Histochemistry and Cell Biology

, Volume 120, Issue 2, pp 111–119 | Cite as

Dissociation of mammalian Polycomb-group proteins, Ring1B and Rae28/Ph1, from the chromatin correlates with configuration changes of the chromatin in mitotic and meiotic prophase

  • Hiro Miyagishima
  • Kyoichi Isono
  • Yuichi Fujimura
  • Masaomi Iyo
  • Yoshihiro Takihara
  • Hiroshi Masumoto
  • Miguel Vidal
  • Haruhiko Koseki
Original Paper


The Polycomb group (PcG) gene products form complexes that regulate chromatin configuration to mediate cellular memory to postmitotic somatic cells and postmeiotic oocytes in Drosophila melanogaster. Structural and functional similarities of PcG proteins between invertebrates and vertebrates suggest mammalian PcG proteins may be involved to imprint transcriptional status at various loci into postmitotic and postmeiotic daughter cells. To address molecular mechanisms underlying PcG-mediated cellular memory, it might be a prerequisite to understand subcellular localization of PcG proteins during mitosis and meiosis. In this study, we analyzed subcellular localization of Rae28/Ph1 and Ring1B by using newly generated monoclonal antibodies in mitotic somatic cells and meiotic mouse oocytes. Results suggest that Rae28/Ph1 and Ring1B dissociate from the chromatin upon its condensation in mitotic prophase in the U2-OS human osteosarcoma cell line. During maturation of oocytes, significant alterations of Rae28/Ph1 and Ring1B localization are concordant with configuration changes of the chromatin at the germinal vesicle stage of meiotic prophase. Importantly, dissociation of Rae28/Ph1 and Ring1B from the chromatin temporally correlates with transcriptional arrest both in mitosis and meiosis. Present and previous observations suggest molecular mechanisms required for mitotic regulation of RNA polymerase II could be involved in dissociation of PcG proteins.


Polycomb Mitosis Oocyte Mammals Chromatin 



We are grateful to Drs. M. Maekawa and Y. Toyama for the valuable suggestions on histological analyses of ovaries, Ms Sanae Takeda and Misao Uchida and Mr. Shozo Sugimori for their technical assistance, and Dr. T. Akasaka for initial instructions for confocal microscopic analyses. This project was supported by Special Coordination Funds for Promoting Science and Technology from the Ministry of Education, Culture, Sports, Science and Technology, the Japanese Government.


  1. Akasaka T, Kanno M, Balling R, Mieza MA, Taniguchi M, Koseki H (1996) A role for mel-18 a Polycomb group-related vertebrate gene, during the anteroposterior specification of the axial skeleton. Development 122:1513–1522PubMedGoogle Scholar
  2. Akasaka T, Takahashi N, Suzuki M, Koseki H, