| 1. |
K Miyamoto, Y Hoshino, Y Nagao, N Minami, M Yamada, H Imai, 53 CELL CYCLE SYNCHRONIZATION OF DONOR CELLS AT G1 PHASE AND DEVELOPMENTAL ABILITY OF NUCLEAR TRANSFER EMBRYOS IN MINIATURE PIGS, Reproduction, Fertility and Development, 17, 2, 176-176, 2004.12. |
| 2. |
宮本 圭, 今井 裕, 無細胞抽出系を用いた培養細胞のリプログラミングとその応用, 日本胚移植学雑誌, 28, 2, 81-87, 2006.05. |
| 3. |
Kei Miyamoto, Tadashi Furusawa, Mari Ohnuki, Sandeep Goel, Tomoyuki Tokunaga, Naojiro Minami, Masayasu Yamada, Keita Ohsumi, Hiroshi Imai, Reprogramming events of mammalian somatic cells induced by Xenopus laevis egg extracts, MOLECULAR REPRODUCTION AND DEVELOPMENT, 10.1002/mrd.20691, 74, 10, 1268-1277, 2007.10, It is known that differentiated cells can be reprogrammed to an undifferentiated state in oocyte cytoplasm after nuclear transfer. Recently, some reports suggested that Xenopus egg extracts have the ability to reprogram mammalian somatic cells. Reprogramming events of mammalian cells after Xenopus egg extract treatment and after cell culture of extract-treated cells have not been elucidated. In this experiment, we examined reprogramming events in reversibly permeabilized or nonpermeabilized porcine fibroblast cells after Xenopus egg extract treatment. The Xenopus egg-specific histone 134 was assembled on porcine chromatin and nuclear lamin LIII was incorporated into nuclei. Deacetylation of histone H3 at lysine 9 in extract-treated cells was detected in nonpermeabilized cells, suggesting that a part of reprogramming may be induced even in nonpermeabilized cells. Following culture of extract-treated cells, the cells began to express the pluripotent marker genes such as POU5F1 (OCT4) and SOX2 and to form colonies. Reactivation of the OCT4 gene in extract-treated cells was also confirmed in bovine fibroblasts transformed with an OCT4-EGFP construct. These results suggest that nuclei of mammalian cells can be partially reprogrammed to an embryonic state by Xenopus egg extracts and the remodeled cells partly dedifferentiate after cell culture. A system using egg extracts may be useful for understanding the mechanisms and processes of dedifferentiation and reprogramming of mammalian somatic cells after nuclear transfer.. |
| 4. |
Kei Miyamoto, Yoichiro Hoshino, Naojiro Minami, Masayasu Yamada, Hiroshi Imai, Effects of synchronization of donor cell cycle on embryonic development and DNA synthesis in porcine nuclear transfer embryos, JOURNAL OF REPRODUCTION AND DEVELOPMENT, 10.1262/jrd.18085, 53, 2, 237-246, 2007.04, The relationship between donor cell cycle and the developmental ability of somatic cell nuclear transfer (SCNT) embryos has not fully been elucidated. Donor cells that are usually prepared by serum starvation or confluent-cell culture for SCNT represent a heterogeneous population that includes mainly G0 phase cells, other cells in different phases of the cell cycle and apoptotic cells. In this study, we compared the developmental ability of porcine SCNT embryos reconstructed from G0 phase cells (GO-SCNT embryos) and strictly synchronized-G1 phase cells (G1-SCNT embryos), and examined the developmental rates and timing of first DNA synthesis. The G0 phase cells were synchronized by confluent culture, and the G1 phase cells were prepared from actively dividing M phase cells. The G1-SCNT embryos showed a significantly higher (P |
| 5. |
Kei Miyamoto, Teruyoshi Yamashita, Tomoyuki Tsukiyama, Naoya Kitamura, Naojiro Minami, Masayasu Yamada, Hiroshi Imai, Reversible Membrane Permeabilization of Mammalian Cells Treated with Digitonin and Its Use for Inducing Nuclear Reprogramming by Xenopus Egg Extracts, CLONING AND STEM CELLS, 10.1089/clo.2008.0020, 10, 4, 535-542, 2008.12, Plasma membranes can be reversibly permeabilized by Streptolysin O. The permeabilized cells can be reprogrammed and partially dedifferentiated in the cell-free system from egg extracts. However, the permeabilizing activity of Streptolysin 0 is not stable, and therefore it is difficult to control its activity. An alternative method for reversible permeabilization is useful for establishing a cell-free system. Here, we used a nonionic detergent, digitonin, for permeabilization. A low concentration of digitonin induced reversible permeabilization of the plasma membrane in bovine, mouse, and porcine somatic cells. The permeabilized cells were treated with Xenoptis laevis egg extracts. The treated cells showed exchange of nuclear proteins from extracts such as incorporation of Xenopus-specific histone B4 and Lamin LIII into nuclei. After resealing of the membrane, the cells showed upregulation of OCT4, SOX2, and NANOG expression. Our results suggest that reversible permeabilization with digitonin can be used to induce nuclear reprogramming and to activate pluripotent genes by a cell-free system.. |
| 6. |
K. Miyamoto, T. Tsukiyama, N. Minami, M. Yamada, H. Imai, Mammalian oocyte extracts induce chromatin remodeling and dedifferentiation of somatic cells, but do not global demethylation, REPRODUCTION IN DOMESTIC ANIMALS, 43, 194-194, 2008.07. |
| 7. |
Kei Miyamoto, Tomoyuki Tsukiyama, Yang Yang, Ning Li, Naojiro Minami, Masayasu Yamada, Hiroshi Imai, Cell-Free Extracts from Mammalian Oocytes Partially Induce Nuclear Reprogramming in Somatic Cells, BIOLOGY OF REPRODUCTION, 10.1095/biolreprod.108.073676, 80, 5, 935-943, 2009.05, Nuclear transfer has been regarded as the only reliable tool for studying nuclear reprogramming of mammalian somatic cells by oocytes. However, nuclear transfer is not well suited for biochemical analyses of the molecular mechanisms of reprogramming. A cell-free system from oocytes is an attractive alternative way to mimic reprogramming in vitro, since a large number of cells can be treated and analyzed. Nevertheless, a cell-free system using oocytes has not been developed in mammals. Here, cell extracts from porcine oocytes were prepared and their ability to induce nuclear reprogramming was evaluated. Extracts from metaphase II (MII) oocytes erased the machinery for regulating gene expression in reversibly permeabilized somatic cells. For example, the extracts caused histone deacetylation and the disappearance of TATA box-binding protein from the nuclei. However, MII-extract-treated cells did not show any obvious changes after cell culture. In contrast, extracts from germinal vesicle (GV) oocytes activated pluripotent marker genes, especially NANOG, and induced partial dedifferentiation after cell culture. The activation of pluripotent marker genes by GV extracts was associated with histone acetylation that was induced during extract treatment. These results indicate that GV- and MII-oocyte extracts have different roles on nuclear reprogramming. Furthermore, both oocyte extracts induced site-specific demethylation in the upstream region of NANOG. These results indicate that cell-free extracts derived from GV- and MII-oocytes could be useful for studying the mechanisms involved in nuclear reprogramming.. |
| 8. |
R. P. Halley-Stott, V. Pasque, C. Astrand, K. Miyamoto, I. Simeoni, J. Jullien, J. B. Gurdon, Mammalian nuclear transplantation to Germinal Vesicle stage Xenopus oocytes - A method for quantitative transcriptional reprogramming, METHODS, 10.1016/j.ymeth.2010.01.035, 51, 1, 56-65, 2010.05, Full-grown Xenopus oocytes in first meiotic prophase contain an immensely enlarged nucleus, the Germinal Vesicle (GV), that can be injected with several hundred somatic cell nuclei. When the nuclei of mammalian somatic cells or cultured cell lines are injected into a GV, a wide range of genes that are not transcribed in the donor cells, including pluripotency genes, start to be transcriptionally activated, and synthesize primary transcripts continuously for several days. Because of the large size and abundance of Xenopus laevis oocytes, this experimental system offers an opportunity to understand the mechanisms by which somatic cell nuclei can be reprogrammed to transcribe genes characteristic of oocytes and early embryos. The use of mammalian nuclei ensures that there is no background of endogenous maternal transcripts of the kind that are induced. The induced gene transcription takes place in the absence of cell division or DNA synthesis and does not require protein synthesis.
