current protocol. We thus expect that the PBAT-mediated TMS will serve as an invaluable method in epigenomics.. |
| 48. |
Hisato Kobayashi, Takayuki Sakurai, Fumihito Miura, Misaki Imai, Kentaro Mochiduki, Eikichi Yanagisawa, Akihiko Sakashita, Takuya Wakai, Yutaka Suzuki, Takashi Ito, Yasuhisa Matsui, Tomohiro Kono, High-resolution DNA methylome analysis of primordial germ cells identifies gender-specific reprogramming in mice, Genome Research, 10.1101/gr.148023.112, 23, 4, 616-627, 2013.04, Dynamic epigenetic reprogramming occurs during mammalian germ cell development, although the targets of this process, including DNA demethylation and de novo methylation, remain poorly understood. We performed genomewide DNA methylation analysis in male and female mouse primordial germ cells at embryonic days 10.5, 13.5, and 16.5 by whole-genome shotgun bisulfite sequencing. Our high-resolution DNA methylome maps demonstrated gender-specific differences in CpG methylation at genome-wide and gene-specific levels during fetal germline progression. There was extensive intra- and intergenic hypomethylation with erasure of methylation marks at imprinted, X-linked, or germlinespecific genes during gonadal sex determination and partial methylation at particular retrotransposons. Following global demethylation and sex determination, CpG sites switched to de novo methylation in males, but the X-linked genes appeared resistant to the wave of de novo methylation. Significant differential methylation at a subset of imprinted loci was identified in both genders, and non-CpG methylation occurred only in male gonocytes. Our data establish the basis for future studies on the role of epigenetic modifications in germline development and other biological processes.. |
| 49. |
Kenjiro Shirane, Hidehiro Toh, Hisato Kobayashi, Fumihito Miura, Hatsune Chiba, Takashi Ito, Tomohiro Kono, Hiroyuki Sasaki, Mouse Oocyte Methylomes at Base Resolution Reveal Genome-Wide Accumulation of Non-CpG Methylation and Role of DNA Methyltransferases, PLoS Genetics, 10.1371/journal.pgen.1003439, 9, 4, 2013.04, DNA methylation is an epigenetic modification that plays a crucial role in normal mammalian development, retrotransposon silencing, and cellular reprogramming. Although methylation mainly occurs on the cytosine in a CG site, non-CG methylation is prevalent in pluripotent stem cells, brain, and oocytes. We previously identified non-CG methylation in several CG-rich regions in mouse germinal vesicle oocytes (GVOs), but the overall distribution of non-CG methylation and the enzymes responsible for this modification are unknown. Using amplification-free whole-genome bisulfite sequencing, which can be used with minute amounts of DNA, we constructed the base-resolution methylome maps of GVOs, non-growing oocytes (NGOs), and mutant GVOs lacking the DNA methyltransferase Dnmt1, Dnmt3a, Dnmt3b, or Dnmt3L. We found that nearly two-thirds of all methylcytosines occur in a non-CG context in GVOs. The distribution of non-CG methylation closely resembled that of CG methylation throughout the genome and showed clear enrichment in gene bodies. Compared to NGOs, GVOs were over four times more methylated at non-CG sites, indicating that non-CG methylation accumulates during oocyte growth. Lack of Dnmt3a or Dnmt3L resulted in a global reduction in both CG and non-CG methylation, showing that non-CG methylation depends on the Dnmt3a-Dnmt3L complex. Dnmt3b was dispensable. Of note, lack of Dnmt1 resulted in a slight decrease in CG methylation, suggesting that this maintenance enzyme plays a role in non-dividing oocytes. Dnmt1 may act on CG sites that remain hemimethylated in the de novo methylation process. Our results provide a basis for understanding the mechanisms and significance of non-CG methylation in mammalian oocytes.. |
| 50. |
