| 1. |
Fumihito Miura, Miki Miura, Yukiko Shibata, Yoshikazu Furuta, Keisuke Miyamura, Yuki Ino, Asmaa M. A. Bayoumi, Utako Oba, Takashi Ito, Identification, expression, and purification of DNA cytosine 5-methyltransferases with short recognition sequences, BMC Biotechnology, 10.1186/s12896-022-00765-3, 22, 1, 33, 2022.11, Abstract
Background
DNA methyltransferases (MTases) are enzymes that induce methylation, one of the representative epigenetic modifications of DNA, and are also useful tools for analyzing epigenomes. However, regarding DNA cytosine 5-methylation, MTases identified so far have drawbacks in that their recognition sequences overlap with those for intrinsic DNA methylation in mammalian cells and/or that the recognition sequence is too long for fine epigenetic mapping. To identify MTases with short recognition sequences that never overlap with the CG dinucleotide, we systematically investigated the 25 candidate enzymes identified using a database search, which showed high similarity to known cytosine 5-MTases recognizing short sequences.
Results
We identified MTases with six new recognition sequences, including TCTG, CC, CNG, TCG, GCY, and GGCA. Because the recognition sequence never overlapped with the CG dinucleotide, MTases recognizing the CC dinucleotide were promising.
Conclusions
In the current study, we established a procedure for producing active CC-methylating MTases and applied it to nucleosome occupancy and methylome sequencing to prove the usefulness of the enzyme for fine epigenetic mapping. MTases that never overlap with CG dinucleotides would allow us to profile multiple epigenomes simultaneously.. |
| 2. |
Keishi Sugimachi, Hiromitsu Araki, Hideyuki Saito, Takaaki Masuda, Fumihito Miura, Kentaro Inoue, Tomonari Shimagaki, Yohei Mano, Tomohiro Iguchi, Masaru Morita, Yasushi Toh, Tomoharu Yoshizumi, Takashi Ito, Koshi Mimori, Persistent epigenetic alterations in transcription factors after a sustained virological response in hepatocellular carcinoma, JGH Open, 10.1002/jgh3.12833, 2022.10. |
| 3. |
Fumihito Miura, Takashi Ito, Post-bisulfite Adaptor Tagging with a Highly Efficient Single-Stranded DNA Ligation Technique, Methods in Molecular Biology, 10.1007/978-1-0716-2815-7_4, 2594, 45-57, 2022.10. |
| 4. |
Fumihito Miura, Yukiko Shibata, Miki Miura, Takashi Ito, Post-bisulfite Adaptor Tagging Based on an ssDNA Ligation Technique (tPBAT), Methods in Molecular Biology, 10.1007/978-1-0716-2724-2_2, 2577, 21-37, 2022.09. |
| 5. |
Manabu Fujisawa, Tran Bich Nguyen, Yoshiaki Abe, Yasuhito Suehara, Kota Fukumoto, Sakurako Suma, Kenichi Makishima, Chihiro Kaneko, Yen Thi Minh Nguyen, Kensuke Usuki, Kentaro Narita, Kosei Matsue, Naoya Nakamura, Shumpei Ishikawa, Fumihito Miura, Takashi Ito, Ayako Suzuki, Yutaka Suzuki, Seiya Mizuno, Satoru Takahashi, Shigeru Chiba, Mamiko Sakata-Yanagimoto, Clonal germinal center B cells function as a niche for T-cell lymphoma, Blood, 10.1182/blood.2022015451, 140, 18, 1937-1950, 2022.08, Angioimmunoblastic T-cell lymphoma (AITL) is proposed to be initiated by age-related clonal hematopoiesis (ACH) with TET2 mutations, whereas the G17V RHOA mutation in immature cells with TET2 mutations promotes development of T follicular helper-like tumor cells. Here, we investigated the mechanism by which TET2-mutant immune cells enable AITL development, using mouse models and human samples. Among the two mouse models, mice lacking Tet2 in all the blood cells (Mx-Cre × Tet2flox/flox× G17V RHOA transgenic mice) spontaneously developed AITL for approximately up to a year, while mice lacking Tet2 only in the T cells (Cd4-Cre × Tet2flox/flox× G17V RHOA transgenic mice) did not. Therefore, Tet2-deficient immune cells function as a niche for AITL development. Single-cell RNA-sequencing (scRNA-seq) of > 50,000 cells from mouse and human AITL samples revealed significant expansion of aberrant B cells (exhibiting properties of activating light zone‒like and proliferative dark zone‒like germinal center B (GCB) cells). The GCB cells in AITL clonally evolved with recurrent mutations in genes related to core histones. In silico network analysis using scRNA-seq data identified Cd40-Cd40lg as a possible mediator of GCB and tumor cell cluster interactions. Treatment of AITL model mice with anti-Cd40lg inhibitory antibody prolonged survival. The genes expressed in aberrantly expanded GCB cells in murine tumors were also broadly expressed in the B-lineage cells of TET2-mutant human AITL. Therefore, ACH-derived GCB cells could undergo independent clonal evolution and support the tumorigenesis in AITL via the CD40-CD40LG axis.. |
| 6. |
Utako Oba, Kenichi Kohashi, Yuhei Sangatsuda, Yoshinao Oda, Koh-Hei Sonoda, Shouichi Ohga, Koji Yoshimoto, Yasuhito Arai, Shinichi Yachida, Tatsuhiro Shibata, Takashi Ito, Fumihito Miura, An efficient procedure for the recovery of DNA from formalin-fixed paraffin-embedded tissue sections, Biology Methods and Protocols, 10.1093/biomethods/bpac014, 7, 1, 2022.07, Abstract
With the advent of new molecular diagnostic techniques, retrieving DNA from the formalin-fixed paraffin-embedded (FFPE) tissues has become an essential yet challenging step for efficient downstream processes. Owing to low quality and quantity of DNA retrieved from the FFPE sections, the process is often impractical and needs significant improvements. Here, we established an efficient method for the purification of DNA from FFPE specimens by optimizing incubation temperature, incubation time, and the concentration of a formalin scavenger tris(hydroxymethyl)aminomethane (Tris) for reverse-crosslinking. The optimized method, named “Highly concentrated Tris-mediated DNA extraction” (HiTE), yielded three times the DNA yield per tissue slice compared with a representative DNA extraction kit. Moreover, the use of HiTE-extracted DNA increased the yield of the sequencing library three times and accordingly yielded a log higher and more reproducible sequencing library compared with that obtained using the commonly used commercial kit. The sequencing library prepared from HiTE-extracted FFPE-DNA had longer inserts and produced reads that evenly covered the reference genome. Successful application of HiTE-extracted FFPE-DNA for whole-genome and targeted gene panel sequencing indicates its practical usability.. |
| 7. |
Shunsuke Matsumoto, Suzuka Ono, Saori Shinoda, Chika Kakuta, Satoshi Okada, Takashi Ito, Tomoyuki Numata, Toshiya Endo, GET pathway mediates transfer of mislocalized tail-anchored proteins from mitochondria to the ER, Journal of Cell Biology, 10.1083/jcb.202104076, 221, 6, e202104076, 2022.06, Tail-anchored (TA) membrane proteins have a potential risk to be mistargeted to the mitochondrial outer membrane (OM). Such mislocalized TA proteins can be extracted by the mitochondrial AAA-ATPase Msp1 from the OM and transferred to the ER for ER protein quality control involving ubiquitination by the ER-resident Doa10 complex. Yet it remains unclear how the extracted TA proteins can move to the ER crossing the aqueous cytosol and whether this transfer to the ER is essential for the clearance of mislocalized TA proteins. Here we show by time-lapse microscopy that mislocalized TA proteins, including an authentic ER-TA protein, indeed move from mitochondria to the ER in a manner strictly dependent on Msp1 expression. The Msp1-dependent mitochondria-to-ER transfer of TA proteins is blocked by defects in the GET system, and this block is not due to impaired Doa10 functions. Thus, the GET pathway facilitates the transfer of mislocalized TA proteins from mitochondria to the ER.. |
| 8. |
Yoshikazu Furuta, Fumihito Miura, Takahiro Ichise, Shouta M. M. Nakayama, Yoshinori Ikenaka, Tuvshinzaya Zorigt, Mai Tsujinouchi, Mayumi Ishizuka, Takashi Ito, Hideaki Higashi, A GCDGC-specific DNA (cytosine-5) methyltransferase that methylates the GCWGC sequence on both strands and the GCSGC sequence on one strand, PLOS ONE, 10.1371/journal.pone.0265225, 17, 3, e0265225-e0265225, 2022.03, 5-Methylcytosine is one of the major epigenetic marks of DNA in living organisms. Some bacterial species possess DNA methyltransferases that modify cytosines on both strands to produce fully-methylated sites or on either strand to produce hemi-methylated sites. In this study, we characterized a DNA methyltransferase that produces two sequences with different methylation patterns: one methylated on both strands and another on one strand. M.BatI is the orphan DNA methyltransferase of Bacillus anthracis coded in one of the prophages on the chromosome. Analysis of M.BatI modified DNA by bisulfite sequencing revealed that the enzyme methylates the first cytosine in sequences of 5ʹ-GCAGC-3ʹ, 5ʹ-GCTGC-3ʹ, and 5ʹ-GCGGC-3ʹ, but not of 5ʹ-GCCGC-3ʹ. This resulted in the production of fully-methylated 5ʹ-GCWGC-3ʹ and hemi-methylated 5ʹ-GCSGC-3ʹ. M.BatI also showed toxicity when expressed in E. coli, which was caused by a mechanism other than DNA modification activity. Homologs of M.BatI were found in other Bacillus species on different prophage like regions, suggesting the spread of the gene by several different phages. The discovery of the DNA methyltransferase with unique modification target specificity suggested unrevealed diversity of target sequences of bacterial cytosine DNA methyltransferase.. |
| 9. |
Osamu Hisano, Takashi Ito, Fumihito Miura, Short single-stranded DNAs with putative non-canonical structures comprise a new class of plasma cell-free DNA, BMC Biology, 10.1186/s12915-021-01160-8, 19, 1, 225-225, 2021.12, Abstract
Background
Cell-free DNA (cfDNA), which is extracellular DNA present in the circulating plasma and other body fluids, is currently investigated as a minimally invasive, highly informative biomarker. While nucleosome-sized cfDNA fragments have been investigated intensively, shorter DNA fragments in the plasma have not been studied due to several technical limitations.
Results
We aimed to investigate the existence of shorter cfDNA fragments in the blood. Using an improved cfDNA purification protocol and a 3′-end-labeling method, we found DNA fragments of approximately 50 nucleotides in length in the human plasma, present at a molar concentration comparable to that of nucleosome-sized fragments. Unfortunately, these short fragments cannot be recovered by widely used cfDNA isolation methods. In addition, they are composed of single-stranded DNA (ssDNA), thus escaping detection in previous studies. Therefore, we established a library-preparation protocol based on our unique ssDNA ligation technique and applied it to the isolated cfDNA. Deep sequencing of these libraries revealed that the short fragments are derived from hundreds of thousands of genomic sites in open chromatin regions and enriched with transcription factor-binding sites. Remarkably, antisense strands of putative G-quadruplex motifs occupy as much as one-third of the peaks by these short fragments.
Conclusions
We propose a new class of plasma cfDNA composed of short single-stranded fragments that potentially form non-canonical DNA structures.
