九州大学-研究者情報 [原田哲仁 (准教授) 生体防御医学研究所附属高深度オミクスサイエンスセンター]

1.	†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, doi: 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..
2.	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, [URL], 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..
3.	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, 2018.12, [URL], 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..
4.	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, [URL], 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..
5.	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, [URL], 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..
6.	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, [URL], 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..
7.	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, [URL], 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..
8.	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, [URL], 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..
9.	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, [URL], 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..

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Akihito Harada, Kazumitsu Maehara, Yasuyuki Ohkawa, High-throughput single cell epigenomic profiling using chromatin integration labelling-sequence, Keystone Symposia on Molecular and Cellular Biology, 2019.03, Analysis of chromatin states is regularly performed in development and disease research of multicellular organisms. The standard method of epigenomic analysis has been chromatin immunoprecipitation followed by sequencing (ChIP-seq) which is a powerful technique capable of unveiling protein-DNA interactions on the whole genome. There has been a recent development of the method allowing sequencing from single cells, which could facilitate the analysis of clinical samples as well as rare cell populations such as stem cells. However, the single-cell ChIP protocol employs microfluidic devices and hence still requires a great number of cells. Recently, we have established a new immunoprecipitation-free epigenomic profiling method based on immunostaining named chromatin integration labeling followed by sequencing (ChIL-seq). While ChIL-seq was demonstrated to detect histone modifications at the single-cell level, the current published protocol is not cost-effective for high-throughput assays because all the reactions need to be performed separately. We report on our progress on the further development of single-cell ChIL-seq at high-throughput..

Harada A, Functional analysis of novel histone variant H3mm13 in skeletal muscle to Histone variants, EMBO Workshop Histone Variants, 2017.06.

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特許出願件数 2件

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学会活動

所属学会名

日本エピジェネティクス研究会

日本農芸化学会

日本分子生物学会

学会大会・会議・シンポジウム等における役割

2022.06.09～2022.06.10, 第15回日本エピジェネティクス研究会年会, 組織委員.

受賞

公益財団法人福岡県すこやか健康事業団平成28年度がん研究助成金優秀賞, 公益財団法人福岡県すこやか健康事業団, 2017.01.

研究資金

科学研究費補助金の採択状況(文部科学省、日本学術振興会)

2023年度～2025年度, 基盤研究(B), 代表, 精子形成における体細胞型H3から精子型H3T/tへのヒストン置換の意義の解明.

2022年度～2024年度, 基盤研究(C), 分担, 無精子症における精巣内内分泌環境に着目した単一細胞トランスクリプトーム解析.

2021年度～2025年度, 学術変革領域研究(A), 代表, 細胞競合における細胞間相互作用を計測するための空間オミクス技術開発.

2021年度～2025年度, 学術変革領域研究(A), 分担, 競合的コミュニケーションから迫る多細胞生命システムの自律性.

2021年度～2022年度, 新学術領域研究, 代表, 革新的Perturb-ChIL法を用いた網羅的なクロマチン構造解析研究.

2020年度～2021年度, 新学術領域研究, 代表, 全能性獲得機構の解明のためのmulti-omics技術の開発.

2019年度～2020年度, 新学術領域研究, 代表, 単一細胞multi-omicsによるシンギュラリティー細胞同定技術の開発.

2019年度～2021年度, 基盤研究(B), 代表, 精子特異的ヒストンバリアントH3T/tを起点としたクロマチンダイナミクスの解明.

2019年度～2021年度, 基盤研究(C), 分担, 単一細胞遺伝子解析によるヒト造精機能障害の分子機構の解明.

2018年度～2019年度, 挑戦的研究（萌芽）, 代表, 単一細胞超高解像度解析を可能にするゲノム3次元構造解析技術の開発.

2016年度～2017年度, 新学術領域研究, 代表, 生殖細胞におけるヒストンバリアントによるゲノムマーキング機構の解明.

2015年度～2016年度, 若手研究(B), 代表, 骨格筋分化における新規ヒストンH3バリアントH3mm7の遺伝子選択機構の解明.

競争的資金(受託研究を含む)の採択状況

2020年度～2022年度, 国立研究開発法人日本医療研究開発機構, 分担, 単一細胞解析によるヘルペスウイルス慢性感染の分子基盤の解明.

2019年度～2022年度, 戦略的創造研究推進事業 (文部科学省), 代表, 組織特異的ゲノム構造の再構築技術の開発.

2016年度～2018年度, 公益財団法人福岡県すこやか健康事業団平成28年度がん研究助成金, 代表, エピジェネティック破綻により引き起こされた筋肉腫の解析.

九大関連コンテンツ

pure2017年10月2日から、「九州大学研究者情報」を補完するデータベースとして、Elsevier社の「Pure」による研究業績の公開を開始しました。

QIR　九州大学学術情報リポジトリシステム情報科学研究院

生体防御医学研究所


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