SADO Takashi

Researcher Information

Degree

• Ph. D.

URL

https://jglobal.jst.go.jp/detail?JGLOBAL_ID=200901025435256075

ORCID ID

•  https://orcid.org/0000-0002-1232-0250

J-Global ID

• 200901025435256075

Research Interests

• クロマチン   ノンコーディングRNA   エピジェネティクス   X染色体不活性化   Epigenetics   DNA Methylation   X-inactivation   

Research Areas

• Life sciences / Genetics

Academic & Professional Experience

• 2014/04 - Today  Kindai UniversityFaculty of Agriculture教授
• 2009/05 - 2014/03  Medical Institute of Bioregulation, Kyushu Univ.Associate professor
• 2000/04 - 2009/04  The Graduate University for Advanced Studies
• 1999/05 - 2009/04  National Institute of Genetics

Education

• 1989/04 - 1995/06  北海道大学大学院理学研究科動物学専攻
• 1985/04 - 1989/03  Hokkaido University  School of Science  生物学科（動物）
•        -   Hokkaido University

Association Memberships

• 日本エピジェネティクス研究会   日本RNA学会   日本分子生物学会   日本遺伝学会   The Molecular Biology Society of Japan   The Genetics Society of Japan   

Published Papers

• Replication dynamics identifies the folding principles of the inactive X chromosome

  Rawin Poonperm; Saya Ichihara; Hisashi Miura; Akie Tanigawa; Koji Nagao; Chikashi Obuse; Takashi Sado; Ichiro Hiratani

  Nature Structural & Molecular Biology 30 (8) 1224 - 1237 2023/08 [Refereed]
   

  Chromosome-wide late replication is an enigmatic hallmark of the inactive X chromosome (Xi). How it is established and what it represents remains obscure. By single-cell DNA replication sequencing, here we show that the entire Xi is reorganized to replicate rapidly and uniformly in late S-phase during X-chromosome inactivation (XCI), reflecting its relatively uniform structure revealed by 4C-seq. Despite this uniformity, only a subset of the Xi became earlier replicating in SmcHD1-mutant cells. In the mutant, these domains protruded out of the Xi core, contacted each other and became transcriptionally reactivated. 4C-seq suggested that they constituted the outermost layer of the Xi even before XCI and were rich in escape genes. We propose that this default positioning forms the basis for their inherent heterochromatin instability in cells lacking the Xi-binding protein SmcHD1 or exhibiting XCI escape. These observations underscore the importance of 3D genome organization for heterochromatin stability and gene regulation.
• SmcHD1 underlies the formation of H3K9me3 blocks on the inactive X chromosome in mice

  Saya Ichihara; Koji Nagao; Takehisa Sakaguchi; Chikashi Obuse; Takashi Sado

  Development 2022/08 [Refereed]
  
• Ectopic Splicing Disturbs the Function of Xist RNA to Establish the Stable Heterochromatin State

  Ruka Matsuura; Tatsuro Nakajima; Saya Ichihara; Takashi Sado

  Frontiers in Cell and Developmental Biology Frontiers Media SA 9 2021/10 [Refereed]
   

  Non-coding Xist RNA plays an essential role in X chromosome inactivation (XCI) in female mammals. It coats the X chromosome in cis and mediates the recruitment of many proteins involved in gene silencing and heterochromatinization. The molecular basis of how Xist RNA initiates chromosomal silencing and what proteins participate in this process has been extensively studied and elucidated. Its involvement in the establishment and maintenance of the X-inactivated state is, however, less understood. The Xist^IVS allele we previously reported is peculiar in that it can initiate XCI but fails to establish the inactive state that is stably maintained and, therefore, may provide an opportunity to explore how Xist RNA contributes to establish a robust heterochromatin state. Here we demonstrate that ectopic splicing taking place to produce Xist^IVS RNA disturbs its function to properly establish stable XCI state. This finding warrants the potential of Xist^IVS RNA to provide further insight into our understanding of how Xist RNA contributes to establish sustainable heterochromatin.
• Does XIST safeguard against sex-biased human diseases?

  Takashi Sado

  Molecular cell 81 (8) 1598 - 1600 2021/04 [Invited]
   

  Yu et al. (2021) demonstrate that a subset of X-linked immune genes is repressed on the inactive X chromosome (Xi) in a manner dependent on XIST RNA in B cells, and derepression of these genes upon XIST depletion could bias differentiation of naive B cells and be involved in etiology of female-biased autoimmune diseases.
• Characterization of genetic-origin-dependent monoallelic expression in mouse embryonic stem cells.

  Hiroaki Ohishi; Wan Kin Au Yeung; Motoko Unoki; Kenji Ichiyanagi; Kei Fukuda; Shoji Maenohara; Kenjiro Shirane; Hatsune Chiba; Takashi Sado; Hiroyuki Sasaki

  Genes to cells : devoted to molecular & cellular mechanisms 25 (1) 54 - 64 2020/01 [Refereed]
   

  Monoallelic gene expression occurs in various mammalian cells and can be regulated genetically, epigenetically and/or stochastically. We identified 145 monoallelically expressed genes (MoEGs), including seven known imprinted genes, in mouse embryonic stem cells (ESCs) derived from reciprocal F1 hybrid blastocysts and cultured in 2i/LIF. As all MoEGs except for the imprinted genes were expressed in a genetic-origin-dependent manner, we focused on this class of MoEGs for mechanistic studies. We showed that a majority of the genetic-origin-dependent MoEGs identified in 2i/LIF ESCs remain monoallelically expressed in serum/LIF ESCs, but become more relaxed or even biallelically expressed upon differentiation. These MoEGs and their regulatory regions were highly enriched for single nucleotide polymorphisms. In addition, some MoEGs were associated with retrotransposon insertions/deletions, consistent with the fact that certain retrotransposons act as regulatory elements in pluripotent stem cells. Interestingly, most MoEGs showed allelic differences in enrichment of histone H3K27me and H3K4me marks, linking allelic epigenetic differences and monoallelic expression. In contrast, there was little or no allelic difference in CpG methylation or H3K9me. Taken together, our study highlights the impact of genetic variation including single nucleotide polymorphisms and retrotransposon insertions/deletions on monoallelic epigenetic marks and expression in ESCs.
• Publisher Correction: Female mice lacking Ftx lncRNA exhibit impaired X-chromosome inactivation and a microphthalmia-like phenotype.

  Hosoi Y; Soma M; Shiura H; Sado T; Hasuwa H; Abe K; Kohda T; Ishino F; Kobayashi S

  Nature communications 9 (1) 4618  2018/10 [Refereed]
  
• Female mice lacking Ftx lncRNA exhibit impaired X-chromosome inactivation and a microphthalmia-like phenotype.

  Hosoi, Y; Soma M; Shiura, H; Sado, T; Hasuwa, H; Abe, K; Kohda, K; Ishino, F; Kobayashi, S

  Nat. Commun. 9: 3829 2018/09 [Refereed]
  
• Role of SmcHD1 in establishment of epigenetic states required for the maintenance of the X-inactivated state in mice.

  Sakakibara Y; Nagao K; Blewitt M; Sasaki H; Obuse C; Sado T

  Development (Cambridge, England) 145: dev166462 doi: 10.1242/dev.166462 0950-1991 2018/08 [Refereed]
  
• The 5 ' region of Xist RNA has the potential to associate with chromatin through the A-repeat

  Yuta Chigi; Hiroyuki Sasaki; Takashi Sado

  RNA COLD SPRING HARBOR LAB PRESS, PUBLICATIONS DEPT 23 (12) 1894 - 1901 1355-8382 2017/12 [Refereed]
   

  X inactive-specific transcript (Xist) is a long noncoding RNA that plays an essential role in X chromosome inactivation. Although Xist RNA, like common protein-coding mRNAs, is transcribed by RNA polymerase II, spliced and polyadenylated, it is retained in the nucleus and associates with the X chromosome it originates from. It has been assumed that Xist RNA recruits proteins involved in epigenetic modifications and chromatin compaction to the X chromosome. One of the major proteins constituting the nuclear matrix, hnRNP U, has been shown to be required for the association of Xist RNA with the inactive X chromosome (Xi). In this study, we found that the first 950-nt sequence of Xist RNA had the potential to associate with chromatin in a manner independent of hnRNP U. Furthermore, its chromatin association is apparently dependent on the presence of an intact A-repeat sequence, which is one of the repeats in Xist/XIST RNA conserved among many mammalian species, and has been shown to be important for Xist RNA-mediated silencing. Taking this unexpected finding and a previous study demonstrating the effect of Xist RNA lacking the A-repeat on the formation of the silent heterochromatin domain together, we suggest that the A-repeat captures chromatin near the initial loading site of Xist RNA and relocates it into the core of the heterochromatin domain.
• What makes the maternal X chromosome resistant to undergoing imprinted X inactivation?

  Takashi Sado

  PHILOSOPHICAL TRANSACTIONS OF THE ROYAL SOCIETY B-BIOLOGICAL SCIENCES ROYAL SOC 372 (1733) 0962-8436 2017/11 [Refereed]
   

  In the mouse, while either X chromosome is chosen for inactivation in a random fashion in the embryonic tissue, the paternally derived X chromosome is preferentially inactivated in the extraembryonic tissues. It has been shown that the maternal X chromosome is imprinted so as not to undergo inactivation in the extraembryonic tissues. X-linked noncoding Xist RNA becomes upregulated on the X chromosome that is to be inactivated. An antisense noncoding RNA, Tsix, which occurs at the Xist locus and has been shown to negatively regulate Xist expression in cis, is imprinted to be expressed from the maternal X in the extraembryonic tissues. Although Tsix appears to be responsible for the imprint laid on the maternal X, those who disagree with this idea would point out the fact that Tsix has not yet been expressed from the maternal X when Xist becomes upregulated on the paternal but not the maternal X at the onset of imprinted X-inactivation in preimplantation embryos. Recent studies have demonstrated, however, that there is a prominent difference in the chromatin structure at the Xist locus depending on the parental origin, which I suggest might account for the repression of maternal Xist in the absence of maternal Tsix at the preimplantation stages. This article is part of the themed issue 'X-chromosome inactivation: a tribute to Mary Lyon'.
• Defects in dosage compensation impact global gene regulation in the mouse trophoblast

  Yuka Sakata; Koji Nagao; Yuko Hoki; Hiroyuki Sasaki; Chikashi Obuse; Takashi Sado

  DEVELOPMENT COMPANY OF BIOLOGISTS LTD 144 (15) 2784 - 2797 0950-1991 2017/08 [Refereed]
   

  Xist RNA, which is responsible for X inactivation, is a key epigenetic player in the embryogenesis of female mammals. Of the several repeats conserved in Xist RNA, the A-repeat has been shown to be essential for its silencing function in differentiating embryonic stem cells. Here, we introduced a new Xist allele into mouse that produces mutated Xist RNA lacking the A-repeat (Xist(CAG Delta 5')). Xist(CAG Delta 5') RNA expressed in the embryo coated the X chromosome but failed to silence it. Although imprinted X inactivation was substantially compromised upon paternal transmission, allele-specific RNA-seq in the trophoblast revealed that Xist(CAG Delta 5') RNA still retained some silencing ability. Furthermore, the failure of imprinted X inactivation had more significant impacts than expected on genome-wide gene expression. It is likely that dosage compensation is required not only for equalizing X-linked gene expression between the sexes but also for proper global gene regulation in differentiated female somatic cells.
• Maintenance of Xist Imprinting Depends on Chromatin Condensation State and Rnf12 Dosage in Mice

  Atsushi Fukuda; Atsushi Mitani; Toshiyuki Miyashita; Takashi Sado; Akihiro Umezawa; Hidenori Akutsu

  PLOS GENETICS PUBLIC LIBRARY SCIENCE 12 (10) 1553-7404 2016/10 [Refereed]
   

  In female mammals, activation of Xist (X-inactive specific transcript) is essential for establishment of X chromosome inactivation. During early embryonic development in mice, paternal Xist is preferentially expressed whereas maternal Xist (Xm-Xist) is silenced. Unlike autosomal imprinted genes, Xist imprinting for Xm-Xist silencing was erased in cloned or parthenogenetic but not fertilized embryos. However, the molecular mechanism underlying the variable nature of Xm-Xist imprinting is poorly understood. Here, we revealed that Xm-Xist silencing depends on chromatin condensation states at the Xist/Tsix genomic region and on Rnf12 expression levels. In early preimplantation, chromatin decondensation via H3K9me3 loss and histone acetylation gain caused Xm-Xist derepression irrespective of embryo type. Although the presence of the paternal genome during pronuclear formation impeded Xm-Xist derepression, Xm-Xist was robustly derepressed when the maternal genome was decondensed before fertilization. Once Xm-Xist was derepressed by chromatin alterations, the derepression was stably maintained and rescued XmXp. lethality, indicating that loss of Xm-Xist imprinting was irreversible. In late preimplantation, Oct4 served as a chromatin opener to create transcriptional permissive states at Xm-Xist/Tsix genomic loci. In parthenogenetic embryos, Rnf12 overdose caused Xm-Xist derepression via Xm-Tsix repression; physiological Rnf12 levels were essential for Xm-Xist silencing maintenance in fertilized embryos. Thus, chromatin condensation and fine-tuning of Rnf12 dosage were crucial for Xist imprint maintenance by silencing Xm-Xist.
• Control of Chromosomal Localization of Xist by hnRNP U Family Molecules

  Takehisa Sakaguchi; Yuko Hasegawa; Neil Brockdorff; Ken Tsutsui; Kimiko M. Tsutsui; Takashi Sado; Shinichi Nakagawa

  DEVELOPMENTAL CELL CELL PRESS 39 (1) 11 - 12 1534-5807 2016/10 [Refereed]
  
• Maintenance of Xist Imprinting Depends on Chromatin Condensation State and Rnf12 Dosage in Mice

  Atsushi Fukuda; Atsushi Mitani; Toshiyuki Miyashita; Takashi Sado; Akihiro Umezawa; Hidenori Akutsu

  PLoS Genetics Public Library of Science 12 (10) e1006375  1553-7404 2016/10 [Refereed]
   

  In female mammals, activation of Xist (X-inactive specific transcript) is essential for establishment of X chromosome inactivation. During early embryonic development in mice, paternal Xist is preferentially expressed whereas maternal Xist (Xm-Xist) is silenced. Unlike autosomal imprinted genes, Xist imprinting for Xm-Xist silencing was erased in cloned or parthenogenetic but not fertilized embryos. However, the molecular mechanism underlying the variable nature of Xm-Xist imprinting is poorly understood. Here, we revealed that Xm-Xist silencing depends on chromatin condensation states at the Xist/Tsix genomic region and on Rnf12 expression levels. In early preimplantation, chromatin decondensation via H3K9me3 loss and histone acetylation gain caused Xm-Xist derepression irrespective of embryo type. Although the presence of the paternal genome during pronuclear formation impeded Xm-Xist derepression, Xm-Xist was robustly derepressed when the maternal genome was decondensed before fertilization. Once Xm-Xist was derepressed by chromatin alterations, the derepression was stably maintained and rescued XmXpΔ lethality, indicating that loss of Xm-Xist imprinting was irreversible. In late preimplantation, Oct4 served as a chromatin opener to create transcriptional permissive states at Xm-Xist/Tsix genomic loci. In parthenogenetic embryos, Rnf12 overdose caused Xm-Xist derepression via Xm-Tsix repression physiological Rnf12 levels were essential for Xm-Xist silencing maintenance in fertilized embryos. Thus, chromatin condensation and fine-tuning of Rnf12 dosage were crucial for Xist imprint maintenance by silencing Xm-Xist.
• A new Xist allele driven by a constitutively active promoter is dominated by Xist locus environment and exhibits the parent-of-origin effects

  Yuko Amakawa; Yuka Sakata; Yuko Hoki; Satoru Arata; Seiji Shioda; Tatsuo Fukagawa; Hiroyuki Sasaki; Takashi Sado

  DEVELOPMENT COMPANY OF BIOLOGISTS LTD 142 (24) 4299 - 4308 0950-1991 2015/12 [Refereed]
   

  The dosage difference of X-linked genes between the sexes in mammals is compensated for by genetic inactivation of one of the X chromosomes in XX females. A noncoding RNA transcribed from the Xist gene at the onset of X chromosome inactivation coats the X chromosome in cis and induces chromosome-wide heterochromatinization. Here, we report a new Xist allele (Xist(CAG)) driven by a CAG promoter, which is known to be constitutively active in many types of cells. The paternal transmission of Xist(CAG) resulted in the preferential inactivation of the targeted paternal X (Xp) not only in the extra-embryonic but also the embryonic lineage, whereas maternal transmission ended with embryonic lethality at the early postimplantation stage with a phenotype that resembled mutant embryos carrying a maternal deficiency in Tsix, an antisense negative regulator of Xist, in both sexes. Interestingly, we found that the upregulation of Xist(CAG) in preimplantation embryos temporally differed depending on its parental origin: its expression started at the 4- to 8-cell stages when paternally inherited, and Xist(CA G) was upregulated at the blastocyst stage when maternally inherited. This might indicate that the Xist locus on Xp is permissive to transcription, but the Xist locus on the maternal X (Xm) is not. We extrapolated from these findings that the maternal Xist allele might manifest a chromatin structure inaccessible by transcription factors relative to the paternal allele. This might underlie the mechanism for the maternal repression of Xist at the early cleavage stage when Tsix expression has not yet occurred on Xm.
• An Xist-activating antisense RNA required for X-chromosome inactivation

  Mrinal K. Sarkar; Srimonta Gayen; Surinder Kumar; Emily Maclary; Emily Buttigieg; Michael Hinten; Archana Kumari; Clair Harris; Takashi Sado; Sundeep Kalantry

  NATURE COMMUNICATIONS NATURE PUBLISHING GROUP 6 8564 - ST008028 2041-1723 2015/10 [Refereed]
   

  The transcriptional imbalance due to the difference in the number of X chromosomes between male and female mammals is remedied through X-chromosome inactivation, the epigenetic transcriptional silencing of one of the two X chromosomes in females. The X-linked Xist long non-coding RNA functions as an X inactivation master regulator; Xist is selectively upregulated from the prospective inactive X chromosome and is required in cis for X inactivation. Here we discover an Xist antisense long non-coding RNA, XistAR (Xist Activating RNA), which is encoded within exon 1 of the mouse Xist gene and is transcribed only from the inactive X chromosome. Selective truncation of XistAR, while sparing the overlapping Xist RNA, leads to a deficiency in Xist RNA expression in cis during the initiation of X inactivation. Thus, the Xist gene carries within its coding sequence an antisense RNA that drives Xist expression.
• X Chromosome Reactivation Dynamics Reveal Stages of Reprogramming to Pluripotency

  Vincent Pasque; Jason Tchieu; Rahul Karnik; Molly Uyeda; Anupama Sadhu Dimashkie; Dana Case; Bernadett Papp; Giancarlo Bonora; Sanjeet Patel; Ritchie Ho; Ryan Schmidt; Robin McKee; Takashi Sado; Takashi Tada; Alexander Meissner; Kathrin Plath

  CELL CELL PRESS 159 (7) 1681 - 1697 0092-8674 2014/12 [Refereed]
   

  Reprogramming to iPSCs resets the epigenome of somatic cells, including the reversal of X chromosome inactivation. We sought to gain insight into the steps underlying the reprogramming process by examining the means by which reprogramming leads to X chromosome reactivation (XCR). Analyzing single cells in situ, we found that hallmarks of the inactive X (Xi) change sequentially, providing a direct readout of reprogramming progression. Several epigenetic changes on the Xi occur in the inverse order of developmental X inactivation, whereas others are uncoupled from this sequence. Among the latter, DNA methylation has an extraordinary long persistence on the Xi during reprogramming, and, like Xist expression, is erased only after pluripotency genes are activated. Mechanistically, XCR requires both DNA demethylation and Xist silencing, ensuring that only cells undergoing faithful reprogramming initiate XCR. Our study defines the epigenetic state of multiple sequential reprogramming intermediates and establishes a paradigm for studying cell fate transitions during reprogramming.
• The CENP-O complex requirement varies among different cell types

  Naoko Kagawa; Tetsuya Hori; Yuko Hoki; Osamu Hosoya; Kimiko Tsutsui; Yumiko Saga; Takashi Sado; Tatsuo Fukagawa

  CHROMOSOME RESEARCH SPRINGER 22 (3) 293 - 303 0967-3849 2014/09 [Refereed]
   

  CENP-U (CENP-50) is a component of the CENP-O complex, which includes CENP-O, CENP-P, CENP-Q, CENP-R, and CENP-U and is constitutively localized at kinetochores throughout the cell cycle in vertebrates. Although CENP-U deficiency results in some mitotic defects in chicken DT40 cells, CENP-U-deficient chicken DT40 cells are viable. To examine the functional roles of CENP-U in an organism-dependent context, we generated CENP-U-deficient mice. The CENP-U-deficient mice died during early embryogenesis (approximately E7.5). Thus, conditional CENP-U-deficient mouse ES cells were generated to analyze CENP-U-deficient phenotypes at the cell level. When CENP-U was disrupted in the mouse ES cells, all CENP-O complex proteins disappeared from kinetochores. In contrast, other kinetochore proteins were recruited in CENP-U-deficient mouse ES cells as CENP-U-deficient DT40 cells. However, the CENP-U-deficient ES cells died after exhibiting abnormal mitotic behavior. Although CENP-U was essential for cell viability during mouse early embryogenesis, CENP-U-deficient mouse embryonic fibroblast cells were viable, similar to the DT40 cells. Thus, although both DT40 and ES cells with CENP-U deficiency have similar mitotic defects, cellular responses to mitotic defects vary among different cell types.
• Current view of the potential roles of proteins enriched on the inactive X chromosome

  Tatsuro Nakajima; Takashi Sado

  GENES & GENETIC SYSTEMS GENETICS SOC JAPAN 89 (4) 151 - 157 1341-7568 2014/08 [Refereed]
   

  X chromosome inactivation (X-inactivation) is triggered by X-linked noncoding Xist RNA, which is expressed asymmetrically from one of the two X chromosomes in females and coats it in cis to induce chromosome-wide silencing. Xist RNA is thought to play a role as a platform in recruiting proteins involved in gene silencing and heterochromatinization, which mediate serial changes in epigenetic modification of the chromatin. During the last two decades, many proteins have been shown to be enriched on the inactivated X chromosome in mouse and human. Although the biological significance of most of them for X-inactivation has not been fully established, extensive studies of these proteins should provide a better understanding of the molecular basis of how X-inactivation mediated by Xist RNA is regulated. Here, we review the potential roles of some of these proteins in the stepwise process of Xist RNA-mediated chromosome silencing.
• Histone Variants Enriched in Oocytes Enhance Reprogramming to Induced Pluripotent Stem Cells

  Toshie Shinagawa; Tsuyoshi Takagi; Daisuke Tsukamoto; Chinatsu Tomaru; Linh My Huynh; Padavattan Sivaraman; Thirumananseri Kumarevel; Kimiko Inoue; Ryuichiro Nakato; Yuki Katou; Takashi Sado; Satoru Takahashi; Atsuo Ogura; Katsuhiko Shirahige; Shunsuke Ishii

  CELL STEM CELL CELL PRESS 14 (2) 217 - 227 1934-5909 2014/02 [Refereed]
   

  Expression of Oct3/4, Sox2, Klf4, and c-Myc (OSKM) can reprogram somatic cells into induced pluripotent stem cells (iPSCs). Somatic cell nuclear transfer (SCNT) can also be used for reprogramming, suggesting that factors present in oocytes could potentially augment OSKM-mediated induction of pluripotency. Here, we report that two histone variants, TH2A and TH2B, which are highly expressed in oocytes and contribute to activation of the paternal genome after fertilization, enhance OSKM-dependent generation of iPSCs and can induce reprogramming with Klf4 and Oct3/4 alone. TH2A and TH2B are enriched on the X chromosome during the reprogramming process, and their expression in somatic cells increases the DNase I sensitivity of chromatin. In addition, Xist deficiency, which was reported to enhance SCNT reprogramming efficiency, stimulates iPSC generation using TH2A/TH2B in conjunction with OSKM, but not OSKM alone. Thus, TH2A/TH2B may enhance reprogramming by introducing processes that normally operate in zygotes and during SCNT.
• Species-specific differences in X chromosome inactivation in mammals

  Takashi Sado; Takehisa Sakaguchi

  REPRODUCTION BIOSCIENTIFICA LTD 146 (4) R131 - R139 1470-1626 2013/10 [Refereed]
   

  In female mammals, the dosage difference in X-linked genes between XX females and XY males is compensated for by inactivating one of the two X chromosomes during early development. Since the discovery of the X inactive-specific transcript (XIS I) gene in humans and its subsequent isolation of the mouse homolog, Xist, in the early 1990s, the molecular basis of X chromosome inactivation (X-inactivation) has been more fully elucidated using genetically manipulated mouse embryos and embryonic stem cells. Studies on X-inactivation in other mammals, although limited when compared with those in the mice, have revealed that, while their inactive X chromosome shares many features with those in the mice, there are marked differences in not only some epigenetic modifications of the inactive X chromosome but also when and how X-inactivation is initiated during early embryonic development. Such differences raise the issue about what extent of the molecular basis of X-inactivation in the mice is commonly shared among others. Recognizing similarities and differences in X-inactivation among mammals may provide further insight into our understanding of not only the evolutionary but also the molecular aspects for the mechanism of X-inactivation. Here, we reviewed species-specific differences in X-inactivation and discussed what these differences may reveal.
• Targeted gene silencing in mouse germ cells by insertion of a homologous DNA into a piRNA generating locus.