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| 10. |
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. |
| 11. |
Tomonori KAMEDA, Hideyuki NAKASHIMA, Takumi TAKIZAWA, Fumihito MIURA, Takashi ITO, Kinichi NAKASHIMA, Takuya IMAMURA, Neuronal activation modulates enhancer activity of genes for excitatory synaptogenesis through de novo DNA methylation, Journal of Reproduction and Development, 10.1262/jrd.2021-106, Online ahead of print., 2021.10. |
| 12. |
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. |
| 13. |
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, 2021.09. |
| 14. |
Satoshi Okada, Goro Doi, Shitomi Nakagawa, Emiko Kusumoto, Takashi Ito, Simple-to-use CRISPR-SpCas9/SaCas9/AsCas12a vector series for genome editing in Saccharomyces cerevisiae, G3 Genes|Genomes|Genetics, 10.1093/g3journal/jkab304, 11, jkab304, 2021.08, Abstract
Genome editing using the CRISPR/Cas system has been implemented for various organisms and becomes increasingly popular even in the genetically tractable budding yeast Saccharomyces cerevisiae. Since each CRISPR/Cas system recognizes only the sequences flanked by its unique protospacer adjacent motif (PAM), a certain single system often fails to target a region of interest due to the lack of PAM, thus necessitating the use of another system with a different PAM. Three CRISPR/Cas systems with distinct PAMs, namely SpCas9, SaCas9, and AsCas12a, have been successfully used in yeast genome editing. Their combined use should expand the repertoire of editable targets. However, currently available plasmids for these systems were individually developed under different design principles, thus hampering their seamless use in the practice of genome editing. Here we report a series of Golden Gate Assembly-compatible backbone vectors designed under a unified principle to exploit the three CRISPR/Cas systems in yeast genome editing. We also created a program to assist the design of genome-editing plasmids for individual target sequences using the backbone vectors. Genome editing with these plasmids demonstrated practically sufficient efficiency in the insertion of gene fragments to essential genes (median 52.1%), the complete deletion of an open reading frame (median 78.9%), and the introduction of single amino acid substitutions (median 79.2%). The backbone vectors with the program would provide a versatile toolbox to facilitate the seamless use of SpCas9, SaCas9, and AsCas12a in various types of genome manipulation, especially those that are difficult to perform with conventional techniques in yeast genetics.. |
| 15. |
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, eabd7924-eabd7924, 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.. |
| 16. |
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, 10.1242/dev.194605, 148, 10, 2021.05, ABSTRACT
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.. |
| 17. |
Goro Doi, Satoshi Okada, Takehiro Yasukawa, Yuki Sugiyama, Siqin Bala, Shintaro Miyazaki, Dongchon Kang, Takashi Ito, Catalytically inactive Cas9 impairs DNA replication fork progression to induce focal genomic instability, Nucleic Acids Research, 10.1093/nar/gkaa1241, 2021.01, Abstract
Catalytically inactive Cas9 (dCas9) has become an increasingly popular tool for targeted gene activation/inactivation, live-cell imaging, and base editing. While dCas9 was reported to induce base substitutions and indels, it has not been associated with structural variations. Here, we show that dCas9 impedes replication fork progression to destabilize tandem repeats in budding yeast. When targeted to the CUP1 array comprising ∼16 repeat units, dCas9 induced its contraction in most cells, especially in the presence of nicotinamide. Replication intermediate analysis demonstrated replication fork stalling in the vicinity of dCas9-bound sites. Genetic analysis indicated that while destabilization is counteracted by the replisome progression complex components Ctf4 and Mrc1 and the accessory helicase Rrm3, it involves single-strand annealing by the recombination proteins Rad52 and Rad59. Although dCas9-mediated replication fork stalling is a potential risk in conventional applications, it may serve as a novel tool for both mechanistic studies and manipulation of genomic instability.. |
| 18. |
Teruhiko Kawamura, Hiroyuki Tomari, Ichiro Onoyama, Hiromitsu Araki, Masafumi Yasunaga, Cui Lin, Keiko Kawamura, Natsuko Yokota, Sachiko Yoshida, Hiroshi Yagi, Kazuo Asanoma, Kenzo Sonoda, Katsuko Egashira, Takashi Ito, Kiyoko Kato, Identification of genes associated with endometrial cell ageing, Molecular Human Reproduction, 10.1093/molehr/gaaa078, 2020.12, Abstract
Ageing of the uterine endometrium is a critical factor that affects reproductive success, but the mechanisms associated with uterine ageing are unclear. In this study, we conducted a qualitative examination of age-related changes in endometrial tissues and identified candidate genes as markers for uterine ageing. Gene expression patterns were assessed by two RNA-sequencing experiments using uterine tissues from wild type (WT) C57BL/6 mice. Gene expression data obtained by RNA-sequencing were validated by real-time PCR. Genes expressing the pro-inflammatory cytokines Il17rb and chemokines Cxcl12 and Cxcl14 showed differential expression between aged WT mice and a group of mice composed of 5- and 8-week-old WT (young) animals. Protein expression levels of the above-mentioned genes and of IL8, which functions downstream of IL17RB, were analysed by quantitative immunohistochemistry of unaffected human endometrium tissue samples from patients in their 20s and 40s (10 cases each). In the secretory phase samples, 3,3ʹ- diaminobenzidine staining intensities of IL17RB, CXCL12 and CXCL14 for patients in their 40s were significantly higher than that for patients in their 20s, as detected by a Mann–hitney U test. These results suggest that these genes are candidate markers for endometrial ageing and for prediction of age-related infertility, although confirmation of these findings is needed in larger studies involving fertile and infertile women.. |
| 19. |
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, 2020.12. |
| 20. |
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, Journal of Experimental Medicine, 10.1084/jem.20192283, 218, 3, e20192283, 2020.11, 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.. |
| 21. |
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, 2020.09, Abstract
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.. |
| 22. |
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, 2020.09. |
| 23. |
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, 2020.08. |
| 24. |
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, 2020.06. |
| 25. |
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, 445-458.e6, 2020.05. |
| 26. |
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. |
| 27. |
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, Abstract
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.. |
| 28. |
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, 2019.02, Minimal sets of transcription factors can directly reprogram somatic cells into neurons. However, epigenetic remodeling during neuronal reprogramming has not been well reconciled with transcriptional regulation. Here we show that NeuroD1 achieves direct neuronal conversion from mouse microglia both in vitro and in vivo. Exogenous NeuroD1 initially occupies closed chromatin regions associated with bivalent trimethylation of histone H3 at lysine 4 (H3K4me3) and H3K27me3 marks in microglia to induce neuronal gene expression. These regions are resolved to a monovalent H3K4me3 mark at later stages of reprogramming to establish the neuronal identity. Furthermore, the transcriptional repressors Scrt1 and Meis2 are induced as NeuroD1 target genes, resulting in a decrease in the expression of microglial genes. In parallel, the microglial epigenetic signature in promoter and enhancer regions is erased. These findings reveal NeuroD1 pioneering activity accompanied by global epigenetic remodeling for two sequential events: onset of neuronal property acquisition and loss of the microglial identity during reprogramming.. |
| 29. |
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, 2018.12. |
| 30. |
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, 1436, 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.. |
| 31. |
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, 2018.09, 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.. |
| 32. |
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. |
| 33. |
Taichi Umeyama, Takashi Ito, DMS-seq for In Vivo Genome-Wide Mapping of Protein-DNA Interactions and Nucleosome Centers, Current Protocols in Molecular Biology, 10.1002/cpmb.60, 123, 1, e60-e60, 2018.07.  |
| 34. |
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. |
| 35. |
Fumihito Miura, Takashi Ito, Post-bisulfite adaptor tagging for PCR-free whole-genome bisulfite sequencing., Methods in Molecular Biology, 1708, 123-136, 2018.01. |
| 36. |
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, 2017.12. |
| 37. |
Taichi Umeyama, Takashi Ito, DMS-Seq for In Vivo Genome-wide Mapping of Protein-DNA Interactions and Nucleosome Centers, Cell Reports, 10.1016/j.celrep.2017.09.035, 21, 1, 289-300, 2017.10. |
| 38. |
Tsukasa Sanosaka, Takuya Imamura, Nobuhiko Hamazaki, MuhChyi 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. |
| 39. |
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.. |
| 40. |
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. |
| 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, 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 (P = 0.0045 and P = 0.0116). 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. |
Keiji Kito, Mitsuhiro Okada, Yuko Ishibashi, Satoshi Okada, Takashi Ito, A strategy for absolute proteome quantification with mass spectrometry by hierarchical use of peptide-concatenated standards, PROTEOMICS, 10.1002/pmic.201500414, 16, 10, 1457-1473, 2016.05, The accurate and precise absolute abundance of proteins can be determined using mass spectrometry by spiking the sample with stable isotope-labeled standards. In this study, we developed a strategy of hierarchical use of peptide-concatenated standards (PCSs) to quantify more proteins over a wider dynamic range. Multiple primary PCSs were used for quantification of many target proteins. Unique "ID-tag peptides" were introduced into individual primary PCSs, allowing us to monitor the exact amounts of individual PCSs using a "secondary PCS" in which all "ID-tag peptides" were concatenated. Furthermore, we varied the copy number of the "ID-tag peptide" in each PCS according to a range of expression levels of target proteins. This strategy accomplished absolute quantification over a wider range than that of the measured ratios. The quantified abundance of budding yeast proteins showed a high reproducibility for replicate analyses and similar copy numbers per cell for ribosomal proteins, demonstrating the accuracy and precision of this strategy. A comparison with the absolute abundance of transcripts clearly indicated different post-transcriptional regulation of expression for specific functional groups. Thus, the approach presented here is a faithful method for the absolute quantification of proteomes and provides insights into biological mechanisms, including the regulation of expressed protein abundance.. |
| 43. |
Futoshi Kuribayashi, Hiroyuki Nunoi, Kaori Wakamatsu, Shohko Tsunawaki, Kazuki Sato, Takashi Ito, Hideki Sumimoto, The adaptor protein p40(phox) as a positive regulator of the superoxide-producing phagocyte oxidase (vol 21, pg 6312, 2002), EMBO JOURNAL, 10.15252/embj.201593689, 35, 3, 369-370, 2016.02. |
| 44. |
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, 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.. |
| 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, 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 (R-2 = 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 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. |
Kazuhisa Ota, Shu-Ying Feng, Takashi Ito, Detecting protein-DNA interactions using a modified yeast one-hybrid system, METHODS IN MOLECULAR BIOLOGY, 1164, 39-50, 2014.07, The yeast one-hybrid (Y1H) system has been among the methods of choice to detect protein-DNA interactions. However, conventional Y1H systems with a single auxotrophic reporter gene often suffer from high incidence of false positives to demonstrate a limited power in large-scale screenings. Here we describe a refined Y1H system that uses two independent bait sequences, each controlling a distinct reporter gene integrated in the host genome. With these modifications and a method of targeted DNA methylation, we succeeded in efficient isolation of clones for methylated DNA-binding proteins from mammalian cDNA libraries.. |
| 48. |
Taichi Umeyama, Satoshi Okada, Takashi Ito, Synthetic Gene Circuit-Mediated Monitoring of Endogenous Metabolites: Identification of GAL11 as a Novel Multicopy Enhancer of S-Adenosylmethionine Level in Yeast, ACS SYNTHETIC BIOLOGY, 10.1021/sb300115n, 2, 8, 425-430, 2013.08, Monitoring levels of key metabolites in living cells comprises a critical step in various investigations. The simplest approach to this goal is a fluorescent reporter gene using an endogenous promoter responsive to the metabolite. However, such a promoter is often not identified or even present in the species of interest. An alternative can be a synthetic gene circuit based on a heterologous pair consisting of a promoter and a transcription factor known to respond to the metabolite. We exploited the met operator and MetJ repressor of Escherichia coli, the interaction between which depends on S-adenosylmethionine (SAM), to construct synthetic gene circuits that report SAM levels in Saccharomyces cerevisiae. Using a dual-input circuit that outputs selection marker genes in a doxycycline-tunable manner, we screened a genomic library to identify GAL11 as a novel multicopy enhancer of SAM levels. These results demonstrate the potential and utility of synthetic gene circuit-mediated metabolite monitoring.. |
| 49. |
Kei Fukuda, Kenji Ichiyanagi, Yoichi Yamada, Yasuhiro Go, Toshifumi Udono, Seitaro Wada, Toshiyuki Maeda, Hidenobu Soejima, Naruya Saitou, Takashi Ito, Hiroyuki Sasaki, Regional DNA methylation differences between humans and chimpanzees are associated with genetic changes, transcriptional divergence and disease genes, JOURNAL OF HUMAN GENETICS, 10.1038/jhg.2013.55, 58, 7, 446-454, 2013.07, Changes in gene expression have been proposed to have an important role in the evolutionary changes in phenotypes. Interspecific changes in gene expression can result not only from genetic changes in regulatory regions but also from epigenetic changes in such regions. Here we report the identification of genomic regions showing differences in DNA methylation between humans and chimpanzees (termed S-DMRs for species-specific differentially methylated regions) on chromosomes 21 and 22. These regional methylation differences are frequently associated with genes, including those relevant to a disease, such as Alzheimer's disease, diabetes mellitus or cancer. Methylation differences are often correlated with changes in promoter activity or alternative splicing. Comparative studies including other great ape species provide evidence for the contribution of genetic changes to some of these S-DMRs. Genetic changes responsible for the S-DMRs include gain or loss of CTCF-binding site and changes in CpG density in microsatellite repeats. Our results suggest that DNA methylation changes, often caused by small sequence changes, contribute to transcriptional and phenotypic diversification in hominid evolution.. |
| 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 genome-wide 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 germline-specific 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.. |
| 51. |
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, e136, 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.. |
| 52. |
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-U511, 2011.10, The 5'-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 5'-UTRs. We studied these pre-AUG poly(A) tracts in a set of 3274 recently identified 5'-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.. |
| 53. |
Shu-Ying Feng, Kazuhisa Ota, Takashi Ito, A yeast one-hybrid system to screen for methylated DNA-binding proteins, NUCLEIC ACIDS RESEARCH, 10.1093/nar/gkq757, 38, 20, e189, 2010.11, We had previously exploited a method for targeted DNA methylation in budding yeast to succeed in one-hybrid detection of methylation-dependent DNA-protein interactions. Based on this finding, we developed a yeast one-hybrid system to screen cDNA libraries for clones encoding methylated DNA-binding proteins. Concurrent use of two independent bait sequences in the same cell, or dual-bait system, effectively reduced false positive clones, which were derived from methylation-insensitive sequence-specific DNA-binding proteins. We applied the dual-bait system to screen cDNA libraries and demonstrated efficient isolation of clones for methylated DNA-binding proteins. This system would serve as a unique research tool for epigenetics.. |
| 54. |
Hiroshi Tanahashi, Keiji Kito, Takashi Ito, Katsuji Yoshioka, MafB protein stability is regulated by the JNK and ubiquitin-proteasome pathways, ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS, 10.1016/j.abb.2009.11.018, 494, 1, 94-100, 2010.02, MafB is a basic leucine zipper transcription factor that plays important roles in development and differentiation processes. During osteoclastogenesis, its expression is downregulated at the transcriptional level via the JNK and p38 MAP kinase pathways. In the present Study, we demonstrated that MafB protein stability is regulated by JNK and identified a phosphorylation site, Thr62. The expression of a constitutively active form of JNK (a fusion protein MKK7 alpha 1-JNK1 beta 1) promoted the degradation of MafB in COS7 cells, and a T62A substitution significantly reduced the instability of MafB. The introduction of a fourfold (T58A/T62A/S70A/S74A) Substitution in an acidic transcription-activating domain almost protected the instability resulting from the activation of JNK. Furthermore, treatment with proteasome inhibitors increased the MafB level, and a high-molecular-weight smear, characteristic of polyubiquitination was observed in lysates from cells in which MafB, ubiquitin, and MKK7 alpha 1-JNK1 beta 1 were coexpressed. These results suggest that phosphorylation of MafB by JNK confers Susceptibility to proteasomal degradation. (C) 2009 Elsevier Inc. All rights reserved.. |
| 55. |
Mitsunori Kato, Keiji Kito, Kazuhisa Ota, Takashi Ito, Remodeling of the SCF complex-mediated ubiquitination system by compositional alteration of incorporated F-box proteins, PROTEOMICS, 10.1002/pmic.200900497, 10, 1, 115-123, 2010.01, Ubiquitination regulates not only the stability but the localization and activity of substrate proteins involved in a plethora of cellular processes, The Skp1-Cullin-F-box protein (SCF) complexes constitute a major family of ubiquitin protein ligases, in each member of which an F-box protein serves as the variable component responsible for substrate recognition, thereby defining the function of each complex. Here we studied whether the composition of F-box proteins in the SCF complexes is remodeled under different conditions. We exploited stable isotope labeling and MS for relative quantification of F-box proteins in the SCF complexes affinity-purified en masse from budding yeast cells at log and post-diauxic phases, and revealed an increment of Saf1, an F-box protein involved in entry into quiescence, during the diauxic shift. Similarly, we found that Met4 overexpression induces a specific increment of Met30, the F-box protein responsible for ubiquitination of Met4. These results illustrate a cellular response to environmental and genetic perturbations through remodeling of the SCF complex-mediated ubiquitination system. Compositional alteration of incorporated F-box proteins may redirect the activity of this system toward appropriate substrates to be ubiquitinated under individual conditions for the maintenance of cellular homeostasis.. |
| 56. |
Satoshi Okada, Kazuhisa Ota, Takashi Ito, Circular permutation of ligand-binding module improves dynamic range of genetically encoded FRET-based nanosensor, PROTEIN SCIENCE, 10.1002/pro.266, 18, 12, 2518-2527, 2009.12, Quantitative measurement of small molecules with high spatiotemporal resolution provides a solid basis for correct understanding and accurate modeling of metabolic regulation. A promising approach toward this goal is the FLIP (fluorescent indicator protein) nanosensor based on bacterial periplasmic binding proteins (PBPs) and fluorescence resonance energy transfer (FRET) between the yellow and cyan variants of green fluorescent protein (GFP). Each FLIP has a PBP module that specifically binds its ligand to induce a conformation change, leading to a change in FRET between the two GFP variant modules attached to the N- and C-termini of the PBP. The larger is the dynamic range the more reliable is the measurement. Thus, we attempted to expand the dynamic range of FLIP by introducing a circular permutation with a hinge loop deletion to the PBP module. All the six circularly permutated PBPs tested, including structurally distinct Type I and Type 11 PBPs, showed larger dynamic ranges than their respective native forms when used for FLIP. Notably, the circular permutation made three PBPs, which totally failed to show FRET change when used as their native forms, fully capable of functioning as a ligand binding module of FLIP. These FLIPs were successfully used for the determination of amino acid concentration in complex solutions as well as real-time measurement of amino acid influx in living yeast cells. Thus, the circular permutation strategy would not only improve the performance of each nanosensor but also expand the repertoire of metabolites that can be measured by the FLIP nanosensor technology.. |
| 57. |
Kenji Ogura, Tsubasa Tandai, Sosuke Yoshinaga, Yoshihiro Kobashigawa, Hiroyuki Kumeta, Takashi Ito, Hideki Sumimoto, Fuyuhiko Inagaki, NMR Structure of the Heterodimer of Bem1 and Cdc24 PB1 Domains from Saccharomyces Cerevisiae, JOURNAL OF BIOCHEMISTRY, 10.1093/jb/mvp075, 146, 3, 317-325, 2009.09, Bem1 and Cdc24 of the budding yeast Saccharomyces cerevisiae interact with each other through PB1-PB1 heterodimer formation to regulate the establishment of cell polarity. Here we present the tertiary structure of the heterodimer of Bem1 and Cdc24 PB1 domains determined by NMR spectroscopy. To avoid ambiguity in the NMR spectral analysis, we first prepared a mutant of the Cdc24 PB1 domain that had truncated loops. The mutant provided well dispersed spectra without spectral overlapping, thus allowing unambiguous spectral assignments for structure determination. We confirmed that the loop deletion-mutant was quite similar to the wildtype in both 3D structure and binding affinity. The NMR structure of the heterodimer of the deletion-mutant of Cdc24 PB1 and Bem1 PB1 was determined using a variety of isotope labelled samples including perdeuteration. The interface between the Bem1/Cdc24 PB1 heterodimer was analysed at atomic resolution. Through a comparison with the tertiary structures of other PB1-PB1 heterodimers, we found that conserved electrostatic properties on the molecular surface were commonly used for PB1-PB1 interaction, but hydrophobic interactions were important for cognate interaction in Bem1/Cdc24 PB1 heterodimer formation.. |
| 58. |
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.. |
| 59. |
Kazuhisa Ota, Keiji Kito, Satoshi Okada, Takashi Ito, A proteomic screen reveals the mitochondrial outer membrane protein Mdm34p as an essential target of the F-box protein Mdm30p, GENES TO CELLS, 10.1111/j.1365-2443.2008.01228.x, 13, 10, 1075-1085, 2008.10, Ubiquitination plays various critical roles in eukaryotic cellular regulation and is medated by a cascade of enzymes including ubiquitin protein ligase (E3). The Skp1-Cullin-F-box protein complex comprises the largest E3 family, in each member of which a unique F-box protein binds its targets to define substrate specificity. Although genome sequencing uncovers a growing number of F-box proteins, most of them have remained as "orphans" because of the difficulties in identification of their substrates. To address this issue, we tested a quantitative proteomic approach by combining the stable isotope labeling by amino acids in cell culture (SILAC), parallel affinity purification (PAP) that we had developed for efficient enrichment of ubiquitinated proteins, and mass spectrometry (MS). We applied this SILAC-PAP-MS approach to compare ubiquitinated proteins between yeast cells with and without over-expressed Mdm30p, an F-box protein implicated in mitochondrial morphology. Consequently, we identified the mitochondrial outer membrane protein Mdm34p as a target of Mdm30p. Furthermore, we found that mitochondrial defects induced by deletion of MDM30 are not only recapitulated by a mutant Mdm34p defective in interaction with Mdm30p but alleviated by ubiquitination-mimicking forms of Mdm34p. These results indicate that Mdm34p is a physiologically important target of Mdm30p.. |
| 60. |
Kazuhisa Ota, Keiji Kito, Shun-ichiro Iemura, Tohru Natsume, Takashi Ito, A parallel affinity purification method for selective isolation of polyubiquitinated proteins, PROTEOMICS, 10.1002/pmic.200800271, 8, 15, 3004-3007, 2008.08, We developed a parallel affinity purification (PAP) procedure, in which ubiquitinated proteins are purified from the cells that coexpress two affinity-tagged ubiquitins by sequential use of affinity chromatography specific to each tag. In contrast with previous procedures using a single affinity-tagged ubiquitin, the PAP eliminates highly abundant ubiquitin monomers and mono-ubiquitinated proteins to selectively enrich proteins bearing both affinity-tags, or poly- and multi-ubiquitinated proteins. Accordingly, it would serve as a powerful method to facilitate mass-spectrometric identification of ubiquitinated proteins.. |
| 61. |
Keiji Kito, Noriko Kawaguchi, Satoshi Okada, Takashi Ito, Discrimination between stable and dynamic components of protein complexes by means of quantitative proteomics, PROTEOMICS, 10.1002/pmic.200800182, 8, 12, 2366-2370, 2008.06, To discriminate between stable and dynamic protein-protein interactions, we propose a strategy in which cells with and without tagged bait are differentially labeled with stable isotope and combined prior to complex purification. Mass-spectrometric analysis of the purified complexes identifies stable and dynamic components as those derived exclusively from the tagged cells and those from both cells, respectively. We successfully applied this strategy to analyze two yeast protein complexes, eIF2B-eIF2 and cyclin-Cdc28.. |
| 62. |
Keiji Kito, Takashi Ito, Mass spectrometry-based approaches toward absolute quantitative proteomics, CURRENT GENOMICS, 10.2174/138920208784533647, 9, 4, 263-274, 2008.06, Mass spectrometry has served as a major tool for the discipline of proteomics to catalogue proteins in an unprecedented scale. With chemical and metabolic techniques for stable isotope labeling developed over the past decade, it is now routinely used as a method for relative quantification to provide valuable information on alteration of protein abundance in a proteome-wide scale. More recently, absolute or stoichiometric quantification of proteome is becoming feasible, in particular, with the development of strategies with isotope-labeled standards composed of concatenated peptides. On the other hand, remarkable progress has been also made in label-free quantification methods based on the number of identified peptides. Here we review these mass spectrometry-based approaches for absolute quantification of proteome and discuss their implications.. |
| 63. |
Kazue Kakiuchi, Yoshio Yamauchi, Masato Taoka, Maki Iwago, Tomoko Fujita, Takashi Ito, Si-Young Song, Akira Sakai, Toshiaki Isobe, Tohru Ichimura, Proteomic analysis of in vivo 14-3-3 interactions in the yeast Saccharomyces cerevisiae, BIOCHEMISTRY, 10.1021/bi700501t, 46, 26, 7781-7792, 2007.07, The yeast Saccharomyces cerevisiae produces two 14-3-3 proteins, Bmh1 and Bmh2, whose exact functions have remained unclear. Here, we performed a comprehensive proteomic analysis using multistep immunoaffinity purification and mass spectrometry and identified 271 yeast proteins that specifically bind to Bmh1 and -2 in a phosphorylation-dependent manner. The identified proteins have diverse biochemical functions and cellular roles, including cell signaling, metabolism, and cell cycle regulation. Importantly, there are a number of protein subsets that are involved in the regulation of yeast physiology through a variety of cell signaling pathways, including stress-induced transcription, cell division, and chitin synthesis at the cell wall. In fact, we found that a yeast mutant deficient in Bmh1 and -2 had defects in signal-dependent response of the MAPK (Hog1 and Mpk1) cascade and exhibited an abnormal accumulation of chitin at the bud neck. We propose that Bmh1 and -2 are common regulators of many cell signaling modules and pathways mediated by protein phosphorylation and regulate a variety of biological events by coordinately controlling the identified multiplex phosphoprotein components.. |
| 64. |
Keiji Kito, Kazuhisa Ota, Tomoko Fujita, Takashi Ito, A synthetic protein approach toward accurate mass spectrometric quantification of component stoichiometry of multiprotein complexes, JOURNAL OF PROTEOME RESEARCH, 10.1021/pr060447s, 6, 2, 792-800, 2007.02, Quantitative description of protein interactions is crucial to understand and model molecular systems regulating various cellular activities. Here, we developed a novel peptide-concatenated standard (PCS) strategy for accurate mass spectrometric quantification of component stoichiometry of multiprotein complexes. In this strategy, tryptic peptides suitable for quantification are selected with their natural flanking sequences from each component of multiprotein complex and concatenated into a single synthetic protein called PCS. The concatenation guarantees equimolarity among the peptides added to the sample to obviate the need for preparation of accurately known amounts of individual peptides. The flanking sequences would equalize the excision efficiency of each peptide between the PCS and the target protein to improve the accuracy of quantification. To validate this strategy, we quantified the budding yeast eIF2B gamma, the gamma subunit of eukaryotic initiation factor 2B, using a PCS composed of tryptic peptides from eIF2B gamma with their flanking sequences. An identical sample-to-standard signal ratio was obtained within 5% measured error for these peptides, including the one prone to incomplete digestion, thereby proving the principle of PCS strategy. We applied the strategy to reveal the stoichiometry of the eIF2B-eIF2 complex using a PCS covering the 5 eIF2B and 3 eIF2 components. While the complex contained equimolar amounts of the eIF2B subunits, the ratio of each eIF2 subunit to eIF2B was 30-40%. The PCS strategy would provide a versatile method to quantitatively analyze compositional alteration of multiprotein complexes or dynamics of protein-protein interactions in response to various stimuli.. |
| 65. |
Munkhuu Bayarsaikhan, Takahisa Takino, Davaakhuu Gantulga, Hiroshi Sato, Takashi Ito, Katsuji Yoshioka, Regulation of N-cadherin-based cell-cell interaction by JSAP1 scaffold in PC12h cells, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 10.1016/j.bbrc.2006.12.029, 353, 2, 357-362, 2007.02, We previously reported that the level of c-Jun NH2-terminal kinase (JNK)/stress- activated protein kinase-associated protein I (JSAP1), a scaffold protein for JNK signaling, increases dramatically during nerve growth factor (NGF)-induced differentiation of PC12h cells. In the present study, we investigated the function of JSAP1 during PC12h cell differentiation by knocking down the level of JSAP1. The depletion of JSAP1 caused NGF-treated PC12h cells to form aggregates and impaired their differentiation. The aggregation was not observed in JSAP1-depleted cells that were untreated or treated with epidermal growth factor. Immunocytochemical studies indicated that N-cadherin, but not E-cadherin, was localized to sites of cell-cell contact in the aggregated cells. Furthermore, an inhibitory anti-N-cadherin antibody completely blocked the aggregation. Taken together, these results suggest that JSAP1 regulates cell-cell interactions in PC12h cells specifically in the NGF-induced signaling pathway, and does so by modulating N-cadherin. (c) 2006 Elsevier Inc. All rights reserved.. |
| 66. |
Yoshihiro Yamaguchi, Kazuhisa Ota, Takashi Ito, A novel Cdc42-interacting domain of the yeast polarity establishment protein Bem1 - Implications for modulation of mating pheromone signaling, JOURNAL OF BIOLOGICAL CHEMISTRY, 10.1074/jbc.M609308200, 282, 1, 29-38, 2007.01, In Saccharomyces cerevisiae, the Rho-type small GTPase Cdc42 is activated by its guanine-nucleotide exchange factor Cdc24 to polarize the cell for budding and mating. A multidomain protein Bem1 interacts not only with Cdc42 but also with Cdc24 and the effectors of Cdc42, including the p21-activated kinase Ste20, to function as a scaffold for cell polarity establishment. Although Bem1 interacts with Cdc24 and Ste20 via its PB1 and the second SH3 domains (SH3b), respectively, it is unclear how Bem1 binds Cdc42. Here we show that a region comprising the SH3b and its C-terminal flanking segment termed CI (SH3b-CI) directly interacts, with Cdc42. A dual-bait reverse two-hybrid approach revealed that the CI is critical to the interaction: N253D substitution in the CI abolishes the binding of the SH3b-CI to Cdc42 but not to the proline-rich region of Ste20, whereas W192K substitution in the SH3b has the opposite effect. Nevertheless, the SH3b-CI interacts with Ste20 proline-rich region and Cdc42 in a mutually exclusive manner. The N253D substitution renders cellular growth temperature-sensitive and suppresses mating. The W192K-induced mating defect is exacerbated by the N253D substitution and suppressed by increasing the dosage of Ste20 provided that the CI is intact. Intriguingly, Cdc42 can mediate an indirect interaction of the SH3b-CI to the CRIB domain of Ste20. These results suggest that the SH3b and the CI collaborate in tethering of Ste20 to Bem1 to ensure efficient mating pheromone signaling.. |
| 67. |
Yuko Kawahashi, Nobuhide Doi, Yo Oishi, Chizuru Tsuda, Hideaki Takashima, Tomoya Baba, Hirotada Mori, Takashi Ito, Hiroshi Yanagawa, High-throughput fluorescence labelling of full-length cDNA products based on a reconstituted translation system, JOURNAL OF BIOCHEMISTRY, 10.1093/jb/mvm003, 141, 1, 19-24, 2007.01, Although recent advances in fluorescence-based technologies, such as protein microarrays, have made it possible to analyse more than 10,000 proteins at once, there is a bottleneck in the step of preparation of large numbers of fluorescently labelled proteins for the comprehensive analysis of protein-protein interactions. Here we describe two independent methods for high-throughput fluorescence-labelling of fun-length cDNA products at their C-termini using a reconstituted translation system containing fluorescent puromycin. For the first method, release factor-free systems were used. For the second method, stop codons were excluded from cDNAs by using a common mismatch primer in mutagenic PCR These methods yielded large numbers of labelled proteins from cDNA sets of various organisms, such as mouse, yeast and Escherichia coli.. |
| 68. |
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.. |
| 69. |
Yoichi Yamada, Tomoyo Shirakawa, Todd D. Taylor, Kohji Okamura, Hidenobu Soejima, Michiko Uchiyama, Tsuyoshi Iwasaka, Tsunehiro Mukai, Ken-Ichiro Muramoto, Yoshiyuki Sakaki, Takashi Ito, A comprehensive analysis of allelic methylation status of CpG islands on human chromosome 11q: Comparison with chromosome 21q, DNA SEQUENCE, 10.1080/10425170600886128, 17, 4, 300-306, 2006.08, It was generally believed that autosomal CpG islands (CGIs) escape methylation. However, our comprehensive analysis of allelic methylation status of 149 CGIs on human chromosome 21q revealed that a sizable fraction of them are methylated on both alleles even in normal blood cells. Here, we performed a similar analysis of 656 CGIs on chromosome 11q, which is gene-rich in contrast with 21q. The results indicate that 11q contains less methylated CGIs, especially those with tandem repeats and those in the coding or 3'-untranslated regions (UTRs), than 21q. Thus, methylation status of CGIs may substantially differ from one chromosome to another.. |
| 70. |
Jieun Cheong, Yoichi Yamada, Riu Yamashita, Takuma Irie, Akinori Kanai, Hiroyuki Wakaguri, Kenta Nakai, Takashi Ito, Izumu Saito, Smuio Sugano, Yutaka Suzuki, Diverse DNA methylation statuses at alternative promoters of human genes in various tissues, DNA RESEARCH, 10.1093/dnares/ds1008, 13, 4, 155-167, 2006.08, We characterized the DNA methylation status at 144 tissue-biased and 37 non-tissue-biased alternative promoters of 61 human genes in five normal tissues. Analysis of the collected data revealed that (i) DNA methylation status differed greatly among alternative promoters belonging to the same gene; (ii) DNA methylation status differed between tissues for the majority of the individual promoters, and (iii) 80-90% of CpG-island-containing promoters were not methylated on either allele throughout the five tissues examined. Furthermore, although the statistical significance was not as clear as for the above features, we also found that (iv) the DNA methylation patterns of tissue-biased promoters changed more drastically than those of non-tissue-biased promoters; (v) tissue-biased promoters tended to be less methylated than their respective alternative promoters in the tissues where they were preferentially expressed, and (vi) the 'null' methylation pattern of a given promoter was enriched in the tissues where the transcription was most active. These findings together indicate that there are dynamic physiological changes of DNA methylation. DNA methylation appears to play a significant role in differential usage of alternative promoters and may be related to functional diversification between CpG-island-containing promoters and CpG-island-less promoters.. |
| 71. |
F Miura, C Uematsu, Y Sakaki, T 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 the 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.. |
| 72. |
K Okamura, Y Sakaki, T Ito, Comparative genomics approach toward critical determinants for the imprinting of an evolutionarily conserved gene Impact, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 10.1016/j.bbrc.2005.02.048, 329, 3, 824-830, 2005.04, The Impact is an evolutionarily conserved gene subjected to genomic imprinting in mouse but not in human. A characteristic tandem repeat similar to those found in many other imprinted genes and an elevated expression level, both observed only for the mouse gene, are implicated in the evolution of imprinting, to which the repeat might have contributed via enhancement of the expression. To pursue the possibility further, we examined the correlation among the repeat, expression level, and imprinting of Impact in various mammals ranging from rodents, lagomorphs, carnivores, artiodactyls to primates. Intriguingly, rabbit Impact is abundantly expressed and imprinted like those of rodents, but is missing the repeat from its first intron like those of other mammals that express both alleles weakly. It thus seems that lineage-specific enhancement of gene expression rather than the tandem repeat per se played a critical role in the evolution of imprinting of Impact. (c) 2005 Elsevier Inc. All rights reserved.. |
| 73. |
R Matsuo, H Kubota, T Obata, K Kito, K Ota, T Kitazono, S Ibayashi, T Sasaki, M Iida, T Ito, The yeast eIF4E-associated protein Eap1p attenuates GCN4 translation upon TOR-inactivation, FEBS LETTERS, 10.1016/j.febslet.2004.03.043, 579, 11, 2433-2438, 2005.04, Amino acid-starved yeast activates the eIF2a kinase Gcn2p to suppress general translation and to selectively derepress the transcription factor Gcn4p, which induces various biosynthetic genes to elicit general amino acid control (GAAC). Well-fed yeast activates the target of rapamycin (TOR) to stimulate translation via the eIF4F complex. A crosstalk was demonstrated between the pathways for GAAC and TOR signaling: the TOR-specific inhibitor rapamycin activates Gcn2p. Here we demonstrate that, upon TOR-inactivation, the putative TOR-regulated eIF4E-associated protein Eap1p likely functions downstream of Gcn2p to attenuate GCN4 translation via a mechanism independent of eIF4E-binding, thereby constituting another interface between the two pathways. 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.. |
| 74. |
K Okamura, Y Yamada, Y Sakaki, T Ito, An evolutionary scenario for genomic imprinting of Impact lying between nonimprinted neighbors, DNA RESEARCH, 10.1093/dnares/11.6.381, 11, 6, 381-390, 2004.12, Mouse Impact is the sole imprinted gene mapped to chromosome 18 to date. Despite its remarkable evolutionary conservation, human IMPACT was shown to escape genomic imprinting. Here we identified Hrh4 and Osbpl1 as the distal and proximal nearest neighbors of Impact, respectively, and found that both genes are expressed biallelically. Thus, in contrast with most imprinted genes, Impact fails to show apparent physical clustering with other imprinted genes. Since Impact not only lies in an intergenic region but also consists of 11 exons, it does not seem to be an imprinted gene generated by a retrotransposition. Hazardous effects of overexpressed Impact, a genomic segment containing paralogues of Hrh4 and Osbpl1 but not of Impact, and enhanced promoter activity in the mouse led us to propose an alternative model. This model assumes that segmental duplication followed by enhancement of the promoter activity in the lineage to mouse is responsible for the species-specific imprinting of Impact.. |
| 75. |
K Kito, T Ito, Mass spectrometry-based proteomics for quantitative description of cellular events, CURRENT GENOMICS, 10.2174/1389202043348616, 5, 8, 629-635, 2004.12, For system-level understanding of various cellular events, it is vital to identify all molecules participating in each process and understand their interactions in a quantitative manner with spatiotemporal resolution. The advent of recent proteomics approaches has enabled large scale analyses of the quantities and interactions of proteins, the most important biomolecules, in the budding yeast Saccharomyces cerevisiae. In particular, differential protein expression analysis can be achieved by mass spectrometry with stable isotope labeling either in vitro or in vivo to quantify relative difference in protein abundance. Furthermore, researchers are further developing these techniques for absolute quantification of proteins as well as their modifications and interactions, aiming to grasp more precise Pictures of the underlying molecular mechanisms for the system-level understanding. Here we review recent advance of quantitative proteomic approaches. focusing on those using mass spectrometry.. |
| 76. |
M Onda, K Ota, T Chiba, Y Sakaki, T Ito, Analysis of gene network regulating yeast multidrug resistance by artificial activation of transcription factors: involvement of Pdr3 in salt tolerance, GENE, 10.1016/j.gene.2004.02.003, 332, 1-2, 51-59, 2004.05, We established a strategy to constitutively activate Zn(2)Cys(6)-type protein by fusing its DNA-binding domain with the VP16 transactivation domain. To explore gene network regulating yeast multidrug resistance, the strategy was applied to Pdr1, Pdr3 and Yrr1, known to regulate multidrug resistance. as well as three uncharacterized Yrr1-related transcription factors. DNA microarray analysis revealed that all of the six mutants induce typical drug transporter genes including SNQ2 and YOR1, suggesting redundancy in regulation. On the other hand, each displays a unique spectrum of targets, which is coincident with the phylogenetic tree of the transcription factors and presumably reflects their functional specification. Indeed, careful analysis of target genes specific to each transcription factor led us to reveal an unexpected role for Pdr3 in salt tolerance. The strategy would thus contribute not only to identify target genes but to reveal redundancy and specificity in complex gene regulatory networks. (C) 2004 Elsevier B.V. All rights reserved.. |
| 77. |
T Ichimura, H Kubota, T Goma, N Mizushima, Y Ohsumi, M Iwago, K Kakiuchi, HU Shekhar, T Shinkawa, M Taoka, T Ito, T Isobe, Transcriptomic and proteomic analysis of a 14-3-3 gene-deficient yeast, BIOCHEMISTRY, 10.1021/bi03542li, 43, 20, 6149-6158, 2004.05, BMH1 and BMH2 encode Saccharomyces cerevisiae 14-3-3 homologues whose exact functions have remained unclear. The present work compares the transcriptomic and proteomic profiles of the wild type and a BMH1/2-deficient S. cerevisiae mutant (bmhDelta) using DNA microarrays and two-dimensional polyacrylamide gel electrophoresis. It is reported here that, although the global patterns of gene and protein expression are very similar between the two types of yeast cells, a subset of genes and proteins (a total of 220 genes) is significantly induced or reduced in the absence of Bmh1/2p. These genes include approximately 60 elements that could be linked to the reported phenotypes of the bmhDelta mutant (e.g., accumulation of glycogen and hypersensitivity to environmental stress) and/or could be the potential downstream targets of interacting partners of Bmh1/2p such as Msn2p and Rtg3p. Importantly, > 30% of the identified genes (71 genes) were found to be associated with carbon (C) and nitrogen (N) metabolism and transport, thereby suggesting that Bmh1/2p may play a major role in the regulation of C/N-responsive cellular processes. This study presents the first comprehensive overview of the genes and proteins that are affected by the depletion of Bmh1/2p and extends the scope of knowledge of the regulatory roles of Bmh1/2p in S. cerevisiae.. |
| 78. |
K Nagai, T Yamaguchi, T Takami, A Kawasumi, M Aizawa, N Masuda, M Shimizu, S Tominaga, T Ito, T Tsukamoto, T Osumi, SKIP modifies gene expression by affecting both transcription and splicing, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 10.1016/j.bbrc.2004.02.077, 316, 2, 512-517, 2004.04, SKIP has been described as a transcriptional coregulator as well as a spliceosome component, but the relationship between these functions is not clear. We found that SKIP activated reporter gene expression from the basal promoters of viral origin. SKIP exhibited more prominent effect on the promoters with stronger activities, in an experiment employing a series of reporter constructs carrying different numbers of GC boxes. We also found that SKIP suppressed aberrant splicing at a cryptic splice donor site in the luciferase reporter gene. In addition, SKIP suppressed splicing of an extra intron created by a beta-thalassemia mutation in the human beta-globin gene. In the transfection experiment, an intronless reporter exhibited a higher level of expression, but was less significantly activated by SKIP, than the intron-containing reporter. These results indicate that SKIP affects gene expression by both transcriptional activation and regulation of pre-mRNA splicing. (C) 2004 Elsevier Inc. All rights reserved.. |
| 79. |
S Sato, M Ito, T Ito, K Yoshioka, Scaffold protein JSAP1 is transported to growth cones of neurites independent of JNK signaling pathways in PC12h cells, GENE, 10.1016/j.gene.2003.12.034, 329, 1-2, 51-60, 2004.03, The c-Jun NH2-terminal kinase (JNK)/stress-activated protein kinase-associated protein 1 [JSAP1; also known as JNK-interacting protein 3 (JIP3)] has been identified as a scaffold protein for INK mitogen-activated protein kinase signal transduction pathways and as a cargo adapter in the conventional kinesin-mediated transport system. Furthermore, a functional relationship between UNC-16, the C. elegans ortholog of JSAP1, and JNK signaling has been established genetically. In this study, we first demonstrated that the kinesin light chain is required for the targeting and localization of JSAP1 to the tips of neurites in PC12h cells. Furthermore, to understand whether JNK signaling is involved in kinesin-mediated JSAP1 trafficking, we established stable PC12h cell lines that expressed wild-type JSAP1 or its mutant lacking the JNK-binding domain (JBD). Immunocytochemical studies of the cell lines indicated that the mutant JSAP1 was localized to the growth cones of differentiating PC12h cells in a similar manner to wild-type JSAP1. Taken together, these results suggest that the proper subcellular localization of JSAP1 along microtubules probably does not require JNK signaling. (C) 2004 Elsevier B.V. All rights reserved.. |
| 80. |
Y Yamada, H Watanabe, F Miura, H Soejima, M Uchiyama, T Iwasaka, T Mukai, Y Sakaki, T 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.. |
| 81. |
SY Feng, K Ota, Y Yamada, N Sawabu, T Ito, A yeast one-hybrid system to detect methylation-dependent DNA-protein interactions, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 10.1016/j.bbrc.2003.12.027, 313, 4, 922-925, 2004.01, We developed a method for site-selective CpG methylation of the budding yeast genome. The method recruits LexA-fused M.SssI DNA methyltransferase to LexA operator sequences integrated adjacent to the target site. Microarray analysis of methylated DNAs indicated that the tethered enzyme selectively methylates the region around the target site. Exploiting this method to methylate bait DNA in the one-hybrid system, we demonstrated methylation-dependent DNA binding of methyl-CpG binding proteins, MBD1 and Kaiso, in vivo. This methylation-dependent one-hybrid system would provide a versatile tool for the search and analysis of proteins that recognize methylated DNA to participate in epigenetic regulation. (C) 2003 Elsevier Inc. All rights reserved.. |
| 82. |
A Shakoori, G Fujii, S Yoshimura, M Kitamura, K Nakayama, T Ito, H Ohno, N Nakamura, Identification of a five-pass transmembrane protein family localizing in the Golgi apparatus and the ER, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 10.1016/j.bbrc.2003.10.197, 312, 3, 850-857, 2003.12, A family of five-pass transmembrane proteins (FinGERs) were identified from the protein sequence database. The family includes yeast Yip1p, Yip4p Yip5p, and Yif1p, and also their plant, insects, nematode, and mammalian homologues, suggesting their conserved function in a broad range of species. Eight family members were found in human. Multiple sequence alignment revealed three regions conserved among all family members. All of the human family members were expressed widely in various tissues. The human proteins were localized in and around the Golgi apparatus and may also be in the ER to some extent. The Golgi apparatus was fragmented by overexpression of the five of the family members. Some of the members were found to interact by yeast two-hybrid analysis, suggesting the formation of a complex. These results suggest that FinGERs function in maintenance of the Golgi structure and/or transport between the ER and the Golgi apparatus. (C) 2003 Elsevier Inc. All rights reserved.. |
| 83. |
Y Noda, M Kohjima, T Izaki, K Ota, S Yoshinaga, F Inagaki, T Ito, H Sumimoto, Molecular recognition in dimerization between PB1 domains, JOURNAL OF BIOLOGICAL CHEMISTRY, 10.1074/jbc.M306330200, 278, 44, 43516-43524, 2003.10, The PB1 ( Phox and Bem 1) domain is a recently identified module that mediates formation of a heterodimeric complex with other PB1 domain, e. g. the complexes between the phagocyte oxidase activators p67(phox) and p40(phox) and between the yeast polarity proteins Bem1p and Cdc24p. These PB1 domains harbor either a conserved lysine residue on one side or an acidic OPCA (OPR/PC/AID) motif around the other side; the lysine of p67phox or Bem1p likely binds to the OPCA of p40phox or Cdc24p, respectively, via electrostatic interactions. To further understand molecular recognition by PB1 domains, here we investigate the interactions mediated by proteins presenting both the lysine and OPCA on a single PB1 domain, namely Par6, atypical protein kinase C ( aPKC), and ZIP. Par6 and aPKC form a complex via the interaction of the Par6 lysine with aPKC-OPCA but not via that between the aPKC lysine and Par6-OPCA, thereby localizing to the tight junction of epithelial cells. aPKC also uses its OPCA to interact with ZIP, another protein that has a PB1 domain presenting both the lysine and OPCA, whereas aPKC binds via the conserved lysine to MEK5 in the same manner as ZIP interacts with MEK5. In addition, ZIP can form a homotypic complex via the conserved electrostatic interactions. Thus the PB1 domain appears to be a protein module that fully exploits its two mutually interacting elements in molecular recognition to expand its repertoire of protein-protein interactions.. |
| 84. |
S Yoshinaga, M Kohjima, K Ogura, M Yokochi, R Takeya, T Ito, H Sumimoto, F Inagaki, The PB1 domain and the PC motif-containing region are structurally similar protein binding modules, EMBO JOURNAL, 10.1093/emboj/cdg475, 22, 19, 4888-4897, 2003.10, The PC motif is evolutionarily conserved together with the PB1 domain, a binding partner of the PC motif-containing protein. For interaction with the PB1 domain, the PC motif-containing region (PCCR) comprising the PC motif and its flanking regions is required. Because the PB1 domain and the PCCR are novel binding modules found in a variety of signaling proteins, their structural and functional characterization is crucial. Bem1p and Cdc24p interact through the PB1-PCCR interaction and regulate cell polarization in budding yeast. Here, we determined a tertiary structure of the PCCR of Cdc24p by NMR. The tertiary structure of the PCCR is similar to that of the PB1 domain of Bem1p, which is classified into a ubiquitin fold. The PC motif portion takes a compact betabetaalpha-fold, presented on the ubiquitin scaffold. Mutational studies indicate that the PB1-PCCR interaction is mainly electrostatic. Based on the structural information, we group the PB1 domains and the PCCRs into a novel family, named the PB1 family. Thus, the PB1 family proteins form a specific dimer with each other.. |
| 85. |
Kubota H, Obata T, Ota K, Sasaki T, Ito T, Rapamycin-induced translational derepression of GCN4 mRNA involves a novel mechanism for activation of the eIF2α kinase GCN2, Journal of Biological Chemistry, 10.1074/jbc.C300133200, 278, 23, 20457-20460, 2003.06. |
| 86. |
T Ago, F Kuribayashi, H Hiroaki, R Takeya, T Ito, D Kohda, H Sumimoto, Phosphorylation of p47(phox) directs phox homology domain from SH3 domain toward phosphoinositides, leading to phagocyte NADPH oxidase activation, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 10.1073/pnas.0735712100, 100, 8, 4474-4479, 2003.04, Protein-phosphoinositide interaction participates in targeting proteins to membranes where they function correctly and is often modulated by phosphorylation of lipids. Here we show that protein phosphorylation of p47(phox), a cytoplasmic activator of the microbicidal phagocyte. oxidase (phox), elicits interaction of p47(phox) with phosphoinositides. Although the isolated phox homology (PX) domain of p47(phox) can interact directly with phosphoinositides, the lipid-binding activity of this protein is normally suppressed by intramolecular interaction of the PX domain with the C-terminal Src homology 3 (SH3) domain, and hence the wild-type full-length p47(phox) is incapable of binding to the lipids. The W263R substitution in this SH3 domain, abrogating the interaction with the PX domain, leads to a binding of p47(phox) to phosphoinositides. The findings indicate that disruption of the intramolecular interaction renders the PX domain accessible to the lipids. This conformational change is likely induced by phosphorylation of p47(phox), because protein kinase C treatment of the wild-type p47(phox) but not of a mutant protein with the S303/304/328A substitution culminates in an interaction with phosphoinositides. Furthermore, although the wild-type p47(phox) translocates upon cell stimulation to membranes to activate the oxidase, neither the kinase-insensitive p47(phox) nor lipid-binding-defective proteins, one lacking the PX domain and the other carrying the R90K substitution in this domain, migrates. Thus the protein phosphorylation-driven conformational change of p47(phox) enables its PX domain to bind to phosphoinositides, the interaction of which plays a crucial role in recruitment of p47(phox) from the cytoplasm to membranes and subsequent activation of the phagocyte oxidase.. |
| 87. |
LJ Yue, Y Saikawa, K Ota, M Tanaka, R Nishimura, T Uehara, H Maeba, T Ito, T Sasaki, S Koizumi, A functional single-nucleotide polymorphism in the human cytidine deaminase gene contributing to ara-C sensitivity, PHARMACOGENETICS, 10.1097/00008571-200301000-00005, 13, 1, 29-38, 2003.01, To test the hypothesis that analyses of drug targets for polymorphism will help to establish gene-based information for the treatment of cancer patients, we investigated the functional single-nucleotide polymorphisms in the human cytidine deaminase (HCDA) gene. The cDNAs from 52 leukaemia/lymphoma samples and 169 control blood samples were direct-sequenced and analysed for the polymorphisms. Three different polymorphisms (A79C, G208A and T435C) were identified in the coding region of the HCDA gene and displayed allelic frequencies of 20.1%, 4.3% and 70.1%, respectively. No association with susceptibility to disease was observed. A novel polymorphism, G208A produced an alanine to threonine substitution (A70T) within the conserved catalytic domain. By introduction of the polymorphic HCDA genes into the yeast CDA-null mutants, the HCDA-70T showed 40% and 32% activity of prototype for cytidine and ara-C substrates, respectively (P |
| 88. |
F Kuribayashi, H Nunoi, K Wakamatsu, S Tsunawaki, K Sato, T Ito, H Sumimoto, The adaptor protein p40(phox) as a positive regulator of the superoxide-producing phagocyte oxidase, EMBO JOURNAL, 10.1093/emboj/cdf642, 21, 23, 6312-6320, 2002.12, Activation of the superoxide-producing phagocyte NADPH oxidase, crucial in host defense, requires the cytosolic proteins p67(phox) and p47(phox). They translocate to the membrane upon cell stimulation and activate flavocytochrome b(558), the membrane-integrated catalytic core of this enzyme system. The activators p67(phox) and p47(phox) form a ternary complex together with p40(phox), an adaptor protein with unknown function, comprising the PX/PB2, SH3 and PC motif-containing domains: p40(phox) associates with p67(phox) via binding of the p40(phox) PC motif to the p67(phox) PB1 domain, while p47(phox) directly interacts with p67(phox) but not with p40(phox). Here we show that p40(phox) enhances membrane translocation of p67(phox) and p47(phox) in stimulated cells, which leads to facilitated production of superoxide. The enhancement cannot be elicited by a mutant p40(phox) carrying the D289A substitution in PC or a p67(phox) with the K355A substitution in PB1, each being defective in binding to its respective partner. Thus p40(phox) participates in activation of the phagocyte oxidase by regulating membrane recruitment of p67(phox) and p47(phox) via the PB1-PC interaction with p67(phox).. |
| 89. |
T Oyama, K Kitano, K Satou, T Ito, Extraction of knowledge on protein-protein interaction by association rule discovery (vol 18, pg 705, 2002), BIOINFORMATICS, 18, 8, 1151-1151, 2002.08. |
| 90. |
T Ito, K Ota, H Kubota, Y Yamaguchi, T Chiba, K Sakuraba, M Yoshida, Roles for the two-hybrid system in exploration of the yeast protein interactome, MOLECULAR & CELLULAR PROTEOMICS, 10.1074/mcp.R200005-MCP200, 1, 8, 561-566, 2002.08, Comprehensive analysis of protein-protein interactions is a challenging endeavor of functional proteomics and has been best explored in the budding yeast. The yeast protein interactome analysis was achieved first by using the yeast two-hybrid system in a proteome-wide scale and next by large-scale mass spectrometric analysis of affinity-purified protein complexes. While these interaction data have led to a number of novel findings and the emergence of a single huge network containing thousands of proteins, they suffer many false signals and fall short of grasping the entire interactome. Thus, continuous efforts are necessary in both bioinformatics and experimentation to fully exploit these data and to proceed another step forward to the goal. Computational tools to integrate existing biological knowledge buried in literature and various functional genomic data with the interactome data are required for biological interpretation of the huge protein interaction network. Novel experimental methods have to be developed to detect weak, transient interactions involving low abundance proteins as well as to obtain clues to the biological role for each interaction. Since the yeast two-hybrid system can be used for the mapping of the interaction domains and the isolation of interaction-defective mutants, it would serve as a technical basis for the latter purpose, thereby playing another important role in the next phase of protein interactome research.. |
| 91. |
T Oyama, K Kitano, K Satou, T Ito, Extraction of knowledge on protein-protein interaction by association rule discovery, BIOINFORMATICS, 10.1093/bioinformatics/18.5.705, 18, 5, 705-714, 2002.05, Motivation: Protein--protein interactions are systematically examined using the yeast two-hybrid method. Consequently, a lot of protein--protein interaction data are currently being accumulated. Nevertheless, general information or knowledge on protein--protein interactions is poorly extracted from these data. Thus we have been trying to extract the knowledge from the protein--protein interaction data using data mining.
Results: A data mining method is proposed to discover association rules related to protein--protein interactions. To evaluate the detected rules by the method, a new scoring measure of the rules is introduced. The method allowed us to detect popular interaction rules such as 'An SH3 domain binds to a proline-rich region.' These results indicate that the method may detect novel knowledge on protein--protein interactions.. |
| 92. |
CP Ponting, T Ito, J Moscat, MT Diaz-Meco, F Inagaki, H Sumimoto, OPR, PC and AM: all in the PB1 family, TRENDS IN BIOCHEMICAL SCIENCES, 10.1016/S0968-0004(01)02006-0, 27, 1, 10-10, 2002.01. |
| 93. |
T Ito, T Chiba, M Yoshida, Exploring the protein interactome using comprehensive two-hybrid projects, TRENDS IN BIOTECHNOLOGY, 10.1016/S0167-7799(01)01790-5, 19, 10, S23-S27, 2001.10, Large-scale two-hybrid projects were used in an approach to examine protein-protein interactions. Despite the various limitations of this approach, these projects revealed a wealth of novel interactions, and the protein interactome may be much larger than expected.. |
| 94. |
F Miura, T Yada, K Nakai, Y Sakaki, T 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. (C) 2001 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.. |
| 95. |
T Ago, R Takeya, H Hiroaki, F Kuribayashi, T Ito, D Kohda, H Sumimoto, The PX domain as a novel phosphoinositide-binding module, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 10.1006/bbrc.2001.5629, 287, 3, 733-738, 2001.09, The phox (phagocyte oxidase) homology (PX) domain occurs in the mammalian phox proteins p40(phox) and p47(phox). the polarity establishment protein Bem1p in budding yeast, and a variety of proteins involved in membrane trafficking. Here we show that the PX domains of p40(phox) and p47(phox) directly bind to phosphoinositides: P40(phox) prefers Ptdlns(3)P, while p47(phox) does Ptdlns(4)P and Ptdlns(3,4)P-2. In addition, the Bem1p PX domain also interacts with Ptdlns(4)P. When the P40(phox) PX domain is expressed as a fusion to green fluorescent protein in HeLa cells, it exists at early endosomes where Ptdlns(3)P is enriched. Furthermore, a mutant P40(phox) PX carrying the substitution of Lys for Arg105 only weakly binds to phosphoinositides in vitro, and fails to locate to early endosomes. Thus the PX domain functions as a novel phosphoinositide-binding module and likely participates in targeting of proteins to membranes. (C) 2001 Academic Press.. |
| 96. |
Y Nakagawa-Yagi, DK Choi, N Ogane, S Shimada, M Seya, T Momoi, T Ito, Y Sakaki, Discovery of a novel compound: insight into mechanisms for acrylamide-induced axonopathy and colchicine-induced apoptotic neuronal cell death, BRAIN RESEARCH, 10.1016/S0006-8993(01)02608-7, 909, 1-2, 8-19, 2001.08, The exposure of humans and experimental animals to certain industrial toxins such as acrylamide is known to cause nerve damage classified as axonopathy, but the mechanisms involved are poorly understood. Here we show that acrylamide induces morphological changes and tyrosine phosphorylation of focal adhesion kinase (FAK) and proline-rich tyrosine kinase 2 (Pyk2), a member of the FAK subfamily, in human differentiating neuroblastoma SH-SY5Y cells. Furthermore, we identified a novel molecule designated 'compound-1' that inhibits the morphological and biochemical events. Daily oral administrations of the compound also effectively alleviated behavioral deficits in animals elicited by acrylamide in inclined plane testing, landing foot spread testing and rota-rod performance testing. The, compound also effectively inhibited the biological and biochemical responses caused by another axonopathy inducer, colchicine, including tyrosine phosphorylation of Pyk2, formation of an 85-kDa poly(ADP-ribose)polymerase (PARP) fragment and apoptosis-associated induction of the NAPOR gene as well as neuronal cell death. Our findings not only provide insight into FAX and Pyk2 functions in neuronal cells, but may also be important in the development of therapeutic agents for peripheral neuropathy and neurodegeneration (C) 2001 Elsevier Science B.V. All rights reserved.. |
| 97. |
C Uematsu, J Nishida, K Okano, F Miura, T 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, 84, 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.. |
| 98. |
T Ito, Y Matsui, T Ago, K Ota, H Sumimoto, Novel modular domain PB1 recognizes PC motif to mediate functional protein-protein interactions, EMBO JOURNAL, 10.1093/emboj/20.15.3938, 20, 15, 3938-3946, 2001.08, Modular domains mediating specific protein-protein interactions play central roles in the formation of complex regulatory networks to execute various cellular activities. Here we identify a novel domain PB1 in the budding yeast protein Bem1p, which functions in polarity establishment, and mammalian p67(phox) which activates the microbicidal phagocyte NADPH oxidase. Each of these specifically recognizes an evolutionarily conserved PC motif to interact directly with Cdc24p (an essential protein for cell polarization) and p40(phox) (a component of the signaling complex for the oxidase), respectively. Swapping the PB1 domain of Bem1p with that of p67(phox), which abolishes its interaction with Cdc24p, confers on cells temperature-sensitive growth and a bilateral mating defect. These phenotypes are suppressed by a mutant Cdc24p harboring the PC motif-containing region of p40(phox), which restores the interaction with the altered Bem1p. This domain-swapping experiment demonstrates that Bem1p function requires interaction with Cdc24p, in which the PB1 domain and the PC motif participate as responsible modules.. |
| 99. |
H Terasawa, Y Noda, T Ito, H Hatanaka, S Ichikawa, K Ogura, H Sumimoto, F Inagaki, Structure and ligand recognition of the PB1 domain: a novel protein module binding to the PC motif, EMBO JOURNAL, 10.1093/emboj/20.15.3947, 20, 15, 3947-3956, 2001.08, PB1 domains are novel protein modules capable of binding to target proteins that contain PC motifs. We report here the NMR structure and ligand-binding site of the PB1 domain of the cell polarity establishment protein, Bem1p. In addition, we identify the topology of the PC motif-containing region of Cdc24p by NMR, another cell polarity establishment protein that interacts with Bem1p. The PC motif-containing region is a structural domain offering a scaffold to the PC motif. The chemical shift perturbation experiment and the mutagenesis study show that the PC motif is a major structural element that binds to the PB1 domain. A structural database search reveals close similarity between the Bem1p PBI domain and the c-Raf1 Ras-binding domain. However, these domains are functionally distinct from each other.. |
| 100. |
H Hiroaki, T Ago, T Ito, H Sumimoto, D Kohda, Solution structure of the PX domain, a target of the SH3 domain, NATURE STRUCTURAL BIOLOGY, 10.1038/88591, 8, 6, 526-530, 2001.06, The phox homology (PX) domain is a novel protein module containing a conserved proline-rich motif, We have shown that the PX domain isolated from the human p47(phox) protein, a soluble subunit of phagocyte NADPH oxidase, binds specifically to the C-terminal SH3 domain derived from the same protein. The solution structure of p47 PX has an alpha + beta structure with a novel folding moth topology and reveals that the proline-rich motif is presented on the molecular surface for easy recognition by the SH3 domain. The proline-rich moth of p47 PX in the free state adopts a distorted left-handed polyproline type II helix conformation.. |
| 101. |
Kubota H, Ota K, Sakaki Y, Ito T, Budding Yeast GCN1 Binds the GI Domain to Activate the eIF2α Kinase GCN2, Journal of Biological Chemistry, 10.1074/jbc.M011793200, 276, 20, 17591-17596, 2001.05. |
| 102. |
T Ito, T Chiba, R Ozawa, M Yoshida, M Hattori, Y Sakaki, A comprehensive two-hybrid analysis to explore the yeast protein interactome, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 10.1073/pnas.061034498, 98, 8, 4569-4574, 2001.04, Protein-protein interactions play crucial roles in the execution of various biological functions. Accordingly, their comprehensive description would contribute considerably to the functional interpretation of fully sequenced genomes, which are flooded with novel genes of unpredictable functions. We previously developed a system to examine two-hybrid interactions in all possible combinations between the approximate to6,000 proteins of the budding yeast Saccharomyces cerevisiae. Here we have completed the comprehensive analysis using this system to identify 4,549 two-hybrid interactions among 3,278 proteins. Unexpectedly, these data do not largely overlap with those obtained by the other project [Uetz, P,, et al. (2000) Nature (London) 403, 623-627] and hence have substantially expanded our knowledge on the protein interaction space or interactome of the yeast. Cumulative connection of these binary interactions generates a single huge network linking the vast majority of the proteins. Bioinformatics-aided selection of biologically relevant interactions highlights various intriguing subnetworks. They include, for instance, the one that had successfully foreseen the involvement of a novel protein in spindle pole body function as well as the one that may uncover a hitherto unidentified multiprotein complex potentially participating in the process of vesicular transport. Our data would thus significantly expand and improve the protein interaction map for the exploration of genome functions that eventually leads to thorough understanding of the cell as a molecular system.. |
| 103. |
Y Noda, R Takeya, S Ohno, S Naito, T Ito, H Sumimoto, Human homologues of the Caenorhabditis elegans cell polarity protein PAR6 as an adaptor that links the small GTPases Rac and Cdc42 to atypical protein kinase C, GENES TO CELLS, 10.1046/j.1365-2443.2001.00404.x, 6, 2, 107-119, 2001.02, Background: Asymmetric cell division in the Caenorhabditis elegans embryos requires products of par (partitioning defective) genes 1-6 and atypical protein kinase C (aPKC), whereas Cdc42 and Rac, members of the Rho family GTPases, play an essential role in cell polarity establishment in yeast and mammalian cells. However, little is known about a link between PAR proteins and the GTPases in cell polarization.