Here we summarize new as well as established results that characterize this experimental system. In particular, we describe optimal conditions for transplanting somatic nuclei to oocytes and for the efficient activation of transcription by transplanted nuclei. We make a quantitative determination of transcript numbers for pluripotency and housekeeping genes, comparing cultured somatic cell nuclei with those of embryonic stem cells. Surprisingly we find that the transcriptional activation of somatic nuclei differs substantially from one donor cell-type to another and in respect of different pluripotency genes. We also determine the efficiency of an injected mRNA translation into protein. (C) 2010 Elsevier Inc. All rights reserved.. |
| 9. |
V. Pasque, K. Miyamoto, J.B. Gurdon, Efficiencies and mechanisms of nuclear reprogramming, Cold Spring Harb Symp Quant Biol, 10.1101/sqb.2010.75.002, 75, 189-200, 2010.11. |
| 10. |
Kei Miyamoto, Vincent Pasque, Jerome Jullien, John B. Gurdon, Nuclear actin polymerization is required for transcriptional reprogramming of Oct4 by oocytes, GENES & DEVELOPMENT, 10.1101/gad.615211, 25, 9, 946-958, 2011.05, Amphibian oocytes can rapidly and efficiently reprogram the transcription of transplanted somatic nuclei. To explore the factors and mechanisms involved, we focused on nuclear actin, an especially abundant component of the oocyte's nucleus (the germinal vesicle). The existence and significance of nuclear actin has long been debated. Here, we found that nuclear actin polymerization plays an essential part in the transcriptional reactivation of the pluripotency gene Oct4 (also known as Pou5f1). We also found that an actin signaling protein, Toca-1, enhances Oct4 reactivation by regulating nuclear actin polymerization. Toca-1 overexpression has an effect on the chromatin state of transplanted nuclei, including the enhanced binding of nuclear actin to gene regulatory regions. This is the first report showing that naturally stored actin in an oocyte nucleus helps transcriptional reprogramming in a polymerization-dependent manner.. |
| 11. |
Kei Miyamoto, Vincent Pasque, John B. Gurdon, Nuclear actin in transcriptional reprogramming by oocytes Are actin nucleators key players?, CELL CYCLE, 10.4161/cc.10.18.16946, 10, 18, 3040-3041, 2011.09. |
| 12. |
Miyamoto K, Gurdon JB, Nuclear actin and transcriptional activation., Communicative & integrative biology, 10.4161/cib.4.5.16491, 4, 5, 582-583, 2011.09. |
| 13. |
Jerome Jullien, Vincent Pasque, Richard P. Halley-Stott, Kei Miyamoto, J. B. Gurdon, Mechanisms of nuclear reprogramming by eggs and oocytes: a deterministic process?, NATURE REVIEWS MOLECULAR CELL BIOLOGY, 10.1038/nrm3140, 12, 7, 453-459, 2011.07, Differentiated cells can be experimentally reprogrammed back to pluripotency by nuclear transfer, cell fusion or induced pluripotent stem cell technology. Nuclear transfer and cell fusion can lead to efficient reprogramming of gene expression. The egg and oocyte reprogramming process includes the exchange of somatic proteins for oocyte proteins, the post-translational modification of histones and the demethylation of DNA. These events occur in an ordered manner and on a defined timescale, indicating that reprogramming by nuclear transfer and by cell fusion rely on deterministic processes.. |
| 14. |
Kei Miyamoto, Kouhei Nagai, Naoya Kitamura, Tomoaki Nishikawa, Haruka Ikegami, Nguyen T. Binh, Satoshi Tsukamoto, Mai Matsumoto, Tomoyuki Tsukiyama, Naojiro Minami, Masayasu Yamada, Hiroyoshi Ariga, Masashi Miyake, Tatsuo Kawarasaki, Kazuya Matsumoto, Hiroshi Imai, Identification and characterization of an oocyte factor required for development of porcine nuclear transfer embryos, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 10.1073/pnas.1013634108, 108, 17, 7040-7045, 2011.04, Nuclear reprogramming of differentiated cells can be induced by oocyte factors. Despite numerous attempts, these factors and mechanisms responsible for successful reprogramming remain elusive. Here, we identify one such factor, necessary for the development of nuclear transfer embryos, using porcine oocyte extracts in which some reprogramming events are recapitulated. After incubating somatic nuclei in oocyte extracts from the metaphase II stage, the oocyte proteins that were specifically and abundantly incorporated into the nuclei were identified by mass spectrometry. Among 25 identified proteins, we especially focused on a multifunctional protein, DJ-1. DJ-1 is present at a high concentration in oocytes from the germinal vesicle stage until embryos at the four-cell stage. Inhibition of DJ-1 function compromises the development of nuclear transfer embryos but not that of fertilized embryos. Microarray analysis of nuclear transfer embryos in which DJ-1 function is inhibited shows perturbed expression of P53 pathway components. In addition, embryonic arrest of nuclear transfer embryos injected with anti-DJ-1 antibody is rescued by P53 inhibition. We conclude that DJ-1 is an oocyte factor that is required for development of nuclear transfer embryos. This study presents a means for identifying natural reprogramming factors in mammalian oocytes and a unique insight into the mechanisms underlying reprogramming by nuclear transfer.. |
| 15. |
Vincent Pasque, Jerome Jullien, Kei Miyamoto, Richard P. Halley-Stott, J. B. Gurdon, Epigenetic factors influencing resistance to nuclear reprogramming, TRENDS IN GENETICS, 10.1016/j.tig.2011.08.002, 27, 12, 516-525, 2011.12, Patient-specific somatic cell reprogramming is likely to have a large impact on medicine by providing a source of cells for disease modelling and regenerative medicine. Several strategies can be used to reprogram cells, yet they are generally characterised by a low reprogramming efficiency, reflecting the remarkable stability of the differentiated state. Transcription factors, chromatin modifications, and noncoding RNAs can increase the efficiency of reprogramming. However, the success of nuclear reprogramming is limited by epigenetic mechanisms that stabilise the state of gene expression in somatic cells and thereby resist efficient reprogramming. We review here the factors that influence reprogramming efficiency, especially those that restrict the natural reprogramming mechanisms of eggs and oocytes. We see this as a step towards understanding the mechanisms by which nuclear reprogramming takes place.. |
| 16. |
Patrick Narbonne, Kei Miyamoto, J. B. Gurdon, Reprogramming and development in nuclear transfer embryos and in interspecific systems, CURRENT OPINION IN GENETICS & DEVELOPMENT, 10.1016/j.gde.2012.09.002, 22, 5, 450-458, 2012.10, Nuclear transfer (NT) remains the most effective method to reprogram somatic cells to totipotency. Somatic cell nuclear transfer (SCNT) efficiency however remains low, but recurrent problems occurring in partially reprogrammed cloned embryos have recently been identified and some remedied. In particular, the trophectoderm has been identified as a lineage whose reprogramming success has a large influence on SCNT embryo development. Several interspecific hybrid and cybrid reprogramming systems have been developed as they offer various technical advantages and potential applications, and together with SCNT, they have led to the identification of a series of reprogramming events and responsible reprogramming factors. Interspecific incompatibilities hinder full exploitation of cross-species reprogramming systems, yet recent findings suggest that these may not constitute insurmountable obstacles.. |
| 17. |
Miyamoto K, Gurdon JB, Transcriptional regulation and nuclear reprogramming: roles of nuclear actin and actin-binding proteins., Cellular and molecular life sciences : CMLS, 10.1007/s00018-012-1235-7, 70, 18, 3289-3302, 2013.09. |
| 18. |
Teperek M, Miyamoto K, Nuclear reprogramming of sperm and somatic nuclei in eggs and oocytes., Reproductive medicine and biology, 10.1007/s12522-013-0155-z, 12, 4, 133-149, 2013.06. |
| 19. |
Kei Miyamoto, Marta Teperek, Kosuke Yusa, George E. Allen, Charles R. Bradshaw, J. B. Gurdon, Nuclear Wave1 Is Required for Reprogramming Transcription in Oocytes and for Normal Development, SCIENCE, 10.1126/science.1240376, 341, 6149, 1002-1005, 2013.08, Eggs and oocytes have a remarkable ability to induce transcription of sperm after normal fertilization and in somatic nuclei after somatic cell nuclear transfer. This ability of eggs and oocytes is essential for normal development. Nuclear actin and actin-binding proteins have been shown to contribute to transcription, although their mode of action is elusive. Here, we find that Xenopus Wave1, previously characterized as a protein involved in actin cytoskeleton organization, is present in the oocyte nucleus and is required for efficient transcriptional reprogramming. Moreover, Wave1 knockdown in embryos results in abnormal development and defective hox gene activation. Nuclear Wave1 binds by its WHD domain to active transcription components, and this binding contributes to the action of RNA polymerase II. We identify Wave1 as a maternal reprogramming factor that also has a necessary role in gene activation in development.. |
| 20. |
Kei Miyamoto, Maternal factors involved in nuclear reprogramming by eggs and oocytes, Journal of Mammalian Ova Research, 10.1274/jmor.30.68, 30, 3, 68-78, 2013.10, When a somatic cell nucleus is transplanted into an egg or an oocyte, the transplanted nucleus can be reprogrammed to support early embryonic development so that the reconstructed embryo gives rise to a cloned animal. Nuclear reprogramming of somatic nuclei is induced by maternal components stored in eggs and oocytes. These endogenous reprogramming factors and mechanisms have been explored for decades in mammals and amphibia. There are several ways of investigating reprogramming mechanisms, including nuclear transfer to eggs/oocytes and incubation in egg/oocyte extracts. In this review I describe the type of reprogramming events induced in each system and what factors in eggs and oocytes are responsible for these. Based on our current knowledge, I propose a model for the early phase of nuclear reprogramming in eggs and oocytes.