  Yasuhiro Yamamoto; Toshiaki Watanabe; Yuko Hoki; Kenjiro Shirane; Yufeng Li; Kenji Ichiiyanagi; Satomi Kuramochi-Miyagawa; Atsushi Toyoda; Asao Fujiyama; Masayuki Oginuma; Hitomi Suzuki; Takashi Sado; Toru Nakano; Hiroyuki Sasaki

  Genome research 2 23 (2) 292 - 9 1088-9051 2013/02 [Refereed]
   

  In germ cells, early embryos, and stem cells of animals, PIWI-interacting RNAs (piRNAs) have an important role in silencing retrotransposons, which are vicious genomic parasites, through transcriptional and post-transcriptional mechanisms. To examine whether the piRNA pathway can be used to silence genes of interest in germ cells, we have generated knock-in mice in which a foreign DNA fragment was inserted into a region generating pachytene piRNAs. The knock-in sequence was transcribed, and the resulting RNA was processed to yield piRNAs in postnatal testes. When reporter genes possessing a sequence complementary to portions of the knock-in sequence were introduced, they were greatly repressed after the time of pachytene piRNA generation. This repression mainly occurred at the post-transcriptional level, as degradation of the reporter RNAs was accelerated. Our results show that the piRNA pathway can be used as a tool for sequence-specific gene silencing in germ cells and support the idea that the piRNA generating regions serve as traps for retrotransposons, enabling the host cell to generate piRNAs against active retrotransposons.
• Advances in understanding chromosome silencing by the long non-coding RNA Xist

  Takashi Sado; Neil Brockdorff

  PHILOSOPHICAL TRANSACTIONS OF THE ROYAL SOCIETY B-BIOLOGICAL SCIENCES ROYAL SOC 368 (1609) 20110325  0962-8436 2013/01 [Refereed]
   

  In female mammals, one of the two X chromosomes becomes genetically silenced to compensate for dosage imbalance of X-linked genes between XX females and XY males. X chromosome inactivation (X-inactivation) is a classical model for epigenetic gene regulation in mammals and has been studied for half a century. In the last two decades, efforts have been focused on the X inactive-specific transcript (Xist) locus, discovered to be the master regulator of X-inactivation. The Xist gene produces a non-coding RNA that functions as the primary switch for X-inactivation, coating the X chromosome from which it is transcribed in cis. Significant progress has been made towards understanding how Xist is regulated at the onset of X-inactivation, but our understanding of the molecular basis of silencing mediated by Xist RNA has progressed more slowly. A picture has, however, begun to emerge, and new tools and resources hold out the promise of further advances to come. Here, we provide an overview of the current state of our knowledge, what is known about Xist RNA and how it may trigger chromosome silencing.
• Human inactive X chromosome is compacted through a PRC2-independent SMCHD1-HBiX1 pathway

  Ryu-Suke Nozawa; Koji Nagao; Ken-Taro Igami; Sachiko Shibata; Natsuko Shirai; Naohito Nozaki; Takashi Sado; Hiroshi Kimura; Chikashi Obuse

  Nature Structural and Molecular Biology Nature Publishing Group 20 (5) 566 - 573 1545-9985 2013 [Refereed]
   

  Human inactive X chromosome (Xi) forms a compact structure called the Barr body, which is enriched in repressive histone modifications such as trimethylation of histone H3 Lys9 (H3K9me3) and Lys27 (H3K27me3). These two histone marks are distributed in distinct domains, and X-inactive specific transcript (XIST) preferentially colocalizes with H3K27me3 domains. Here we show that Xi compaction requires HBiX1, a heterochromatin protein 1 (HP1)-binding protein, and structural maintenance of chromosomes hinge domain-containing protein 1 (SMCHD1), both of which are enriched throughout the Xi chromosome. HBiX1 localization to H3K9me3 and XIST-associated H3K27me3 (XIST-H3K27me3) domains was mediated through interactions with HP1 and SMCHD1, respectively. Furthermore, HBiX1 was required for SMCHD1 localization to H3K9me3 domains. Depletion of HBiX1 or SMCHD1, but not Polycomb repressive complex 2 (PRC2), resulted in Xi decompaction, similarly to XIST depletion. Thus, the molecular network involving HBiX1 and SMCHD1 links the H3K9me3 and XIST-H3K27me3 domains to organize the compact Xi structure. © 2013 Nature America, Inc. All rights reserved.
• Incomplete X-inactivation initiated by a hypomorphic Xist allele in the mouse

  Yuko Hoki; Rieko Ikeda; Nathan Mise; Yuka Sakata; Tatsuya Ohhata; Hiroyuki Sasaki; Kuniya Abe; Takashi Sado

  DEVELOPMENT COMPANY OF BIOLOGISTS LTD 138 (13) 2649 - 2659 0950-1991 2011/07 [Refereed]
   

  X chromosome inactivation (X-inactivation) in female mammals is triggered by differential upregulation of the Xist gene on one of the two X chromosomes and subsequent coating of the X in cis with its non-coding transcripts. Although targeted mutation has clearly shown that Xist is essential for X-inactivation in cis, the molecular mechanism by which Xist RNA induces chromosome silencing is largely unknown. Here, we demonstrate that an Xist mutant generated previously in mouse by gene targeting, Xist(IVS), is unique in that it partially retains the capacity to silence the X chromosome. Although Xist(IVS) is differentially upregulated and its mutated transcript coats the X chromosome in cis in embryonic and extra-embryonic tissues, X-inactivation thus initiated does not seem to be fully established. The state of such incomplete inactivation is probably unstable and the mutated X is apparently reactivated in a subset of extra-embryonic tissues and, perhaps, early epiblastic cells. Xist(IVS), which can be referred to as a partial loss-of-function mutation, would provide an opportunity to dissect the molecular mechanism of Xist RNA-mediated chromosome silencing.
• Role for piRNAs and noncoding RNA in de novo DNA methylation of the imprinted mouse Rasgrf1 locus.

  Toshiaki Watanabe; Shin-ichi Tomizawa; Kohzoh Mitsuya; Yasushi Totoki; Yasuhiro Yamamoto; Satomi Kuramochi-Miyagawa; Naoko Iida; Yuko Hoki; Patrick J Murphy; Atsushi Toyoda; Kengo Gotoh; Hitoshi Hiura; Takahiro Arima; Asao Fujiyama; Takashi Sado; Tatsuhiro Shibata; Toru Nakano; Haifan Lin; Kenji Ichiyanagi; Paul D Soloway; Hiroyuki Sasaki

  Science (New York, N.Y.) 6031 332 (6031) 848 - 52 0036-8075 2011/05 [Refereed]
   

  Genomic imprinting causes parental origin-specific monoallelic gene expression through differential DNA methylation established in the parental germ line. However, the mechanisms underlying how specific sequences are selectively methylated are not fully understood. We have found that the components of the PIWI-interacting RNA (piRNA) pathway are required for de novo methylation of the differentially methylated region (DMR) of the imprinted mouse Rasgrf1 locus, but not other paternally imprinted loci. A retrotransposon sequence within a noncoding RNA spanning the DMR was targeted by piRNAs generated from a different locus. A direct repeat in the DMR, which is required for the methylation and imprinting of Rasgrf1, served as a promoter for this RNA. We propose a model in which piRNAs and a target RNA direct the sequence-specific methylation of Rasgrf1.
• MITOPLD is a mitochondrial protein essential for nuage formation and piRNA biogenesis in the mouse germline.

  Toshiaki Watanabe; Shinichiro Chuma; Yasuhiro Yamamoto; Satomi Kuramochi-Miyagawa; Yasushi Totoki; Atsushi Toyoda; Yuko Hoki; Asao Fujiyama; Tatsuhiro Shibata; Takashi Sado; Toshiaki Noce; Toru Nakano; Norio Nakatsuji; Haifan Lin; Hiroyuki Sasaki

  Developmental cell 3 20 (3) 364 - 75 1534-5807 2011/03 [Refereed]
   

  MITOPLD is a member of the phospholipase D superfamily proteins conserved among diverse species. Zucchini (Zuc), the Drosophila homolog of MITOPLD, has been implicated in primary biogenesis of Piwi-interacting RNAs (piRNAs). By contrast, MITOPLD has been shown to hydrolyze cardiolipin in the outer membrane of mitochondria to generate phosphatidic acid, which is a signaling molecule. To assess whether the mammalian MITOPLD is involved in piRNA biogenesis, we generated Mitopld mutant mice. The mice display meiotic arrest during spermatogenesis, demethylation and derepression of retrotransposons, and defects in primary piRNA biogenesis. Furthermore, in mutant germ cells, mitochondria and the components of the nuage, a perinuclear structure involved in piRNA biogenesis/function, are mislocalized to regions around the centrosome, suggesting that MITOPLD may be involved in microtubule-dependent localization of mitochondria and these proteins. Our results indicate a conserved role for MITOPLD/Zuc in the piRNA pathway and link mitochondrial membrane metabolism/signaling to small RNA biogenesis.
• Impeding Xist Expression from the Active X Chromosome Improves Mouse Somatic Cell Nuclear Transfer

  Kimiko Inoue; Takashi Kohda; Michihiko Sugimoto; Takashi Sado; Narumi Ogonuki; Shogo Matoba; Hirosuke Shiura; Rieko Ikeda; Keiji Mochida; Takashi Fujii; Ken Sawai; Arie P. Otte; X. Cindy Tian; Xiangzhong Yang; Fumitoshi Ishino; Kuniya Abe; Atsuo Ogura

  SCIENCE AMER ASSOC ADVANCEMENT SCIENCE 330 (6003) 496 - 499 0036-8075 2010/10 [Refereed]
   

  Cloning mammals by means of somatic cell nuclear transfer (SCNT) is highly inefficient because of erroneous reprogramming of the donor genome. Reprogramming errors appear to arise randomly, but the nature of nonrandom, SCNT-specific errors remains elusive. We found that Xist, a noncoding RNA that inactivates one of the two X chromosomes in females, was ectopically expressed from the active X (Xa) chromosome in cloned mouse embryos of both sexes. Deletion of Xist on Xa showed normal global gene expression and resulted in about an eight-to ninefold increase in cloning efficiency. We also identified an Xist-independent mechanism that specifically down-regulated a subset of X-linked genes through somatic-type repressive histone blocks. Thus, we have identified nonrandom reprogramming errors in mouse cloning that can be altered to improve the efficiency of SCNT methods.
• Sex-reversed somatic cell cloning in the mouse

  Kimiko Inoue; Narumi Ogonuki; Kazuyuki Mekada; Atsushi Yoshiki; Takashi Sado; Atsuo Ogura

  Journal of Reproduction and Development 5 55 (5) 566 - 569 0916-8818 2009 [Refereed]
   

  Somatic cell nuclear transfer has many potential applications in the fields of basic and applied sciences. However, it has a disadvantage that can never be overcome technically-the inflexibility of the sex of the offspring. Here, we report an accidental birth of a female mouse following nuclear transfer using an immature Sertoli cell. We produced a batch of 27 clones in a nuclear transfer experiment using Sertoli cells collected from neonatal male mice. Among them, one pup was female. This "male-derived female" clone grew into a normal adult and produced offspring by natural mating with a littermate. Chromosomal analysis revealed that the female clone had a 39,X karyotype, indicating that the Y chromosome had been deleted in the donor cell or at some early step during nuclear transfer. This finding suggests the possibility of resuming sexual reproduction after a single male is cloned, which should be especially useful for reviving extinct or endangered species.
• A proximal conserved repeat in the Xist gene is essential as a genomic element for X-inactivation in mouse.

  Hoki Y; Kimura N; Kanbayashi M; Amakawa Y; Ohhata T; Sasaki H; Sado T

  Development 136, 139 - 146 2009 [Refereed]
  
• De Novo DNA Methylation Independent Establishment of Maternal Imprint on X Chromosome in Mouse Oocytes

  Hatsune Chiba; Ryutaro Hirasawa; Masahiro Kaneda; Yuko Amakawa; En Li; Takashi Sado; Hiroyuki Sasaki

  GENESIS WILEY-LISS 46 (12) 768 - 774 1526-954X 2008/12 [Refereed]
   

  In female mouse embryos, the paternal X chromosome (Xp) is preferentially inactivated during preimplantation development and trophoblast differentiation. This imprinted X-chromosome inactivation (XCI) is partly due to an activating imprint on the maternal X chromosome (Xm), which is set during oocyte growth. However, the nature of this imprint is unknown. DNA methylation is one candidate, and therefore we examined whether disruptions of the two de novo, DNA methyltransferases in growing oocytes affect imprinted XCI. We found that accumulation of histone H3 lysine-27 trimethylation, a hallmark of XCI, occurs normally on the Xp, and not on the Xm, in female blastocysts developed from the mutant oocytes. Furthermore, the allelic expression patterns of X-linked genes including Xist and Tsix were unchanged in preimplantation embryos and also in the trophoblast. These results show that a maternal disruption of the DNA methyltransferases has no effect on imprinted XCI and argue that de novo DNA methylation is dispensable for Xm imprinting. This underscores the difference between imprinted XCI and autosomal imprinting. genesis 46:768-774, 2008. (C) 2008 Wiley-Liss, Inc.
• De Novo DNA Methylation Independent Establishment of Maternal Imprint on X Chromosome in Mouse Oocytes

  Hatsune Chiba; Ryutaro Hirasawa; Masahiro Kaneda; Yuko Amakawa; En Li; Takashi Sado; Hiroyuki Sasaki

  GENESIS WILEY-LISS 46 (12) 768 - 774 1526-954X 2008/12 [Refereed]
   

  In female mouse embryos, the paternal X chromosome (Xp) is preferentially inactivated during preimplantation development and trophoblast differentiation. This imprinted X-chromosome inactivation (XCI) is partly due to an activating imprint on the maternal X chromosome (Xm), which is set during oocyte growth. However, the nature of this imprint is unknown. DNA methylation is one candidate, and therefore we examined whether disruptions of the two de novo, DNA methyltransferases in growing oocytes affect imprinted XCI. We found that accumulation of histone H3 lysine-27 trimethylation, a hallmark of XCI, occurs normally on the Xp, and not on the Xm, in female blastocysts developed from the mutant oocytes. Furthermore, the allelic expression patterns of X-linked genes including Xist and Tsix were unchanged in preimplantation embryos and also in the trophoblast. These results show that a maternal disruption of the DNA methyltransferases has no effect on imprinted XCI and argue that de novo DNA methylation is dispensable for Xm imprinting. This underscores the difference between imprinted XCI and autosomal imprinting. genesis 46:768-774, 2008. (C) 2008 Wiley-Liss, Inc.
• Crucial role of antisense transcription across the Xist promoter in Tsix-mediated Xist chromatin modification

  Tatsuya Ohhata; Yuko Hoki; Hiroyuki Sasaki; Takashi Sado

  DEVELOPMENT COMPANY OF BIOLOGISTS LTD 135 (2) 227 - 235 0950-1991 2008/01 [Refereed]
   

  Expression of Xist, which triggers X inactivation, is negatively regulated in cis by an antisense gene, Tsix, transcribed along the entire Xist gene. We recently demonstrated that Tsix silences Xist through modification of the chromatin structure in the Xist promoter region. This finding prompted us to investigate the role of antisense transcription across the Xist promoter in Tsix-mediated silencing. Here, we prematurely terminated Tsix transcription before the Xist promoter and addressed its effect on Xist silencing in mouse embryos. We found that although 93% of the region encoding Tsix was transcribed, truncation of Tsix abolished the antisense regulation of Xist. This resulted in a failure to establish the repressive chromatin configuration at the Xist promoter on the mutated X, including DNA methylation and repressive histone modifications, especially in extraembryonic tissues. These results suggest a crucial role for antisense transcription across the Xist promoter in Xist silencing.
• Dicer regulates Xist promoter methylation in ES cells indirectly through transcriptional control of Dnmt3a

  Tatyana B. Nesterova; Bilyana C. Popova; Bradley S. Cobb; Sara Norton; Claire E. Senner; Y. Amy Tang; Thomas Spruce; Tristan A. Rodriguez; Takashi Sado; Matthias Merkenschlager; Neil Brockdorff