Results: Here we have cloned cDNAs for three human homologues of PAR6, designated PAR6 alpha, beta and gamma, comprising 345, 372 and 376 amino acids, respectively. The PAR6 proteins harbour a PDZ domain and a CRIB-like motif, and directly interact with GTP-bound Rac and Cdc42 via this motif and with the aPKC isoforms PKC iota/lambda and PKC zeta via the N-terminal head-to-head association. These interactions are not mutually exclusive, thereby allowing the PAR6 proteins to form a ternary complex with the GTPases and aPKC, both in vitro and in vivo. When PAR6 and aPKC are expressed with a constitutively active form of Rac in HeLa or COS-7 cells, these proteins co-localize to membrane ruffles, which are known to occur at the leading edge of polarized cells during cell. movement.
Conclusion: Human PAR6 homologues most likely play an important role in the cell polarization of mammalian cells, by functioning as an adaptor protein that links activated Rac and Cdc42 to aPKC signalling, aPKC isoforms PKC iota/lambda and PKC zeta via the N-terminal head-to-head association. These interactions are not mutually exclusive, thereby allowing the PAR6 proteins to form a ternary complex with the GTPases and aPKC, both in vitro and in vivo. When PAR6 and aPKC are expressed with a constitutively active form of Rac in HeLa or COS-7 cells, these proteins co-localize to membrane ruffles, which are known to occur at the leading edge of polarized cells during cell. movement. Conclusion: Human PAR6 homologues most likely play an important role in the cell polarization of mammalian cells, by functioning as an adaptor protein that links activated Rac and Cdc42 to aPKC signalling.. |
| 104. |
K Okamura, Y Hagiwara-Takeuchi, T Li, TH Vu, M Hirai, M Hattori, Y Sakaki, AR Hoffman, T Ito, Comparative genome analysis of the mouse imprinted gene IMPACT and its nonimprinted human homolog IMPACT: Toward the structural basis for species-specific imprinting, GENOME RESEARCH, 10.1101/gr.139200, 10, 12, 1878-1889, 2000.12, Mouse Impact is a paternally expressed gene encoding an evolutionarily conserved protein of unknown function. Here we identified IMPACT, the human homolog of Impact, on chromosome 18q11.2-12.1, a region syntenic to the mouse Impact locus. IMPACT was expressed biallelically in brain and in various tissues from two informative Fetuses and in peripheral blood from an informative adult. To reveal the structural basis for the difference in allelic expression between the two species, we elucidated complete genome sequences For both mouse Impact (-38 kb) and human IMPACT (-30 kb). Sequence comparison revealed that the two genes share a well-conserved exon-intron organization but bear significantly different CpG islands. The mouse island lies in the first intron and contains characteristic tandem repeats. Furthermore, this island serves as a differentially methylated region (DMR) consisting of a hypermethylated maternal allele and an unmethylated paternal allele. Intriguingly, this intronic island is missing from the nonimprinted human IMPACT; whose sole CpG island spans the First exon, lacks any apparent repeats, and escapes methylation on both chromosomes. These results suggest that the intronic DMR prays a role in the imprinting of Impact.. |
| 105. |
K Mizushima, Y Miyamoto, F Tsukahara, M Hirai, Y Sakaki, T Ito, A novel G-protein-coupled receptor gene expressed in striatum, GENOMICS, 10.1006/geno.2000.6340, 69, 3, 314-321, 2000.11, Differential display screening for region-specific transcripts in rat brain revealed a novel striatum-specific transcript encoding an orphan G-protein-coupled receptor (GPCR) designated Strg/Gpr88 for striatum-specific GPCR, We isolated its homologues from human (HGMW-approved symbol GPR88) and mouse and mapped them to chromosomes 1p21.3 and 3G1, respectively. These loci are syntenic to each other, thereby suggesting their orthology, The predicted primary sequences of Strg/Gpr88 proteins are highly conserved between human and rodents and show the highest level of homology to receptors for biogenic amines, However, Strg/Gpr88 lacks some residues conserved in all known biogenic amine receptors and hence may represent a novel subtype of GPCR. Northern blot and in situ hybridization analyses revealed that Strg/Gpr88 transcripts are expressed almost exclusively in striatum in both human and rodents. Remarkable conservation in primary structure and a unique expression pattern may indicate a role for Strg/Gpr88 in the fundamental functions of striatum such as the control of motor behavior, (C) 2000 Academic Press.. |
| 106. |
Kubota H, Sakaki Y, Ito T, GI domain-mediated association of the eukaryotic initiation factor 2α kinase GCN2 with its activator GCN1 is required for general amino acid control in budding yeast, Journal of Biological Chemistry, 10.1074/jbc.C000262200, 275, 27, 20243-20246, 2000.07. |
| 107. |
Y Iijima, T Ito, T Oikawa, M Eguchi, M Eguchi-Ishimae, N Kamada, K Kishi, S Asano, Y Sakaki, Y Sato, A new ETV6/TEL partner gene, ARG (ABL-related gene or ABL2), identified in an AML-M3 cell line with a t(1;12)(q25;p13) translocation, BLOOD, 95, 6, 2126-2131, 2000.03, The ETV6/TEL gene has been reported to fuse to PDGFR beta b MDS1/EVI1, BTL, ACS2, STL, JAK2, ASL, CDX2, TRKC, AML1, and MN1. Among them, PDGFR beta, ASL, JAK2, and TRKC are tyrosine kinases (TK), We identified a novel ETV6 partner gene, ARG (ABL-related gene or ASL2), another TK gene in a cell line established from a patient with acute myelogenous leukemia (AML-MB) with a t(15;17)(q22;q11.2) and a t(1;12)(q25;p13), which has the remarkable feature to differentiate to mature eosinophils in culture with all-trans retinoic acid and cytokines, The ETV6/ARG transcripts consisted of exon 1 to 5 of ETV6 and the 3' portion of ARG starting from exon IB or exon 2, resulting in an open reading frame for a fusion protein consisting of the entire PNT oligomerization domain of ETV6 and all of the functional domains of ARG including the TK domain. This is the same protein structure as identified In the other ETV6 TK fusion proteins. The reciprocal ARG/ETV6 transcript was not expressed, and the normal ETV6 allele was not deleted or rearranged. Although the ABL Is known to be involved in various human malignancies, ARG has not been involved in human malignancies despite its high homology to ASL, Thus, this Is the first report showing involvement of ARG in human leukemia, The ETV6/ARG protein may be involved in the unique differentiation capacity of this cell line. (C) 2000 by The American Society of Hematology.. |
| 108. |
T Ito, K Tashiro, S Muta, R Ozawa, T Chiba, M Nishizawa, K Yamamoto, S Kuhara, Y Sakaki, Toward a protein-protein interaction map of the budding yeast: A comprehensive system to examine two-hybrid interactions in all possible combinations between the yeast proteins, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 10.1073/pnas.97.3.1143, 97, 3, 1143-1147, 2000.02, Protein-protein interactions play pivotal roles in various aspects of the structural and functional organization of the cell, and their complete description is indispensable to thorough understanding of the cell. As an approach toward this goal, here we report a comprehensive system to examine two-hybrid interactions in all of the possible combinations between proteins of Saccharomyces cerevisiae, We cloned all of the yeast ORFs individually as a DNA-binding domain fusion ("bait") in a MATa strain and as an activation domain fusion ("prey") in a MAT alpha strain, and subsequently divided them into pools, each containing 96 clones. These bait and prey clone pools were systematically mated with each other, and the transformants were subjected to strict selection for the activation of three reporter genes followed by sequence tagging. Our initial examination of approximate to 4 x 10(6) different combinations, constituting approximate to 10% of the total to be tested, has revealed 183 independent two-hybrid interactions, more than half of which are entirely novel. Notably, the obtained binary data allow us to extract more complex interaction networks, including the one that may explain a currently unsolved mechanism for the connection between distinct steps of vesicular transport. The approach described here thus will provide many leads for integration of various cellular functions and serve as a major driving force in the completion of the protein-protein interaction map.. |
| 109. |
T Ago, H Nunoi, T Ito, H Sumimoto, Mechanism for phosphorylation-induced activation of the phagocyte NADPH oxidase protein p47(phox) - Triple replacement of serines 303, 304, and 328 with aspartates disrupts the SH3 domain-mediated intramolecular interaction in p47phox, thereby activating the oxidase, JOURNAL OF BIOLOGICAL CHEMISTRY, 10.1074/jbc.274.47.33644, 274, 47, 33644-33653, 1999.11, Activation of the superoxide-producing phagocyte NADPH oxidase requires interaction between p47(phox) and p22(phox), which is mediated via the SH3 domains of the former protein. This interaction is considered to be induced by exposure of the domains that are normally masked by an intramolecular interaction with the C-terminal region of p47(phox). Here we locate the intramolecular SH3-binding site at the region of amino acid residues 286-340, where Ser-303, Ser-304, and Ser-328 that are among several serines known to become phosphorylated upon cell stimulation exist. Simultaneous replacement of the three serines in p47(phox) with aspartates or glutamates, each mimicking phosphorylated residues, is sufficient for disruption of the intramolecular interaction and resultant access to p22(phox). Th, triply mutated proteins are also capable of activating the NADPH oxidase without in vitro activators such as arachidonate under cell-free conditions. In a whole-cell system where expression of the wild-type p47(phox) reconstitutes the stimulus-dependent oxidase activity, substitution of the kinase-insensitive residue alanine for Ser-328 as well as for Ser-303/Ser-304 leads to a defective production of superoxide. These findings suggest that phosphorylation of the three serines in p47(phox) induces a conformational change to a state accessible to p22(phox), thereby activating the NADPH oxidase.. |
| 110. |
DK Choi, T Ito, F Tsukahara, M Hirai, Y Sakaki, Developmentally-regulated expression of mNapor encoding an apoptosis-induced ELAV-type RNA binding protein, GENE, 10.1016/S0378-1119(99)00312-1, 237, 1, 135-142, 1999.09, Proteins with RNA recognition motifs (RRMs) participate in many aspects of RNA metabolism, and some of them are required for the accomplishment of normal development. The neuroblastoma apoptosis-related RNA binding protein (NAPOR) is an ELAV-type RNA-binding protein with three characteristic RNP2/RNP1-type RRMs, which we identified as a gene induced during apoptosis of neuroblastoma cells. Here we isolated and characterized the cDNA for mNapor, the mouse homolog of NAPOR. The mNapor encodes mRNA sharing striking homology with that of NAPOR, not only in its open reading frame (98.5%) but also in the 3'-untranslated region (80.1%), and is mapped to chromosome 2 A2-A3, a region syntenic to the human NAPOR locus. In situ hybridization analysis revealed that the expression pattern of mNapor is spatially and temporally coincident with the occurrence of programmed cell death, suggesting its involvement in the development of the central nervous system in which apoptosis plays a crucial role. (C) 1999 Elsevier Science B.V. All rights reserved.. |
| 111. |
Y Yamada, Y Hagiwara, K Shiokawa, Y Sakaki, T Ito, Spatiotemporal, allelic, and enforced expression of Ximpact, the Xenopus homolog of mouse imprinted gene Impact, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 10.1006/bbrc.1999.0297, 256, 1, 162-169, 1999.03, Mouse Impact is an imprinted gene encoding an evolutionarily conserved protein of unknown function. We isolated cDNA for the Xenopus homolog of Impact (Ximpact), since the clawed frog not only provides an excellent model for the study of gene function in early development but also allows the generation of interspecific F1 hybrids required for the examination of allelic expression status. The predicted product of Ximpact shows an extreme sequence similarity to those of mouse Impact and its homologs in nematoda, fission yeast, and budding yeast. The transcript of Ximpact is present in oocytes as well as in early embryos, and its spatial distribution is ubiquitous in both embryonic and adult stages. An RT-PCR-RFLP assay using the reciprocal interspecific F1 hybrids and a DNA polymorphism between X. laevis and X. borealis showed that Ximpact is expressed bialletically when analyzed as a whole embryo. Overexpression of Ximpact by RNA microinjection resulted in a higher than normal rate of gastrulation defects, suggesting the need for tight control of its dosage in early development, (C) 1999 Academic Press.. |
| 112. |