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| 21. |
Marta Teperek, Kei Miyamoto, Angela Simeone, Renata Feret, Michael J. Deery, John B. Gurdon, Jerome Jullien, Sperm and Spermatids Contain Different Proteins and Bind Distinct Egg Factors, INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES, 10.3390/ijms150916719, 15, 9, 16719-16740, 2014.09, Spermatozoa are more efficient at supporting normal embryonic development than spermatids, their immature, immediate precursors. This suggests that the sperm acquires the ability to support embryonic development during spermiogenesis (spermatid to sperm maturation). Here, using Xenopus laevis as a model organism, we performed 2-D Fluorescence Difference Gel Electrophoresis (2D-DIGE) and mass spectrometry analysis of differentially expressed proteins between sperm and spermatids in order to identify factors that could be responsible for the efficiency of the sperm to support embryonic development. Furthermore, benefiting from the availability of egg extracts in Xenopus, we also tested whether the chromatin of sperm could attract different egg factors compared to the chromatin of spermatids. Our analysis identified: (1) several proteins which were present exclusively in sperm; but not in spermatid nuclei and (2) numerous egg proteins binding to the sperm (but not to the spermatid chromatin) after incubation in egg extracts. Amongst these factors we identified many chromatin-associated proteins and transcriptional repressors. Presence of transcriptional repressors binding specifically to sperm chromatin could suggest its preparation for the early embryonic cell cycles, during which no transcription is observed and suggests that sperm chromatin has a unique protein composition, which facilitates the recruitment of egg chromatin remodelling factors. It is therefore likely that the acquisition of these sperm-specific factors during spermiogenesis makes the sperm chromatin suitable to interact with the maternal factors and, as a consequence, to support efficient embryonic development.. |
| 22. |
Jerome Jullien, Kei Miyamoto, Vincent Pasque, George E. Allen, Charles R. Bradshaw, Nigel J. Garrett, Richard P. Halley-Stott, Hiroshi Kimura, Keita Ohsumi, John B. Gurdon, Hierarchical Molecular Events Driven by Oocyte-Specific Factors Lead to Rapid and Extensive Reprogramming, MOLECULAR CELL, 10.1016/j.molcel.2014.06.024, 55, 4, 524-536, 2014.08, Nuclear transfer to oocytes is an efficient way to transcriptionally reprogram somatic nuclei, but its mechanisms remain unclear. Here, we identify a sequence of molecular events that leads to rapid transcriptional reprogramming of somatic nuclei after transplantation to Xenopus oocytes. RNA-seq analyses reveal that reprogramming by oocytes results in a selective switch in transcription toward an oocyte rather than pluripotent type, without requiring new protein synthesis. Time-course analyses at the single-nucleus level show that transcriptional reprogramming is induced in most transplanted nuclei in a highly hierarchical manner. We demonstrate that an extensive exchange of somatic-for oocyte-specific factors mediates reprogramming and leads to robust oocyte RNA polymerase II binding and phosphorylation on transplanted chromatin. Moreover, genome-wide binding of oocyte-specific linker histone B4 supports its role in transcriptional reprogramming. Thus, our study reveals the rapid, abundant, and stepwise loading of oocyte-specific factors onto somatic chromatin as important determinants for successful reprogramming.. |
| 23. |
塚口 智将, 守田 昴太郎, 宮本 圭, 松本和也, 初期胚発生におけるDNA及びヒストンメチル化修飾の重要性, 近畿大学先端技術総合研究所紀要, 6号, 20, 1-8, 2015.03, [要旨] 哺乳類の初期胚では, 分化全能性を獲得するために, 受精直後に終末分化した精子と卵子のエピゲノムの情報がリセットされる. この現象では, 遺伝子の転写制御に関与するDNA やヒストンのメチル化及びアセチル化修飾がダイナミックに変化することが知られている. 受精後, 遺伝子の転写抑制に関わるDNAのメチル化修飾は, 酵素及びDNA 複製によって取り除かれ, その後再び, DNA メチル基転移酵素（DNAmethyltransferases, DNMTs）が働くことにより, 特定の遺伝子の転写制御が行われる. ヒストンにおいては, histone methyltransferase（HMT）によるメチル化, histone acetyltransferase（HAT）によるアセチル化などの修飾がクロマチン構造の変化を誘起させ, 転写の活性あるいは抑制に関わっている. 以上のように遺伝子の発現は, DNA やヒストン修飾の相互の働きかけによって制御されていると考えられる. したがって, 初期胚で起こるDNA 及びヒストン修飾の機構やそれらの修飾の持つ役割を知ることで, エピジェネティックリプログラミングの詳細な分子機構を深く理解することができる. さらに, それらの知見をiPS細胞作製効率の向上や分化誘導の改善に応用させることで, 再生医療や創薬分野への貢献が期待される. そこで本稿では, 哺乳類の初期胚において発生に必要な遺伝子の転写制御に関与しているDNA 及びヒストンのメチル化修飾について概説する. [Abstract] In mammalian early embryo, epigenome information of sperm and oocytes is reset to acquire the developmental totipotency. This phenomenon is accompanied by the dynamic changes in DNA and histone modifications. DNA methylation involved in transcriptional repression is removed by the enzymes or DNA replication after fertilization. The genome is remethylated by DNA methyltransferases（DNMTs） and the specific set of genes is transcriptionally repressed. Histone modifications such as methylation by histone methyltransferases（HMTs） and acetylation by histone acetyltransferases（HATs） also induce changes in chromatin structure and are involved in transcriptional regulation. Therefore, gene expression regulation seems to be achieved by the combination of DNA and histone modifications. Consequently, understanding mechanisms and roles of DNA and histone modifications can be a clue to understand detailed mechanism of epigenetic reprogramming. Moreover, the basic knowledge about epigenetic reprogramming to improve iPS cell conversion and induction of differentiation would contribute to the fields of regenerative medicine and innovative drug development. Here, we will review DNA and histone methylation related totranscriptional regulation of genes necessary for development in mammalian early embryo.近畿大学先端技術総合研究所紀要編集委員会. |
| 24. |
Kei Miyamoto, Ken-ichi T. Suzuki, Miyuki Suzuki, Yuto Sakane, Tetsushi Sakuma, Sarah Herberg, Angela Simeone, David Simpson, Jerome Jullien, Takashi Yamamoto, J. B. Gurdon, The Expression of TALEN before Fertilization Provides a Rapid Knock-Out Phenotype in Xenopus laevis Founder Embryos, PLOS ONE, 10.1371/journal.pone.0142946, 10, 11, e0142946, 2015.11, Recent advances in genome editing using programmable nucleases have revolutionized gene targeting in various organisms. Successful gene knock-out has been shown in Xenopus, a widely used model organism, although a system enabling less mosaic knock-out in founder embryos (F0) needs to be explored in order to judge phenotypes in the F0 generation. Here, we injected modified highly active transcription activator-like effector nuclease (TALEN) mRNA to oocytes at the germinal vesicle (GV) stage, followed by in vitro maturation and intracytoplasmic sperm injection, to achieve a full knock-out in F0 embryos. Unlike conventional injection methods to fertilized embryos, the injection of TALEN mRNA into GV oocytes allows expression of nucleases before fertilization, enabling them to work from an earlier stage. Using this procedure, most of developed embryos showed full knock-out phenotypes of the pigmentation gene tyrosinase and/or embryonic lethal gene pax6 in the founder generation. In addition, our method permitted a large 1 kb deletion. Thus, we describe nearly complete gene knock-out phenotypes in Xenopus laevis F0 embryos. The presented method will help to accelerate the production of knock-out frogs since we can bypass an extra generation of about 1 year in Xenopus laevis. Meantime, our method provides a unique opportunity to rapidly test the developmental effects of disrupting those genes that do not permit growth to an adult able to reproduce. In addition, the protocol shown here is considerably less invasive than the previously used host transfer since our protocol does not require surgery. The experimental scheme presented is potentially applicable to other organisms such as mammals and fish to resolve common issues of mosaicism in founders.. |