Hisato Kobayashi, Takayuki Sakurai, Fumihito Miura, Misaki Imai, Kentaro Mochiduki, Eikichi Yanagisawa, Akihiko Sakashita, Takuya Wakai, Yutaka Suzuki, Takashi Ito, Yasuhisa Matsui, Tomohiro Kono, High-resolution DNA methylome analysis of primordial germ cells identifies gender-specific reprogramming in mice, Genome Research, 10.1101/gr.148023.112, 23, 4, 616-627, 2013.04, Dynamic epigenetic reprogramming occurs during mammalian germ cell development, although the targets of this process, including DNA demethylation and de novo methylation, remain poorly understood. We performed genomewide DNA methylation analysis in male and female mouse primordial germ cells at embryonic days 10.5, 13.5, and 16.5 by whole-genome shotgun bisulfite sequencing. Our high-resolution DNA methylome maps demonstrated gender-specific differences in CpG methylation at genome-wide and gene-specific levels during fetal germline progression. There was extensive intra- and intergenic hypomethylation with erasure of methylation marks at imprinted, X-linked, or germlinespecific genes during gonadal sex determination and partial methylation at particular retrotransposons. Following global demethylation and sex determination, CpG sites switched to de novo methylation in males, but the X-linked genes appeared resistant to the wave of de novo methylation. Significant differential methylation at a subset of imprinted loci was identified in both genders, and non-CpG methylation occurred only in male gonocytes. Our data establish the basis for future studies on the role of epigenetic modifications in germline development and other biological processes. © 2013, Published by Cold Spring Harbor Laboratory Press.. |
| 51. |
Kenjiro Shirane, Hidehiro Toh, Hisato Kobayashi, Fumihito Miura, Hatsune Chiba, Takashi Ito, Tomohiro Kono, Hiroyuki Sasaki, Mouse Oocyte Methylomes at Base Resolution Reveal Genome-Wide Accumulation of Non-CpG Methylation and Role of DNA Methyltransferases, PLoS Genetics, 10.1371/journal.pgen.1003439, 9, 4, e1003439-e1003439, 2013.04, DNA methylation is an epigenetic modification that plays a crucial role in normal mammalian development, retrotransposon silencing, and cellular reprogramming. Although methylation mainly occurs on the cytosine in a CG site, non-CG methylation is prevalent in pluripotent stem cells, brain, and oocytes. We previously identified non-CG methylation in several CG-rich regions in mouse germinal vesicle oocytes (GVOs), but the overall distribution of non-CG methylation and the enzymes responsible for this modification are unknown. Using amplification-free whole-genome bisulfite sequencing, which can be used with minute amounts of DNA, we constructed the base-resolution methylome maps of GVOs, non-growing oocytes (NGOs), and mutant GVOs lacking the DNA methyltransferase Dnmt1, Dnmt3a, Dnmt3b, or Dnmt3L. We found that nearly two-thirds of all methylcytosines occur in a non-CG context in GVOs. The distribution of non-CG methylation closely resembled that of CG methylation throughout the genome and showed clear enrichment in gene bodies. Compared to NGOs, GVOs were over four times more methylated at non-CG sites, indicating that non-CG methylation accumulates during oocyte growth. Lack of Dnmt3a or Dnmt3L resulted in a global reduction in both CG and non-CG methylation, showing that non-CG methylation depends on the Dnmt3a-Dnmt3L complex. Dnmt3b was dispensable. Of note, lack of Dnmt1 resulted in a slight decrease in CG methylation, suggesting that this maintenance enzyme plays a role in non-dividing oocytes. Dnmt1 may act on CG sites that remain hemimethylated in the de novo methylation process. Our results provide a basis for understanding the mechanisms and significance of non-CG methylation in mammalian oocytes. © 2013 Shirane et al.. |
| 52. |
Kenjiro Shirane, Hidehiro Toh, Hisato Kobayashi, Fumihito Miura, Hatsune Chiba, Takashi Ito, Tomohiro Kono, Hiroyuki Sasaki, Mouse Oocyte Methylomes at Base Resolution Reveal Genome-Wide Accumulation of Non-CpG Methylation and Role of DNA Methyltransferases, PLOS GENETICS, 10.1371/journal.pgen.1003439, 9, 4, 2013.04, DNA methylation is an epigenetic modification that plays a crucial role in normal mammalian development, retrotransposon silencing, and cellular reprogramming. Although methylation mainly occurs on the cytosine in a CG site, non-CG methylation is prevalent in pluripotent stem cells, brain, and oocytes. We previously identified non-CG methylation in several CG-rich regions in mouse germinal vesicle oocytes (GVOs), but the overall distribution of non-CG methylation and the enzymes responsible for this modification are unknown. Using amplification-free whole-genome bisulfite sequencing, which can be used with minute amounts of DNA, we constructed the base-resolution methylome maps of GVOs, non-growing oocytes (NGOs), and mutant GVOs lacking the DNA methyltransferase Dnmt1, Dnmt3a, Dnmt3b, or Dnmt3L. We found that nearly two-thirds of all methylcytosines occur in a non-CG context in GVOs. The distribution of non-CG methylation closely resembled that of CG methylation throughout the genome and showed clear enrichment in gene bodies. Compared to NGOs, GVOs were over four times more methylated at non-CG sites, indicating that non-CG methylation accumulates during oocyte growth. Lack of Dnmt3a or Dnmt3L resulted in a global reduction in both CG and non-CG methylation, showing that non-CG methylation depends on the Dnmt3a-Dnmt3L complex. Dnmt3b was dispensable. Of note, lack of Dnmt1 resulted in a slight decrease in CG methylation, suggesting that this maintenance enzyme plays a role in non-dividing oocytes. Dnmt1 may act on CG sites that remain hemimethylated in the de novo methylation process. Our results provide a basis for understanding the mechanisms and significance of non-CG methylation in mammalian oocytes.. |
| 53. |
Fumihito Miura, Yusuke Enomoto, Ryo Dairiki, Takashi Ito, Amplification-free whole-genome bisulfite sequencing by post-bisulfite adaptor tagging, Nucleic Acids Research, 10.1093/nar/gks454, 40, 17, 2012.09, DNA methylation plays a key role in epigenetic regulation of eukaryotic genomes. Hence the genome-wide distribution of 5-methylcytosine, or the methylome, has been attracting intense attention. In recent years, whole-genome bisulfite sequencing (WGBS) has enabled methylome analysis at single-base resolution. However, WGBS typically requires microgram quantities of DNA as well as global PCR amplification, thereby precluding its application to samples of limited amounts. This is presumably because bisulfite treatment of adaptor-tagged templates, which is inherent to current WGBS methods, leads to substantial DNA fragmentation. To circumvent the bisulfite-induced loss of intact sequencing templates, we conceived an alternative method termed Post-Bisulfite Adaptor Tagging (PBAT) wherein bisulfite treatment precedes adaptor tagging by two rounds of random primer extension. The PBAT method can generate a substantial number of unamplified reads from as little as subnanogram quantities of DNA. It requires only 100 ng of DNA for amplification-free WGBS of mammalian genomes. Thus, the PBAT method will enable various novel applications that would not otherwise be possible, thereby contributing to the rapidly growing field of epigenomics.. |
| 54. |
Fumihito Miura, Yusuke Enomoto, Ryo Dairiki, Takashi Ito, Amplification-free whole-genome bisulfite sequencing by post-bisulfite adaptor tagging, Nucleic Acids Research, 10.1093/nar/gks454, 40, 17, 136, 2012.09, DNA methylation plays a key role in epigenetic regulation of eukaryotic genomes. Hence the genome-wide distribution of 5-methylcytosine, or the methylome, has been attracting intense attention. In recent years, whole-genome bisulfite sequencing (WGBS) has enabled methylome analysis at single-base resolution. However, WGBS typically requires microgram quantities of DNA as well as global PCR amplification, thereby precluding its application to samples of limited amounts. This is presumably because bisulfite treatment of adaptor-tagged templates, which is inherent to current WGBS methods, leads to substantial DNA fragmentation. To circumvent the bisulfite-induced loss of intact sequencing templates, we conceived an alternative method termed Post-Bisulfite Adaptor Tagging (PBAT) wherein bisulfite treatment precedes adaptor tagging by two rounds of random primer extension. The PBAT method can generate a substantial number of unamplified reads from as little as subnanogram quantities of DNA. It requires only 100 ng of DNA for amplification-free WGBS of mammalian genomes. Thus, the PBAT method will enable various novel applications that would not otherwise be possible, thereby contributing to the rapidly growing field of epigenomics. © 2012 The Author(s).. |
| 55. |
Xuhua Xia, Vivian Mackay, Xiaoquan Yao, Jianhua Wu, Fumihito Miura, Takashi Ito, David R. Morris, Translation initiation
A regulatory role for poly(A) tracts in front of the AUG codon in saccharomyces cerevisiae, Genetics, 10.1534/genetics.111.132068, 189, 2, 469-478, 2011.10, The 59-UTR serves as the loading dock for ribosomes during translation initiation and is the key site for translation regulation. Many genes in the yeast Saccharomyces cerevisiae contain poly(A) tracts in their 59-UTRs. We studied these pre-AUG poly(A) tracts in a set of 3274 recently identified 59-UTRs in the yeast to characterize their effect on in vivo protein abundance, ribosomal density, and protein synthesis rate in the yeast. The protein abundance and the protein synthesis rate increase with the length of the poly(A), but exhibit a dramatic decrease when the poly(A) length is ≥12. The ribosomal density also reaches the lowest level when the poly(A) length is ≥12. This supports the hypothesis that a pre-AUG poly(A) tract can bind to translation initiation factors to enhance translation initiation, but a long (≥12) pre-AUG poly(A) tract will bind to Pab1p, whose binding size is 12 consecutive A residues in yeast, resulting in repression of translation. The hypothesis explains why a long pre-AUG poly(A) leads to more efficient translation initiation than a short one when PABP is absent, and why pre-AUG poly(A) is short in the early genes but long in the late genes of vaccinia virus.. |
| 56. |
Xuhua Xia, Vivian Mackay, Xiaoquan Yao, Jianhua Wu, Fumihito Miura, Takashi Ito, David R. Morris, Translation initiation: A regulatory role for poly(A) tracts in front of the AUG codon in saccharomyces cerevisiae, Genetics, 10.1534/genetics.111.132068, 189, 2, 469-478, 2011.10, The 59-UTR serves as the loading dock for ribosomes during translation initiation and is the key site for translation regulation. Many genes in the yeast Saccharomyces cerevisiae contain poly(A) tracts in their 59-UTRs. We studied these pre-AUG poly(A) tracts in a set of 3274 recently identified 59-UTRs in the yeast to characterize their effect on in vivo protein abundance, ribosomal density, and protein synthesis rate in the yeast. The protein abundance and the protein synthesis rate increase with the length of the poly(A), but exhibit a dramatic decrease when the poly(A) length is ≥12. The ribosomal density also reaches the lowest level when the poly(A) length is ≥12. This supports the hypothesis that a pre-AUG poly(A) tract can bind to translation initiation factors to enhance translation initiation, but a long (≥12) pre-AUG poly(A) tract will bind to Pab1p, whose binding size is 12 consecutive A residues in yeast, resulting in repression of translation. The hypothesis explains why a long pre-AUG poly(A) leads to more efficient translation initiation than a short one when PABP is absent, and why pre-AUG poly(A) is short in the early genes but long in the late genes of vaccinia virus. © 2011 by the Genetics Society of America.. |
| 57. |
Takashi Ito, Fumihito Miura, Miyuki Onda, Unexpected complexity of the budding yeast transcriptome, IUBMB Life, 10.1002/iub.121, 60, 12, 775-781, 2008.12, The genome of the budding yeast Saccharomyces cerevisiae was sequenced over a decade ago and has been annotated to encode ∼6,000 genes. However, recent high throughput studies using tiling array hybridization and cDNA sequencing have revealed an unexpectedly large number of previously undescribed transcripts. They largely lack protein-coding capacity and are transcribed from both strands of intragenic and intergenic regions in the genome. Accordingly, pervasive transcription leading to a plethora of noncoding RNAs, which was first revealed for mammalian genomes to attract intense attentions, is likely an intrinsic feature of eukaryotic genomes. Although it is not clear what fraction of these transcription events are functional, some were shown to induce transcriptional interference or histone modifications to regulate gene expression. The budding yeast may serve as an excellent model to study pervasive transcription and noncoding RNAs.. |
| 58. |
Takashi Ito, Fumihito Miura, Miyuki Onda, Unexpected Complexity of the Budding Yeast Transcriptome, IUBMB LIFE, 10.1002/iub.121, 60, 12, 775-781, 2008.12, The genome of the budding yeast Saccharomyces cerevisiae was sequenced over a decade ago and has been annotated to encode similar to 6,000 genes. However, recent high throughput studies using tiling array hybridization and cDNA sequencing have revealed an unexpectedly large number of previously undescribed transcripts. They largely lack protein-coding capacity and are transcribed from both strands of intragenic and intergenic regions in the genome. Accordingly, pervasive transcription leading to a plethora of noncoding RNAs, which was first revealed for mammalian genomes to attract intense attentions, is likely an intrinsic feature of eukaryotic genomes. Although it is not clear what fraction of these transcription events are functional, some were shown to induce transcriptional interference or histone modifications to regulate gene expression. The budding yeast may serve as an excellent model to study pervasive transcription and noncoding RNAs. (C) 2008 IUBMB. |
| 59. |
Fumihito Miura, Noriko Kawaguchi, Mikio Yoshida, Chihiro Uematsu, Keiji Kito, Yoshiyuki Sakaki, Takashi Ito, Absolute quantification of the budding yeast transcriptome by means of competitive PCR between genomic and complementary DNAs, BMC Genomics, 10.1186/1471-2164-9-574, 9, 2008.11, Background: An ideal format to describe transcriptome would be its composition measured on the scale of absolute numbers of individual mRNAs per cell. It would help not only to precisely grasp the structure of the transcriptome but also to accelerate data exchange and integration. Results: We conceived an idea of competitive PCR between genomic DNA and cDNA. Since the former contains every gene exactly at the same copy number, it can serve as an ideal normalization standard for the latter to obtain stoichiometric composition data of the transcriptome. This data can then be easily converted to absolute quantification data provided with an appropriate calibration. To implement this idea, we improved adaptor-tagged competitive PCR, originally developed for relative quantification of the 3′-end restriction fragment of each cDNA, such that it can be applied to any restriction fragment. We demonstrated that this "generalized" adaptor-tagged competitive PCR (GATC-PCR) can be performed between genomic DNA and cDNA to accurately measure absolute expression level of each mRNA in the budding yeast Saccharomyces cerevisiae. Furthermore, we constructed a large-scale GATC-PCR system to measure absolute expression levels of 5,038 genes to show that the yeast contains more than 30,000 copies of mRNA molecules per cell. Conclusion: We developed a GATC-PCR method to accurately measure absolute expression levels of mRNAs by means of competitive amplification of genomic and cDNA copies of each gene. A large-scale application of GATC-PCR to the budding yeast transcriptome revealed that it is twice or more as large as previously estimated. This method is flexibly applicable to both targeted and genome-wide analyses of absolute expression levels of mRNAs.. |
| 60. |
Fumihito Miura, Noriko Kawaguchi, Mikio Yoshida, Chihiro Uematsu, Keiji Kito, Yoshiyuki Sakaki, Takashi Ito, Absolute quantification of the budding yeast transcriptome by means of competitive PCR between genomic and complementary DNAs, BMC Genomics, 10.1186/1471-2164-9-574, 9, 574, 2008.11, Background: An ideal format to describe transcriptome would be its composition measured on the scale of absolute numbers of individual mRNAs per cell. It would help not only to precisely grasp the structure of the transcriptome but also to accelerate data exchange and integration. Results: We conceived an idea of competitive PCR between genomic DNA and cDNA. Since the former contains every gene exactly at the same copy number, it can serve as an ideal normalization standard for the latter to obtain stoichiometric composition data of the transcriptome. This data can then be easily converted to absolute quantification data provided with an appropriate calibration. To implement this idea, we improved adaptor-tagged competitive PCR, originally developed for relative quantification of the 3′-end restriction fragment of each cDNA, such that it can be applied to any restriction fragment. We demonstrated that this "generalized" adaptor-tagged competitive PCR (GATC-PCR) can be performed between genomic DNA and cDNA to accurately measure absolute expression level of each mRNA in the budding yeast Saccharomyces cerevisiae. Furthermore, we constructed a large-scale GATC-PCR system to measure absolute expression levels of 5,038 genes to show that the yeast contains more than 30,000 copies of mRNA molecules per cell. Conclusion: We developed a GATC-PCR method to accurately measure absolute expression levels of mRNAs by means of competitive amplification of genomic and cDNA copies of each gene. A large-scale application of GATC-PCR to the budding yeast transcriptome revealed that it is twice or more as large as previously estimated. This method is flexibly applicable to both targeted and genome-wide analyses of absolute expression levels of mRNAs. © 2008 Miura et al; licensee BioMed Central Ltd.. |
| 61. |
Fumihito Miura, Noriko Kawaguchi, Jun Sese, Atsushi Toyoda, Masahira Hattori, Shinichi Morishita, Takashi Ito, A large-scale full-length cDNA analysis to explore the budding yeast transcriptome, Proceedings of the National Academy of Sciences of the United States of America, 10.1073/pnas.0605645103, 103, 47, 17846-17851, 2006.11, We performed a large-scale cDNA analysis to explore the transcriptome of the budding yeast Saccharomyces cerevisiae. We sequenced two cDNA libraries, one from the cells exponentially growing in a minimal medium and the other from meiotic cells. Both libraries were generated by using a vector-capping method that allows the accurate mapping of transcription start sites (TSSs). Consequently, we identified 11,575 TSSs associated with 3,638 annotated genomic features, including 3,599 ORFs, to suggest that most yeast genes have two or more TSSs. In addition, we identified 45 previously undescribed introns, including those affecting current ORF annotations and those spliced alternatively. Furthermore, the analysis revealed 667 transcription units in the intergenic regions and transcripts derived from antisense strands of 367 known features. We also found that 348 ORFs carry TSSs in their 3′-halves to generate sense transcripts starting from inside the ORFs. These results indicate that the budding yeast transcriptome is considerably more complex than previously thought, and it shares many recently revealed characteristics with the transcriptomes of mammals and other higher eukaryotes. Thus, the genome-wide active transcription that generates novel classes of transcripts appears to be an intrinsic feature of the eukaryotic cells. The budding yeast will serve as a versatile model for the studies on these aspects of transcriptome, and the full-length cDNA clones can function as an invaluable resource in such studies.. |
| 62. |
Fumihito Miura, Noriko Kawaguchi, Jun Sese, Atsushi Toyoda, Masahira Hattori, Shinichi Morishita, Takashi Ito, A large-scale full-length cDNA analysis to explore the budding yeast transcriptome, Proceedings of the National Academy of Sciences of the United States of America, 10.1073/pnas.0605645103, 103, 47, 17846-17851, 2006.11, We performed a large-scale cDNA analysis to explore the transcriptome of the budding yeast Saccharomyces cerevisiae. We sequenced two cDNA libraries, one from the cells exponentially growing in a minimal medium and the other from meiotic cells. Both libraries were generated by using a vector-capping method that allows the accurate mapping of transcription start sites (TSSs). Consequently, we identified 11,575 TSSs associated with 3,638 annotated genomic features, including 3,599 ORFs, to suggest that most yeast genes have two or more TSSs. In addition, we identified 45 previously undescribed introns, including those affecting current ORF annotations and those spliced alternatively. Furthermore, the analysis revealed 667 transcription units in the intergenic regions and transcripts derived from antisense strands of 367 known features. We also found that 348 ORFs carry TSSs in their 3′-halves to generate sense transcripts starting from inside the ORFs. These results indicate that the budding yeast transcriptome is considerably more complex than previously thought, and it shares many recently revealed characteristics with the transcriptomes of mammals and other higher eukaryotes. Thus, the genome-wide active transcription that generates novel classes of transcripts appears to be an intrinsic feature of the eukaryotic cells. The budding yeast will serve as a versatile model for the studies on these aspects of transcriptome, and the full-length cDNA clones can function as an invaluable resource in such studies. © 2006 by The National Academy of Sciences of the USA.. |
| 63. |
Fumihito Miura, Chihiro Uematsu, Yoshiyuki Sakaki, Takashi Ito, A novel strategy to design highly specific PCR primers based on the stability and uniqueness of 3′-end subsequences, Bioinformatics, 10.1093/bioinformatics/bti716, 21, 24, 4363-4370, 2005.12, Motivation: In contrast with conventional PCR using a pair of specific primers, some applications utilize a single unique primer in combination with a common primer, thereby relying solely on the former for specificity. These applications include rapid amplification of cDNA ends (RACE), adaptor-tagged competitive PCR (ATAC-PCR), PCR-mediated genome walking and so forth. Since the primers designed by conventional methods often fail to work in these applications, an improved strategy is required, particularly, for a large-scale analysis. Results: Based on t he structure of 'off-target' products in the ATAC-PCR, we reasoned that the practical determinant of the specificity of primers may not be the uniqueness of entire sequence but that of the shortest 3′-end subsequence that exceeds a threshold of duplex stability. We termed such a subsequence as a 'specificity-determining subsequence' (SDSS) and developed a simple algorithm to predict the performance of the primer: the algorithm identifies the SDSS of each primer and examines its uniqueness in the target genome. The primers designed using this algorithm worked much better than those designed using a conventional method in both ATAC-PCR and 5′-RACE experiments. Thus, the algorithm will be generally useful for improving various PCR-based applications.. |
| 64. |
Fumihito Miura, Chihiro Uematsu, Yoshiyuki Sakaki, Takashi Ito, A novel strategy to design highly specific PCR primers based on the stability and uniqueness of 3′-end subsequences, Bioinformatics, 10.1093/bioinformatics/bti716, 21, 24, 4363-4370, 2005.12, Motivation: In contrast with conventional PCR using a pair of specific primers, some applications utilize a single unique primer in combination with a common primer, thereby relying solely on the former for specificity. These applications include rapid amplification of cDNA ends (RACE), adaptor-tagged competitive PCR (ATAC-PCR), PCR-mediated genome walking and so forth. Since the primers designed by conventional methods often fail to work in these applications, an improved strategy is required, particularly, for a large-scale analysis. Results: Based on t he structure of 'off-target' products in the ATAC-PCR, we reasoned that the practical determinant of the specificity of primers may not be the uniqueness of entire sequence but that of the shortest 3′-end subsequence that exceeds a threshold of duplex stability. We termed such a subsequence as a 'specificity-determining subsequence' (SDSS) and developed a simple algorithm to predict the performance of the primer: the algorithm identifies the SDSS of each primer and examines its uniqueness in the target genome. The primers designed using this algorithm worked much better than those designed using a conventional method in both ATAC-PCR and 5′-RACE experiments. Thus, the algorithm will be generally useful for improving various PCR-based applications. © The Author 2005. Published by Oxford University Press. All rights reserved.. |
| 65. |
