研究者データベース

村上 洋太(ムラカミ ヨウタ)
理学研究院 化学部門 有機・生命化学分野
教授

基本情報

所属

  • 理学研究院 化学部門 有機・生命化学分野

職名

  • 教授

学位

  • 理学博士(京都大学)

ホームページURL

J-Global ID

研究キーワード

  • 染色体複製   エピジェネティクス   ヘテロクロマチン   セントロメア   染色体分配   クロマチン構造   Heterochromatin   Centromere   Chromosome segregation   Chromatin structure   Chromosomal replication   

研究分野

  • ライフサイエンス / 遺伝学 / クロマチン
  • ライフサイエンス / 遺伝学 / エピジェネティクス

職歴

  • 2009年04月 - 現在 北海道大学大学院 理学研究院 教授
  • 1993年10月 - 現在 Associate Professor,
  • 1993年10月 - 2009年03月 京都大学 ウイルス研究所 准教授(助教授)
  • 1990年10月 - 1993年10月 米国スローン・ケタリング記念がんセンター
  • 1990年10月 - 1993年10月 Memorial Sloan-Keetering Cancer Center(USA),
  • 1987年10月 - 1990年10月 博士研究員京都大学ウィルス研究所
  • 1987年10月 - 1990年10月 Institute for Virus Research,
  • 博士研究員
  • 京都大学 ウイルス研究所
  • Postdoctoral Fellow
  • Postdoctoral Fellow

学歴

  •         -   京都大学理学部
  •         -   京都大学理学研究科
  •         -   Kyoto University, Faculty of Science
  •         -   Kyoto University, Graduate School of Science

所属学協会

  • エピジェネティクス研究会   日本細胞生物学会   分子生物学会   The Molecular Biology Society of Japan   

研究活動情報

論文

  • Kuragano M, Uyeda TQ, Kamijo K, Murakami Y, Takahashi M
    Molecular biology of the cell 2018年02月 [査読有り][通常論文]
  • Kuragano M, Murakami Y, Takahashi M
    Biochemical and biophysical research communications 2018年02月 [査読有り][通常論文]
  • DNA replication machinery is required for development in Drosophila.
    Kohzaki H, Asano M, Murakami Y
    Frontiers in bioscience (Landmark edition) 23 493 - 505 2018年01月 [査読有り][通常論文]
  • DNA replication machinery contributes to development of eye in Drosophila.
    Kohzaki H, Murakami Y
    Frontiers in bioscience (Landmark edition) 23 506 - 511 2018年01月 [査読有り][通常論文]
  • Takuya Kajitani, Hiroaki Kato, Yuji Chikashige, Chihiro Tsutsumi, Yasushi Hiraoka, Hiroshi Kimura, Yasuyuki Ohkawa, Chikashi Obuse, Damien Hermand, Yota Murakami
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 114 52 E11208 - E11217 2017年12月 [査読有り][通常論文]
     
    Some long noncoding RNAs (ncRNAs) transcribed by RNA polymerase II (RNAPII) are retained on chromatin, where they regulate RNAi and chromatin structure. The molecular basis of this retention remains unknown. We show that in fission yeast serine 7 (Ser7) of the C-terminal domain (CTD) of RNAPII is required for efficient siRNA generation for RNAi-dependent heterochromatin formation. Surprisingly, Ser7 facilitates chromatin retention of nascent heterochromatic RNAs (hRNAs). Chromatin retention of hRNAs and siRNA generation requires both Ser7 and an RNA-binding activity of the chromodomain of Chp1, a subunit of the RNA-induced transcriptional silencing (RITS) complex. Furthermore, RITS associates with RNAPII in a Ser7-dependent manner. We propose that Ser7 promotes cotranscriptional chromatin retention of hRNA by recruiting the RNA-chromatin connector protein Chp1, which facilitates RNAi-dependent hetero-chromatin formation. Our findings reveal a function of the CTD code: linking ncRNA transcription to RNAi for heterochromatin formation.
  • Yuta Sato, Yota Murakami, Masayuki Takahashi
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 488 4 614 - 620 2017年07月 [査読有り][通常論文]
     
    A variety of biochemical fractionation methods are available for the quantification of cytoskeletal components. However, each method is designed to target only one cytoskeletal network, either the micro tubule (MT) or actin cytoskeleton, and non-targeted cytoskeletal networks are ignored. Considering the importance of MT actin crosstalk, the organization of both the targeted and non-targeted cytoskeletal networks must be retained intact during fractionation for the accurate analysis of cytoskeletal organization. In this study, we reveal that existing fractionation methods, represented by the MT sedimentation-method for MTs and the Triton X-100 solubility assay-method for actin cytoskeletons, disrupt the organizations of the non-targeted cytoskeletons. We demonstrate a novel fractionation method for the accurate analysis of the cytoskeletal organizations using a taxol-containing PEM-based permeabilization buffer, which we name "semi-retentive cytoskeletal fractionation (SERCYF)-method". The organizations of both MTs and actin cytoskeletons were retained intact even after permeabilization with this buffer. By using the SERCYF-method, we analyzed the effects of nocodazole on the cytoskeletal organizations biochemically and showed promotion of the actin cytoskeletal organization by MT depolymerization. (C) 2017 Elsevier Inc. All rights reserved.
  • Suzuki S, Murakami Y, Takahata S
    Transcription 8 1 26 - 31 2017年01月 [査読有り][通常論文]
  • Shota Suzuki, Hiroaki Kato, Yutaka Suzuki, Yuji Chikashige, Yasushi Hiraoka, Hiroshi Kimura, Koji Nagao, Chikashi Obuse, Shinya Takahata, Yota Murakami
    NUCLEIC ACIDS RESEARCH 44 9 4147 - 4162 2016年05月 [査読有り][通常論文]
     