| 25. |
Kei Miyamoto, David Simpson, John B. Gurdon, Manipulation and In Vitro Maturation of Xenopus laevis Oocytes, Followed by Intracytoplasmic Sperm Injection, to Study Embryonic Development, JOVE-JOURNAL OF VISUALIZED EXPERIMENTS, 10.3791/52496, 96, e52496, 2015.02, Amphibian eggs have been widely used to study embryonic development. Early embryonic development is driven by maternally stored factors accumulated during oogenesis. In order to study roles of such maternal factors in early embryonic development, it is desirable to manipulate their functions from the very beginning of embryonic development. Conventional ways of gene interference are achieved by injection of antisense oligonucleotides (oligos) or mRNA into fertilized eggs, enabling under-or over-expression of specific proteins, respectively. However, these methods normally require more than several hours until protein expression is affected, and, hence, the interference of gene functions is not effective during early embryonic stages. Here, we introduce an experimental system in which expression levels of maternal proteins can be altered before fertilization. Xenopus laevis oocytes obtained from ovaries are defolliculated by incubating with enzymes. Antisense oligos or mRNAs are injected into defolliculated oocytes at the germinal vesicle (GV) stage. These oocytes are in vitro matured to eggs at the metaphase II (MII) stage, followed by intracytoplasmic sperm injection (ICSI). By this way, up to 10% of ICSI embryos can reach the swimming tadpole stage, thus allowing functional tests of specific gene knockdown or overexpression. This approach can be a useful way to study roles of maternally stored factors in early embryonic development.. |
| 26. |
Sarah Herberg, Angela Simeone, Mami Oikawa, Jerome Jullien, Charles R. Bradshaw, Marta Teperek, John Gurdon, Kei Miyamoto, Histone H3 lysine 9 trimethylation is required for suppressing the expression of an embryonically activated retrotransposon in Xenopus laevis, SCIENTIFIC REPORTS, 10.1038/srep14236, 5, 14236, 2015.09, Transposable elements in the genome are generally silenced in differentiated somatic cells. However, increasing evidence indicates that some of them are actively transcribed in early embryos and the proper regulation of retrotransposon expression is essential for normal development. Although their developmentally regulated expression has been shown, the mechanisms controlling retrotransposon expression in early embryos are still not well understood. Here, we observe a dynamic expression pattern of retrotransposons with three out of ten examined retrotransposons (1a11, lambda-olt 2-1 and xretpos( L)) being transcribed solely during early embryonic development. We also identified a transcript that contains the long terminal repeat (LTR) of lambda-olt 2-1 and shows a similar expression pattern to lambda-olt 2-1 in early Xenopus embryos. All three retrotransposons are transcribed by RNA polymerase II. Although their expression levels decline during development, the LTRs are marked by histone H3 lysine 4 trimethylation. Furthermore, retrotransposons, especially lambda-olt 2-1, are enriched with histone H3 lysine 9 trimethylation (H3K9me3) when their expression is repressed. Overexpression of lysine-specific demethylase 4d removes H3K9me3 marks from Xenopus embryos and inhibits the repression of lambda-olt 2-1 after gastrulation. Thus, our study shows that H3K9me3 is important for silencing the developmentally regulated retrotransposon in Xenopus laevis.. |
| 27. |
Marta Teperek, Angela Simeone, Vincent Gaggioli, Kei Miyamoto, George E. Allen, Serap Erkek, Taejoon Kwon, Edward M. Marcotte, Philip Zegerman, Charles R. Bradshaw, Antoine H. F. M. Peters, John B. Gurdon, Jerome Jullien, Sperm is epigenetically programmed to regulate gene transcription in embryos, GENOME RESEARCH, 10.1101/gr.201541.115, 26, 8, 1034-1046, 2016.08, For a long time, it has been assumed that the only role of sperm at fertilization is to introduce the male genome into the egg. Recently, ideas have emerged that the epigenetic state of the sperm nucleus could influence transcription in the embryo. However, conflicting reports have challenged the existence of epigenetic marks on sperm genes, and there are no functional tests supporting the role of sperm epigenetic marking on embryonic gene expression. Here, we show that sperm is epigenetically programmed to regulate embryonic gene expression. By comparing the development of sperm-and spermatid-derived frog embryos, we show that the programming of sperm for successful development relates to its ability to regulate transcription of a set of developmentally important genes. During spermatid maturation into sperm, these genes lose H3K4me2/3 and retain H3K27me3 marks. Experimental removal of these epigenetic marks at fertilization de-regulates gene expression in the resulting embryos in a paternal chromatin-dependent manner. This demonstrates that epigenetic instructions delivered by the sperm at fertilization are required for correct regulation of gene expression in the future embryos. The epigenetic mechanisms of developmental programming revealed here are likely to relate to the mechanisms involved in transgenerational transmission of acquired traits. Understanding how parental experience can influence development of the progeny has broad potential for improving human health.. |
| 28. |
奥野 智美, 山口 壮輝, 濱田 歩花, 竹林 真理愛, 守田 昂太郎, 樋口 智香, 神谷 拓磨, 安齋 政幸, 山縣 一夫, 細井 美彦, Jerome Jullien, 松本 和也, 宮本 圭, 初期胚特異的レトロトランスポゾンMERVLの活性化に伴う核内変化, 生命科学系学会合同年次大会, 2017年度, [2P-0708], 2017.12. |
| 29. |
Higuchi C, Shimizu N, Shin SW, Morita K, Nagai K, Anzai M, Kato H, Mitani T, Yamagata K, Hosoi Y, Miyamoto K, Matsumoto K, Ubiquitin-proteasome system modulates zygotic genome activation in early mouse embryos and influences full-term development., The Journal of reproduction and development, 10.1262/jrd.2017-127, 64, 1, 65-74, 2017.12. |
| 30. |
Kei Miyamoto, Yosuke Tajima, Koki Yoshida, Mami Oikawa, Rika Azuma, George E. Allen, Tomomi Tsujikawa, Tomomasa Tsukaguchi, Charles R. Bradshaw, Jerome Jullien, Kazuo Yamagata, Kazuya Matsumoto, Masayuki Anzai, Hiroshi Imai, John B. Gurdon, Masayasu Yamada, Reprogramming towards totipotency is greatly facilitated by synergistic effects of small molecules, BIOLOGY OPEN, 10.1242/bio.023473, 6, 4, 415-424, 2017.04, Animal cloning has been achieved in many species by transplanting differentiated cell nuclei to unfertilized oocytes. However, the low efficiencies of cloning have remained an unresolved issue. Here we find that the combination of two small molecules, trichostatin A (TSA) and vitamin C (VC), under culture condition with bovine serum albumin deionized by ion-exchange resins, dramatically improves the cloning efficiency in mice and 15% of cloned embryos develop to term by means of somatic cell nuclear transfer (SCNT). The improvement was not observed by adding the non-treated, rather than deionized, bovine serum. RNA-seq analyses of SCNT embryos at the two-cell stage revealed that the treatment with TSA and VC resulted in the upregulated expression of previously identified reprogramming-resistant genes. Moreover, the expression of early-embryo-specific retroelements was upregulated by the TSA and VC treatment. The enhanced gene expression was relevant to the VC-mediated reduction of histone H3 lysine 9 methylation in SCNT embryos. Our study thus shows a simply applicable method to greatly improve mouse cloning efficiency, and furthers our understanding of how somatic nuclei acquire totipotency.. |