DK Choi, T Ito, Y Mitsui, Y Sakaki, Fluorescent differential display analysis of gene expression in apoptotic neuroblastoma cells, GENE, 10.1016/S0378-1119(98)00364-3, 223, 1-2, 21-31, 1998.11, Identification of differentially expressed genes will provide leads in the elucidation of the molecular mechanisms underlying neuronal cell death associated with neurodegenerative disorders. Using a high-throughput fluorescent differential display (FDD) system based on an automated DNA sequencer, we analyzed global patterns of gene expression during the apoptosis of neuroblastoma SH-SY5Y cells induced by a neurotoxin, colchicine. Initial screening of similar to 24 000 cDNA bands displayed with 320 primer combinations has revealed 263 fragments showing differential expression patterns, suggesting that similar to 1% of transcripts are modulated in their expression level. Of these differentially displayed bands, we cloned 18 fragments composed of 17 distinct species and confirmed differential expression of each species by reverse transcription-PCR or Northern blot hybridization, thereby proving the reliability of the approach. These include eight derived from seven known genes, five homologous to expressed sequence tags (ESTs), and five totally lacking any homology to those deposited in the database. Among these, a novel transcript SAII induced prominently was characterized further and revealed to encode a putative RNA-binding protein NAPOR (neuroblastoma apoptosis-related RNA-binding protein), containing three copies of evolutionarily conserved RNA recognition motif. Since several RNA binding proteins have been known to play crucial roles in other apoptosis systems, it is conceivable that NAPOR is also involved in the process of neuronal cell death. (C) 1998 Elsevier Science B.V. All rights reserved.. |
| 113. |
M Oka, C Furihata, K Kitoh, M Yamamoto, M Tatematsu, M Ichinose, K Miki, T Ito, Y Sakaki, K Reske, Involvement of dendritic cell response to resistance of stomach carcinogenesis caused by N-methyl-N '-nitro-N-nitrosoguanidine in rats, CANCER RESEARCH, 58, 18, 4107-4112, 1998.09, The involvement of immune response in the resistance of chemically induced stomach cancer was studied in a resistant rat strain (Buffalo) and a sensitive rat strain (ACI). Groups of 10 male Buffalo and ACI rats, 6 weeks of age, were given drinking water with or without N-methyl-N-nitro-N-nitrosoguanidine (MNNG; 100 mg/l) for 14 days. Total RNA was isolated from the stomach pyloric mucosa from five rats, and cDNA was prepared with reverse transcriptase. Tissue sections of the stomach pyloric mucosa from five rats were stained with antibodies recognizing molecules expressed by various immune cells. Reverse transcription-PCR (RT-PCR), competitive RT-PCR, and Northern blot demonstrated that the expression of MHC class II group genes [MHC class II, MHC class II-associated invariant chain (Ii), CD4 and IgM (B cell marker)], MHC class I group genes (MHC class I and CD8), B7-1 (costimulator on dendritic cells), and CD28 (receptor to B7 on T cells) in the pyloric mucosa was elevated by MNNG in both rat strains but was elevated to a 4-7-fold greater extent in Buffalo rats than in ACI rats. These genes were scarcely expressed in control rats. Histochemical antibody staining after MNNG exposure showed a greater number of cells stained with monoclonal antibody to Ii, OX-62 (dendritic cell marker), and ED-1 (dendritic cell and macrophage common marker) in the interstitial tissue of the pyloric mucosa of Buffalo rats compared with ACI rats. Cell proliferation, as measured by 5-bromo-2-deoxyuridine (BrdUrd)-labeling indices, revealed the presence of BrdUrd-labeled cells only among epithelial cells in the proliferative zone; cells in the interstitial tissue were not labeled with BrdUrd. The results suggest the involvement of dendritic cell response in the resistance to the MNNG induction of stomach carcinogenesis in rats.. |
| 114. |
K Hata, T Ito, K Takeshige, H Sumimoto, Anionic amphiphile-independent activation of the phagocyte NADPH oxidase in a cell-free system by p47(phox) and p67(phox), both in C terminally truncated forms - Implication for regulatory Src homology 3 domain-mediated interactions, JOURNAL OF BIOLOGICAL CHEMISTRY, 10.1074/jbc.273.7.4232, 273, 7, 4232-4236, 1998.02, Anionic amphiphiles, such as arachidonate, activate the superoxide-producing phagocyte NADPH oxidase in a cell-free system with human neutrophil membrane, which contains cytochrome b(558) comprising gp91(phox) and p22(phox), and three cytosolic proteins: p47(phox) and p67(phox), each harboring two SH3 domains, and the small GTPase Rac. Here we show that, even without the amphiphiles, the oxidase is activated in vitro by a C terminally truncated p47(phox)., retaining the N-terminal and the two SH3 domains, and the N terminus of p67(phox). When either truncated p47(phox) or, p67(phox) is replaced by the respective full-length one, the activation absolutely requires the amphiphiles. The results indicate that both p47(phox) and p67(phox) are the primary targets of the amphiphiles, and that their C-terminal regions play negative regulatory roles. We also find that the truncated p47(phox), but not the full-length one, can bind to p22(phox), a binding required for the oxidase activation. The N-terminal SH3 domain of p47(phox) is responsible for the binding not only to p22(phox), but also to the p47(phox) C terminus. Thus the SH3 domain is accessible in the active p47(phox) but is normally masked in the full-length one probably via intramolecularly interacting with the C terminus. The present findings support our previous proposal of regulatory SH3 domain-mediated interactions.. |
| 115. |
K Mizuki, K Kadomatsu, K Hata, T Ito, QW Fan, Y Kage, Y Fukumaki, Y Sakaki, K Takeshige, H Sumimoto, Functional modules and expression of mouse p40(phox) and p67(phox), SH3-domain-containing proteins - Involved in the phagocyte NADPH oxidase complex, EUROPEAN JOURNAL OF BIOCHEMISTRY, 10.1046/j.1432-1327.1998.2510573.x, 251, 3, 573-582, 1998.02, The phagocyte NADPH oxidase is activated during phagocytosis to produce superoxide, a precursor of microbicidal oxidants. The formation of the active oxidase complex at the membrane requires translocation of the Rac GTPase and two specialized cytosolic proteins that harbor SH3 domains, p67(phox) and p47(phox). Another SH3-domain-containing protein p40(phox), which is constitutively associated with p67(phox) in phagocytes, also enters the complex upon cell stimulation. Here we describe how we cloned mouse cDNAs encoding p40(phox) and its partner in phagocytes, p67(phox). Both p40(phox) and p67(phox) comprise several protein-binding modules that are structurally and functional well conserved between mouse and human, indicating their nature as adaptor proteins. We have also systematically investigated expression of the gene for p40(phox) in comparison with those for p67(phox) and p47(phox). Distributions of the mRNAs for the three proteins among tissues are similar, with the most abundant: expression in the spleen. The messages are abundant not only in phagocytic cells, but also in B cell lineage. The p40(phox) gene, but not the other two, is expressed in some types of cells such as plasma cells and T lymphocytes, Furthermore, in situ hybridization analysis shows that the p40(phox) mRNA is distributed in neuronal cells of mouse brain, providing evidence that one of the genes for the specialized oxidase factors is expressed in neurons. These observations raise the possibility that the adaptor protein p40(phox) plays a heretofore unsuspected role via interacting with other proteins in the cells that do not express p67(phox)or p47(phox).. |
| 116. |
R Nakamura, H Sumimoto, K Mizuki, K Hata, T Ago, S Kitajima, K Takeshige, Y Sakaki, T Ito, The PC motif: a novel and evolutionarily conserved sequence involved in interaction between p40(phox) and p67(phox), SH3 domain-containing cytosolic factors of the phagocyte NADPH oxidase, EUROPEAN JOURNAL OF BIOCHEMISTRY, 10.1046/j.1432-1327.1998.2510583.x, 251, 3, 583-589, 1998.02, The superoxide-generating NADPH oxidase, dormant in resting phagocytes, is activated during phagocytosis following assembly of the membrane-integrated protein cytochrome b(558) and cytosolic factors. Among the latter are the three proteins containing Src homology 3(SH3) domains,p67(phox),p47(phox) and p40(phox). While the first two factors are indispensable for the activity, p40(phox) is tightly associated with p67(phox) in resting cells and is suggested to have some modulatory role. Here we describe a systematic analysis of the interaction between p40(phox) and p67(phox) using the yeast two-hybrid system and in vitro binding assays with recombinant proteins. Both methods unequivocally showed that the minimum requirements for stable interaction are the C-terminal region of p40(phox) and the region between the two SH3 domains of p67(phox). This interaction is maintained even in the presence of anionic amphiphiles used for the activation of the NADPH oxidase, raising a possibility that it mediates constitutive association of the two factors in both resting and activated cells. The C-terminal region of p40(phox) responsible for the interaction contains a characteristic stretch of amino acids designated as the PC motif, that also exists in other signal-transducing proteins from yeast to human. Intensive site-directed mutagenesis to the motif in p40(phox) revealed that it plays a critical role in the binding to p67(phox). Thus the PC motif appears to represent a novel module for protein-protein interaction used in a variety of signaling pathways.. |
| 117. |
N Adati, T Ito, Y Sakaki, K Shiokawa, Isolation and expression study of a maternally expressed novel Xenopus gene Xem1 encoding a putative evolutionarily conserved membrane protein, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 10.1006/bbrc.1997.7215, 238, 3, 899-904, 1997.09, A novel Xenopus maternally expressed gene, Xem1, was isolated by differential display PCR and 5'-RACE. Xem1 coded for a putative transmembrane protein of 172 amino acids. Sequence analysis, including the clustering and reconstruction of ESTs (Expressed Sequence Tags), revealed that homologs of Xem1 are widely distributed in eukaryotic phyla, suggesting that Xem1 is a member of evolutionarily conserved proteins. Expression of Xem1 mRNA occurred from the previtellogenic stage and its level increased during oogenesis, maintained throughout oocyte maturation to blastula stage and then decreased in post gastrula stages. In cleavage stage, Xem1 RNA was distributed uniformly, and in adult, occurred predominantly in ovary and testis. We assume that Xenopus Xem1 may have its function in gametogenesis and ire early phase of embryogenesis, whose function may he related to transport mechanism of small molecular weight substances like metal ions, from analogy to the function of its homologs in other organisms. (C) 1997 Academic Press.. |
| 118. |
Y Hagiwara, M Hirai, K Nishiyama, Kanazawa, I, T Ueda, Y Sakaki, T Ito, Screening for imprinted genes by allelic message display: Identification of a paternally expressed gene Impact on mouse chromosome 18, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 10.1073/pnas.94.17.9249, 94, 17, 9249-9254, 1997.08, A systematic screen termed the allelic message display (AMD) was developed for the hunting of imprinted genes, In AMD, differential display PCR is adopted to image allelic expression status of multiple polymorphic transcripts in two parental mouse strains, reciprocal Fl hybrids and pooled backcross progenies, From the displayed patterns, paternally and maternally expressed transcripts can be unequivocally identified, The effectiveness of AMD screening was clearly demonstrated by the identification of a paternally expressed gene Impact on mouse chromosome 18, the predicted product of which belongs to the YCR59c/yigZ hypothetical protein family composed of yeast and bacterial proteins with currently unknown function, In contrast with previous screening methods necessitating positional cloning efforts or generation of parthenogenetic embryos, this approach requires nothing particular but appropriately crossed mice and can be readily applied to any tissues at various developmental stages, Hence, AMD would considerably accelerate the identification of imprinted genes playing pivotal roles in mammalian development and the pathogenesis of various diseases.. |
| 119. |
C Furihata, M Oka, M Yamamoto, T Ito, M Ichinose, K Miki, M Tatematsu, Y Sakaki, K Reske, Differentially expressed MHC class II-associated invariant chain in rat stomach pyloric mucosa with N-methyl-N'-nitro-N-nitrosoguanidine exposure, CANCER RESEARCH, 57, 8, 1416-1418, 1997.04, Administration of N-methyl-N'-nitro-N-nitrosoguanidine, a glandular stomach carcinogen, at the concentration of 100 mu g/ml in drinking water for 8 days induced the appearance of a MHC class II-associated invariant chain in the target organ of stomach pyloric mucosa of male Lewis rats, The up-regulation of the MHC class II-associated invariant chain was revealed by fluorescent differential display analysis, reverse transcription-PCR, Northern blot, and histochemical staining, The appearance of MHC class II and MHC class I was also demonstrated by reverse transcription-PCR and Northern blot. The results suggest the involvement of MHC-controlled immune reactions in chemically-induced stomach carcinogenesis.. |
| 120. |
C Furihata, M Tatematsu, M Saito, S Ishida, H Nakanishi, K Inada, H Tei, M Hattori, T Ito, Y Sakaki, Rare occurrence of ras and p53 gene mutations in mouse stomach tumors induced by N-methyl-N-nitrosourea, JAPANESE JOURNAL OF CANCER RESEARCH, 10.1111/j.1349-7006.1997.tb00390.x, 88, 4, 363-368, 1997.04, The incidence of point mutations of H-, K- and N-ras and p53 oncogenes in male BALB/c mouse stomach tumors induced with N-methyl-N-nitrosourea (MNU) was examined by direct sequencing and PCR single-strand conformation polymorphism (PCR-SSCP). A mutation of GGT to AGT at K-ras codon 12 was found by SSCP in one adenocarcinoma from a total of 19 specimens including 5 adenocarcinomas, 9 adenomatous hyperplastic regions, 1 squamous cell carcinoma and 4 normal-like stomach regions from 4 mice. No mutations were detected by direct sequencing of H-, K- and N-ras oncogenes at exons 1 (codons 12 and 13) and 2 (codon 61) in a total of 26 specimens comprising 10 adenocarcinomas, 10 adenomatous hyperplastic regions, 2 squamous cell carcinomas and 4 normal-like stomach regions from 6 mice. No mutations were detected by direct sequencing of p53 oncogene at exons 5, 6, 7 and 8 in a total of 30 specimens including 13 adenocarcinomas, 8 adenomatous hyperplastic regions, 2 squamous cell carcinomas, 1 papilloma and 6 normal-like stomach regions from 7 mice. These results suggest that ras and p53 oncogenes do not play a role in mouse stomach carcinogenesis induced by MNU.. |