    In budding yeast, Set2 catalyzes di- and trimethylation of H3K36 (H3K36me2 and H3K36me3) via an interaction between its Set2-Rpb1 interaction (SRI) domain and C-terminal repeats of RNA polymerase II (Pol2) phosphorylated at Ser(2) and Ser(5) (CTD-S2,5-P). H3K36me2 is sufficient for recruitment of the Rpd3S histone deacetylase complex to repress cryptic transcription from transcribed regions. In fission yeast, Set2 is also responsible for H3K36 methylation, which represses a subset of RNAs including heterochromatic and subtelomeric RNAs, at least in part via recruitment of Clr6 complex II, a homolog of Rpd3S. Here, we show that CTD-S2P-dependent interaction of fission yeast Set2 with Pol2 via the SRI domain is required for formation of H3K36me3, but not H3K36me2. H3K36me3 silenced heterochromatic and subtelomeric transcripts mainly through post-transcriptional and transcriptional mechanisms, respectively, whereas H3K36me2 was not enough for silencing. Clr6 complex II appeared not to be responsible for heterochromatic silencing by H3K36me3. Our results demonstrate that H3K36 methylation has multiple outputs in fission yeast; these findings provide insights into the distinct roles of H3K36 methylation in metazoans, which have different enzymes for synthesis of H3K36me1/2 and H3K36me3.
  • Tange, Y., Chikashige, Y., Takahata, S., Kawakami, K., Higashi, M., Mori, C., Kojidani, T., Hirano, Y., Asakawa, H., Murakami, Y., Haraguchi, T., Hiraoka, Y.
    Genes to Cells 21 8 812 - 832 2016年 [査読有り][通常論文]
  • Shota Suzuki, Koji Nagao, Chikashi Obuse, Yota Murakami, Shinya Takahata
    PROTEIN EXPRESSION AND PURIFICATION 97 44 - 49 2014年05月 [査読有り][通常論文]
     
    Chromatin-associated proteins are heterogeneously and dynamically composed. To gain a complete understanding of DNA packaging and basic nuclear functions, it is important to generate a comprehensive inventory of these proteins. However, biochemical purification of chromatin-associated proteins is difficult and is accompanied by concerns over complex stability, protein solubility and yield. Here, we describe a new method for optimized purification of the endogenously expressed fission yeast Set2 complex, histone H3K36 methyltransferase. Using the standard centrifugation procedure for purification, approximately half of the Set2 protein separated into the insoluble chromatin pellet fraction, making it impossible to recover the large amounts of soluble Set2. To overcome this poor recovery, we developed a novel protein purification technique termed the filtration/immunoaffinity purification/mass spectrometry (FIM) method, which eliminates the need for centrifugation. Using the FIM method, in which whole cell lysates were filtered consecutively through eight different pore sizes (53-0.8 mu m), a high yield of soluble FLAG-tagged Set2 was obtained from fission yeast. The technique was suitable for affinity purification and produced a low background. A mass spectrometry analysis of anti-FLAG immunoprecipitated proteins revealed that Rpb1, Rpb2 and Rpb3, which have all been reported previously as components of the budding yeast Set2 complex, were isolated from fission yeast using the FIM method. In addition, other subunits of RNA polymerase II and its phosphatase were also identified. In conclusion, the FIM method is valid for the efficient purification of protein complexes that separate into the insoluble chromatin pellet fraction during centrifugation. (C) 2014 Elsevier Inc. All rights reserved.
  • Yota Murakami
    EMBO JOURNAL 32 17 2301 - 2303 2013年08月 [査読有り][通常論文]
  • Takayuki Kiboku, Tsuyoshi Katoh, Akio Nakamura, Akira Kitamura, Masataka Kinjo, Yota Murakami, Masayuki Takahashi
    GENES TO CELLS 18 2 90 - 109 2013年02月 [査読有り][通常論文]
     
    Nonmuscle myosin II forms a folded conformation (10S form) in the inactivated state; however, the physiological importance of the 10S form is still unclear. To investigate the role of 10S form, we generated a chimeric mutant of nonmuscle myosin IIB (IIB-SK1 center dot 2), in which S1462-R1490 and L1551-E1577 were replaced with the corresponding portions of skeletal muscle myosin heavy chain. The IIB-SK1 center dot 2 mutant did not fold into a 10S form under physiological condition in vitro. IIB-SK1 center dot 2 was less dynamic by stabilizing the filamentous form and accumulated in the posterior region of migrating cells. IIB-SK1 center dot 2 functioned properly in cytokinesis but altered migratory properties; the rate and directional persistence were increased by IIB-SK1 center dot 2 expression. Surprisingly, endogenous nonmuscle myosin IIA was excluded from the posterior region of migrating cells expressing IIB-SK1 center dot 2, which may underlie the change of the cellular migratory properties. These results suggest that the 10S form is necessary for maintaining nonmuscle myosin II in an unassembled state and for recruitment of nonmuscle myosin II to a specific region of the cell.
  • Kato H, Okazaki K, Iida T, Nakayama J, Murakami Y, Urano T
    Scientific reports 3 2186  2013年 [査読有り][通常論文]
  • Oya E, Kato H, Chikashige Y, Tsutsumi C, Hiraoka Y, Murakami Y
    PLoS genetics 9 8 e1003677  2013年 [査読有り][通常論文]
  • Hirotsugu Hino, Kana Arimoto, Michio Yazawa, Yota Murakami, Akiko Nakatomi
    MARINE BIOTECHNOLOGY 14 4 479 - 490 2012年08月 [査読有り][通常論文]
     
    Calcineurin is a calcium/calmodulin-dependent protein phosphatase that plays important roles in the transduction of calcium signals in a variety of tissues. In addition, calcineurin has been implicated in the process of spermatogenesis. A novel calcineurin-binding protein, CaNBP75, has been identified in scallop testis. The C-terminal region of CaNBP75 is homologous to the C-terminal region of RanBP3, a Ran-binding domain-containing protein. A small G protein Ran has been involved in spermiogenesis by virtue of the fact that its localization in spermatids changes during spermiogenesis. The current study was performed to investigate the functions of Ran and CaNBP75 in the regulation of calcineurin in testis to further understand the basic functions of calcineurin during spermatogenesis. First, cloning and sequencing of a scallop Ran cDNA isolated from testis revealed that scallop Ran is well-conserved at the amino acid level. Secondly, direct binding of Ran to CaNBP75 was demonstrated in an in vitro pull-down assay. Thirdly, analysis of the tissue distribution of Ran, CaNBP75, and calcineurin showed that these proteins are abundantly expressed in testis. Fourthly, comparison of the expression profiles of Ran and CaNBP75 with that of calcineurin in scallop testis during the maturation cycle revealed that Ran and CaNBP75 mRNA levels increase during meiosis and spermiogenesis, similar to calcineurin. Finally, co-immunoprecipitation analysis suggests that Ran, CaNBP75, and calcineurin interact in scallop testis during maturation. These results suggest that Ran, CaNBP75, and calcineurin may act in a coordinated manner to regulate spermatogenesis.
  • Kei Kawakami, Aki Hayashi, Jun-ichi Nakayama, Yota Murakami
    GENES & DEVELOPMENT 26 16 1811 - 1824 2012年08月 [査読有り][通常論文]
     
    In fission yeast, siRNA is generated from pericentromeric noncoding RNA by the RNAi machinery. siRNA synthesis and heterochromatin formation are interdependent, forming a self-reinforcing loop on chromatin. In this system, siRNA is amplified by the RNA-dependent RNA polymerase complex (RDRC) and the endoribonuclease Dcr1, which synthesizes dsRNA and processes the dsRNA, respectively. The amplification is essential for stable heterochromatin formation. Here, a novel gene, dsh1(+) (defect of the gene silencing at centromeric heterochromatin), is identified as an essential component of RNAi-directed heterochromatin assembly. Loss of dsh1(+) abolishes normal RNAi function and heterochromatic gene silencing at pericentromeres. Dsh1 interacts with Dcr1 and RDRC and couples the reactions of both proteins to the effective production of siRNA in vivo. Dsh1 binds to heterochromatin in the absence of RDRC, while RDRC requires Dsh1 for its chromatin-binding activity, suggesting that Dsh1 recruits RDRC to chromatin. Immunofluorescence analysis shows that Dsh1 forms foci at the nuclear periphery, and some Dsh1 foci colocalize with Dcr1 and RDRC. Dsh1 is required for the colocalization of Dcr1 and RDRC. Moreover, loss of the nuclear periphery localization of Dsh1 abolishes Dsh1 function. Taken together, these results suggest that Dsh1 assembles the RNAi machinery on heterochromatin and forms a perinuclear compartment for amplification of heterochromatic siRNA.
  • Aki Hayashi, Mayumi Ishida, Rika Kawaguchi, Takeshi Urano, Yota Murakami, Jun-ichi Nakayama
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 109 16 6159 - 6164 2012年04月 [査読有り][通常論文]
     
    In fission yeast, the RNAi pathway is required for centromeric heterochromatin assembly. siRNAs derived from centromeric transcripts are incorporated into the RNA-induced transcriptional silencing (RITS) complex and direct it to nascent homologous transcripts. The RNA-induced transcriptional silencing-bound nascent transcripts further recruit the RNA-directed RNA polymerase complex (RDRC) to promote dsRNA synthesis and siRNA production. Heterochromatin coated with Swi6/Heterochromain Protein 1 is then formed following recruitment of chromatin modification machinery. Swi6 is also required for the upstream production of siRNA, although the mechanism for this has remained obscure. Here, we demonstrate that Swi6 recruits RDRC to heterochromatin through Ers1, an RNAi factor intermediate. An ers1(+) mutant allele (ers1-C62) was identified in a genetic screen for mutants that alleviate centromeric silencing, and this phenotype was suppressed by overexpression of either the Hrr1 RDRC subunit or Clr4 histone H3-K9 methyltransferase. Ers1 physically interacts with Hrr1, and loss of Ers1 impairs RDRC centromeric localization. Although Ers1 failed to bind Clr4, a direct interaction with Swi6 was detected, and centromeric localization of Swi6 was enhanced by Clr4 overexpression in ers1-C62 cells. Consistent with this, deletion of swi6(+) reduced centromeric localization of Ers1 and RDRC. Moreover, tethering of Ers1 or Hrr1 to centromeric heterochromatin partially bypassed Swi6 function. These findings demonstrate an alternative mechanism for RDRC recruitment and explain the essential role of Swi6/Heterochromain Protein 1 in RNAi-directed heterochromatin assembly.
  • Mina Nakama, Kei Kawakami, Takuya Kajitani, Takeshi Urano, Yota Murakami
    GENES TO CELLS 17 3 218 - 233 2012年03月 [査読有り][通常論文]
     
    Certain noncoding RNAs (ncRNAs) implicated in the regulation of chromatin structure associate with chromatin. During the formation of RNAi-directed heterochromatin in fission yeast, ncRNAs transcribed from heterochromatin are thought to recruit the RNAi machinery to chromatin for the formation of heterochromatin; however, the molecular details of this association are not clear. Here, using RNA immunoprecipitation assay, we showed that the heterochromatic ncRNA was associated with chromatin via the formation of a DNARNA hybrid and bound to the RNA-induced transcriptional silencing (RITS) complex. The presence of DNARNA hybrid in the cell was also confirmed by immunofluorescence analysis using anti-DNARNA hybrid antibody. Over-expression and depletion of RNase H in vivo decreased and increased the amount of DNARNA hybrid formed, respectively, and both disturbed heterochromatin. Moreover, DNARNA hybrid was formed on, and over-expression of RNase H inhibited the formation of, artificial heterochromatin induced by tethering of RITS to mRNA. These results indicate that heterochromatic ncRNAs are retained on chromatin via the formation of DNARNA hybrids and provide a platform for the RNAi-directed heterochromatin assembly and suggest that DNARNA hybrid formation plays a role in chromatic ncRNA function.
  • Kitagawa C, Nakatomi A, Hwang D, Osaka I, Fujimori H, Kawasaki H, Arakawa R, Murakami Y, Ohki S
    Biophysics (Nagoya-shi, Japan) 7 35 - 49 2011年 [査読有り][通常論文]
  • Atsushi Shimada, Yota Murakami
    EPIGENETICS 5 1 30 - 33 2010年01月 [査読有り][通常論文]
     
    Heterochromatin is characterized by methylation of histone H3 at lysine 9, which is recognized by well-conserved HP1-family proteins. Heterochromatin participates in various chromosome functions, which include transcriptional gene silencing and sister-chromatid cohesion. These heterochromatic functions are carried out by various effector proteins that associate with HP1-family proteins; however, the regulation of this association with the effectors is not well understood. Recently, we showed that phosphorylation of the fission-yeast HP1 homolog Swi6 regulates the association of the transcriptional regulators differentially and changes the transcriptional activity of heterochromatin, without affecting sister-chromatid cohesion. This study, together with another study performed using other systems, indicates that phosphorylation of HP1/Swi6 provides a dynamic pathway for the differential regulation of heterochromatin in response to inter-and intracellular signals.
  • [Heterochromatin as a dynamic higher order chromatin structure].
    Murakami Y
    Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme 54 4 Suppl 488 - 495 2009年03月 [査読有り][通常論文]
  • Atsushi Shimada, Kohei Dohke, Mahito Sadaie, Kaori Shinmyozu, Jun-Ichi Nakayama, Takeshi Urano, Yota Murakami
    GENES & DEVELOPMENT 23 1 18 - 23 2009年01月 [査読有り][通常論文]
     
    Heterochromatin protein 1 (HP1) recruits various effectors to heterochromatin for multiple functions, but its regulation is unclear. In fission yeast, a HP1 homolog Swi6 recruits SHREC, Epe1, and cohesin, which are involved in transcriptional gene silencing (TGS), transcriptional activation, and sister chromatid cohesion, respectively. We found that casein kinase II (CK2) phosphorylated Swi6. Loss of CK2-dependent Swi6 phosphorylation alleviated heterochromatic TGS without affecting heterochromatin structure. This was due to the inhibited recruitment of SHREC to heterochromatin, accompanied by an increase in Epe1. Interestingly, loss of phosphorylation did not affect cohesion. These results indicate that CK2-dependent Swi6 phosphorylation specifically controls TGS in heterochromatin.
  • Kohei Dohke, Shota Miyazaki, Katsunori Tanaka, Takeshi Urano, Shiv I. S. Grewal, Yota Murakami
    GENES TO CELLS 13 10 1027 - 1043 2008年10月 [査読有り][通常論文]
     
    Chromatin assembly factor-1 (CAF1) is a well-conserved histone chaperone that loads the histone H3-H4 complex onto newly synthesized DNA in vitro through interaction with the replication factor PCNA. CAF1 is considered to be involved in heterochromatin maintenance in several organisms, but the evidence is circumstantial and functional details have not been established. We identified fission yeast CAF-1 (spCAF1), which interacts with PCNA in S phase. Depletion of spCAF1 caused defects in silencing at centromeric and mating locus heterochromatin, accompanied with a decrease in Swi6, the fission yeast HP1 homologue. Loss of spCAF1 destabilized both the silent and active states of chromatin at the meta-stable heterochromatic region, with a more pronounced effect on the silent state, indicating that spCAF1 is involved in the maintenance of heterochromatin. Swi6 dissociated from heterochromatin during G1/S phase appears to associate with spCAF1. In early S phase, spCAF1 localized to replicating heterochromatin as well as euchromatin and remained associated with Swi6, and Swi6 then bound to heterochromatin. Taken together, we propose that spCAF1 functions in heterochromatin maintenance by recruiting dislocated Swi6 during replication to replicated heterochromatin at the replication fork.
  • Schizosaccharomyces pombe Orc5 plays multiple roles in the maintenance of genome stability throughout the cell cycle.
    Kato H, Matsunaga F, Miyazaki S, Yin L, D'Urso G, Tanaka K, Murakami Y
    Cell cycle (Georgetown, Tex.) 7 8 1085 - 1096 2008年04月 [査読有り][通常論文]
  • Mika Yokoyama, Hirokazu Inoue, Chizu Ishii, Yota Murakami
    DNA REPAIR 6 6 770 - 780 2007年06月 [査読有り][通常論文]
     