| 31. |
Shinji Misu, Marina Takebayashi, Kei Miyamoto, Nuclear Actin in Development and Transcriptional Reprogramming, FRONTIERS IN GENETICS, 10.3389/fgene.2017.00027, 8, 27, 2017.03, Actin is a highly abundant protein in eukaryotic cells and dynamically changes its polymerized states with the help of actin-binding proteins. Its critical function as a constituent of cytoskeleton has been well-documented. Growing evidence demonstrates that actin is also present in nuclei, referred to as nuclear actin, and is involved in a number of nuclear processes, including transcriptional regulation and chromatin remodeling. The contribution of nuclear actin to transcriptional regulation can be explained by its direct interaction with transcription machineries and chromatin remodeling factors and by controlling the activities of transcription factors. In both cases, polymerized states of nuclear actin affect the transcriptional outcome. Nuclear actin also plays an important role in activating strongly silenced genes in somatic cells for transcriptional reprogramming. When these nuclear functions of actin are considered, it is plausible to speculate that nuclear actin is also implicated in embryonic development, in which numerous genes need to be activated in a well-coordinated manner. In this review, we especially focus on nuclear actin's roles in transcriptional activation, reprogramming and development, including stem cell differentiation and we discuss how nuclear actin can be an important player in development and cell differentiation.. |
| 32. |
Jerome Jullien, Munender Vodnala, Vincent Pasque, Mami Oikawa, Kei Miyamoto, George Allen, Sarah Anne David, Vincent Brochard, Stan Wang, Charles Bradshaw, Haruhiko Koseki, Vittorio Sartorelli, Nathalie Beaujean, John Gurdon, Gene Resistance to Transcriptional Reprogramming following Nuclear Transfer Is Directly Mediated by Multiple Chromatin-Repressive Pathways, MOLECULAR CELL, 10.1016/j.molcel.2017.01.030, 65, 5, 873-+, 2017.03, Understanding the mechanism of resistance of genes to reactivation will help improve the success of nuclear reprogramming. Using mouse embryonic fibroblast nuclei with normal or reduced DNA methylation in combination with chromatin modifiers able to erase H3K9me3, H3K27me3, and H2AK119ub1 from transplanted nuclei, we reveal the basis for resistance of genes to transcriptional reprogramming by oocyte factors. A majority of genes is affected by more than one type of treatment, suggesting that resistance can require repression through multiple epigenetic mechanisms. We classify resistant genes according to their sensitivity to 11 chromatin modifier combinations, revealing the existence of synergistic as well as adverse effects of chromatin modifiers on removal of resistance. We further demonstrate that the chromatin modifier USP21 reduces resistance through its H2AK119 deubiquitylation activity. Finally, we provide evidence that H2A ubiquitylation also contributes to resistance to transcriptional reprogramming in mouse nuclear transfer embryos.. |
| 33. |
Christian Baarlink, Matthias Plessner, Alice Sherrard, Kohtaro Morita, Shinji Misu, David Virant, Eva-Maria Kleinschnitz, Robert Harniman, Dominic Alibhai, Stefan Baumeister, Kei Miyamoto, Ulrike Endesfelder, Abderrahmane Kaidi, Robert Grosse, A transient pool of nuclear F-actin at mitotic exit controls chromatin organization, NATURE CELL BIOLOGY, 10.1038/ncb3641, 19, 12, 1389-+, 2017.12, Re-establishment of nuclear structure and chromatin organization after cell division is integral for genome regulation or development and is frequently altered during cancer progression. The mechanisms underlying chromatin expansion in daughter cells remain largely unclear. Here, we describe the transient formation of nuclear actin filaments (F-actin) during mitotic exit. These nuclear F-actin structures assemble in daughter cell nuclei and undergo dynamic reorganization to promote nuclear protrusions and volume expansion throughout early G1 of the cell cycle. Specific inhibition of this nuclear F-actin assembly impaired nuclear expansion and chromatin decondensation after mitosis and during early mouse embryonic development. Biochemical screening for mitotic nuclear F-actin interactors identified the actin-disassembling factor cofilin-1. Optogenetic regulation of cofilin-1 revealed its critical role for controlling timing, turnover and dynamics of F-actin assembly inside daughter cell nuclei. Our findings identify a cell-cycle-specific and spatiotemporally controlled form of nuclear F-actin that reorganizes the mammalian nucleus after mitosis.. |
| 34. |
池上春香, 松橋珠子, 永井宏平, 宮本圭, 大林賢伍, 坂口慎一, 松本和也, 黒毛和種去勢牛の脂肪交雑を生体評価するバイオマーカー候補タンパク質の血清プロテオーム 解析による探索, 関西畜産学会報, 第175号, 2018.03. |
| 35. |
奥野智美, 松本和也, 宮本 圭, 受精卵およびクローン胚におけるエピジェネティックリプログラミング, 日本胚移植学雑誌, 40, 3, 117-122, 2018.09. |
| 36. |
Takashi Ikeda, Takafusa Hikichi, Hisashi Miura, Hirofumi Shibata, Kanae Mitsunaga, Yosuke Yamada, Knut Woltjen, Kei Miyamoto, Ichiro Hiratani, Yasuhiro Yamada, Akitsu Hotta, Takuya Yamamoto, Keisuke Okita, Shinji Masui, Srf destabilizes cellular identity by suppressing cell-type-specific gene expression programs., Nature communications, 10.1038/s41467-018-03748-1, 9, 1, 1387-1387, 2018.04, Multicellular organisms consist of multiple cell types. The identity of these cells is primarily maintained by cell-type-specific gene expression programs; however, mechanisms that suppress these programs are poorly defined. Here we show that serum response factor (Srf), a transcription factor that is activated by various extracellular stimuli, can repress cell-type-specific genes and promote cellular reprogramming to pluripotency. Manipulations that decrease β-actin monomer quantity result in the nuclear accumulation of Mkl1 and the activation of Srf, which downregulate cell-type-specific genes and alter the epigenetics of regulatory regions and chromatin organization. Mice overexpressing Srf exhibit various pathologies including an ulcerative colitis-like symptom and a metaplasia-like phenotype in the pancreas. Our results demonstrate an unexpected function of Srf via a mechanism by which extracellular stimuli actively destabilize cell identity and suggest Srf involvement in a wide range of diseases.. |
| 37. |
