Kyushu University Academic Staff Educational and Research Activities Database
Researcher information (To researchers) Need Help? How to update
Yasuyuki Ohkawa Last modified date:2024.05.08

Professor / Division of Medical Molecular Cell Biology
Medical Research Center for High Depth Omics
Medical Institute of Bioregulation

Graduate School
Other Organization
Administration Post

E-Mail *Since the e-mail address is not displayed in Internet Explorer, please use another web browser:Google Chrome, safari.
 Reseacher Profiling Tool Kyushu University Pure
Academic Degree
Field of Specialization
chromatin, cell differentiation
ORCID(Open Researcher and Contributor ID)
Total Priod of education and research career in the foreign country
Research Interests
  • Development of epigenomic/trascriptomic technology
    keyword : epigenome transcriptome technology
  • Analysis of selective gene expression in organogenesis
    keyword : epigenetics chromatin deep sequencer epigenome transcriptome
  • Epigenetics Analysis in Stem Cell biology
    keyword : epigenetics chromatin deep sequencer epigenome transcriptome
Current and Past Project
  • The analysis of Genomic re-organization in myogenesis
Academic Activities
1. OhkawaY., Mallappa C., Dacwag CS., Imbalzano AN. , An improved restriction enzyme accessibility assay (REAA) for analyzing changes in chromatin structure in samples of limited cell number.
, 2011.10.
2. Ohkawa Y., Mallappa C., Dacwag CS., Imbalzano AN. , Isolation of nuclei from skeletal muscle satellite cells and myofibers for use in chromatin immunoprecipitation assays.
, 2011.10.
1. de la Serna IL, Ohkawa Y, Imbalzano AN., Chromatin remodelling in mammalian differentiation: lessons from ATP-dependent remodellers., Nat Rev Genet., 2006.06.
1. Masahiro Oka, Mayumi Otani, Yoichi Miyamoto, Rieko Oshima, Jun Adachi, Takeshi Tomonaga, Munehiro Asally, Yuya Nagaoka, Kaori Tanaka, Atsushi Toyoda, Kazuki Ichikawa, Shinichi Morishita, Kyoichi Isono, Haruhiko Koseki, Ryuichiro Nakato, Yasuyuki Ohkawa, Yoshihiro Yoneda, Phase-separated nuclear bodies of nucleoporin fusions promote condensation of MLL1/CRM1 and rearrangement of 3D genome structure., Cell reports, 10.1016/j.celrep.2023.112884, 42, 8, 112884-112884, 2023.07, NUP98 and NUP214 form chimeric fusion proteins that assemble into phase-separated nuclear bodies containing CRM1, a nuclear export receptor. However, these nuclear bodies' function in controlling gene expression remains elusive. Here, we demonstrate that the nuclear bodies of NUP98::HOXA9 and SET::NUP214 promote the condensation of mixed lineage leukemia 1 (MLL1), a histone methyltransferase essential for the maintenance of HOX gene expression. These nuclear bodies are robustly associated with MLL1/CRM1 and co-localized on chromatin. Furthermore, whole-genome chromatin-conformation capture analysis reveals that NUP98::HOXA9 induces a drastic alteration in high-order genome structure at target regions concomitant with the generation of chromatin loops and/or rearrangement of topologically associating domains in a phase-separation-dependent manner. Collectively, these results show that the phase-separated nuclear bodies of nucleoporin fusion proteins can enhance the activation of target genes by promoting the condensation of MLL1/CRM1 and rearrangement of the 3D genome structure..
2. Victoria Moiseeva, Andrés Cisneros, Valentina Sica, Oleg Deryagin, Yiwei Lai, Sascha Jung, Eva Andrés, Juan An, Jessica Segalés, Laura Ortet, Vera Lukesova, Giacomo Volpe, Alberto Benguria, Ana Dopazo, Salvador Aznar-Benitah, Yasuteru Urano, Antonio Del Sol, Miguel A Esteban, Yasuyuki Ohkawa, Antonio L Serrano, Eusebio Perdiguero, Pura Muñoz-Cánoves., Senescence atlas reveals an aged-like inflamed niche that blunts muscle regeneration., Nature, doi: 10.1038/s41586-022-05535-x., 163, 7942, 169-178, 2023.01, [URL], Tissue regeneration requires coordination between resident stem cells and local niche cells1,2. Here we identify that senescent cells are integral components of the skeletal muscle regenerative niche that repress regeneration at all stages of life. The technical limitation of senescent-cell scarcity3 was overcome by combining single-cell transcriptomics and a senescent-cell enrichment sorting protocol. We identified and isolated different senescent cell types from damaged muscles of young and old mice. Deeper transcriptome, chromatin and pathway analyses revealed conservation of cell identity traits as well as two universal senescence hallmarks (inflammation and fibrosis) across cell type, regeneration time and ageing. Senescent cells create an aged-like inflamed niche that mirrors inflammation associated with ageing (inflammageing4) and arrests stem cell proliferation and regeneration. Reducing the burden of senescent cells, or reducing their inflammatory secretome through CD36 neutralization, accelerates regeneration in young and old mice. By contrast, transplantation of senescent cells delays regeneration. Our results provide a technique for isolating in vivo senescent cells, define a senescence blueprint for muscle, and uncover unproductive functional interactions between senescent cells and stem cells in regenerative niches that can be overcome. As senescent cells also accumulate in human muscles, our findings open potential paths for improving muscle repair throughout life..
3. Mizuki Honda, Ryuichi Kimura, Akihito Harada, Kazumitsu Maehara, Kaori Tanaka, Yasuyuki Ohkawa, Shinya Oki., Photo-isolation chemistry for high-resolution and deep spatial transcriptome with mouse tissue sections., STAR Protoc., doi: 10.1016/j.xpro.2022., 3, 2, 101346, 2022.04, [URL], Photo-isolation chemistry (PIC) enables isolation of transcriptome information from locally defined areas by photo-irradiation. Here, we present an optimized PIC protocol for formalin-fixed frozen and paraffin mouse sections and fresh-frozen mouse sections. We describe tissue section preparation and permeabilization, followed by in situ reverse transcription using photo-caged primers. We then detail immunostaining and UV-mediated uncaging to the target areas, followed by linear amplification of uncaged cDNAs, library preparation, and quantification. This protocol can be applied to various animal tissue types. For complete details on the use and execution of this protocol, please refer to Honda et al. (2021)..
4. Kazumitsu Maehara, Kosuke Tomimatsu, Akihito Harada, Kaori Tanaka, Shoko Sato, Megumi Fukuoka, Seiji Okada, Tetsuya Handa, Hitoshi Kurumizaka, Noriko Saitoh, Hiroshi Kimura, and Yasuyuki Ohkawa*, Modeling population size independent tissue epigenomes by ChIL-seq with single thin sections, Molecular Systems Biology, 2021.11, Recent advances in genome-wide technologies have enabled analyses using small cell numbers of even single cells. However, obtaining tissue epigenomes with cell-type resolution from large organs and tissues still remains challenging, especially when the available material is limited. Here, we present a ChIL-based approach for analyzing the diverse cellular dynamics at the tissue level using high-depth epigenomic data. "ChIL for tissues" allows the analysis of a single tissue section and can reproducibly generate epigenomic profiles from several tissue types, based on the distribution of target epigenomic states, tissue morphology, and number of cells. The proposed method enabled the independent evaluation of changes in cell populations and gene activation in cells from regenerating skeletal muscle tissues, using a statistical model of RNA polymerase II distribution on gene loci. Thus, the integrative analyses performed using ChIL can elucidate in vivo cell-type dynamics of tissues..
5. †Mizuki Honda, Shinya †Oki, Ryuichi Kimura, Akihito Harada, Kazumitsu Maehara, Kaori Tanaka, Chikara Meno, Yasuyuki Ohkawa., High-depth spatial transcriptome analysis by photo-isolation chemistry., Nature Communications, 10.1038/s41467-021-24691-8, 12, 1, 4416, 2021.07, In multicellular organisms, expression profiling in spatially defined regions is crucial to elucidate cell interactions and functions. Here, we establish a transcriptome profiling method coupled with photo-isolation chemistry (PIC) that allows the determination of expression profiles specifically from photo-irradiated regions of interest. PIC uses photo-caged oligodeoxynucleotides for in situ reverse transcription. PIC transcriptome analysis detects genes specifically expressed in small distinct areas of the mouse embryo. Photo-irradiation of single cells demonstrated that approximately 8,000 genes were detected with 7 × 104 unique read counts. Furthermore, PIC transcriptome analysis is applicable to the subcellular and subnuclear microstructures (stress granules and nuclear speckles, respectively), where hundreds of genes can be detected as being specifically localised. The spatial density of the read counts is higher than 100 per square micrometre. Thus, PIC enables high-depth transcriptome profiles to be determined from limited regions up to subcellular and subnuclear resolutions..
6. Tetsuya Handa, Akihito Harada, Kazumitsu Maehara, Shoko Sato, Masaru Nakao, Naoki Goto, Hitoshi Kurumizaka, Yasuyuki Ohkawa, Hiroshi Kimura, Chromatin integration labeling for mapping DNA-binding proteins and modifications with low input., Nature protocols, 10.1038/s41596-020-0375-8, 15, 10, 3334-3360, 2020.10, Cell identity is determined by the selective activation or silencing of specific genes via transcription factor binding and epigenetic modifications on the genome. Chromatin immunoprecipitation (ChIP) has been the standard technique for mapping the sites of transcription factor binding and histone modification. Recently, alternative methods to ChIP have been developed for addressing the increasing demands for low-input epigenomic profiling. Chromatin integration labeling (ChIL) followed by sequencing (ChIL-seq) has been demonstrated to be particularly useful for epigenomic profiling of low-input samples or even single cells because the technique amplifies the target genomic sequence before cell lysis. After labeling the target protein or modification in situ with an oligonucleotide-conjugated antibody (ChIL probe), the nearby genome sequence is amplified by Tn5 transposase-mediated transposition followed by T7 RNA polymerase-mediated transcription. ChIL-seq enables the detection of the antibody target localization under a fluorescence microscope and at the genomic level. Here we describe the detailed protocol of ChIL-seq with assessment methods for the key steps, including ChIL probe reaction, transposition, in situ transcription and sequencing library preparation. The protocol usually takes 3 d to prepare the sequencing library, including overnight incubations for the ChIL probe reaction and in situ transcription. The ChIL probe can be separately prepared and stored for several months, and its preparation and evaluation protocols are also documented in detail. An optional analysis for multiple targets (multitarget ChIL-seq) is also described. We anticipate that the protocol presented here will make the ChIL technique more widely accessible for analyzing precious samples and facilitate further applications..
7. Akihito Harada, Kazumitsu Maehara, Tetsuya Handa, Yasuhiro Arimura, Jumpei Nogami, Yoko Hayashi-Takanaka, Katsuhiko Shirahige, Hitoshi Kurumizaka, Hiroshi Kimura, Yasuyuki Ohkawa, A chromatin integration labelling method enables epigenomic profiling with lower input, Nature Cell Biology, 10.1038/s41556-018-0248-3, 21, 2, 287-296, 2019.02, Chromatin plays a crucial role in gene regulation, and chromatin immunoprecipitation followed by sequencing (ChIP–seq) has been the standard technique for examining protein–DNA interactions across the whole genome. However, it is difficult to obtain epigenomic information from limited numbers of cells by ChIP–seq because of sample loss during chromatin preparation and inefficient immunoprecipitation. In this study, we established an immunoprecipitation-free epigenomic profiling method named chromatin integration labelling (ChIL), which enables the amplification of genomic sequences closely associated with the target molecules before cell lysis. Using ChIL followed by sequencing (ChIL–seq), we reliably detected the distributions of histone modifications and DNA-binding factors in 100–1,000 cells. In addition, ChIL–seq successfully detected genomic regions associated with histone marks at the single-cell level. Thus, ChIL–seq offers an alternative method to ChIP–seq for epigenomic profiling using small numbers of cells, in particular, those attached to culture plates and after immunofluorescence..
8. Akihito Harada, Kazumitsu Maehara, Yusuke Ono, Hiroyuki Taguchi, Kiyoshi Yoshioka, Yasuo Kitajima, Yan Xie, Yuko Sato, Takeshi Iwasaki, Jumpei Nogami, Seiji Okada, Tetsuro Komatsu, Yuichiro Semba, Tatsuya Takemoto, Hiroshi Kimura, Hitoshi Kurumizaka, Yasuyuki Ohkawa, Histone H3.3 sub-variant H3mm7 is required for normal skeletal muscle regeneration, Nature Communications, 10.1038/s41467-018-03845-1, 9, 1, 2018.04, Regulation of gene expression requires selective incorporation of histone H3 variant H3.3 into chromatin. Histone H3.3 has several subsidiary variants but their functions are unclear. Here we characterize the function of histone H3.3 sub-variant, H3mm7, which is expressed in skeletal muscle satellite cells. H3mm7 knockout mice demonstrate an essential role of H3mm7 in skeletal muscle regeneration. Chromatin analysis reveals that H3mm7 facilitates transcription by forming an open chromatin structure around promoter regions including those of myogenic genes. The crystal structure of the nucleosome containing H3mm7 reveals that, unlike the S57 residue of other H3 proteins, the H3mm7-specific A57 residue cannot form a hydrogen bond with the R40 residue of the cognate H4 molecule. Consequently, the H3mm7 nucleosome is unstable in vitro and exhibited higher mobility in vivo compared with the H3.3 nucleosome. We conclude that the unstable H3mm7 nucleosome may be required for proper skeletal muscle differentiation..
9. Yukari Kondo, Shinichiro Higa, Takeshi Iwasaki, Tomoya Matsumoto, Kazumitsu Maehara, Akihito Harada, Yoshihiro Baba, Masatoshi Fujita, Yasuyuki Ohkawa, Sensitive detection of fluorescence in western blotting by merging images, PLoS One, 10.1371/journal.pone.0191532, 13, 1, 2018.01, The western blotting technique is widely used to analyze protein expression levels and protein molecular weight. The chemiluminescence method is mainly used for detection due to its high sensitivity and ease of manipulation, but it is unsuitable for detailed analyses because it cannot be used to detect multiple proteins simultaneously. Recently, more attention has been paid to the fluorescence detection method because it is more quantitative and is suitable for the detection of multiple proteins simultaneously. However, fluorescence detection can be limited by poor image resolution and low detection sensitivity. Here, we describe a method to detect fluorescence in western blots using fluorescence microscopy to obtain high-resolution images. In this method, filters and fluorescent dyes are optimized to enhance detection sensitivity to a level similar to that of the chemiluminescence method..
10. Akihito Harada, Yasuyuki Ohkawa, Anthony N. Imbalzano, Temporal regulation of chromatin during myoblast differentiation, Seminars in Cell and Developmental Biology, 10.1016/j.semcdb.2017.10.022, 72, 77-86, 2017.12, The commitment to and execution of differentiation programmes involves a significant change in gene expression in the precursor cell to facilitate development of the mature cell type. In addition to being regulated by lineage-determining and auxiliary transcription factors that drive these changes, the structural status of the chromatin has a considerable impact on the transcriptional competence of differentiation-specific genes, which is clearly demonstrated by the large number of cofactors and the extraordinary complex mechanisms by which these genes become activated. The terminal differentiation of myoblasts to myotubes and mature skeletal muscle is an excellent system to illustrate these points. The MyoD family of closely related, lineage-determining transcription factors directs, largely through targeting to chromatin, a cascade of cooperating transcription factors and enzymes that incorporate or remove variant histones, post-translationally modify histones, and alter nucleosome structure and positioning via energy released by ATP hydrolysis. The coordinated action of these transcription factors and enzymes prevents expression of differentiation-specific genes in myoblasts and facilitates the transition of these genes from transcriptionally repressed to activated during the differentiation process. Regulation is achieved in both a temporal as well as spatial manner, as at least some of these factors and enzymes affect local chromatin structure at myogenic gene regulatory sequences as well as higher-order genome organization. Here we discuss the transition of genes that promote myoblast differentiation from the silenced to the activated state with an emphasis on the changes that occur to individual histones and the chromatin structure present at these loci..
11. Yuichiro Semba, Akihito Harada, Kazumitsu Maehara, Shinya Oki, Chikara Meno, Jun Ueda, Kazuo Yamagata, Atsushi Suzuki, Mitsuho Onimaru, Jumpei Nogami, Seiji Okada, Koichi Akashi, Yasuyuki Ohkawa, Chd2 regulates chromatin for proper gene expression toward differentiation in mouse embryonic stem cells, Nucleic Acids Research, 10.1093/nar/gkx475, 45, 15, 8758-8772, 2017.09, Chromatin reorganization is necessary for pluripotent stem cells, including embryonic stem cells (ESCs), to acquire lineage potential. However, it remains unclear how ESCs maintain their characteristic chromatin state for appropriate gene expression upon differentiation. Here, we demonstrate that chromodomain helicase DNA-binding domain 2 (Chd2) is required to maintain the differentiation potential of mouse ESCs. Chd2-depleted ESCs showed suppressed expression of developmentally regulated genes upon differentiation and subsequent differentiation defects without affecting gene expression in the undifferentiated state. Furthermore, chromatin immunoprecipitation followed by sequencing revealed alterations in the nucleosome occupancy of the histone variant H3.3 for developmentally regulated genes in Chd2-depleted ESCs, which in turn led to elevated trimethylation of the histone H3 lysine 27. These results suggest that Chd2 is essential in preventing suppressive chromatin formation for developmentally regulated genes and determines subsequent effects on developmental processes in the undifferentiated state..
12. Kensuke Kudou, Tetsuro Komatsu, Jumpei Nogami, Kazumitsu Maehara, Akihito Harada, Hiroshi Saeki, Eiji Oki, Yoshihiko Maehara, Yasuyuki Ohkawa, The requirement of Mettl3-promoted MyoD mRNA maintenance in proliferative myoblasts for skeletal muscle differentiation., Open biology, 10.1098/rsob.170119, 7, 9, 2017.09, Myogenic progenitor/stem cells retain their skeletal muscle differentiation potential by maintaining myogenic transcription factors such as MyoD. However, the mechanism of how MyoD expression is maintained in proliferative progenitor cells has not been elucidated. Here, we found that MyoD expression was reduced at the mRNA level by cell cycle arrest in S and G2 phases, which in turn led to the absence of skeletal muscle differentiation. The reduction of MyoD mRNA was correlated with the reduced expression of factors regulating RNA metabolism, including methyltransferase like 3 (Mettl3), which induces N6-methyladenosine (m6A) modifications of RNA. Knockdown of Mettl3 revealed that MyoD RNA was specifically downregulated and that this was caused by a decrease in processed, but not unprocessed, mRNA. Potential m6A modification sites were profiled by m6A sequencing and identified within the 5' untranslated region (UTR) of MyoD mRNA. Deletion of the 5' UTR revealed that it has a role in MyoD mRNA processing. These data showed that Mettl3 is required for MyoD mRNA expression in proliferative myoblasts..