    The progression of replication forks is often impeded by obstacles that cause them to stall or collapse, and appropriate responses to replication- associated DNA damage are important for genome integrity. Here we identified a new gene, mus7(+), that is involved in the repair of replication- associated DNA damage in the fission yeast Schizosaccharomyces pombe. The Delta mus7 mutant shows enhanced sensitivity to methyl methanesulfonate (MMS), camptothecin, and hydroxyurea, agents that cause replication fork stalling or collapse, but not to ultraviolet light or X-rays. Epistasis analysis of MMS sensitivity indicates that Mus7 functions in the same pathway as Mus81, a subunit of the Mus81-Eme1 structure -specific endonuclease, which has been implicated in the repair of the replication-associated DNA damage. In Delta mus7 and Delta mus81 cells, the repair of MMS-induced DNA double-strand breaks (DSBs) is severely impaired. Moreover, some cells with either mutation are hyper-elongated or enlarged, and most of these cells accumulate in late G2 phase. Spontaneous Rad22 (recombination mediator protein RAD52 homolog) foci increase in S phase to late G2 phase in Delta mus7 and Delta mus81 cells. These results suggest that replication-associated DSBs accumulate in these cells and that Rad22 foci form in the absence of Mus7 or Mus81. We also found that the rate of spontaneous conversion-type recombination is reduced in mitotic Delta mus7 cells, suggesting that Rhp51- (RAD51 homolog) dependent homologous recombination is disturbed in this mutant. From these data, we propose that Mus7 functions in the repair of replication- associated DSBs by promoting RAD51-dependent conversion-type recombination downstream of Rad22 and Mus81. (c) 2007 Elsevier B.V All rights reserved.
  • [Regulation of chromosomal DNA replication by transcription factors].
    Kohzaki H, Murakami Y
    Seikagaku. The Journal of Japanese Biochemical Society 79 5 458 - 462 2007年05月 [査読有り][通常論文]
  • Yota Murakami, Ling-Feng Chen, Noriyuki Sanechika, Hidetsugu Kohzaki, Yoshiaki Ito
    JOURNAL OF CELLULAR BIOCHEMISTRY 100 5 1313 - 1323 2007年04月 [査読有り][通常論文]
     
    Eukaryotic DNA replication takes place in the replication factories, where replication proteins are properly assembled to form replication forks. Thus, recruitment of DNA replication origins to the replication factories must be the key step for the regulation of DNA replication. The transcription factor Runx1 associates with the nuclear matrix, the putative substructure of DNA replication factories. An earlier report from our laboratory showed that Runx1 activates polyomavirus DNA replication, and that this requires its nuclear matrix-binding activity. Here, we show that Runx1 activates polyomavirus DNA replication by stimulating the binding of the viral-encoded replication initiator/helicase, large T antigen, to its replication origin. We found that newly replicated polyomavirus DNA is associated with the nuclear matrix and that large T antigen is targeted to replication factories, suggesting that polyomavirus is replicated in replication factories on the nuclear matrix. Although Runx1 did not co-localize with large T antigen-containing foci by itself, it co-localized with large T antigen-containing replication factories during Runx1-dependent polyomavirus DNA replication. These observations together suggest that Runx1 recruits the polyomavirus replication origin to the replication factory on the nuclear matrix, and that this requires the nuclear matrix-binding activity of Runx1.
  • Hidetsugu Kohzaki, Yota Murakami
    PROTEOMICS 7 1 10 - 14 2007年01月 [査読有り][通常論文]
     
    Chromatin immunoprecipitation (ChIP) assays are widely used to investigate where chromatin-binding proteins bind to the genome. The standard assay is very time consuming. We have developed a rapid ChIP assay in which the immunoprecipitates serve directly as PCR templates. This assay eliminates the step to reverse the crosslinking, shortening the assay by 1 day. It also requires a less immunoprecipitating antibody, permits many samples to be tested simultaneously, and is more sensitive than the standard ChIP assay.
  • [Regulation of higher order chromatin structure by RNA].
    Murakami Y
    Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme 51 16 Suppl 2456 - 2462 2006年12月 [査読有り][通常論文]
  • Hiromitsu Yamamoto, Kosei Ito, Mariko Kawai, Yota Murakami, Kazuhisa Bessho, Yoshiaki Ito
    ANATOMICAL RECORD PART A-DISCOVERIES IN MOLECULAR CELLULAR AND EVOLUTIONARY BIOLOGY 288A 7 695 - 699 2006年07月 [査読有り][通常論文]
     
    Studies of molecular mechanisms underlying the development of the mammalian oral mucosa have revealed a major involvement of transforming growth factor beta (TGF-beta) and bone morphologic protein (BMP) signaling pathways. Here, we examined the expression of a downstream target of TGF-beta and BMPs, Runx3, in oral mucosa. Runx3 is a runt-related transcription factor that acts as a gastric tumor suppressor and regulator of growth and differentiation in mammalian gastric epithelial cells. Another member of the Runx family in C. elegans, run, is invoflved in the development of a functional hypodermis and gut. In this report, we examined Runx3 expression using reverse transcription-polymerase chain reaction, immnunohistochemistry, and in situ hybridization and found that Runx3 is expressed in the tongue and palate epithelium of mouse embryos from embryonic day 12.5 to 16.5. The functional relationship between Runx3 and TGF-beta/BMPs signaling in tongue and palate development is discussed.
  • MA Marchetti, M Weinberger, Y Murakami, WC Burhans, JA Huberman
    JOURNAL OF CELL SCIENCE 119 1 124 - 131 2006年01月 [査読有り][通常論文]
     
    Previous studies have indicated that replication stress can trigger apoptosis-like cell death, accompanied (where tested) by production of reactive oxygen species (ROS), in mammalian cells and budding yeast (Saccharomyces cerevisiae). In mammalian cells, inappropriate entry into mitosis also leads to cell death. Here, we report similar responses in fission yeast (Schizosaccharomyces pombe). We used ROS- and death-specific fluorescent stains to measure the effects of mutations in replication initiation and checkpoint genes in fission yeast on the frequencies of ROS production and cell death. We found that certain mutant alleles of each of the four tested replication initiation genes caused elevated ROS and cell death. Where tested, these effects were not enhanced by checkpoint-gene mutations. Instead, when cells competent for replication but defective in both the replication and damage checkpoints were treated with hydroxyurea, which slows replication fork movement, the frequencies of ROS production and cell death were greatly increased. This was a consequence of elevated CDK activity, which permitted inappropriate entry into mitosis. Thus, studies in fission yeast are likely to prove helpful in understanding the pathways that lead from replication stress and inappropriate mitosis to cell death in mammalian cells.
  • [Role of RNA polymerase II in RNAi-dependent heterochromatin formation].
    Murakami Y
    Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme 51 1 54 - 60 2006年01月 [査読有り][通常論文]
  • Alexandra M. Locovei, Maria-Grazia Spiga, Katsunori Tanaka, Yota Murakami, Gennaro D'Urso
    CELL DIVISION 1 27  2006年 [査読有り][通常論文]
     
    Abp1, and the closely related Cbh1 and Cbh2 are homologous to the human centromere-binding protein CENP-B that has been implicated in the assembly of centromeric heterochromatin. Fission yeast cells lacking Abp1 show an increase in mini-chromosome instability suggesting that Abp1 is important for chromosome segregation and/or DNA synthesis. Here we show that Abp1 interacts with the DNA replication protein Cdc23 (MCM10) in a two-hybrid assay, and that the Delta abp1 mutant displays a synthetic phenotype with a cdc23 temperature-sensitive mutant. Moreover, genetic interactions were also observed between abp1(+) and four additional DNA replication initiation genes cdc18(+), cdc21(+), orc1(+), and orc2(+). Interestingly, we find that S phase is delayed in cells deleted for abp1(+) when released from a G1 block. However, no delay is observed when cells are released from an early S phase arrest induced by hydroxyurea suggesting that Abp1 functions prior to, or coincident with, the initiation of DNA replication.
  • H Kohzaki, Y Murakami
    BIOESSAYS 27 11 1107 - 1116 2005年11月 [査読有り][通常論文]
     
    tThe chromosomes of eukaryotic cells possess many potential DNA replication origins, of which a subset is selected in response to the cellular environment, such as the developmental stage, to act as active replication start sites. The mechanism of origin selection is not yet fully understood. In this review, we summarize recent observations regarding replication origins and initiator proteins in various organisms. These studies suggest that the DNA-binding specificities of the initiator proteins that bind to the replication origins and promote DNA replication are primarily responsible for origin selection. We particularly focus on the importance of transcription factors in the origin selection process. We propose that transcription factors are general regulators of the formation of functional complexes on the chromosome, including the replication initiation complex. We discuss the possible mechanisms by which transcription factors influence the selection of particular origins. (c) 2005 Wiley Periodicals, Inc.
  • H Kato, DB Goto, RA Martienssen, T Urano, K Furukawa, Y Murakami
    SCIENCE 309 5733 467 - 469 2005年07月 [査読有り][通常論文]
     
    In Schizosaccharomyces pombe, the RNA interference (RNAi) machinery con. verts pericentromeric transcripts into small interfering RNAs (siRNAs) and is, required for the assembly of pericentromeric heterochromatin. Here we describe a. mutation in the second largest subunit of RNA polymerase II (RNAPII). Both wild-type and mutant RNAPII localized to the pericentromere. However, the mutation resulted in the loss of heterochromatic histone modifications and in the accumulation of pericentromeric transcripts, accompanied by the loss of siRNAs. This phenotype resembles mutants in RNAi and suggests that RNAPII couples pericentromeric transcription with siRNA processing and heterochromatin assembly.
  • [Heterochromatin and centromere: heterochromatin meets RNAi].
    Kato H, Murakami Y
    Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme 49 12 1990 - 1997 2004年09月 [査読有り][通常論文]
  • M Ueno, T Murase, T Kibe, N Ohashi, K Tomita, Y Murakami, M Uritani, T Ushimaru, M Harata
    NUCLEIC ACIDS RESEARCH 32 2 736 - 741 2004年01月 [査読有り][通常論文]
     
    The actin-related proteins (Arps), which are subdivided into at least eight subfamilies, are conserved from yeast to humans. A member of the Arp6 subfamily in Drosophila, Arp4/Arp6, co-localizes with heterochromatin protein 1 (HP1) in pericentric heterochromatin. Fission yeast Schizosaccharomyces pombe possesses both an HP1 homolog and an Arp6 homolog. However, the function of S.pombe Arp6 has not been characterized yet. We found that deletion of arp6(+) impaired telomere silencing, but did not affect centromere silencing. Chromatin immunoprecipitation assays revealed that Arp6 bound to the telomere region. However, unlike Drosophila Arp4/Arp6, S.pombe Arp6 was distributed throughout nuclei. The binding of Arp6 to telomere DNA was not affected by deletion of swi6(+). Moreover, the binding of Swi6 to telomere ends was not affected by deletion of arp6(+). These results suggest that Arp6 and Swi6 function independently at telomere ends. We propose that the Arp6-mediated repression mechanism works side by side with Swi6-based telomere silencing in S.pombe.
  • J Kikuchi, J Iwahara, T Kigawa, Y Murakami, T Okazaki, S Yokoyama
    JOURNAL OF BIOMOLECULAR NMR 22 4 333 - 347 2002年04月 [査読有り][通常論文]
     
    We have solved the solution structure of the N-terminal region of the fission yeast centromere protein, Abp1, bound to a 21-base pair DNA fragment bearing its recognition site (Mw = 30 kDa). Although the two DNA-binding domains in the Abp1 protein were defined well by a conventional NOE-based NMR methodology, the overall structure of the Abp1 protein was poorly defined, due to the lack of interdomain distance restraints. Therefore, we additionally used residual dipolar couplings measured in a weakly aligned state, and rotational diffusion anisotropies. Neither the NH residual dipolar couplings nor the backbone (1)5N T-1/T-2 data were sufficient to determine the overall structure of the Abp1 protein, due to spectral overlap. We used a combination of these two orientational restraints (residual dipolar coupling and rotational diffusion anisotropy), which significantly improved the convergence of the overall structures. The range of the observed T-1/T-2 ratios was wider (20-50 for the secondary structure regions of Abp1) than the previously reported data for several globular proteins, indicating that the overall shape of the Abp1.DNA complex is ellipsoid. This extended form would facilitate the recognition of the two separate sites in the relatively long DNA sequence by the DNA-binding domains of Apb1.

その他活動・業績

  • H Nakagawa, JK Lee, J Hurwitz, RC Allshire, J Nakayama, SIS Grewal, K Tanaka, Y Murakami GENES & DEVELOPMENT 16 (14) 1766 -1778 2002年07月 [査読無し][通常論文]
     
    Heterochromatin is a functionally important chromosomal component, especially at centromeres. In fission yeast, conserved heterochromatin-specific modifications of the histone H3 tail, involving deacetylation of Lys 9 and Lys 14 and subsequent methylation of Lys 9, promote the recruitment of a heterochromatin protein, Swi6, a homolog of the Drosophila heterochromatin protein 1. However, the primary determinants of the positioning of heterochromatin are still unclear. The fission yeast proteins Abp1, Cbh1, and Cbh2 are homologs of the human protein CENP-B that bind to centromeric a-satellite DNA and associate with centromeric heterochromatin. We show that the CENP-B homologs are functionally redundant at centromeres, and that Abp1 binds specifically to centromeric heterochromatin. In the absence of Abp1 or Cbh1, the centromeric association of Swi6 is diminished, resulting in a decrease in silencing of the region. CENP-B-homolog double disruptants show a synergistic reduction of Swi6 at centromeric heterochromatin, indicating that the three proteins are functionally redundant in the recruitment of Swi6. Furthermore, using chromatin immunoprecipitation assays, we show that disruption of CENP-B homologs causes a decrease in heterochromatin-specific modifications of histone H3. These results indicate that the CENP-B homologs act as site-specific nucleation factors for the formation of centromeric heterochromatin by heterochromatin-specific modifications of histone tails.
  • Context-dependent modulation of replication activity of Saccharomyces cerevisiae autonomously replicating sequences by transcription factors
    Mol. Cell. Biol. 19: 7428-7435 1999年 [査読無し][通常論文]
  • Characterization of a fission yeast SUMO-1 homologue, Pmt3p, required for multiple nuclear events, including the control of telomere lenghth and chromosome segregation.
    Mol. Cell. Biol. 19: 8660-8672 1999年 [査読無し][通常論文]

共同研究・競争的資金等の研究課題

  • 真核細胞の染色体複製制御機構の解析
    研究期間 : 1996年 -2006年
  • 真核細胞の染色体機能におけるクロマチン・核の高次構造の役割の解明
    研究期間 : 2000年
  • Structure and Funtion of higher order chromatin structure in the nuclei
  • Regulation of eukariotic chromosome replication