Rika Azuma, Kei Miyamoto, Mami Oikawa, Masayasu Yamada, Masayuki Anzai, Combinational Treatment of Trichostatin A and Vitamin C Improves the Efficiency of Cloning Mice by Somatic Cell Nuclear Transfer., Journal of visualized experiments : JoVE, 10.3791/57036, 134, e57036, 2018.04, Somatic cell nuclear transfer (SCNT) provides a unique opportunity to directly produce a cloned animal from a donor cell, and it requires the use of skillful techniques. Additionally, the efficiencies of cloning have remained low since the successful production of cloned animals, especially mice. There have been many attempts to improve the cloning efficiency, and trichostatin A (TSA), a histone deacetylase inhibitor, has been widely used to enhance the efficiency of cloning. Here, we report a dramatically improved cloning method in mice. This somatic cell nuclear transfer method involves usage of Hemagglutinating virus of Japan Envelope (HVJ-E), which enables easy manipulation. Moreover, the treatment using two small molecules, TSA and vitamin C (VC), with deionized bovine serum albumin (dBSA), is highly effective for embryonic development. This approach requires neither additional injection nor genetic manipulation, and thus presents a simple, suitable method for practical use. This method could become a technically feasible approach for researchers to produce genetically modified animals from cultured cells. Furthermore, it might be a useful way for the rescue of endangered animals via cloning.. |
| 38. |
Miyamoto K, Nguyen KT, Allen GE, Jullien J, Kumar D, Otani T, Bradshaw CR, Livesey FJ, Kellis M, Gurdon JB, Chromatin Accessibility Impacts Transcriptional Reprogramming in Oocytes., Cell reports, 10.1016/j.celrep.2018.06.030, 24, 2, 304-311, 2018.07. |
| 39. |
Miyamoto K, Various nuclear reprogramming systems using egg and oocyte materials., The Journal of reproduction and development, 10.1262/jrd.2019-002, 65, 3, 203-208, 2019.02. |
| 40. |
Yamagata K, Nagai K, Miyamoto H, Anzai M, Kato H, Miyamoto K, Kurosaka S, Azuma R, Kolodeznikov II, Protopopov AV, Plotnikov VV, Kobayashi H, Kawahara-Miki R, Kono T, Uchida M, Shibata Y, Handa T, Kimura H, Hosoi Y, Mitani T, Matsumoto K, Iritani A, Signs of biological activities of 28,000-year-old mammoth nuclei in mouse oocytes visualized by live-cell imaging., Scientific reports, 10.1038/s41598-019-40546-1, 9, 1, 4050-4050, 2019.03. |
| 41. |
Higuchi C, Yamamoto M, Shin SW, Miyamoto K, Matsumoto K, Perturbation of maternal PIASy abundance disrupts zygotic genome activation and embryonic development via SUMOylation pathway., Biology open, 10.1242/bio.048652, 8, 10, bio048652-bio048652, 2019.10. |
| 42. |
Almonacid M, Al Jord A, El-Hayek S, Othmani A, Coulpier F, Lemoine S, Miyamoto K, Grosse R, Klein C, Piolot T, Mailly P, Voituriez R, Genovesio A, Verlhac MH, Active Fluctuations of the Nuclear Envelope Shape the Transcriptional Dynamics in Oocytes., Developmental cell, 10.1016/j.devcel.2019.09.010, 2019.10. |
| 43. |
永井 宏平, 宮本 裕史, 安齋 政幸, 東 里香, 西端 智也, 山縣 一夫, 加藤 博己, 宮本 圭, Kolodeznikov Igor I., Protopopov Albert V., Plotnikov Valerii V., 細井 美彦, 三谷 匡, 松本 和也, 入谷 明, 2万8千年前のケナガマンモスの筋肉組織と骨髄組織のプロテオーム解析, 電気泳動, 63, Suppl., 196-196, 2019.07. |
| 44. |
西端 智也, 永井 宏平, 山縣 一夫, 宮本 裕史, 安齋 政幸, 加藤 博己, 宮本 圭, 東 里香, Kolodeznikov Igor I., Protopopov Albert V., Plotnikov Valerii V., 細井 美彦, 三谷 匡, 松本 和也, 入谷 明, 2万8千年前のケナガマンモス組織から得られたタンパク質の翻訳後修飾の解析, 電気泳動, 63, Suppl., 195-195, 2019.07. |
| 45. |
Tomomi Okuno, Wayne Yang Li, Yu Hatano, Atsushi Takasu, Yuko Sakamoto, Mari Yamamoto, Zenki Ikeda, Taiki Shindo, Matthias Plessner, Kohtaro Morita, Kazuya Matsumoto, Kazuo Yamagata, Robert Grosse, Kei Miyamoto, Zygotic Nuclear F-Actin Safeguards Embryonic Development., Cell reports, 10.1016/j.celrep.2020.107824, 31, 13, 107824-107824, 2020.06, After fertilization, sperm and oocyte nuclei are rapidly remodeled to form swollen pronuclei (PN) in mammalian zygotes, and the proper formation and function of PN are key to producing totipotent zygotes. However, how mature PN are formed has been unclear. We find that filamentous actin (F-actin) assembles in the PN of mouse zygotes and is required for fully functional PN. The perturbation of nuclear actin dynamics in zygotes results in the misregulation of genes related to genome integrity and abnormal development of mouse embryos. We show that nuclear F-actin ensures DNA damage repair, thus preventing the activation of a zygotic checkpoint. Furthermore, optogenetic control of cofilin nuclear localization reveals the dynamically regulated F-actin nucleoskeleton in zygotes, and its timely disassembly is needed for developmental progression. Nuclear F-actin is a hallmark of totipotent zygotic PN, and the temporal regulation of its polymerized state is necessary for normal embryonic development.. |
| 46. |
Taiki Shindo, Shunya Ihashi, Yuko Sakamoto, Tomomi Okuno, Junko Tomikawa, Kei Miyamoto, Visualization of endogenous nuclear F-actin in mouse embryos reveals abnormal actin assembly after somatic cell nuclear transfer., Journal of biochemistry, 10.1093/jb/mvaa125, 2020.11, Actin in the nucleus, referred to as nuclear actin, is involved in a variety of nuclear events. Nuclear actin is present as a globular (G-actin) and filamentous form (F-actin), and dynamic assembly/disassembly of nuclear actin profoundly affects nuclear functions. However, it is still challenging to observe endogenous nuclear F-actin. Here, we present a condition to visualize endogenous nuclear F-actin of mouse zygotes using different fixation methods. Zygotes fixed with paraformaldehyde and treated with fluorescently conjugated phalloidin show both short and long actin filaments in their pronuclei. Short nuclear actin filaments are characteristic of phalloidin staining, rather than the consequence of severing actin filaments by the fixation process, since long nuclear actin filaments probed with the nuclear actin chromobody are not disassembled into short filaments after fixation with paraformaldehyde. Furthermore, we find that nuclear actin assembly is impaired after somatic cell nuclear transfer (SCNT), suggesting abnormal nucleoskeleton structures in SCNT embryos. Taken together, our presented method for visualizing nuclear F-actin with phalloidin can be used to observe the states of nuclear actin assembly, and revealed improper reprogramming of actin nucleoskeleton structures in cloned mouse embryos.. |
| 47. |
Shota Yamazaki, Christian Gerhold, Koji Yamamoto, Yuya Ueno, Robert Grosse, Kei Miyamoto, Masahiko Harata, The Actin-Family Protein Arp4 Is a Novel Suppressor for the Formation and Functions of Nuclear F-Actin., Cells, 10.3390/cells9030758, 9, 3, 2020.03, The crosstalk between actin and actin-related proteins (Arps), namely Arp2 and Arp3, plays a central role in facilitating actin polymerization in the cytoplasm and also in the nucleus. Nuclear F-actin is required for transcriptional regulation, double-strand break repair, and nuclear organization. The formation of nuclear F-actin is highly dynamic, suggesting the involvement of positive and negative regulators for nuclear actin polymerization. While actin assembly factors for nuclear F-actin have been recently described, information about inhibitory factors is still limited. The actin-related protein Arp4 which is predominantly localized in the nucleus, has been previously identified as an integral subunit of multiple chromatin modulation complexes, where it forms a heterodimer with monomeric actin. Therefore, we tested whether Arp4 functions as a suppressor of nuclear F-actin formation. The knockdown of Arp4 (Arp4 KD) led to an increase in nuclear F-actin formation in NIH3T3 cells, and purified Arp4 potently inhibited F-actin formation in mouse nuclei transplanted into Xenopus laevis oocytes. Consistently, Arp4 KD facilitated F-actin-inducible gene expression (e.g., OCT4) and DNA damage repair. Our results suggest that Arp4 has a critical role in the formation and functions of nuclear F-actin.. |
| 48. |