  EPIGENETICS & CHROMATIN BIOMED CENTRAL LTD 1 (1) 2  1756-8935 2008 [Refereed]
   

  Background: X chromosome inactivation is the mechanism used in mammals to achieve dosage compensation of X-linked genes in XX females relative to XY males. Chromosome silencing is triggered in cis by expression of the non-coding RNA Xist. As such, correct regulation of the Xist gene promoter is required to establish appropriate X chromosome activity both in males and females. Studies to date have demonstrated co-transcription of an antisense RNA Tsix and low-level sense transcription prior to onset of X inactivation. The balance of sense and antisense RNA is important in determining the probability that a given Xist allele will be expressed, termed the X inactivation choice, when X inactivation commences. Results: Here we investigate further the mechanism of Xist promoter regulation. We demonstrate that both sense and antisense transcription modulate Xist promoter DNA methylation in undifferentiated embryonic stem (ES) cells, suggesting a possible mechanistic basis for influencing X chromosome choice. Given the involvement of sense and antisense RNAs in promoter methylation, we investigate a possible role for the RNA interference (RNAi) pathway. We show that the Xist promoter is hypomethylated in ES cells deficient for the essential RNAi enzyme Dicer, but that this effect is probably a secondary consequence of reduced levels of de novo DNA methyltransferases in these cells. Consistent with this we find that Dicer-deficient XY and XX embryos show appropriate Xist expression patterns, indicating that Xist gene regulation has not been perturbed. Conclusion: We conclude that Xist promoter methylation prior to the onset of random X chromosome inactivation is influenced by relative levels of sense and antisense transcription but that this probably occurs independent of the RNAi pathway. We discuss the implications for this data in terms of understanding Xist gene regulation and X chromosome choice in random X chromosome inactivation.
• Tsix defective in splicing is competent to establish Xist silencing

  Takashi Sado; Yuko Hoki; Hiroyuki Sasaki

  DEVELOPMENT COMPANY OF BIOLOGISTS LTD 133 (24) 4925 - 4931 0950-1991 2006/12 [Refereed]
   

  Dosage differences of X-linked genes between male and female mammals are compensated for by a mechanism known as X-inactivation, and the noncoding Xist gene plays a crucial role in this process. The expression of Xist is regulated in cis by its noncoding antisense gene, Tsix, whose transcripts (though a fraction of them stay unspliced), are processed like common protein-coding RNAs. It has been suggested that certain classes of sense-antisense pairs of RNA are causally involved in not only gene regulation but also higher order chromatin structure in various organisms. In fact, recent studies demonstrated that Tsix modulates Xist expression through modi. cation of the chromatin structure. It is still unknown, however, whether the RNA product is important for the function of Tsix or whether the antisense transcription is sufficient. To obtain insight into this issue, we eliminated the splicing products of Tsix in the mouse and explored the effects of this elimination on Tsix-mediated Xist silencing. To our surprise, the Xist locus was stably repressed on the X carrying the splicing-defective Tsix allele. Moreover, the repressive chromatin configuration was properly established at the Xist locus. These unexpected results indicate that the splicing products are dispensable for Tsix-mediated Xist silencing.
• Tsix-deficient X chromosome does not undergo inactivation in the embryonic lineage in males: implications for Tsix-independent silencing of Xist

  T Ohhata; Y Hoki; H Sasaki; T Sado

  CYTOGENETIC AND GENOME RESEARCH KARGER 113 (1-4) 345 - 349 1424-8581 2006 [Refereed]
   

  Differential induction of the X-linked non-coding Xist gene is a key event in the process of X inactivation occurring in female mammalian embryos. Xist is negatively regulated in cis by its antisense gene Tsix through modification of the chromatin structure. The maternal Xist allele, which is normally silent in the extraembryonic lineages, is ectopically activated when Tsix is disrupted on the same chromosome, and subsequently the maternal X chromosome undergoes inactivation in the extraembryonic lineages even in males. However, it is still unknown whether the single Tsix-deficient X chromosome (X-Delta Tsix) in males is also inactivated in the embryonic lineage. Here, we show that both male and female embryos carrying a maternally derived X-Delta Tsix could survive if the extraembryonic tissues were complemented by wild-type tetraploid cells. In addition, Xist on the X-Delta Tsix was properly silenced and methylated at CpG sites in adult male somatic cells. These results indicate that the embryonic lethality caused by the maternal X-Delta Tsix is solely attributable to the defects in the extraembryonic lineages. X-Delta Tsix does not seem to undergo inactivation in the embryonic lineage in males, suggesting the presence of a Tsix-independent silencing mechanism for Xist in the embryonic lineage.
• Tsix silences Xist through modification of chromatin structure

  T Sado; Y Hoki; H Sasaki

  DEVELOPMENTAL CELL CELL PRESS 9 (1) 159 - 165 1534-5807 2005/07 [Refereed]
   

  X inactivation is controlled by Xist and its antisense gene, Tsix, neither of which encodes a protein. Xist is essential for X inactivation to occur in cis, and its differential expression is a key event in the initiation of X inactivation. Xist and Tsix are imprinted in the extraembryonic tissues of mouse embryos so that they are expressed from the paternal and maternal X, respectively, resulting in the preferential inactivation of the paternal X. Targeted disruption of Tsix causes ectopic expression of Xist, suggesting that Tsix negatively regulates Xist in cis. However, the molecular mechanism of this antisense regulation remains unknown. Here, we demonstrate that Tsix transcriptionally silences Xist in both embryonic and extraembryonic tissues of mouse embryos. Moreover, we show that disruption of Tsix impairs establishment of repressive epigenetic modifications and chromatin structure at the Xist locus. We propose that Tsix silences Xist through modification of the chromatin structure.
• Imprinted X inactivation and reprogramming in the preimplantation mouse embryo

  T Sado; AC Ferguson-Smith

  HUMAN MOLECULAR GENETICS OXFORD UNIV PRESS 14 R59 - R64 0964-6906 2005/04 [Refereed]
   

  X chromosome inactivation is a developmentally regulated process that causes one of the two X chromosomes in normal female mammals to become transcriptionally silenced, thus equalizing the expression of X-linked genes between the sexes. Such dosage compensation depends upon dynamic genetic and epigenetic events occurring very early in development. X inactivation is controlled by an X inactivation centre that is associated with the expression of non-coding RNAs required for the silencing. Also associated with the inactive X are repressive histone modifications and polycomb protein-mediated states, which are progressively acquired during the inactivation process. In mouse, two forms of X inactivation have been described. Random X inactivation happens in the derivatives of the inner cell mass (ICM) giving rise to embryos where the maternally inherited X(Xm) is inactive in some cells and the paternally derived X (Xp) is inactive in others. Random X inactivation occurs around the time of implantation. Imprinted X inactivation, the preferential inactivation of the Xp chromosome, occurs earlier and, although there has been some debate as to the precise timing of initiation of this event, is apparent in all cells early in preimplantation development, then is subsequently confined to the cells of the extraembryonic lineages. A picture is emerging whereby initial epigenetic asymmetry between the two parental X chromosomes is reprogrammed in a lineage specific manner resulting in a switch from imprinted to random inactivation in embryonic derivatives. Neither the underlying reason nor the full extent of these early lineage specific epigenetic changes is known, but they may be correlated with more genome-wide reprogramming events essential for normal development.
• Large-scale identification and mapping of nuclear matrix-attachment regions in the distal imprinted domain of mouse chromosome 7

  N Purbowasito; C Suda; T Yokomine; M Zubair; T Sado; K Tsutsui; H Sasaki

  DNA RESEARCH UNIVERSAL ACADEMY PRESS INC 11 (6) 391 - 407 1340-2838 2004/12 [Refereed]
   

  Mammalian imprinted genes, which are expressed from only one of the parental alleles, have a tendency to form clusters and are regulated by long-range mechanisms. Nuclear matrix-attachment regions (MARs), the anchor points of loop domains, are involved in coordination of gene expression and could play a role in regulation of imprinted domains. We have identified and mapped a total of 52 MARs in a 1-Mb imprinted domain on mouse distal chromosome 7 using our cosmid contigs and an in vitro MAR assay. We find two MAR clusters (comprising 20 and 19 MARs), one of which is mapped in the Th-Ins2 intergenic region, coincident with the boundary between the two imprinted subdomains. However, the imprinted/nonimprinted boundaries are not associated with a MAR. Based on the sequence information, we find that many of the MARs are rich in long interspersed nuclear elements. In addition, comparisons of the results obtained with several MAR-prediction software programs reveal good performance of ChrClass in terms of both sensitivity and specificity. This study presents the first large-scale mapping of MARs in an imprinted domain and provides a platform for understanding the roles of MARS in imprinting.
• X-inactivation is stably maintained in mouse embryos deficient for histone methyl transferase G9a

  T Ohhata; M Tachibana; M Tada; T Tada; H Sasaki; Y Shinkai; T Sado

  GENESIS WILEY-LISS 40 (3) 151 - 156 1526-954X 2004/11 [Refereed]
   

  One of the two X chromosomes becomes inactivated during early development of female mammals. Recent studies demonstrate that the inactive X chromosome is rich in histone H3 methylated at Lys-9 and Lys-27, suggesting an important role for these modifications in X-inactivation. It has been shown that in the mouse Eed is required for maintenance of X-inactivation in the extraembryonic lineages. Interestingly, Eed associates with Ezh2 to form a complex possessing histone methyltransferase activity predominantly for H3 Lys-27. We previously showed that G9a is one of the histone methyltransferases specific for H3 Lys-9 and is essential for embryonic development. Here we examined X-inactivation in mouse embryos deficient for G9a. Expression of Xist, which is crucial for the initiation of X-inactivation, was properly regulated and the inactivated X chromosome was stably maintained even in the absence of G9a. These results demonstrate that G9a is not essential for X-inactivation. genesis 40:151-156, 2004. 0 2004 Wiley-Liss, Inc.
• Essential role for de novo DNA methyltransferase Dnmt3a in paternal and maternal imprinting

  M Kaneda; M Okano; K Hata; T Sado; N Tsujimoto; E Li; H Sasaki

  NATURE NATURE PUBLISHING GROUP 429 (6994) 900 - 903 0028-0836 2004/06 [Refereed]
   

  Imprinted genes are epigenetically marked during gametogenesis so that they are exclusively expressed from either the paternal or the maternal allele in offspring(1). Imprinting prevents parthenogenesis in mammals and is often disrupted in congenital malformation syndromes, tumours and cloned animals(1). Although de novo DNA methyltransferases of the Dnmt3 family are implicated in maternal imprinting(2), the lethality of Dnmt3a and Dnmt3b knockout mice(3) has precluded further studies. We here report the disruption of Dnmt3a and Dnmt3b in germ cells, with their preservation in somatic cells, by conditional knockout technology(4). Offspring from Dnmt3a conditional mutant females die in utero and lack methylation and allele-specific expression at all maternally imprinted loci examined. Dnmt3a conditional mutant males show impaired spermatogenesis and lack methylation at two of three paternally imprinted loci examined in spermatogonia. By contrast, Dnmt3b conditional mutants and their offspring show no apparent phenotype. The phenotype of Dnmt3a conditional mutants is indistinguishable from that of Dnmt3L knockout mice2,5, except for the discrepancy in methylation at one locus. These results indicate that both Dnmt3a and Dnmt3L are required for methylation of most imprinted loci in germ cells, but also suggest the involvement of other factors.
• De novo DNA methylation is dispensable for the initiation and propagation of X chromosome inactivation

  T Sado; M Okano; E Li; H Sasaki

  DEVELOPMENT COMPANY OF BIOLOGISTS LTD 131 (5) 975 - 982 0950-1991 2004/03 [Refereed]
   

  Xist (X-inactive specific transcript) plays a crucial role in X-inactivation. This non-coding RNA becomes upregulated on the X chromosome that is to be inactivated upon differentiation. Previous studies have revealed that although maintenance-type DNA methyllation is not essential for X-inactivation to occur, it is required for the stable repression of Xist in differentiated cells. However, it is unknown whether differential de novo methyllation at the Xist promoter, which is mediated by Dnmt3a and/or Dnmt3b, is a cause or a consequence of monoallelic expression of Xist. We show that Xist expression is appropriately regulated in the absence of Dnmt3a and Dnmt3b and that a single X chromosome undergoes proper inactivation in mutant females. Our results indicate that a mechanism(s) other than DNA methylation plays a principal role in initiating X-inactivation. We also demonstrate that delayed upregulation of Xist does not induce X-inactivation, consistent with a crucial developmental window for the chromosomal silencing.
• Role of de novo DNA methyltransferases in initiation of genomic imprinting and X-chromosome inactivation

  M Kaneda; T Sado; K Hata; M Okano; N Tsujimoto; E Li; H Sasaki

  COLD SPRING HARBOR SYMPOSIA ON QUANTITATIVE BIOLOGY COLD SPRING HARBOR LAB PRESS, PUBLICATIONS DEPT 69 125 - 129 0091-7451 2004 [Refereed]
  
• Three novel DNMT3B mutations in Japanese patients with ICF syndrome

  H Shirohzu; T Kubota; A Kumazawa; T Sado; T Chijiwa; K Inagaki; Suetake, I; S Tajima; K Wakui; Y Miki; M Hayashi; Y Fukushima; H Sasaki

  AMERICAN JOURNAL OF MEDICAL GENETICS WILEY-LISS 112 (1) 31 - 37 0148-7299 2002/09 [Refereed]
   