Yoichi Yamada, Hidemi Watanabe, Fumihito Miura, Hidenobu Soejima, Michiko Uchiyama, Tsuyoshi Iwasaka, Tsunehiro Mukai, Yoshiyuki Sakaki, Takashi Ito, A comprehensive analysis of allelic methylation status of CpG islands on human chromosome 21q, Genome Research, 10.1101/gr.1351604, 14, 2, 247-266, 2004.02, Approximately half of all human genes have CpG islands (CGIs) around their promoter regions. Although CGIs usually escape methylation, those on Chromosome X in females and those in the vicinity of imprinted genes are exceptions: They have both methylated and unmethylated alleles to display a "composite" pattern in methylation analysis. In addition, aberrant methylation of CGIs is known to often occur in cancer cells. Here we developed a simple Hpall-McrBC PCR method for discrimination of full, null, incomplete, and composite methylation patterns, and applied it to all computationally identified CGIs on human Chromosome 21q. This comprehensive analysis revealed that, although most CGIs (103 out of 149) escape methylation, a sizable fraction (31 out of 149) are fully methylated even in normal peripheral blood cells. Furthermore, we identified seven CGIs showing the composite methylation, and demonstrated that three of them are indeed methylated monoallelically. Further analyses using informative pedigrees revealed that two of the three are subject to maternal allele-specific methylation. Intriguingly, the other CGI is methylated in an allele-specific but parental-origin-independent manner. Thus, the cell seems to have a broader repertoire of methylating CGIs than previously thought, and our approach may contribute to uncover novel modes of allelic methylation.. |
| 66. |
Oyama, Takuya
Yoshida, Mikio
Kamegai, Satoshi
Kitano, Kagehiko
Miura, Fumihito
Kawaguchi, Noriko
Onda, Miyuki
Satou, Kenji
Ito, Takashi, Automatic Extraction of Expression-Related Features Shared by a Given Group of Genes, Genome Informatics, 10.11234/gi1990.14.312, 14, 312-313, 2003.01. |
| 67. |
Fumihito Miura, Tetsushi Yada, Kenta Nakai, Yoshiyuki Sakaki, Takashi Ito, Differential display analysis of mutants for the transcription factor Pdr1p regulating multidrug resistance in the budding yeast, FEBS Letters, 10.1016/S0014-5793(01)02792-2, 505, 1, 103-108, 2001.09, The transcription factor Pdr1p recognizes Pdr1p/Pdr3p-response element (PDRE) to activate genes involved in multidrug resistance of the budding yeast. To identify novel targets of Pdr1p, we compared transcriptomes among the yeast cells bearing wild, disrupted and gain-of-function alleles of PDR1 using a high-throughput fluorescent differential display PCR. Consequently, we identified 20 transcripts apparently regulated by Pdr1p, which are derived from well-known target genes as well as those that have never been described in the context of drug resistance. Intriguingly, among the latter, a previously unrecognized gene bearing a small putative open reading frame preceded by a functional PDRE was found.. |
| 68. |
C. Uematsu, J. Nishida, K. Okano, Fumihito Miura, Takashi Ito, Y. Sakaki, H. Kambara, Multiplex polymerase chain reaction (PCR) with color-tagged module-shuffling primers for comparing gene expression levels in various cells, Nucleic Acids Research, 29, 16, 2001.08, A method based on the multiplex polymerase chain reaction (PCR) and gel electrophoresis for the comparative analysis of gene expression levels was developed. Using the method many cDNA fragments from different sources can be compared simultaneously. Competitive PCR amplification of expressed genes from different sources was performed by using 'module-shuffling primers' (MPSs). The MPSs (labeled with different fluorophores) consist of sequence modules of 3 or 4 nt. The modules are arranged in different orders in each primer; therefore, the base sequences of the primers are different but their melting temperatures are identical. The genes expressed in different sources are ligated with tags complementary with the MPSs. Tag-ligated fragments are mixed in one tube and amplified at the same amplification efficiency by the MPSs. Amplified fragments are detected separately by multiple-color gel electrophoresis. This method can detect different amounts of each expressed gene, up to a difference in amounts of 30%, and its detection limit is 0.1 amol per assay.. |
| 69. |
Uematsu C, Nishida J, Okano K, Kambara H, Miura F, Ito T, Sakaki Y., Comparative analysis for expressed genes by polymerase chain reaction using module-shuffling primers., Nucleic Acids Res. Suppl., 10.1093/nass/1.1.91, 2001, 91-92, 2001.01. |