Yuto Takahashi, Shogo Hiratsuka, Nanako Machida, Daisuke Takahashi, Junpei Matsushita, Pavel Hozak, Tom Misteli, Kei Miyamoto, Masahiko Harata, Impairment of nuclear F-actin formation and its relevance to cellular phenotypes in Hutchinson-Gilford progeria syndrome., Nucleus (Austin, Tex.), 10.1080/19491034.2020.1815395, 11, 1, 250-263, 2020.12, Hutchinson-Gilford progeria syndrome (HGPS) is a premature aging disorder caused by a mutation of lamin A, which contributes to nuclear architecture and the spatial organization of chromatin in the nucleus. The expression of a lamin A mutant, named progerin, leads to functional and structural disruption of nuclear organization. Since progerin lacks a part of the actin-binding site of lamin A, we hypothesized that nuclear actin dynamics and function are altered in HGPS cells. Nuclear F-actin is required for the organization of nuclear shape, transcriptional regulation, DNA damage repair, and activation of Wnt/β-catenin signaling. Here we show that the expression of progerin decreases nuclear F-actin and impairs F-actin-regulated transcription. When nuclear F-actin levels are increased by overexpression of nuclear-targeted actin or by using jasplakinolide, a compound that stabilizes F-actin, the irregularity of nuclear shape and defects in gene expression can be reversed. These observations provide evidence for a novel relationship between nuclear actin and the etiology of HGPS.. |
| 49. |
Kohtaro Morita, Yuki Hatanaka, Shunya Ihashi, Masahide Asano, Kei Miyamoto, Kazuya Matsumoto, Symmetrically dimethylated histone H3R2 promotes global transcription during minor zygotic genome activation in mouse pronuclei., Scientific reports, 10.1038/s41598-021-89334-w, 11, 1, 10146, 2021.05, Paternal genome reprogramming, such as protamine-histone exchange and global DNA demethylation, is crucial for the development of fertilised embryos. Previously, our study showed that one of histone arginine methylation, asymmetrically dimethylated histone H3R17 (H3R17me2a), is necessary for epigenetic reprogramming in the mouse paternal genome. However, roles of histone arginine methylation in reprogramming after fertilisation are still poorly understood. Here, we report that H3R2me2s promotes global transcription at the 1-cell stage, referred to as minor zygotic genome activation (ZGA). The inhibition of H3R2me2s by expressing a histone H3.3 mutant H3.3R2A prevented embryonic development from the 2-cell to 4-cell stages and significantly reduced global RNA synthesis and RNA polymerase II (Pol II) activity. Consistent with this result, the expression levels of MuERV-L as minor ZGA transcripts were decreased by forced expression of H3.3R2A. Furthermore, treatment with an inhibitor and co-injection of siRNA to PRMT5 and PRMT7 also resulted in the attenuation of transcriptional activities with reduction of H3R2me2s in the pronuclei of zygotes. Interestingly, impairment of H3K4 methylation by expression of H3.3K4M resulted in a decrease of H3R2me2s in male pronuclei. Our findings suggest that H3R2me2s together with H3K4 methylation is involved in global transcription during minor ZGA in mice.. |
| 50. |
Junko Tomikawa, Kei Miyamoto, Structural alteration of the nucleus for the reprogramming of gene expression., The FEBS journal, 10.1111/febs.15894, 2021.04, The regulation of gene expression is a critical process for establishing and maintaining cellular identity. Gene expression is controlled through a chromatin-based mechanism in the nucleus of eukaryotic cells. Recent studies suggest that chromatin accessibility and the higher-order structure of chromatin affect transcriptional outcome. This is especially evident when cells change their fate during development and nuclear reprogramming. Furthermore, non-chromosomal contents of the cell nucleus, namely nucleoskeleton proteins, can also affect chromatin and nuclear structures, resulting in transcriptional alterations. Here, we review our current mechanistic understanding about how chromatin and nuclear structures impact transcription in the course of embryonic development, cellular differentiation and nuclear reprogramming, and also discuss unresolved questions that remain to be addressed in the field.. |
| 51. |
Kei Miyamoto, Masahiko Harata, Nucleoskeleton proteins for nuclear dynamics., Journal of biochemistry, 10.1093/jb/mvab006, 169, 3, 237-241, 2021.01, The eukaryotic nucleus shows organized structures of chromosomes, transcriptional components and their associated proteins. It has been believed that such a dense nuclear environment prevents the formation of a cytoskeleton-like network of protein filaments. However, accumulating evidence suggests that the cell nucleus also possesses structural filamentous components to support nuclear organization and compartments, which are referred to as nucleoskeleton proteins. Nucleoskeleton proteins including lamins and actin influence nuclear dynamics including transcriptional regulation, chromatin organization and DNA damage responses. Furthermore, these nucleoskeleton proteins play a pivotal role in cellular differentiation and animal development. In this commentary, we discuss how nucleoskeleton-based regulatory mechanisms orchestrate nuclear dynamics.. |
| 52. |
Satoshi Kamimura, Kimiko Inoue, Eiji Mizutani, Jin-Moon Kim, Hiroki Inoue, Narumi Ogonuki, Kei Miyamoto, Shunya Ihashi, Nobuhiko Itami, Teruhiko Wakayama, Akihiro Ito, Norikazu Nishino, Minoru Yoshida, Atsuo Ogura, Improved development of mouse somatic cell nuclear transfer embryos by chlamydocin analogues, class I and IIa histone deacetylase inhibitors†., Biology of reproduction, 10.1093/biolre/ioab096, 105, 2, 543-553, 2021.08, In mammalian cloning by somatic cell nuclear transfer (SCNT), the treatment of reconstructed embryos with histone deacetylase (HDAC) inhibitors improves efficiency. So far, most of those used for SCNT are hydroxamic acid derivatives-such as trichostatin A-characterized by their broad inhibitory spectrum. Here, we examined whether mouse SCNT efficiency could be improved using chlamydocin analogues, a family of newly designed agents that specifically inhibit class I and IIa HDACs. Development of SCNT-derived embryos in vitro and in vivo revealed that four out of five chlamydocin analogues tested could promote the development of cloned embryos. The highest pup rates (7.1-7.2%) were obtained with Ky-9, similar to those achieved with trichostatin A (7.2-7.3%). Thus, inhibition of class I and/or IIa HDACs in SCNT-derived embryos is enough for significant improvements in full-term development. In mouse SCNT, the exposure of reconstructed oocytes to HDAC inhibitors is limited to 8-10 h because longer inhibition with class I inhibitors causes a two-cell developmental block. Therefore, we used Ky-29, with higher selectivity for class IIa than class I HDACs for longer treatment of SCNT-derived embryos. As expected, 24-h treatment with Ky-29 up to the two-cell stage did not induce a developmental block, but the pup rate was not improved. This suggests that the one-cell stage is a critical period for improving SCNT cloning using HDAC inhibitors. Thus, chlamydocin analogues appear promising for understanding and improving the epigenetic status of mammalian SCNT-derived embryos through their specific inhibitory effects on HDACs.. |
| 53. |