  ICF syndrome is a rare autosomal recessive disorder characterized by immunodeficiency, centromeric instability, and facial anomalies. It is caused by mutations in a de novo DNA methyltransferase gene, DNMT3B. We here report the first three Japanese cases of ICF syndrome from two unrelated families. All patients had typical facial dysmorphism and immunoglobulin A (IgA) deficiency, but none of them had apparent mental retardation. Cytogenetic analysis of peripheral blood lymphocytes showed chromosomal abnormalities, including multiradial configurations and a stretching of the pericentromeric heterochromatin of chromosomes 1 and 16. Hypomethylation of classical satellite 2 DNA was also observed. Mutation analyses of DNMT3B revealed three novel mutations: patient 1 from the first family was a compound heterozygote for a nonsense mutation (Q42Term) and a missense mutation (R832Q); patients 2 and 3 from the second family were both homozygous for a missense mutation (S282P). The R832Q mutation occurred within the conserved methyltransferase domain, and thus may affect the enzyme activity directly. The S282P mutation, on the other hand, occurred close to the PWWP domain, which is presumably involved in protein-protein interaction. This is the first missense mutation mapped to the N-terminal half of the protein, suggesting that the region plays an important role in the regulation of the DNMT3B enzyme. (C) 2002 Wiley-Liss, Inc.
• Effect of Tsix disruption on Xist expression in male ES cells

  T Sado; E Li; H Sasaki

  CYTOGENETIC AND GENOME RESEARCH KARGER 99 (1-4) 115 - 118 1424-8581 2002 [Refereed]
   

  Xist and its antisense partner, Tsix, encode non-coding RNAs and play key roles in X chromosome inactivation. Targeted disruption of Tsix causes ectopic expression of Xist in the extraembryonic tissues upon maternal transmission, which subsequently results in embryonic lethality due to inactivation of both X chromosomes in females and a single X chromosome in males. Tsix, therefore, plays a crucial role in maintaining the silenced state of Xist in cis and regulates the imprinted X inactivation in the extraembryonic tissues. In this study, we examined the effect of Tsix disruption on Xist expression in the embryonic lineage using embryonic stem (ES) cells as a model system. Upon differentiation, Xist is ectopically activated in a subset of the nuclei of male ES cells harboring the Tsix-deficient X chromosome. Such ectopic expression, however, eventually ceased during prolonged culture. It is likely that surveillance by the X chromosome counting mechanism somehow shuts off the ectopic expression of Xist before inactivation of the X chromosome. Copyright (C) 2002 S. Karger AG, Basel.
• 5-aza-2 '-deoxycytidine induces histone hyperacetylation of mouse centromeric heterochromatin by a mechanism independent of DNA demethylation

  S Takebayashi; M Nakao; N Fujita; T Sado; M Tanaka; H Taguchi; K Okumura

  BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ACADEMIC PRESS INC 288 (4) 921 - 926 0006-291X 2001/11 [Refereed]
   

  5-Aza-2'-deoxycytidine (5-azadC) is widely used as a potent inhibitor of DNA methyltransferase. Cells treated with this drug show various phenomena such as the reactivation of repressed genes, change in replication timing, and decondensation of heterochromatin. A number of studies using this drug have been reported so far but it is still controversial whether such changes are due to 5-azadC-induced demethylation itself or the side effects of the drug. Here we report that 5-azadC treatment induces histone hyperacetylation in mouse centromeric heterochromatin which normally contains methylated DNA and hypoacetylated histones. Treatment also affects the intranuclear distribution of histone deacetylase 2 (HDAC2). However, histone hyperacetylation was not observed in DNA methyltransferase 1-deficient cells with a reduced level of genomic DNA methylation. Our results suggest that 5-azadC-induced histone hyperacetylation is independent of DNA demethylation and that DNA methylation is not essential for the maintenance of the histone hypoacetylated state in centromeric heterochromatin. (C) 2001 Academic Press.
• Regulation of imprinted X-chromosome inactivation in mice by Tsix

  T Sado; ZJ Wang; H Sasaki; E Li

  DEVELOPMENT COMPANY OF BIOLOGISTS LTD 128 (8) 1275 - 1286 0950-1991 2001/04 [Refereed]
   

  In mammals, X-chromosome inactivation is imprinted in the extra-embryonic lineages with paternal X chromosome being preferentially inactivated. In this study, we investigate the role of Tsix, the antisense transcript from the Xist locus, in regulation of Xist expression and X-inactivation. We show that Tsix is transcribed from two putative promoters and its transcripts are processed, Expression of Tsix is first detected in blastocysts and is imprinted with only the maternal allele transcribed. The imprinted expression of Tsix persists in the extraembryonic tissues after implantation, but is erased in embryonic tissues. To investigate the function of Tsix in X-inactivation, we disrupted Tsix by insertion of an IRES beta geo cassette in the second exon, which blocked transcripts from both promoters. While disruption of the paternal Tsix allele has no adverse effects on embryonic development, inheritance of a disrupted maternal allele results in ectopic Xist expression and early embryonic lethality, owing to inactivation of both X chromosomes in females and single X chromosome in males. Further, early developmental defects of female embryos with maternal transmission of Tsix mutation can be rescued by paternal inheritance of the Xist deletion. These results provide genetic evidence that Tsix plays a crucial role in maintaining Xist silencing in cis and in regulation of imprinted X-inactivation in the extraembryonic tissues.
• Relationship between DNA methylation, histone H4 acetylation and gene expression in the mouse imprinted Igf2-H19 domain

  Grandjean, V; L O'Neill; T Sado; B Turner; A Ferguson-Smith

  FEBS LETTERS ELSEVIER SCIENCE BV 488 (3) 165 - 169 0014-5793 2001/01 [Refereed]
   

  DNA methylation and histone H4 acetylation play a role in gene regulation by modulating the structure of the chromatin, Recently, these two epigenetic modifications have dynamically and physically been linked. Evidence suggests that both modifications are involved in regulating imprinted genes - a subset of genes whose expression depends on their parental origin. Using immunoprecipitation assays, we investigate the relationship between DNA methylation, histone H4 acetylation and gene expression in the well-characterised imprinted Igf2-H19 domain on mouse chromosome 7, A systematic regional analysis of the acetylation status of the domain shows that parental-specific differences in acetylation of the core histone H4 are present in the promoter regions of both lgf2 and H19 genes, with the expressed alleles being more acetylated than the silent alleles, A correlation between DNA methylation, histone hypoacetylation and gene repression is evident only at the promoter region of the H19 gene. Treatment with trichostatin A, a specific inhibitor of histone deacetylase, reduces the expression of the active maternal H19 allele and this can be correlated with regional changes in acetylation within the upstream regulatory domain. The data suggest that histone H4 acetylation and DNA methylation have distinct functions on the maternal and paternal Igf2-H19 domains. (C) 2001 Federation of European Biochemical Societies. Published by Elsevier Science B.V.. All rights reserved.
• X inactivation in the mouse embryo deficient for Dnmt1: Distinct effect of hypomethylation on imprinted and random X inactivation

  T Sado; MH Fenner; SS Tan; P Tam; T Shioda; E Li

  DEVELOPMENTAL BIOLOGY ACADEMIC PRESS INC 225 (2) 294 - 303 0012-1606 2000/09 [Refereed]
   

  It has been suggested that DNA methylation plays a crucial role in genomic imprinting and X inactivation. Using DNA methyltransferase 1 (Dnmt1)-deficient mouse embryos carrying X-linked lacZ transgenes, we studied the effects of genomic demethylation on X inactivation. Based on the expression pattern of lacZ, the imprinted X inactivation in the visceral endoderm, a derivative of the extraembryonic lineage, was unaffected in Dnmt1 mutant embryos at the time other imprinted genes showed aberrant expression. Random X inactivation in the embryonic lineage of Dnmt1 mutant embryos, however, was unstable as a result of hypomethylation, causing reactivation of, at least, one lacZ transgene that had initially been repressed. Our results suggest that maintenance of imprinted X inactivation in the extraembryonic lineage can tolerate extensive demethylation while normal levels of methylation are required for stable maintenance of X inactivation in the embryonic lineage. (C) 2000 Academic Press.
• Effect of CpG methylation on expression of the mouse imprinted gene Mest

  Y Nishita; T Sado; Yoshida, I; N Takagi

  GENE ELSEVIER SCIENCE BV 226 (2) 199 - 209 0378-1119 1999/01 [Refereed]
   

  We previously reported isolation of the mouse gene, Mest (mesoderm-specific transcripts), which is mapped to the proximal part of chromosome 6 and predominantly expressed in the mesoderm and its derivatives during development. Peg1, a paternally expressed gene isolated by a systematic screening of imprinted genes, was recently demonstrated to be identical to Mest. We and others have shown that the human homolog (MEST) of Mest is also imprinted so as to be expressed from the paternal copy and maps to 7q32. lo study transcriptional regulation of Mest/Peg1, we examined the effect of DNA methylation on its expression. In the embryonal carcinoma (EC) cell line, MC12, from which Mest was originally isolated, the 5'-region harboring presumptive promoter of the gene was undermethylated. On the other hand, C4XX, a subclone of MC12 which had lost expression of Mest, was characterized by extremely high levels of methylation in the 5'-region, demethylation of which resulted in activation of Mesa. Furthermore, a methylated reporter construct with the luciferase gene under the control of the putative promoter region of Mest was not competent to produce luciferase activity in MC12 cells. These results suggest a suppressive role for DNA methylation in Mest expression. However, neither methylated nor unmethylated reporter constructs showed luciferase activity in a primary culture from the adult kidney, in which Mest is down-regulated despite apparent unmethylation of the paternal allele. Taken together, the data suggest that there are probably two modes of regulation for the Mest gene; one being a methylation-dependent mechanism that regulates imprinted expression of Mest during development, and the other being a methylation-independent mechanism that is involved in down-regulation of Mest in adult tissues. (C) 1999 Elsevier Science B.V. All rights reserved.
• The mouse Gtl2 gene is differentially expressed during embryonic development, encodes multiple alternatively spliced transcripts, and may act as an RNA

  K Schuster-Gossler; P Bilinski; T Sado; A Ferguson-Smith; A Gossler

  DEVELOPMENTAL DYNAMICS WILEY-LISS 212 (2) 214 - 228 1058-8388 1998/06 [Refereed]
   

  We have isolated a novel mouse gene (Gtl2) from the site of a gene trap integration (Gtl2(lacZ)) that gave rise to developmentally regulated LacZ expression, and a dominant parental origin-dependent phenotype, Heterozygous Gtl2(lacZ) mice that inherited the transgene from the father showed a proportionate dwarfism phenotype, whereas the penetrance and expressivity of the phenotype was strongly reduced in Gtl2(lacZ) mice that inherited the transgene from the mother. Gtl2 expression is highly similar to the beta-galactosidase staining pattern, and is downregulated but not abolished in mice carrying the Gtl2(lacZ) insertion, In early postimplantation embryos, Gtl2 is expressed in the visceral yolk sac and embryonic ectoderm, During subsequent development and organogenesis, Gtl2 transcripts are abundant in the paraxial mesoderm closely correlated with myogenic differentiation, in parts of the central nervous system, and in the epithelial ducts of developing excretory organs. The Gtl2 gene gives rise to various differentially spliced transcripts, which contain multiple small open reading frames (ORF), However, none of the ATG codons of these ORFs is in the context of a strong Kozak consensus sequence for initiation of translation, suggesting that Gtl2 might function as an RNA. Nuclear Gtl2 RNA was detected in a temporally and spatially regulated manner, and partially processed Gtl2 transcripts were readily detected in Northern blot hybridizations of polyadenylated RNA, suggesting that primary Gtl2 transcripts are differently processed in various cell types during development. Gtl2 transcript levels are present in parthenogenic embryos but may be reduced, consistent with the pattern of inheritance of the Gtl2(lacZ) phenotype. (C) 1998 Wiley-Liss, Inc.
• Epigenotype switching of imprintable loci in embryonic germ cells

  T Tada; M Tada; K Hilton; SC Barton; T Sado; N Takagi; MA Surani

  DEVELOPMENT GENES AND EVOLUTION SPRINGER VERLAG 207 (8) 551 - 561 0949-944X 1998/02 [Refereed]
   

  Expression of imprinted genes is dependent on their parental origin. This is reflected in the heritable differential methylation of parental alleles. The gametic imprints are however reversible as they do not endure for more than one generation. To investigate if the epigenetic changes in male and female germ line are similar or not, we derived embryonic germ (EG) cells from primordial germ cells (PGCs) of day 11.5 and 12.5 male and female embryos. The results demonstrate that they have an equivalent epigenotype. First, chimeras made with EG cells derived from both male and female embryos showed comparable fetal overgrowth and skeletal abnormalities, which are similar to but less severe than those induced by androgenetic embryonic stem (ES) cells. Thus, EG cells derived from female embryos resemble androgenetic ES cells more than parthenogenetic cells. Furthermore, the methylation status of both alleles of a number of loci in EG cells was similar to that of the paternal allele in normal somatic cells. Hence, both alleles of Igf2r region 2, Peg1/Mest, Peg3, Nnat were consistently unmethylated in EG cells as well as in the primary embryonic fibroblasts (PEFs) rescued from chimeras. More strikingly, both alleles of p57kip2 that were also unmethylated in EG cells, underwent de novo methylation in PEFs to resemble a paternal allele in somatic cells. The exceptions were the H19 and Igf2 genes that retained the methylation pattern in PEFs as seen in normal somatic tissues. These studies suggest that the initial epigenetic changes in germ cells of male and female embryos are similar.
• Genomic imprinting and chromosomal localization of the human MEST gene

  Y Nishita; Yoshida, I; T Sado; N Takagi

  GENOMICS ACADEMIC PRESS INC JNL-COMP SUBSCRIPTIONS 36 (3) 539 - 542 0888-7543 1996/09 [Refereed]
   

  We have isolated a human homologue (MEST) of the mouse mesoderm-specific transcript (Mest) gene that shares about 70% nucleotide sequence homology. Northern blot analysis showed that the MEST gene was expressed in all major fetal organs and tissues so far examined, i.e., amnion, brain, heart, lung, stomach, gut, adrenal, kidney, muscle, and liver, which does not contradict with mesoderm-specific expression. MEST was abundantly expressed in hydatidiform moles of androgenetic origin, whereas it was barely detectable in dermoid cysts of parthenogenetic origin. Thus, it seems likely that the MEST gene, mapped to 7q32 by fluorescence in situ hybridization, is maternally repressed as the mouse homologue. (C) 1996 Academic Press, Inc.
• Mosaic methylation of Xist gene before chromosome inactivation in undifferentiated female mouse embryonic stem and embryonic germ cells