13. Hiroyuki Taguchi, Yan Xie, Naoki Horikoshi, Kazumitsu Maehara, Akihito Harada, Jumpei Nogami, Koichi Sato, Yasuhiro Arimura, Akihisa Osakabe, Tomoya Kujirai, Takeshi Iwasaki, Yuichiro Semba, Taro Tachibana, Hiroshi Kimura, Yasuyuki Ohkawa, Hitoshi Kurumizaka, Crystal Structure and Characterization of Novel Human Histone H3 Variants, H3.6, H3.7, and H3.8, Biochemistry, 10.1021/acs.biochem.6b01098, 56, 16, 2184-2196, 2017.04, Non-allelic histone variants are considered as epigenetic factors that regulate genomic DNA functions in eukaryotic chromosomes. In this study, we identified three new human histone H3 variants (named H3.6, H3.7, and H3.8), which were previously annotated as pseudogenes. H3.6 and H3.8 conserve the H3.3-specific amino acid residues, but H3.7 shares the specific amino acid residues with H3.1. We successfully reconstituted the nucleosome containing H3.6 in vitro and determined its crystal structure. In the H3.6 nucleosome, the H3.6-specific Val62 residue hydrophobically contacts the cognate H4 molecule, but its contact area is smaller than that of the corresponding H3.3 Ile62 residue. The thermal stability assay revealed that the H3.6 nucleosome is substantially unstable, as compared to the H3.3 nucleosome. Interestingly, mutational analysis demonstrated that the H3.6 Val62 residue is fully responsible for the H3.6 nucleosome instability, probably because of the weakened hydrophobic interaction with H4. We also reconstituted the nucleosome containing H3.8, but its thermal stability was quite low. In contrast, purified H3.7 failed to form nucleosomes in vitro. The identification and characterization of these novel human histone H3 variants provide important new insights into understanding the epigenetic regulation of the human genome..
14. Daiki Kato, Akihisa Osakabe, Yasuhiro Arimura, Yuka Mizukami, Naoki Horikoshi, Kazumi Saikusa, Satoko Akashi, Yoshifumi Nishimura, Sam Yong Park, Jumpei Nogami, Kazumitsu Maehara, Yasuyuki Ohkawa, Atsushi Matsumoto, Hidetoshi Kono, Rintaro Inoue, Masaaki Sugiyama, Hitoshi Kurumizaka, Crystal structure of the overlapping dinucleosome composed of hexasome and octasome, Science, 10.1126/science.aak9867, 356, 6334, 205-208, 2017.04, Nucleosomes are dynamic entities that are repositioned along DNA by chromatin remodeling processes. A nucleosome repositioned by the switch-sucrose nonfermentable (SWI/SNF) remodeler collides with a neighbor and forms the intermediate "overlapping dinucleosome." Here, we report the crystal structure of the overlapping dinucleosome, in which two nucleosomes are associated, at 3.14-angstrom resolution. In the overlapping dinucleosome structure, the unusual "hexasome" nucleosome, composed of the histone hexamer lacking one H2A-H2B dimer from the conventional histone octamer, contacts the canonical "octasome" nucleosome, and they intimately associate. Consequently, about 250 base pairs of DNA are left-handedly wrapped in three turns, without a linker DNA segment between the hexasome and octasome moieties. The overlapping dinucleosome structure may provide important information to understand how nucleosome repositioning occurs during the chromatin remodeling process..
15. Jun Ueda, Akihito Harada, Takashi Urahama, Shinichi Machida, Kazumitsu Maehara, Masashi Hada, Yoshinori Makino, Jumpei Nogami, Naoki Horikoshi, Akihisa Osakabe, Hiroyuki Taguchi, Hiroki Tanaka, Hiroaki Tachiwana, Tatsuma Yao, Minami Yamada, Takashi Iwamoto, Ayako Isotani, Masahito Ikawa, Taro Tachibana, Yuki Okada, Hiroshi Kimura, Yasuyuki Ohkawa, Hitoshi Kurumizaka, Kazuo Yamagata, Testis-Specific Histone Variant H3t Gene Is Essential for Entry into Spermatogenesis, Cell Reports, 10.1016/j.celrep.2016.12.065, 18, 3, 593-600, 2017.01, Cellular differentiation is associated with dynamic chromatin remodeling in establishing a cell-type-specific epigenomic landscape. Here, we find that mouse testis-specific and replication-dependent histone H3 variant H3t is essential for very early stages of spermatogenesis. H3t gene deficiency leads to azoospermia because of the loss of haploid germ cells. When differentiating spermatogonia emerge in normal spermatogenesis, H3t appears and replaces the canonical H3 proteins. Structural and biochemical analyses reveal that H3t-containing nucleosomes are more flexible than the canonical nucleosomes. Thus, by incorporating H3t into the genome during spermatogonial differentiation, male germ cells are able to enter meiosis and beyond..