Junko Tomikawa, Christopher A Penfold, Takuma Kamiya, Risa Hibino, Ayumi Kosaka, Masayuki Anzai, Kazuya Matsumoto, Kei Miyamoto, Cell division- and DNA replication-free reprogramming of somatic nuclei for embryonic transcription., iScience, 10.1016/j.isci.2021.103290, 24, 11, 103290, 2021.11, Nuclear transfer systems represent the efficient means to reprogram a cell and in theory provide a basis for investigating the development of endangered species. However, conventional nuclear transfer using oocytes of laboratory animals does not allow reprogramming of cross-species nuclei owing to defects in cell divisions and activation of embryonic genes. Here, we show that somatic nuclei transferred into mouse four-cell embryos arrested at the G2/M phase undergo reprogramming toward the embryonic state. Remarkably, genome-wide transcriptional reprogramming is induced within a day, and ZFP281 is important for this replication-free reprogramming. This system further enables transcriptional reprogramming of cells from Oryx dammah, now extinct in the wild. Thus, our findings indicate that arrested mouse embryos are competent to induce intra- and cross-species reprogramming. The direct induction of embryonic transcripts from diverse genomes paves a unique approach for identifying mechanisms of transcriptional reprogramming and genome activation from a diverse range of species.. |
| 54. |
Hiroki Takeuchi, Mari Yamamoto, Megumi Fukui, Akihiro Inoue, Tadashi Maezawa, Mikiko Nishioka, Eiji Kondo, Tomoaki Ikeda, Kazuya Matsumoto, Kei Miyamoto, Single-cell profiling of transcriptomic changes during in vitro maturation of human oocytes, Reproductive medicine and biology, 10.1002/rmb2.12464, 21, 1, e12464, 2022.05. |
| 55. |
Junko Tomikawa, Christopher A. Penfold, Rena Hatakeyama, Kei Miyamoto, Nuclear transfer system for the direct induction of embryonic transcripts from intra- and cross-species nuclei using mouse 4-cell embryos, STAR Protocols, 10.1016/j.xpro.2022.101284, 3, 2, 101284-101284, 2022.06. |
| 56. |
Shunya Ihashi, Mizuto Hamanaka, Masaya Kaji, Miki Mori, Yuma Imasato, Misaki Nakamura, Masayuki Anzai, Kazuya Matsumoto, Masahito Ikawa, Kei Miyamoto, Incomplete activation of developmentally required genes Alyref1 and Gabpb1 leads to preimplantation arrest in cloned mouse embryos, bioRxiv, 10.1101/2022.04.14.488417, 2022.04, SUMMARY
Differentiated cell nuclei can be reprogrammed after nuclear transfer (NT) to oocytes and the produced NT embryos can give rise to cloned animals. However, development of NT embryos is often hampered by recurrent reprogramming failures, including the incomplete activation of developmental genes, yet specific genes responsible for the arrest of NT embryos are not well understood. Here, we searched for developmentally important genes among the reprogramming-resistant H3K9me3-repressed genes, and identified Alyref and Gabpb1 by siRNA screening. Gene knockout of Alyref and Gabpb1 by the CRISPR/Cas9 system resulted in early developmental arrest in mice. Single embryo RNA-seq revealed that Alyref is needed for the formation of inner cell mass. The supplement of Alyref and Gabpb1 by mRNA injection supported efficient preimplantation development of cloned embryos. Thus, our study shows that the H3K9me3-repressed genes contain developmentally required genes and the incomplete activation of such genes results in preimplantation arrest of cloned embryos.. |
| 57. |
Sivagami Gunasekaran, Yasuki Miyagawa, Kei Miyamoto, Actin nucleoskeleton in embryonic development and cellular differentiation., Current opinion in cell biology, 10.1016/j.ceb.2022.102100, 76, 102100-102100, 2022.05, Dynamic assembly and disassembly of actin proteins play a key role in the cytoskeleton, but the cellular functions of actin are not only restricted to the cytoplasmic compartment. Recent studies have shown that actin spatiotemporally changes its polymerized state in the nucleus as well and such dynamic nature of actin is relevant to key nuclear events including gene expression, DNA damage response and chromatin organization. In this review, we highlight emerging roles of actin in the nuclear compartment especially in the context of embryonic development and cellular differentiation. We first explain how the actin nucleoskeleton can be formed and function in cells. Notably, nuclear actin dynamics are greatly altered when cell fates change, such as after fertilization and T cell differentiation. We discuss how the dynamic actin nucleoskeleton contributes to accomplishing developmental programs.. |
| 58. |
坂上 凜, 宮川 靖基, 宮本 圭, 核骨格タンパク質の機能と胚発生における役割, 近畿大学生物理工学部紀要, 10.15100/00023805, 50, 33-43, 2023.03. |
| 59. |
Tanaka Masahito, Rin Sakanoue, Atsushi Takasu, Naoko Watanabe, Yuta Shimamoto, Kei Miyamoto, Transition to the structurally vulnerable nuclear state is an integral part of mouse embryonic development, bioRxiv, 10.1101/2023.02.20.529332, 2023.02, Abstract
Upon fertilization, germ cells are reprogrammed to acquire the ability to develop into an entire organism. Whereas extensive studies have focused on epigenetic reprogramming of chromatin states during development, changes of the nucleus that surrounds chromatin are ill-defined. Here, we show that nuclei become structurally and mechanically vulnerable at the 2-cell stage during mouse embryonic development. The 2-cell stage nuclei are extraordinarily plastic and deformable in contrast to those of 1-cell and 4-cell stages. The mechanically vulnerable nuclear state is attained by autophagy-mediated loss of lamin B1 from the nuclear membrane. This developmentally programmed lamin B1 dynamics is required for chromatin organization and major zygotic genome activation. We thus demonstrate that structural reprogramming of nuclei is a major determinant of embryonic gene expression and acquisition of totipotency.. |
| 60. |
Shunya Ihashi, Mizuto Hamanaka, Masaya Kaji, Ryunosuke Mori, Shuntaro Nishizaki, Miki Mori, Yuma Imasato, Kimiko Inoue, Shogo Matoba, Narumi Ogonuki, Atsushi Takasu, Misaki Nakamura, Kazuya Matsumoto, Masayuki Anzai, Atsuo Ogura, Masahito Ikawa, Kei Miyamoto, Incomplete activation of Alyref and Gabpb1 leads to preimplantation arrest in cloned mouse embryos., Life science alliance, 10.26508/lsa.202302296, 6, 11, 2023.11, Differentiated cell nuclei can be reprogrammed after nuclear transfer (NT) to oocytes and the produced NT embryos can give rise to cloned animals. However, development of NT embryos is often hampered by recurrent reprogramming failures, including the incomplete activation of developmental genes, yet specific genes responsible for the arrest of NT embryos are not well understood. Here, we searched for developmentally important genes among the reprogramming-resistant H3K9me3-repressed genes and identified Alyref and Gabpb1 by siRNA screening. Gene knockout of Alyref and Gabpb1 by the CRISPR/Cas9 system resulted in early developmental arrest in mice. Alyref was needed for the proper formation of inner cell mass by regulating Nanog, whereas Gabpb1 deficiency led to apoptosis. The supplement of Alyref and Gabpb1 mRNA supported efficient preimplantation development of cloned embryos. Alyref and Gabpb1 were silenced in NT embryos partially because of the repressed expression of Klf16 by H3K9me3. Thus, our study shows that the H3K9me3-repressed genes contain developmentally required genes, and the incomplete activation of such genes results in preimplantation arrest of cloned embryos.. |
| 61. |
Svenja Ulferts, Massimo Lopes, Kei Miyamoto, Robert Grosse, Nuclear actin dynamics and functions at a glance, Journal of Cell Science, 10.1242/jcs.261630, 2024.03. |