  T Sado; T Tada; N Takagi

  DEVELOPMENTAL DYNAMICS WILEY-LISS 205 (4) 421 - 434 1058-8388 1996/04 [Refereed]
   

  Epigenetic modification is implicated in the choice of the X chromosome to be inactivated in the mouse, In order to gain more insight into the nature of such modification, we carried out a series of experiments using undifferentiated mouse cell lines as a model system, Not only the paternally derived X (X(P)) chromosome, but the maternally derived one (X(M)) was inactivated in the outer layer of the balloon-like cystic embryoid body probably corresponding to the yolk sac endoderm of the post-implantation embryo in which X(P) is preferentially inactivated, Hence, it is likely that the imprint responsible for the nonrandom X(P) inactivation in early mouse development has been erased or masked in female ES cells, CPG sites in the 5' region of the Xist gene were partially methylated in female ES and EC; and parthenogenetic ES cell lines as in the female somatic cell in which the silent Xist allele on the active X is fully methylated, whereas the expressed allele on the inactive X is completely unmethylated, In the case of undifferentiated ES cells, however, methylation was not differential between two Xist alleles, This observation was supported by the demonstration that single-cell clones derived from female ES cell lines were not characterized by either allele specific Xist methylation or nonrandom X inactivation upon cell differentiation, Apparently these findings are at variance with the view that Xist expression and X inactivation are controlled by preemptive methylation in undifferentiated ES cells and probably in epiblast. (C) 1996 Wiley-Liss, Inc.
• Activation of the inactive X chromosome induced by cell fusion between a murine EC and female somatic cell accompanies reproducible changes in the methylation pattern of the Xist gene

  N Mise; T Sado; M Tada; S Takada; N Takagi

  EXPERIMENTAL CELL RESEARCH ACADEMIC PRESS INC JNL-COMP SUBSCRIPTIONS 223 (2) 193 - 202 0014-4827 1996/03 [Refereed]
   

  Mouse embryonal carcinoma (EC) cell lines are divided into two classes with or without the capability of reactivating the inactive X chromosome from a fusion partner of female lymphocyte. The 5' region of Xist was partially methylated in reactivating-competent EC cells but was fully methylated in reactivating-incompetent EC cells having a single X chromosome. Partial or heterogeneous methylation implies methylation of each CpG site in about half of the cell independently of methylation status of neighboring CpG sites. Fusion of the reactivating-competent EC cells with female lymphocytes induced not only de novo methylation in the 5' region of Xist allele on the hitherto inactivated X chromosome, but also demethylation of the same region of Xist on the other X chromosome from the female somatic cell. In contrast, no such changes occurred in hybrid cells involving reactivating-incompetent EC cells. Thus, partial methylation of the 5' region of Xist most probably maintained by low maintenance and high de novo methylation efficiency is correlated with reactivation potential of the EC cell. It is possible that this unique methylation pattern is implicated in random X inactivation in EC-hybrid cells in vitro and in epiblast cells in vivo. (C) 1996 Academic Press, Inc.
• A NOVEL MESODERM-SPECIFIC CDNA ISOLATED FROM A MOUSE EMBRYONAL CARCINOMA CELL-LINE

  T SADO; N NAKAJIMA; M TADA; N TAKAGI

  DEVELOPMENT GROWTH & DIFFERENTIATION BLACKWELL SCIENCE 35 (5) 551 - 560 0012-1592 1993/10 [Refereed]
  
• NUCLEOTIDE-SEQUENCE OF A MOUSE LAMIN A CDNA AND ITS DEDUCED AMINO-ACID-SEQUENCE

  N NAKAJIMA; T SADO

  BIOCHIMICA ET BIOPHYSICA ACTA ELSEVIER SCIENCE BV 1171 (3) 311 - 314 0006-3002 1993/01 [Refereed]
   

  We have cloned and determined the nucleotide sequence of a mouse lamin A cDNA. The clone contained the C-terminal two-thirds of the lamin A coding sequence and a 3' untranslated sequence with a poly(A) stretch. As has been reported for human lamin A/C cDNAs, a large part of the 5' sequence of our mouse lamin A clone was essentially identical with a previously reported mouse lamin C cDNA sequence, and the deduced C-terminal amino acid sequence shared strong homology with the human lamin A sequence. A putative deduced amino acid sequence for mouse lamin A, which was derived from our sequence and the published lamin C sequence, was 665 amino acids long. The degree of overall homology to the human sequence was more than 95%, and relatively more variation was scattered in the C-terminal lamin A-specific region.

Books etc

• X-Chromosome Inactivation

  Takashi Sado (Editor)Human Press (Springer Nature) 2018/09 9781493987658
• Encyclopedia of Reproduction 2nd Edition

  Takashi Sado (ContributorX-chromosome inactivation)Academic Press (Elsevier) 2018/07 9780128118993
• エピジェネティクス実験スタンダード

  中島達郎; 酒田祐佳; 佐渡 敬 (ContributorlncRNAの遺伝学的解析)羊土社 2017/05 9784758101998
• 遺伝子発現制御機構 クロマチン，転写制御，エピジェネティクス

  千木雄太; 佐渡 敬 (ContributorX染色体不活性化)東京化学同人 2017/04 9784807909179

MISC

• Constitution of Higher Order Heterochromatin Structure Based on Epigenetic Code

  Koji Nagao; Shin-Ya Isobe; Yoshi-Nobu Ohkubo; Yoko Hayashi-Takanaka; Hiroshi Kimura; Takashi Sado; Chikashi Obuse  GENES & GENETIC SYSTEMS  91-  (6)  318  -318  2016/12
• X chromosome inactivation in the mouse

  Takashi Sado  GENES & GENETIC SYSTEMS  91-  (6)  320  -320  2016/12
• The defects in X-inactivation by partially dysfunctional Xist allele

  Tatsuro Nakajima; Yuko Hoki; Hiroyuki Sasaki; Takashi Sado  GENES & GENETIC SYSTEMS  90-  (6)  390  -390  2015/12
• Transcriptional regulation of monoallelically expressed genes in ground-state embryonic stem cells

  Hiroaki Ohishi; Motoko Unoki; Kei Fukuda; Shoji Maenohara; Kyohei Hori; Hatsune Chiba; Takashi Sado; Hiroyuki Sasaki  GENES & GENETIC SYSTEMS  90-  (6)  369  -369  2015/12
• X chromosome inactivation in the mouse embryos deficient for SmcHD1

  Yuki Sakakibara; Hiroyuki Sasaki; Marnie Blewitt; Takashi Sado  GENES & GENETIC SYSTEMS  90-  (6)  390  -390  2015/12
• Differential expression of Xist in the mouse preimplantation embryo

  Takashi Sado; Yuka Sakata; Yuko Amakawa; Yuko Hoki; Tatsuo Fukagawa; Hiroyuki Sasaki  GENES & GENETIC SYSTEMS  90-  (6)  390  -390  2015/12
• Epigenetic changes in the X chromosome accumulated with dysfunctional Xist RNA

  Yuka Sakata; Yuko Hoki; Hiroyuki Sasaki; Takashi Sado  GENES & GENETIC SYSTEMS  89-  (6)  300  -300  2014/12
• X chromosome inactivation in mouse embryos deficient for SmcHD1

  Yuki Sakakibara; Hiroyuki Sasaki; Marnie Brewitt; Takashi Sado  GENES & GENETIC SYSTEMS  89-  (6)  308  -308  2014/12
• Zfp57 is Essential for Transcriptional Regulation of Monoallelic Gene Expression in Ground-State Embryonic Stem Cells

  Hiroaki Ohishi; Motoko Unoki; Kei Fukuda; Takashi Sado; Hiroyuki Sasaki  GENES & GENETIC SYSTEMS  89-  (6)  309  -309  2014/12
• The defects in X-inactivation by partially dysfunctional Xist allele

  Tatsuro Nakajima; Yuko Hoki; Hiroyuki Sasaki; Takashi Sado  GENES & GENETIC SYSTEMS  89-  (6)  308  -308  2014/12
• Toward understanding of the mechanism involved in nuclear retention of Xist RNA

  Yuta Chigi; Hiroyuki Sasaki; Takashi Sado  GENES & GENETIC SYSTEMS  89-  (6)  307  -307  2014/12
• Epigenetic changes in the X chromosome accumulated with Xist RNA defective in silencing

  Yuka Sakata; Yuko Hoki; Hiroyuki Sasaki; Takashi Sado  GENES & GENETIC SYSTEMS  88-  (6)  371  -371  2013/12
• The defects in epigenetic regulation caused by partially dysfunctional Xist RNA

  Tatsuro Nakajima; Yuko Hoki; Hiroyuki Sasaki; Takashi Sado  GENES & GENETIC SYSTEMS  88-  (6)  370  -370  2013/12
• CpG methylation on the X-chromosome inactivated by a hypomorphic Xist allele in the mouse embryo

  Tatsuro Nakajima; Yuko Hoki; Hiroyuki Sasaki; Takashi Sado  GENES & GENETIC SYSTEMS  87-  (6)  414  -414  2012/12
• Silent chromatin domain formed by Xist RNA defective in silencing in the mouse embry

  Yuka Sakata; Yuko Hoki; Hiroyuki Sasaki; Takashi Sado  GENES & GENETIC SYSTEMS  87-  (6)  414  -414  2012/12
• Genetic approach towards the understanding of the molecular basis of Xist RNA-mediated chromosome silencing

  Takashi Sado  GENES & GENETIC SYSTEMS  87-  (6)  389  -389  2012/12
• X chromosome reactivation in female germ cells in the mouse

  Takashi Sado  GENES & GENETIC SYSTEMS  86-  (6)  385  -385  2011/12
• X-inactivation triggered by Xist RNA deleted for the critical 5 ' sequence in the mouse embryo

  Yuka Sakata; Yuko Amakawa; Yuko Hoki; Hiroyuki Sasaki; Takashi Sado  GENES & GENETIC SYSTEMS  86-  (6)  414  -414  2011/12
• Epigenetic regulation of the mammalian X chromosome and associated noncoding RNA

  佐渡 敬  医学のあゆみ  238-  (5)  407  -412  2011/07
• Role of the 5 ' region of Xist RNA in X-chromosome inactivation

  Yuka Sakata; Yuko Amakawa; Yuko Hoki; Hiroyuki Sasaki; Takashi Sado  GENES & GENETIC SYSTEMS  85-  (6)  408  -408  2010/12
• Towards the understanding of the molecular mechanism of Xist RNA-mediated silencing

  Takashi Sado  GENES & GENETIC SYSTEMS  85-  (6)  442  -442  2010/12
• 遺伝子欠損マウスを用いたCENP-O複合体の機能解析

  香川 尚子; 堀 哲也; 保木 裕子; 佐渡 敬; 山縣 一夫; 小久保 博樹; 相賀 裕美子; 深川 竜郎  日本生化学会大会・日本分子生物学会年会合同大会講演要旨集  83回・33回-  3P  -0511  2010/12
• Dynamics of the X chromosome activity and the DNA methylation status of X-linked genes during X reactivation in mouse female PGCs

  Michihiko Sugimoto; Takashi Sado; Kuniya Abe  GENES & GENETIC SYSTEMS  84-  (6)  442  -442  2009/12
• 体細胞核移植胚盤胞期胚に観察される遺伝子発現異常の解析

  井上貴美子; 越後貫成美; 幸田尚; 佐渡敬; 石野史敏; 小倉淳郎  J Reprod Dev  55-  (Supplement)  J82  2009/08
• piRNAによるインプリント遺伝子Rasgrf1のDNAメチル化制御

  渡部聡朗; 三ツ矢幸造; 宮川さとみ; 中馬新一郎; 富澤信一; 十時泰; 豊田敦; 山本耕裕; 保木裕子; 佐渡敬; 野瀬俊明; 仲野徹; 佐々木裕之  日本RNA学会年会要旨集  11th-  50  2009/07
• Dynamics of the X chromosome activity and the DNA methylation of X-linked genes during female PGC development

  Michihiko Sugimoto; Takashi Sado; Kuniya Abe  GENES & GENETIC SYSTEMS  83-  (6)  500  -500  2008/12
• An attempt to preventing X chromosome reactivation in Primordial germ cells

  Yuko Amakawa; Yuko Hoki; Hiroyuki Sasaki; Takashi Sado  GENES & GENETIC SYSTEMS  83-  (6)  500  -500  2008/12
• Regulation of Xist in the mouse embryo

  Takashi Sado; Yuko Hoki; Naomi Kimura; Hiroyuki Sasaki  GENES & GENETIC SYSTEMS  83-  (6)  500  -500  2008/12
• CENP-50 is essential for early development in mice

  Tetsuya Hori; Takashi Sado; Tatsuo Fukagawa  GENES & GENETIC SYSTEMS  82-  (6)  515  -515  2007/12
• Inhibition of X chromosome reactivaton in mouse inner cell mass

  Yuko Amakawa; Yuko Hoki; Hiroyuki Sasaki; Takashi Sado  GENES & GENETIC SYSTEMS  82-  (6)  532  -532  2007/12
• Toward understanding of the molecular mechanisms of dosage compensation in mammals

  Takashi Sado  GENES & GENETIC SYSTEMS  82-  (6)  516  -516  2007/12
• クロマチン工学への応用に向けたキネトコアタンパク質複合体の再構成

  堀哲也; 真柳浩太; 前仲勝実; 保木裕子; 佐渡敬; 岡田聖裕; 深川竜郎  生化学  2P-0788  2007
• 機能性RNAによるダイナミックな染色体の活性制御--哺乳類X染色体不活性化のしくみ (特集=RNAが生物の常識を変える)

  佐渡 敬  科学  76-  (5)  512  -516  2006/05
• X染色体不活性化のメカニズムを解く（特集：総研大発の染色体研究 / Part 2 染色体研究の最前線）

  佐渡 敬; Takashi SADO; サド タカシ  総研大ジャーナル = Sokendai journal  (9)  16  -19  2006/03
• 遺伝子に刷り込まれた記憶〜細胞分化とDNAのメチル化・ゲノムインプリンティング〜

  金田正弘; 佐渡敬; 佐々木裕之  遺伝  58-  (1)  69  -74  2004
• A now diagnostic method for functional chromosome abnormalities based on RNA-FISH.