| 121. |
K Kito, T Ito, Y Sakaki, Fluorescent differential display analysis of gene expression in differentiating neuroblastoma cells, GENE, 10.1016/S0378-1119(96)00577-X, 184, 1, 73-81, 1997.01, Identification of differentially-expressed genes provides an important step toward the elucidation of molecular mechanisms underlying a variety of biological processes. A novel PCR-based approach to detect and clone such transcripts is the so-called differential display (DD). We established an improved DD protocol that can be performed on an automated fluorescent DNA sequencer to ensure high throughput as well as operational safety. Using this florescent DD (FDD) technique, we analyzed the gene expression profile in the retinoic acid-induced differentiation of a human neuroblastoma cell line SH-SY5Y. Screening with 102 primer combinations at eight different time points revealed 66 cDNA bands with variously different behaviors out of similar to 6000 bands displayed. Subsequent analyses with 15 cloned species confirmed the differential expression of corresponding transcripts in all the cases, thereby demonstrating the high reliability of FDD analysis. These clones were composed of seven novel and eight known genes, the latter of which included those that had never been described in the context of neuronal differentiation. These results indicate that FDD is an effective approach to obtain not only novel genes but also clues to possible novel functions of known genes involved in various biological phenomena.. |
| 122. |
H Sumimoto, K Hata, K Mizuki, T Ito, Y Kage, Y Sakaki, Y Fukumaki, M Nakamura, K Takeshige, Assembly and activation of the phagocyte NADPH oxidase - Specific interaction of the N-terminal Src homology 3 domain of p47(phox) with p22(phox) is required for activation of the NADPH oxidase, JOURNAL OF BIOLOGICAL CHEMISTRY, 10.1074/jbc.271.36.22152, 271, 36, 22152-22158, 1996.09, The phagocyte NADPH oxidase is activated during phagocytosis to produce superoxide, a precursor of microbicidal oxidants. The activation involves assembly of membrane-integrated cytochrome b(558) comprising gp91(phox) and p22(phox), two specialized cytosolic proteins (p47(phox) and p67(phox)), each containing two Src homology 3 (SH3) domains, and the small G protein Rac. In the present study, we show that the N-terminal SH3 domain of p47(phox) binds to the C-terminal cytoplasmic tail of p22(phox) with high affinity (K-D = 0.34 mu M). The binding is specific to this domain among several SH3 domains including the C-terminal one of p47(phox) and the two of p67(phox) and requires the Pro(156)-containing proline-rich sequence but not other putative SH3 domain-binding sites of p22(phox). Replacement of Trp(193) by Arg in the N-terminal SH3 domain completely abrogates the association with p22(phox). A mutant p47(phox) with this substitution is incapable of supporting superoxide production under cell-free activation conditions. These findings provide direct evidence that the interaction between the N-terminal SH3 domain of p47(phox) and the proline-rich region of p22(phox) is essential for activation of the NADPH oxidase.. |
| 123. |
T Ito, R Nakamura, H Sumimoto, K Takeshige, Y Sakaki, An SH3 domain-mediated interaction between the phagocyte NADPH oxidase factors p40(phox) and p47(phox), FEBS LETTERS, 10.1016/0014-5793(96)00387-0, 385, 3, 229-232, 1996.05, The phagocyte NADPH oxidase is activated during phagocytosis to produce superoxide, following assembly of a membrane-integrated cytochrome b(558) with cytosolic proteins, p47(phox), p67(phox) and p40(phox), each containing Src homology 3 (SH3) domains, While both p47(phox) and p67(phox) are indispensable for the oxidase activity, role of p40(phox) remains obscure, Here we study interaction between p40(phox) and p47(phox) by two independent methods, a two-hybrid system in the yeast and an in vitro binding assay using purified proteins, The present results show that the interaction is mediated via binding of the SH3 domain of p40(phox) to a C-terminal proline-rich region of p47(phox), This proline-rich region is also the target for binding of p67(phox), and the SH3 domain of p40(phox) can inhibit the binding of the C-terminal one of p67(phox) to p47(phox).. |
| 124. |
N ADATI, T ITO, C KOGA, K KITO, Y SAKAKI, K SHIOKAWA, DIFFERENTIAL DISPLAY ANALYSIS OF GENE-EXPRESSION IN DEVELOPING EMBRYOS OF XENOPUS-LAEVIS, BIOCHIMICA ET BIOPHYSICA ACTA-GENE STRUCTURE AND EXPRESSION, 10.1016/0167-4781(95)00049-M, 1262, 1, 43-51, 1995.05, Differential display (DD), an arbitrarily primed RT-PCR fingerprinting technique, is a novel approach for the search of differentially expressed transcripts. Using our improved DD protocol, reproducible cDNA fingerprints were successfully obtained from RNAs of Xenopus laevis embryos at six representative stages. Parallel comparison among the fingerprints revealed a number of bands with differential expression patterns. Analysis with clones of three randomly chosen bands confirmed that their expression patterns were faithfully reflected on fingerprints, thereby proving the reliability and validity of the approach. Nucleotide sequencing of these clones revealed that one is identical with a known transcript (cardiac actin), the second is a novel developmentally regulated gene showing no significant homology with those reported previously, and the last is a close but unique relative of XK endo B gene showing somewhat different spatial expression pattern. These results indicated that the DD analysis provides a rapid and reliable way for the identification of novel differentially expressed genes as well as a unique 'scope' for the survey of the changes in overall gene expression profiles occurring in the early embryonic development of Xenopus as well as of other organisms.. |
| 125. |
ML CHER, T ITO, N WEIDNER, PR CARROLL, RH JENSEN, MAPPING OF REGIONS OF PHYSICAL DELETION ON CHROMOSOME 16Q IN PROSTATE-CANCER CELLS BY FLUORESCENCE IN-SITU HYBRIDIZATION (FISH), JOURNAL OF UROLOGY, 10.1097/00005392-199501000-00086, 153, 1, 249-254, 1995.01, Recent evidence suggests that a tumor suppressor gene important in the progression of prostatic carcinoma may reside on chromosome 16q. The exact location and identity of this gene are unknown. We used fluorescence in situ hybridization in a novel manner to define more clearly the location of this gene. Region-specific chromosome 16 cosmid contig probes were hybridized directly to interphase prostatic carcinoma nuclei in order to measure physical deletion of chromosomal loci. Fifteen of 30 tumors (50%) showed evidence of physical deletion of 16q24. Other probes were used to test for regions of chromosome 16 deletions in the same specimens, and a map showing a region of common deletion was created. This map showed the proximal terminus of the region of common deletion to be located distal to 16q23.1. These data correlate well with loss of heterozygosity data in the literature and provide further evidence for the presence of a prostatic carcinoma tumor suppressor gene on chromosome 16q.. |
| 126. |
T ITO, K KITO, N ADATI, Y MITSUI, H HAGIWARA, Y SAKAKI, FLUORESCENT DIFFERENTIAL DISPLAY - ARBITRARILY PRIMED RT-PCR FINGERPRINTING ON AN AUTOMATED DNA SEQUENCER, FEBS LETTERS, 10.1016/0014-5793(94)00867-1, 351, 2, 231-236, 1994.09, We established robust, reliable protocols for 'Differential Display (DD),' an RNA fingerprinting method originally developed by Liang and Pardee [(1992) Science 257, 967-971] using RT-PCR with arbitrary primers. Our protocols are optimized so that reliable DD analysis can be performed on a fluorescent DNA sequencer to ensure high throughput as well as improved operational safety, compared with the original one using radioactive compounds. Such 'Fluorescent Differential Display (FDD)' techniques will accelerate the identification of differentially expressed as well as polymorphic transcripts to address various biological questions.. |
| 127. |
Higuchi M, Ito T, Imai Y, Iwaki T, Hattori M, Kohsaka S, Niho Y, Sakaki Y, Expression of the α2-macroglobulin-encoding gene in rat brain and cultured astrocytes, Gene, 10.1016/0378-1119(94)90565-7, 141, 2, 155-162, 1994.04. |
| 128. |
M HATTORI, A TOYODA, H ICHIKAWA, T ITO, H OHGUSU, N OISHI, T KANO, S KUHARA, M OHKI, Y SAKAKI, SEQUENCE-TAGGED NOTI SITES OF HUMAN CHROMOSOME-21 - SEQUENCE-ANALYSIS AND MAPPING, GENOMICS, 10.1006/geno.1993.1280, 17, 1, 39-44, 1993.07. |
| 129. |
T ITO, H HOHJOH, Y SAKAKI, PULSED-FIELD POLYACRYLAMIDE-GEL ELECTROPHORESIS - BASIC PHENOMENA AND APPLICATIONS, ELECTROPHORESIS, 10.1002/elps.1150140149, 14, 4, 278-282, 1993.04, Pulsed-field gel electrophoresis (PFGE) using polyacrylamide gels, termed pulsed-field polyacrylamide gel electrophoresis (PF-PAGE), had been developed for the effective separation of linear DNAs from circular ones [1]. The first generation PF-PAGE employed horizontal polyacrylamide gels run in a contour-clamped homogeneous electric field (CHEF) apparatus. The second generation system, using a vertical slab gel in a discontinuous buffer system and field inversion gel electrophoresis (FIGE), was found to be easier to handle and requires a much shorter time for separation than the previous one [2]. In this report, basic aspects of the second generation PF-PAGE, such as the effects of a discontinuous buffer system and field inversion on the DNA migration in polyacrylamide gels, were investigated. The results indicate that the periodic inversion of electric field can broaden the resolving capability of polyacrylamide gels, enabling DNAs that otherwise fail to enter polyacrylamide gels to be resolved in such systems. Successful and possible applications of PF-PAGE techniques are also discussed.. |
| 130. |
T ITO, CL SMITH, CR CANTOR, TRIPLEX AFFINITY CAPTURE OF A SINGLE COPY CLONE FROM A YEAST GENOMIC LIBRARY, NUCLEIC ACIDS RESEARCH, 10.1093/nar/20.13.3524, 20, 13, 3524-3524, 1992.07. |
| 131. |
T ITO, CL SMITH, CR CANTOR, AFFINITY CAPTURE ELECTROPHORESIS FOR SEQUENCE-SPECIFIC DNA PURIFICATION, GENETIC ANALYSIS-BIOMOLECULAR ENGINEERING, 10.1016/1050-3862(92)90005-P, 9, 3, 96-99, 1992.06, A new method, affinity capture electrophoresis (ACE), has been developed for the sequence-specific isolation of DNA. The target DNA is complexed with a biotinylated probe and electrophoresed in a gel equipped with a trap of immobilized streptavidin. This selectively captures the target molecule and its biotinylated probe, while other nontarget molecules pass through the trap. The target DNA is subsequently recovered from the trap by destroying the interaction between the target DNA and the biotinylated probe. Two variations of this technique, one using triple-helix formation and the other using hybridization with a uracil-containing DNA probe at the end of the target fragment, proved effective in model experiments. Since this technique requires no denaturation and handles DNA inside an agarose gel matrix, it is, in principle, applicable to the isolation of very large DNAs.. |
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T ITO, CL SMITH, CR CANTOR, SEQUENCE-SPECIFIC DNA PURIFICATION BY TRIPLEX AFFINITY CAPTURE, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 10.1073/pnas.89.2.495, 89, 2, 495-498, 1992.01, A DNA isolation procedure was developed by using triple-helix formation and magnetic separation. In this procedure, target DNA is captured by a biotinylated oligonucleotide via intermolecular triplex formation, bound to streptavidin-coated magnetic beads, and recovered in double-stranded form by elution with a mild alkaline buffer that destabilizes the triple helix. The effectiveness of the procedure was demonstrated by a model experiment with an artificially reconstructed library and, also, by the isolation of (dT-dC)n.(dG-dA)n dinucleotide repeats from a human genomic library. This procedure provides a prototype for other triplex-mediated DNA isolation technologies.. |
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H TANAHASHI, T ITO, S INOUYE, FI TSUJI, Y SAKAKI, PHOTOPROTEIN AEQUORIN - USE AS A REPORTER ENZYME IN STUDYING GENE-EXPRESSION IN MAMMALIAN-CELLS, GENE, 10.1016/0378-1119(90)90260-X, 96, 2, 249-255, 1990.12. |
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Ito T, Tanahashi H, Musumi Y, Sakaki Y, Nuclear factors interacting with an interleukin-6 responsive element of rat α2-macroglobulin gene, Nucleic Acids Research, 10.1093/nar/17.22.9425, 17, 22, 9425-9435, 1989.11. |
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Hattori M, Kusakabe S, Ohgusu H, Tsuchiya Y, Ito T, Sakaki Y, Structure of the rat α2-macroglobulin-coding gene, Gene, 10.1016/0378-1119(89)90081-4, 77, 2, 333-340, 1989.04. |
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T ITO, Y SAKAKI, A NOVEL PROCEDURE FOR SELECTIVE CLONING OF NOTI LINKING FRAGMENTS FROM MAMMALIAN GENOMES, NUCLEIC ACIDS RESEARCH, 10.1093/nar/16.19.9177, 16, 19, 9177-9184, 1988.10. |
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Ito T, Sakaki Y, Nuclear matrix association regions of rat α2-macroglobulin gene, Biochemical and Biophysical Research Communications, 10.1016/0006-291X(87)90388-3, 149, 2, 449-454, 1987.12. |
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Ito T, Sakaki Y, A nuclear factor which interacts with an AT-cluster in the first intron of rat α2-macroglobulin gene, Biochemical and Biophysical Research Communications, 10.1016/0006-291X(87)91004-7, 147, 2, 824-830, 1987.09. |