16. Kensuke Kudou, Tetsuro Komatsu, Jumpei Nogami, Kazumitsu Maehara, Akihito Harada, Hiroshi Saeki, Eiji Oki, Yoshihiko Maehara, Yasuyuki Ohkawa, The requirement of Mettl3-promoted MyoD mRNA maintenance in proliferative myoblasts for skeletal muscle differentiation, Open Biology, 10.1098/rsob.170119, 7, 9, 2017.01, Myogenic progenitor/stem cells retain their skeletal muscle differentiation potential by maintaining myogenic transcription factors such as MyoD. However, the mechanism of how MyoD expression is maintained in proliferative progenitor cells has not been elucidated. Here, we found that MyoD expression was reduced at the mRNA level by cell cycle arrest in S and G2 phases, which in turn led to the absence of skeletal muscle differentiation. The reduction of MyoD mRNA was correlated with the reduced expression of factors regulating RNA metabolism, including methyltransferase like 3 (Mettl3), which induces N6-methyladenosine (m6A) modifications of RNA. Knockdown of Mettl3 revealed that MyoD RNA was specifically downregulated and that this was caused by a decrease in processed, but not unprocessed, mRNA. Potential m6A modification sites were profiled by m6A sequencing and identified within the 50 untranslated region (UTR) of MyoD mRNA. Deletion of the 50 UTR revealed that it has a role in MyoD mRNA processing. These data showed that Mettl3 is required for MyoD mRNA expression in proliferative myoblasts..
17. Yuki Kuniyoshi, Kazumitsu Maehara, Takeshi Iwasaki, Masayasu Hayashi, Yuichiro Semba, Masatoshi Fujita, Yuko Sato, Hiroshi Kimura, Akihito Harada, Yasuyuki Ohkawa, Identification of immunoglobulin gene sequences from a small read number of mRNA-seq using hybridomas, PLoS One, 10.1371/journal.pone.0165473, 11, 10, 2016.10, Identification of immunoglobulin genes in hybridomas is essential for producing antibodies for research and clinical applications. A couple of methods such as RACE and degenerative PCR have been developed for determination of the Igh and Igl/Igk coding sequences (CDSs) but it has been difficult to process a number of hybridomas both with accuracy and rapidness. Here, we propose a new strategy for antibody sequence determination by mRNA-seq of hybridomas. We demonstrated that hybridomas highly expressed the Igh and Igl/Igk genes and that de novo transcriptome assembly using mRNA-seq data enabled identification of the CDS of both Igh and Igl/Igk accurately. Furthermore, we estimated that only 30,000 sequenced reads are required to identify immunoglobulin sequences from four different hybridoma clones. Thus, our approach would facilitate determining variable CDSs drastically..
18. Kazumitsu Maehara, Akihito Harada, Yuko Sato, Masaki Matsumoto, Keiichi Nakayama, Hiroshi Kimura, Yasuyuki Ohkawa, Tissue-specific expression of histone H3 variants diversified after species separation, Epigenetics and Chromatin, 10.1186/s13072-015-0027-3, 8, 1, 2015.09, Background: The selective incorporation of appropriate histone variants into chromatin is critical for the regulation of genome function. Although many histone variants have been identified, a complete list has not been compiled. Results: We screened mouse, rat and human genomes by in silico hybridization using canonical histone sequences. In the mouse genome, we identified 14 uncharacterized H3 genes, among which 13 are similar to H3.3 and do not have human or rat counterparts, and one is similar to human testis-specific H3 variant, H3T/H3.4, and had a rat paralog. Although some of these genes were previously annotated as pseudogenes, their tissue-specific expression was confirmed by sequencing the 3′-UTR regions of the transcripts. Certain new variants were also detected at the protein level by mass spectrometry. When expressed as GFP-tagged versions in mouse C2C12 cells, some variants were stably incorporated into chromatin and the genome-wide distributions of most variants were similar to that of H3.3. Moreover, forced expression of H3 variants in chromatin resulted in alternate gene expression patterns after cell differentiation. Conclusions: We comprehensively identified and characterized novel mouse H3 variant genes that encoded highly conserved amino acid sequences compared to known histone H3. We speculated that the diversity of H3 variants acquired after species separation played a role in regulating tissue-specific gene expression in individual species. Their biological relevance and evolutionary aspect involving pseudogene diversification will be addressed by further functional analysis..