  T Kubota; M Matsumura; S Morita; Hatada, I; K Okumura; T Sado; H Sasaki; Y Goto  AMERICAN JOURNAL OF HUMAN GENETICS  73-  (5)  333  -333  2003/11

Awards & Honors

• 2006/09 日本遺伝学会 奨励賞
   

  受賞者: 佐渡 敬

Research Grants & Projects

• 生命発動と器官発生・制御に関わるヒト受精胚分子機序の解明

  日本学術振興会：科学研究費助成事業 基盤研究(A)

  Date (from‐to) : 2020/04 -2024/03 

  Author : 阿久津 英憲; 佐渡 敬; 中林 一彦; 高田 修治; 深見 真紀; 福田 篤

   

  発生モデルを応用するとともに分子遺伝学的アプローチを受精胚クロマチン構造解析に対しても進めていく。モデルの発展性として、ヒトへの展開が必須であり、安定した実験系としてヒト多能性幹細胞を用いたX染色体不活化関連解析を実施した。今年度は、試験管内でのXX核型ES/iPS細胞において、X染色体不活化制御を担うXIST遺伝子のプロモーター領域のDNAメチル化が逸脱した遺伝子発現を誘導することを見いだし国際専門誌へ発表した。さらに、X染色体不活化においてXISTと相反する作用として着目されていたXACT遺伝子が、ヒトでは実はX染色体不活化に関連性がないことを初めて明らかにした。女性固有のサイトジェネティクス動態の科学的エビデンスを発生と器官発生・制御の理解を深めるための重要な基盤知見を得ることができた。
• Elucidation of oocyte molecular mechanism on imprinted X chromosome inactivation enabling a sequential development

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (A)

  Date (from‐to) : 2017/04 -2020/03 

  Author : Akutsu Hidenori

   

  During oocyte maturation, the chromatin is condensed, but after fertilization, the chromatin becomes asymmetric condensing. At the early blastocyst stage, each allylic chromatin relaxes and becomes "Neutral" (chromatin initialization). Subsequently, it has been shown that although placental development takes the form of asymmetric chromatin, random inactivation of the X chromosome occurs in ontogenetic systems under the condensation of random chromatin of both sexes during the peri-implantation periods. Oct4 gene, a pluripotent factor, is responsible for controlling the condensation and relaxation of nuclear chromatin that controls X-chromosome inactivation, and the OCT4 protein acts as a chromatin opener that leads to a relaxed state from chromatin condensation, indicating that the spatiotemporal control of OCT4 is associated with totipotency.
• ヘテロクロマチン形成におけるXist RNA作動エレメントの役割

  文部科学省：科学研究費補助金 (新学術領域研究(研究領域提案型))

  Date (from‐to) : 2017/04 -2019/03 

  Author : 佐渡 敬
• ヘテロクロマチン形成とクロマチン環境

  文部科学省: 科学研究費補助金（新学術領域研究(研究領域提案型))：

  Date (from‐to) : 2016 -2017 

  Author : 佐渡 敬
• 染色体活性制御と偽ヘテロクロマチン化の相関

  文部科学省：科学研究費補助金(新学術領域研究(研究領域提案型))

  Date (from‐to) : 2014 -2015 

  Author : 佐渡 敬

   

  変異型Xist RNAが構築する偽ヘテロクロマチンのヒストン修飾についてChIP-seq解析を行うにはマウス初期胚では十分な材料が得られないため、これに代わる材料として変異胚から樹立したES細胞（ESC）の利用を検討した．その中で生じた最も大きな問題は、メスのESCが培養過程でX染色体を1本欠失し、XOとなる細胞が無視できない頻度で出現することであった．そのため、ESCを利用した解析はこれまでのところ期待通りには進展していない．その打開策として、ESCに変わる新たな材料としてEpiSCの樹立する準備を進めている． 一方、変異Xist RNAが引き起こすサイレンシングの異常の程度を明らかにするため、変異胚を用いたRNA-seqを計画した．胎盤を形成する胚体外組織と胎児を形成する胚体組織の間にはX染色体不活性化の仕組みに違いがあるため、それぞれを分けて解析を行っている．胚体外組織については予定しているシーケンシングの大半を終了し、得られたリードの解析を進めている．胚体組織ついては、順次シーケンシングを行っている状態である．これまでに得られた胚体外組織の解析からは、不活性化すべきX染色体を不活性化できないと、X連鎖遺伝子の発現が2倍になるという単純なものではなく、X連鎖遺伝子にも常染色体連鎖遺伝子にも発現レベルが上昇するもの、減少するものが多数観察された．これはX染色体不活性化が単純に雌雄間のX連鎖遺伝子量の差を補償するためだけの機構にとどまらず、ゲノムワイドの遺伝子発現に大きなインパクトを持つものであることを示唆している．このRNA-seq解析を早急に終え、X染色体不活性化の生物学的意義に関するこの新たな考え方を迅速に成果として公表できるよう努める．
• 長鎖ノンコーディングRNAのクロマチンターゲティング

  文部科学省：科学研究費補助金(新学術領域研究(研究領域提案型))

  Date (from‐to) : 2014 -2015 

  Author : 佐渡 敬

   

  当該研究ではクロマチンのエピジェネティック制御に重要な役割を果たすと考えられるlncRNAがどのように標的クロマチン領域をターゲティングし，その効果を発揮するのかを理解することを目的とした．具体的には，哺乳類のメスの細胞でX染色体を覆い，そのヘテロクロマチン化を引き起こすXist RNAとその局在制御に関与することが示されたhnRNP Uに着目して解析を行ってきた．hnRNP Uの完全機能欠損マウスは報告されていなかったので，条件的ノックアウトマウスを作製した．完全機能欠損アレルへ変換後，ヘテロ接合体同士を交配し，ホモ接合体の表現型を解析した．その結果，ホモ接合体は着床前後の時期に致死となることがわかった．また，分離比からヘテロ接合体のうち離乳に達するものは期待される頻度の20%程度であることが示唆された． 胚発生に重篤な影響をもたらす単純な機能阻害では解析が困難と思われたので，タモキシフェン投与により条件的にhnRNP Uの機能を阻害できるコンディショナルアレルを持つメスの胚線維芽細胞（MEF）あるいはメスのES細胞を胚盤胞から樹立し，胚に代わる解析系を確立することを試みた．その結果得られたこれらの細胞で， hnRNP Uの機能を条件的に阻害してみると，いずれの場合も雌雄の別なく細胞は数日で死ぬことから，hnRNP Uは細胞の生存に不可欠であることがわかった．メスの細胞でhnRNP Uを阻害した後，細胞が死ぬまでの間に免疫RNA-FISHを行うと，不活性X染色体に局在するXist RNAは失われ，H3K27me3の集積も観察されなくなったことから，hnRNP UがXist RNAのクロマチンターゲティングに不可欠であることが確認された．hnRNP Uが他のlncRNAのクロマチンターゲティングにも関与するかについては，解析中のため現時点では不明である
• Moelcular baiss for the maintenance of the epigenome and its failure

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2013 -2015 

  Author : SADO Takashi

   

  We attempted to study the molecular mechanism for the maintenance of epigenetic gene regulation by SmcHD1, which was suggested to be independent of more general polycomb group proteins. SmcHD1 was initially assumed to be involved in the maintenance of the inactivated X chromosome. Knockout of both copies of Smchd1 in embryonic fibroblasts, which stably maintain the X-inactivated state, did not affected genes on the inactive X, suggesting that the X-inactivated state could be maintained in the absence of SmcHD1. SmcHD1 may rather play a role in the process of the establishment of X-inactivation by creating an appropriate chromatin environment required for the maintenance, and may be dispensable for the maintenance once such an environment was established.
• Functional machinery for non-coding RNAs

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2009/07 -2014/03 

  Author : TOMARI Yukihide; KAGEYAMA Yuji; SUZUKI Takeo; TEI Kumiko; NAKAZAWA Takanobu; WADA Takeshi; SADO Takashi; SIOMI Mikiko; MIYAGAWA Satomi; NAKAGAWA Shinichi; TAKESHITA Fumitaka; ODA Takeaki; YAMASHITA Akio; NISHIMURA Yukiko

   

  H21～25年度の新学術領域研究「非コードRNA作用マシナリー」領域からは非コードRNA動作原理に関する優れた研究成果が多数生み出され、本領域はホームページや雑誌企画、ワークショップ、一般向けのプレスリリースなど、外部に対して積極的に情報を発信し、得られた研究成果を社会に還元するように努めてきた。これまで、当該分野で顕著な成果を上げている海外の研究者と共に国際シンポジウムを開催することで、国際的にもこの分野を牽引する日本の非コードRNA研究を世界に向けて発信してきた。平成26年度の本研究取りまとめにおいては、過去の実績を踏まえ、本領域の研究期間である５年間を通して得られた研究成果を取りまとめ、その全体像を当該学問領域のみならず、一般国民に分かりやすく還元すると共に情報の発信を継続することを目的として下記の計画を遂行した。 ①本分野をリードする著名な海外の研究者を第37回日本分子生物学会年会シンポジウムおよびワークショップに招聘するとともに、本領域主催の「非コードRNA作用マシナリー」をテーマとした国際シンポジウムの開催し、領域の研究成果を国内外に発信すると共に海外の研究者との情報交換を強化した。②領域の研究成果を広く社会・国民に発信するためのアウトリーチ活動の一環として、領域班員がRNA関連研究を伝える活動を地域の教育・研究機関で行った。③領域の研究成果や上記イベント等を広報するために、引き続きncrna.jpドメインを維持し、ホームページやブログを継続公開・更新した。④研究成果をわかりやすくまとめた成果報告書を作成し、本領域外の科学分野面にも広く配布した。 以上のように、本領域の研究成果を当該分野のみならず、広く一般国民に還元することができたと考える。
• Regulatory mechanism of Xist RNA machinery

  Ministry of Education, Culture, Sports, Science and Technology：Grants-in-Aid for Scientific Research(新学術領域研究(研究領域提案型))

  Date (from‐to) : 2009 -2013 

  Author : Takashi SADO

   

  In this mission, we attempted to figure out the regulatory mechanisms of Xist RNA machinery, which has been known for many years to play a pivotal role in X chromosome inactivation in female mammals. Especially, we focused on the potential role of the proteins, which has been shown to localize to the inactive X, and their interacting partners in Xist RNA-mediated chromatin silencing. The findings indicates the importance of further studies of the potential role of factors involved in replication programming in the Xist RNA machinery. In addition, we have paved the way to our further understanding of the Xist RNA machinery by deriving various cell lines from the mice we established by knocking out a protein involved in the regulation of Xist RNA deposition to the inactive X.
• Establishment of silent chromatin in mammals

  Ministry of Education, Culture, Sports, Science and Technology：Grants-in-Aid for Scientific Research(基盤研究(B))

  Date (from‐to) : 2008 -2010 

  Author : Takashi SADO

   

  X chromosome inactivation (X-inactivation) in female mammals is triggered by differential upregulation of the Xist gene on one of the two X chromosomes and subsequent coating of the X in cis with its noncoding transcripts. Although targeted mutation clearly showed that Xist is essential for X-inactivation to occur in cis, the molecular mechanism by which Xist RNA induces chromosome silencing is largely unknown. Here, we demonstrate that an Xist mutant generated previously by gene targeting, Xist^ is unique in that it retains a partial function to silence the X chromosome. Although Xist^ is differentially upregulated and its mutated transcript coats the X chromosome in cis in embryonic and extraembryonic tissues, X-inactivation thus initiated does not seem to be fully established. The state of such incomplete inactivation is probably unstable and the mutated X apparently ends up reactivated in a subset of extraembryonic tissues and, perhaps, early epiblastic cells. Xist^, which can be referred to as a partial loss of function mutation, would provide an opportunity to dissect the molecular mechanism of Xist RNA-mediated chromosome silencing.
• 配偶子ゲノムの修飾機構と胚発生への影響

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 2003 -2007 

  Author : 佐々木 裕之; 佐渡 敬; 秦 健一郎

   

  最終年度にあたり、研究計画にそって以下の研究を行い、結果を取りまとめた。(1)DNAメチル化酵素Dnmt3aと補助因子Dnmt3Lに加えて、Dnmt3bが雄の配偶子形成過程で反復配列を修飾することが分かったので、その詳細な解析を完成させ報告した。また、生殖細胞特異的に発現するpiRNAに結合するPiwiファミリー蛋白質が、反復配列(とくにレトロトランスポソン)のメチル化に関わることを発見し報告した。(2)一方、Dnmt3bの欠損は雌雄の配偶子形成を阻害せず、仔の発生にも影響がないことが分かった。すなわち、反復配列のメチル化は受精後のDnmt3bの供給により回復される。現在投稿準備中である。(3)ゲノムワイドな脱メチル化が生じる卵割期に(Dnmt3aやDnmt3bではなく)Dnmt1がインプリントを維持することを証明できたので、論文にまとめた(投稿中)。最後に、(4)two-hybrid法にて上記メチル化酵素複合体と相互作用する因子を得るため、生殖細胞特異的ライブラリーを作成した。このライブラリーは、将来の研究において役立てる予定である。
• アンチセンスRNAによるエピジェネティックな修飾の構築機構

  文部科学省：科学研究費補助金(特定領域研究)

  Date (from‐to) : 2005 -2006 

  Author : 佐渡 敬

   