教育活動情報

主要な担当授業

  • 化学研究先端講義
    開講年度 : 2018年
    課程区分 : 修士課程
    開講学部 : 総合化学院
  • 生命分子化学特論
    開講年度 : 2018年
    課程区分 : 修士課程
    開講学部 : 総合化学院
    キーワード : 遺伝情報,タンパク質構造,分子論的理解,生合成機構,動物細胞,二次代謝産物,バイオポリマー,環境浄化
  • 総合化学特別研究第二
    開講年度 : 2018年
    課程区分 : 博士後期課程
    開講学部 : 総合化学院
  • 生命分子化学特論
    開講年度 : 2018年
    課程区分 : 修士課程
    開講学部 : 工学院
    キーワード : 遺伝情報,タンパク質構造,分子論的理解,生合成機構,動物細胞,二次代謝産物,バイオポリマー,環境浄化
  • 大学院共通授業科目(一般科目):自然科学・応用科学
    開講年度 : 2018年
    課程区分 : 修士課程
    開講学部 : 大学院共通科目
    キーワード : 遺伝情報,タンパク質構造,分子論的理解,生合成機構,動物細胞,二次代謝産物,バイオポリマー,環境浄化
  • 化学研究先端講義
    開講年度 : 2018年
    課程区分 : 修士課程
    開講学部 : 総合化学院
    キーワード : 最先端化学研究,外国人講師,英語
  • 総合化学特論Ⅰ(Modern Trends in Physical and Material Chemistry)
    開講年度 : 2018年
    課程区分 : 修士課程
    開講学部 : 総合化学院
    キーワード : 先端物理化学,物質変換化学,光化学,分子理論化学,化学エネルギー変換,分離プロセス工学,プロセス工学,触媒設計,機能解析化学,ナノフォトニクス材料,無機固体化学、ナノ物質化学,界面電子化学,無機物性化学,電子材料化学,機能固体化学,応用材料化学
  • 総合化学特論Ⅱ(Modern Trends in Organic Chemistry and Biological Chemistry)
    開講年度 : 2018年
    課程区分 : 修士課程
    開講学部 : 総合化学院
    キーワード : 有機構造化学,高分子機能科学,有機金属化学,有機物性化学,遺伝情報制御化学,疾病制御化学,有機合成化学,生物物理化学,生合成工学,生命システム工学,生物計測化学,機能性高分子,動物細胞培養工学
  • 大学院共通授業科目(一般科目):自然科学・応用科学
    開講年度 : 2018年
    課程区分 : 修士課程
    開講学部 : 大学院共通科目
    キーワード : 先端物理化学,物質変換化学,光化学,分子理論化学,化学エネルギー変換,分離プロセス工学,プロセス工学,触媒設計,機能解析化学,ナノフォトニクス材料,無機固体化学、ナノ物質化学,界面電子化学,無機物性化学,電子材料化学,機能固体化学,応用材料化学
  • 大学院共通授業科目(一般科目):自然科学・応用科学
    開講年度 : 2018年
    課程区分 : 修士課程
    開講学部 : 大学院共通科目
    キーワード : 有機構造化学,高分子機能科学,有機金属化学,有機物性化学,遺伝情報制御化学,疾病制御化学,有機合成化学,生物物理化学,生合成工学,生命システム工学,生物計測化学,機能性高分子,動物細胞培養工学
  • 基礎生物化学特論
    開講年度 : 2018年
    課程区分 : 修士課程
    開講学部 : 総合化学院
    キーワード : 細胞増殖と分化、遺伝子発現、エピジェネティクス、シグナル伝達、癌遺伝子、 免疫、感染症
  • 大学院共通授業科目(一般科目):自然科学・応用科学
    開講年度 : 2018年
    課程区分 : 修士課程
    開講学部 : 大学院共通科目
    キーワード : 細胞増殖と分化、遺伝子発現、エピジェネティクス、シグナル伝達、癌遺伝子、 免疫、感染症
  • 生物化学A(Ⅰ)
    開講年度 : 2018年
    課程区分 : 修士課程
    開講学部 : 総合化学院
    キーワード : 遺伝情報、DNA、RNA、蛋白質、クロマチン、染色体、筋収縮、モータータンパク質、細胞運動、細胞骨格、細胞形態
  • 生命分子化学特論
    開講年度 : 2018年
    課程区分 : 博士後期課程
    開講学部 : 工学院
    キーワード : 遺伝情報,タンパク質構造,分子論的理解,生合成機構,動物細胞,二次代謝産物,バイオポリマー,環境浄化
  • 先端総合化学特論Ⅰ
    開講年度 : 2018年
    課程区分 : 博士後期課程
    開講学部 : 総合化学院
    キーワード : 先端物理化学,物質変換化学,光化学,分子理論化学,化学エネルギー変換,分離プロセス工学,プロセス工学,触媒設計,機能解析化学,ナノフォトニクス材料,無機固体化学、ナノ物質化学,界面電子化学,無機物性化学,電子材料化学,機能固体化学,応用材料化学
  • 先端総合化学特論Ⅰ
    開講年度 : 2018年
    課程区分 : 博士後期課程
    開講学部 : 総合化学院
    キーワード : 有機構造化学,高分子機能科学,有機金属化学,有機物性化学,遺伝情報制御化学,疾病制御化学,有機合成化学,生物物理化学,生合成工学,生命システム工学,生物計測化学,機能性高分子,動物細胞培養工学
  • 総合化学特別研究第二
    開講年度 : 2018年
    課程区分 : 博士後期課程
    開講学部 : 総合化学院
    キーワード : 物理化学,無機分析化学,有機化学,生物化学,物質化学,有機プロセス工学,生物機能高分子
  • 化学Ⅱ
    開講年度 : 2018年
    課程区分 : 学士課程
    開講学部 : 全学教育
    キーワード : 有機化合物、官能基、分子構造、化学的性質、化学反応、機能性有機物、生体関連有機物質
  • 機能生化学
    開講年度 : 2018年
    課程区分 : 学士課程
    開講学部 : 理学部
    キーワード : 細胞内での物質変換過程、糖代謝、脂質代謝、クエン酸サイクルと酸化的リン酸化、光合成、代謝の調節、シグナル伝達
  • 国際交流Ⅰ
    開講年度 : 2018年
    課程区分 : 学士課程
    開講学部 : 国際本部
    キーワード : 先端物理化学,分子理論化学,集合機能化学,物質変換化学,光化学,材料製造工学,プロセス工学,分離生成工学,触媒材料設計,触媒反応設計,化学エネルギー変換,固体物性化学,機能解析化学,先端無機化学,ナノ物質化学,分子組織化学,固体化学,ナノセラミックス,界面電子化学,無機物性化学,無機プロセシング科学,電子材料化学,セラミック材科学,応用材料化学
  • 国際交流Ⅰ
    開講年度 : 2018年
    課程区分 : 学士課程
    開講学部 : 国際本部
    キーワード : 有機合成化学,有機金属化学,有機構造化学,生物有機化学,高分子合成化学,化学生物学,生物化学,分子生物学,生物物理化学,再生医療工学,生体触媒学,生物無機化学,生物計測化学,遺伝情報制御化学,疾病制御化学
  • 生物化学Ⅱ
    開講年度 : 2018年
    課程区分 : 学士課程
    開講学部 : 理学部
    キーワード : タンパク質化学、タンパク質の機能、酵素触媒、酵素反応速度論


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