19. Akihito Harada, Chandrashekara Mallappa, Seiji Okada, John T. Butler, Stephen P. Baker, Jeanne B. Lawrence, Yasuyuki Ohkawa, Anthony N. Imbalzano, Spatial re-organization of myogenic regulatory sequences temporally controls gene expression, Nucleic Acids Research, 10.1093/nar/gkv046, 43, 4, 2008-2021, 2015.02, During skeletal muscle differentiation, the activation of some tissue-specific genes occurs immediately while others are delayed. The molecular basis controlling temporal gene regulation is poorly understood. We show that the regulatory sequences, but not other regions of genes expressed at late times of myogenesis, are in close physical proximity in differentiating embryonic tissue and in differentiating culture cells, despite these genes being located on different chromosomes. Formation of these inter-chromosomal interactions requires the lineage-determinant MyoD and functional Brg1, the ATPase subunit of SWI/SNF chromatin remodeling enzymes. Ectopic expression of myogenin and a specific Mef2 isoform induced myogenic differentiation without activating endogenous MyoD expression. Under these conditions, the regulatory sequences of late gene loci were not in close proximity, and these genes were prematurely activated. The data indicate that the spatial organization of late genes contributes to temporal regulation of myogenic transcription by restricting late gene expression during the early stages of myogenesis..
20. Akihito Harada, Kazumitsu Maehara, Yuko Sato, Daijiro Konno, Taro Tachibana, Hiroshi Kimura, Yasuyuki Ohkawa, Incorporation of histone H3.1 suppresses the lineage potential of skeletal muscle, Nucleic Acids Research, 10.1093/nar/gku1346, 43, 2, 775-786, 2015.01, Lineage potential is triggered by lineage-specific transcription factors in association with changes in the chromatin structure. Histone H3.3 variant is thought to play an important role in the regulation of lineage-specific genes. To elucidate the function of H3.3 in myogenic differentiation, we forced the expression of GFP-H3.1 to alter the balance between H3.1 and H3.3 in mouse C2C12 cells that could be differentiated into myotubes. GFP-H3.1 replaced H3.3 in the regulatory regions of skeletal muscle (SKM) genes and induced a decrease of H3K4 trimethylation (H3K4me3) and increase of H3K27 trimethylation (H3K27me3). Similar results were obtained by H3.3 knockdown. In contrast, MyoD-dependent H3.3 incorporation into SKM genes in fibroblasts induced an increase of H3K4me3 and H3K27me3. In mouse embryos, a bivalent modification of H3K4me3 and H3K27me3 was formed on H3.3-incorporated SKM genes before embryonic skeletal muscle differentiation. These results suggest that lineage potential is established through a selective incorporation of specific H3 variants that governs the balance of histone modifications..
21. Kazumitsu Maehara, Jun Odawara, Akihito Harada, Tomohiko Yoshimi, Koji Nagao, Chikashi Obuse, Koichi Akashi, Taro Tachibana, Toshio Sakata, Yasuyuki Ohkawa, A co-localization model of paired ChIP-seq data using a large ENCODE data set enables comparison of multiple samples, Nucleic Acids Research, 10.1093/nar/gks1010, 41, 1, 54-62, 2013.01, Deep sequencing approaches, such as chromatin immunoprecipitation by sequencing (ChIP-seq), have been successful in detecting transcription factor-binding sites and histone modification in the whole genome. An approach for comparing two different ChIP-seq data would be beneficial for predicting unknown functions of a factor. We propose a model to represent co-localization of two different ChIP-seq data. We showed that a meaningful overlapping signal and a meaningless background signal can be separated by this model. We applied this model to compare ChIP-seq data of RNA polymerase II C-terminal domain (CTD) serine 2 phosphorylation with a large amount of peak-called data, including ChIP-seq and other deep sequencing data in the Encyclopedia of DNA Elements (ENCODE) project, and then extracted factors that were related to RNA polymerase II CTD serine 2 in HeLa cells. We further analyzed RNA polymerase II CTD serine 7 phosphorylation, of which their function is still unclear in HeLa cells. Our results were characterized by the similarity of localization for transcription factor/histone modification in the ENCODE data set, and this suggests that our model is appropriate for understanding ChIP-seq data for factors where their function is unknown..