  Tsixの転写によって産生されるスプライシングを受けたRNAが,Tsixを介したXistの発現制御機構にどのようなインパクトを持つかについては,前年度の解析から,胚発生においてはTsixのスプライシング欠損アリル(Tsix^<ΔSA>)を持つX染色体上でもXistのエピジェネティック制御は適切に確立されることから,Tsixスプライシング産物はTsixによるXist遺伝子のアンチセンス制御を担うものではないという結論に達していた.そこで,平成18年度開始当初は,その投稿準備を進めるとともに,新たにTsixはRNAポリメラーゼII(polII)によって転写されることが必要なのか検討する計画であった.ところが,論文投稿にあたって,いくつかの追加実験を行ったところ,未分化なオスES細胞においてはTsixのスプライシング欠損を持つX染色体からのXistの発現量が野生型のそれに比べ有意に高いことがわかった.これは胚における前年度からの解析結果と一見矛盾するが,Tsix^<ΔSA>アリルを持つこのES細胞を分化させるとXistの発現は野生型同様消失することから,やはりTsixを介したXistの発現制御機構に大きな影響はないことが改めて示された.さらに以前作製したTsix^<ΔSA>とよく似た構造を持つもののTsixによるアンチセンス転写は正常に認められる改変アリル(Xist^<1lox>)を持つES細胞を用いて同様の解析を行った結果,この場合も未分化なES細胞においてのみXistの発現上昇が認められ,分化誘導後のXistの発現は適切に抑えられることがわかった.これらの結果から,Tsix^<ΔSA>とXist^<1lox>で共通に欠失している領域に未分化状態特異的にXistの発現制御に効果を持つ配列が存在すると考えた.一連の成果はDevelopment誌に掲載された.現在は,新たにその存在が示唆された未分化状態特異的に効果を持つXistの発現制御配列について解析を進めている.polIIによる転写の意義に関しては,現在中断している.
• X染色体不活性化センターにおけるアンチセンス制御機構

  文部科学省：科学研究費補助金(特定領域研究)

  Date (from‐to) : 2005 -2006 

  Author : 佐渡 敬

   

  Xistのアンチセンス遺伝子Tsixを介したXistの発現制御機構において,Xistプロモーター領域がTsixによって転写されることが不可欠であるか調べるため,ポリA付加シグナルを利用してアンチセンス転写の強制的な終結を図った.初年度の引き続きその影響を詳細に解析した結果,Tsixの転写がXistプロモーター領域に及ばないようにしたTsix^<ρA>アリルを持つマウス胚では,変異を導入したX染色体上のXist遺伝子プロモーター領域のクロマチン修飾が正しく構築されず,その結果Xistの異所的な発現亢進を招くことが強く示唆された.Tsix^<ρA>アリルとよく似た構造を持つもののポリA付加シグナルを欠くためTsixによるアンチセンス転写は中断されないアリル(Xist^)を持つマウスを新たに作製し胚におけるXistプロモーター領域のクロマチン修飾を解析したところ目立った異常は認められずXistも適切に制御されていることがわかった.これらの結果は,Tsixによるアンチセンス制御においては,Xistプロモーター領域がアンチセンス方向の転写を受けることが不可欠であることを示している.また,Tsix^<ρA>アリルの影響を胚体組織と胚体外組織で比較したところ,後者はより顕著な影響を受けていることもわかった.これは,胚体組織がTsix非依存なXist発現制御機構も持ち合わせているのに対し,胚体外組織はもっぱらTsixに依存しているとする,我々の従来の報告とも一致する.これらの解析については現在論文を投稿中である.一方,Xist RNAの核外輸送抑制におけるXist配列の効果について検討する新たな試みのために,従来のものとは異なる構造を持つレポーター配列を新たにXist遺伝子座にノックインしたES細胞を複数株作製した.今後これらを用いて詳細な解析を行う.
• 脊椎動物におけるエピジェネティクス機構の比較ゲノム解析

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 2003 -2003 

  Author : 佐々木 裕之; 秦 健一郎; 佐渡 敬

   

  エピジェネティクスはゲノム機能を調節する重要な機構だが、脊椎動物の中でも哺乳類には性染色体の遺伝子量補償やゲノムインプリンティングがあるが、鳥類には存在しないなど、役割や働き方に違いがある。本研究では、脊椎動物のエピジェネティクスで中心的な役割を果たすDNAメチル化酵素(DNMT)ファミリーに注目し、哺乳類と鳥類の間での比較ゲノム学アプローチと標的遺伝子破壊を用いて、その機能と進化を総合的に理解する。まず、これまで鳥類におけるインプリンティングの不在はIGF2とMPRの2つの遺伝子の結果だけに依存していたため、新たにCASH4とINS(ともに哺乳類ではインプリンティングを受ける)を調べ、インプリンティングのないことを確かめた(投稿中)。次に、(1)ニワトリのDNAメチル化酵素DNMT2、DNMT3A,DNMT3BのcDNAを全て単離し解析した(投稿準備中)(DNMT1は既に他研究室で単離済み)。(2)哺乳類のインプリンティングに特異的に関与するDNMT1o(DNMT1のアイソフォーム)、DNMT3A2(DNMT3Aのアイソフォーム)、DNMT3Lのホモログがニワトリに存在するか、サザン法やシークエンスの結果をもとに検討を始めた。また、(3)酵素活性や時間的空間的局在を解析するためのベクター作り、(4)ニワトリDNMTをDT40細胞中でノックアウトするためのベクター作りを開始した。(5)それらの実験により得られる結果をマウスのノックアウトと比較し、脊椎動物のエピジェネティックな機構の普遍性と多様性を知ることについては、来年度以降に持ち越した。
• Molecular Genetic Studies on the Physiological Function and Disorders of DNA Methylation

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2001 -2003 

  Author : SASAKI Hiroyuki; NAKAO Mitsuyoshi; SADO Takashi

   

  Methylation of CpG dinucleotides plays a central role in the epigenetic genome regulation, and its defects cause congenital disorders and cancers. We have studied the functions and disorders of DNA methyltransferases (DNMTs) and methyl-CpG-binding proteins (MBD proteins) and found the followings. 1.By knocking out mouse DNMT3A, a de novo-type. DNMT, in a germline-specific manner, we found that this enzyme is essential for paternal and maternal imprinting. 2.Enzymatic properties and target specificities of DNMT3A and DNMT3B were determined. 3.New mutations of DNMT3B were identified in Japanese families with ICE syndrome. We also found an ICE case with no mutation in DNMT3B, which suggests the heterogeneity of the disease. 4.We reported that paternal disomy 14 is a new imprinting-related disorder characterized by bell-shaped chest and wavy ribs. 5.We found that MBD1 represses transcription and forms repressive chromatin through two independent pathways: 1) one mediated by a complex formed with a transcriptional mediator MCAF/AM and a histone methyltransferase SETDB1/ESET; 2) the other involving a histone methyltransferase Suv39, a histone deacetylase HDAC1/2 and a heterochromatin protein HP1. 6.The tertiary structure of the DNA binding domain of MBD1 complexed with a. methylated-CpG-containing DNA molecule was determined. 7.The mutations of McCP2 causing Rett syndrome were found to impair its ability to repress transcription and to form repressive chromatin. 8.We reported that MBD1 promotes base excision repair in cooperation with a methylated-purine DNA glycosylase. All together, our findings have greatly increased our knowledge on the mechanisms of DNA methylation and transcriptional repression through it and the pathology of DNA methylation-associated disorders.
• X染色体不活性化における機能的RNAと de novoメチル化酵素の相互作用

  文部科学省：科学研究費補助金(特定領域研究)

  Date (from‐to) : 2002 -2002 

  Author : 佐渡 敬

   

  (1)テトラサイクリンによるDnmt3a, Dnmt3bの発現誘導系をde novoメチル化酵素欠損ES細胞へ導入することはできた.しかし,これらの酵素の発現を誘導しても,Xistプロモーターにおけるメチル化の回復は認められなかった.また,分化誘導後Xistを異所的に発現している状況でDnmt3a, Dnmt3bを誘導しても,X染色体不活性化よると思われる細胞死は引き起こされなかった.したがって,Dnmt3a, Dnmt3bはXistの発現制御のみならず,X染色体全域に結合した後のプロセスにも必要であるという当初の仮説は適切ではなかったと思われる.むしろ,X染色体不活性化が細胞分化と密接に関わっていることを考えると,de novoメチル化酵素欠損ES細胞では,異所的なXistの発現は不活性化を誘導するのに重要な分化段階を経過してから起こったため,染色体の不活性化には至らなかったのではないかと考えている.(2)Dnmt3a/Dnmt3b二重欠損マウス胚におけるX染色体不活性化を解析した.雌の正常マウス胚では二者択一的にメチル化されているXistプロモーター領域が,Dnmt3a/Dnmt3b二重欠損マウス胚では極端に低メチル化状態になっているにもかかわらず,大多数の細胞においてXistのディファレンシャルな発現は影響を受けていなかった.また,不活性化も細胞分化にともない正常に進行すると思われた.これらは,Dnmt3aおよびDnmt3bがX染色体不活性化の開始と不活性状態の染色体全体への伝播に必須ではないことを示している.また,不活性化の開始にはXistのmonoallelicな発現亢進が重要であるが,今回の解析からDnmt3a/Dnmt3bによるXistプロモーターの二者択一的なde novoメチル化がそれを担う最初のイベントではないことも強く示唆された.
• ゲノムインプリンティングドメインの構造・制御・進化

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 2001 -2001 

  Author : 佐々木 裕之; 佐渡 敬

   

  ゲノムインプリンティングを受ける哺乳類遺伝子がゲノムの特定の場所にクラスターを形成することは,それがインプリンティングの制御機構や進化に密接に関わることを示唆している。本研究課題では,マウス第7染色体遠位部にあるインプリンティングドメインをモデルとして,機能ドメインの構造,制御,進化について比較ゲノム学とマウス発生工学を用いた総合的な研究を行った。まず,これまでの研究により得られたこのドメイン(およそ1.0Mbに10個余のインプリンティング遺伝子がある)の塩基配列を手掛かりとして,新たなエンハンサー,インスレーター,核マトリクス付着部位などを同定した。すなわち,これらの配列はヒトとマウス間で保存されており,その働きはin vivo,in vitroの機能測定により確かめられた。また,インプリンティングが哺乳類で進化した理由を探るため,上記のドメインに対応するニワトリのドメインをBACにクローン化し,その全塩基配列(およそ0.5Mb)を決定した。また,このドメイン内にあるニワトリIGF2,CASH4などの遺伝子は,その哺乳類のオルソログとは異なり,インプリンティングを受けないことを示した。今後は,哺乳類・鳥類間のさらに詳細な比較ゲノム解析により,インプリンティングドメインの進化についての手掛かりを得たい。
• マウス胚発生におけるDNAメチル化の意義

  文部科学省：科学研究費補助金(奨励研究(A))

  Date (from‐to) : 2000 -2001 

  Author : 佐渡 敬

   

  本研究課題では、ほ乳類発生に不可欠であるX染色体不活性化におけるDNAメチル化の意義を調べるために、その触媒酵素である3種類のDNAメチル化酵素Dnmt1,Dnmt3a,およびDnmt3bの欠損マウス、およびES細胞を材料に研究を行ってきた。Dnmt1ノックアウトマウス胚における染色体複製時期を観察した結果、今後さらに解析が必要であるものの、胎仔が死亡する頃には不活性X染色体のみならず全ての染色体の複製時期が異常になっている可能性が示唆された。また、当初計画していたDnmt1のコンディショナルノックアウトマウスに関しては、ターゲティングに成功したES細胞を用いて多数のキメラマウスを作製したものの、いまだノックアウトマウスの誕生には至っていない。Dnmt3a,Dnmt3bの各欠損ES細胞を用いた解析から、これらの細胞ではX染色体不活性化の過程に異常は生じていないと考えられた。しかし、両者の二重欠損ES細胞においては分化誘導後、不活性化の開始に不可欠なXist遺伝子が異所的に発現されることが分かった。ところが、X染色体連鎖遺伝子の発現解析から、X染色体の不活性化には至っていないことが示唆された。これらの結果は、X染色体不活性化にはXistの発現に引き続き、Dnmt3a,Dnmt3bの機能が関わるプロセスが必要であることを示唆している。
• ゲノムインプリンティングドメインの構造・制御・進化

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 2000 -2000 

  Author : 佐々木 裕之; 佐渡 敬

   

  ゲノムインプリンティングを受ける哺乳類遺伝子がゲノムの特定の場所にクラスターを形成することは、それがインプリンティングの制御機構や進化に密接に関わることを示唆している。本研究ではインプリンティングドメインをモデルとして、今日の分子生物学の重要な課題である染色体の機能ドメインの構造、制御、進化についてゲノム解析を用いた総合的な研究を行う。本年度は、マウス第7染色体遠位部にあるインプリンティングドメイン(およそ1Mbに10個余のインプリンティング遺伝子がある)の全塩基配列を佐賀医大向井ら、理研服部らと共同で決定した。ヒトの当該領域(第11染色体短腕)と比較した結果、例えばKvLQT1のイントロン内に進化的に保存された配列があり、それらが制御配列である可能性が示された。さらに、H19周辺の保存された配列を実際にレポーター測定法やゲルシフト法により調べると、組織特異的エンハンサーやインプリンティングに関わるインスレーター蛋白質の結合配列であることが分かった。現在、このドメインの配列の特性などをコンピュータで詳しく解析中である。また、インプリンティングが哺乳類で進化した理由を探るため鳥類の研究を始め、上記のドメイン内にあるIGF2がニワトリでインプリンティングを受けないことを示した。当該ニワトリ領域をBACにクローン化し、現在その塩基配列の解析中である。鳥類も含めた比較ゲノム学的アプローチで、インプリンティングの進化についての手掛かりを得たい。
• X染色体不活性化の分子機構

  Date (from‐to) : 1999
• Molecular Mechanisms of X-inactivation

  Date (from‐to) : 1999

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