22. Akihito Harada, Seiji Okada, Daijiro Konno, Jun Odawara, Tomohiko Yoshimi, Saori Yoshimura, Hiromi Kumamaru, Hirokazu Saiwai, Toshiaki Tsubota, Hitoshi Kurumizaka, Koichi Akashi, Taro Tachibana, Anthony N. Imbalzano, Yasuyuki Ohkawa, Chd2 interacts with H3.3 to determine myogenic cell fate, EMBO Journal, 10.1038/emboj.2012.136, 31, 13, 2994-3007, 2012.07, Cell differentiation is mediated by lineage-determining transcription factors. We show that chromodomain helicase DNA-binding domain 2 (Chd2), a SNF2 chromatin remodelling enzyme family member, interacts with MyoD and myogenic gene regulatory sequences to specifically mark these loci via deposition of the histone variant H3.3 prior to cell differentiation. Directed and genome-wide analysis of endogenous H3.3 incorporation demonstrates that knockdown of Chd2 prevents H3.3 deposition at differentiation-dependent, but not housekeeping, genes and inhibits myogenic gene activation. The data indicate that MyoD determines cell fate and facilitates differentiation-dependent gene expression through Chd2-dependent deposition of H3.3 at myogenic loci prior to differentiation..
Works, Software and Database
1. .
1. 大川 恭行, The baselines of transcription levels are determined by selective incorporation of histone H3 variants., Transcriptional and Epigenetic Control in Stem Cells (J1), 2017.01, Selective gene expression in cell differentiation is initiated by the binding of tissue-specific transcription factors (TFs).
Chromatin structure is known to tune the function of bound TFs by nucleosome positioning, histone modification, and the
incorporation of histone variants [1]. However the specific contribution of each component of chromatin has been
We recently reported that we identified hitherto unknown fourteen histone H3 variants and that they were detected in a
variety of tissues [2]. Our functional analysis revealed that H3mm7, one of the newly discovered histone H3 variants, was
required for skeletal muscle differentiation. H3mm7 knockout in C2C12 myoblasts resulted in rigid chromatin structure
and in the reduction of transcription levels of a subset of the genes into which H3mm7 was incorporated. The data
suggest that H3mm7 and other variants contribute to set the baselines of transcription levels. I will discuss other recent
advances in understanding the novel histone H3 variants.
[1] Harada, A. et al. (2015) Nucleic Acids Res. 43, 775. Incorporation of histone H3.1 suppresses the lineage potential of
skeletal muscle.
[2] Maehara, K. et al. (2016) Epigenetics Chromatin. 8, 35. Tissue-specific expression of histone H3 variants diversified
after species separation..
2. 大川 恭行, N6-methyladenosine is required for the processing of MyoD pre-mRNA for maintaining skeletal muscle differentiation potential., Keystone Symposia Chromatin and Epigenetics (C2), 2016.03.
3. 大川 恭行, Cell fate decision on chromatin by MyoD, BMB2010(第38回日本分子生物学会年会・第88回日本生化学会大会合同大会), 2015.12.
4. 大川 恭行, The Diversity of Mouse Histone H3 Variants, International Symposium on Chromatin Structure, Dynamics, and Function, 2015.08.
5. 原田 哲仁, 前原 一満, 佐藤 優子, 木村 宏, 大川 恭行, Incorporation of Histone H3 Variants Dictates the Lineage Potential of Skeletal Muscle, FASEB;Science Research Conferences, 2014.07.
6. 前原 一満, 大川 恭行, Exploring nucleosome positioning patterns act as a functional component of chromatin structure, ENBO Workshop Histone varisnts, 2014.06.
7. 原田 哲仁, 前原 一満, 大川 恭行, Incorporation of Histone H3 Variants Dictates Myogenesis, ENBO Workshop Histone varisnts, 2014.06.
8. 大川 恭行, Diversity of histone H3 variants on mouse genome, ENBO Workshop Histone varisnts, 2014.06.
9. 原田 哲仁, 前原 一満, 大川 恭行, The balance of histone H3 variants around transcription start sites dictates cell differential potential, Keystone Symposia , 2014.02.
10. 前原 一満, 大川 恭行, The formation of nucleosome positioning patterns flanked by transcription factor binding site, Keystone Symposia , 2014.02.
11. 大川 恭行, Epigenomic approach unveils cell fate decision, 日本遺伝学会第85回大会, 2013.09.
12. 大川 恭行, High order chromatin regulation in skeletal muscle differentiation, 第86回日本生化学会大会, 2013.09.
13. 大川 恭行, Histone bariants determine the lineage potential of skeletal muscle, Fukuoka Internasional Symposium on Genomics & Epigenomics 2013, 2013.09.
14. Genomic clustering accompanies cellular differentiation to temporally control gene expression.
Membership in Academic Society
  • ​Japan Muscle Society
  • about The Japanese Biochemical Sicuety
  • Structural Epigenomics Association
  • Japanese Society for Epigenetics
  • Japanese Society for bioinformatics
  • Japan Society for Biology
  • The Molecular Biology Society of Japan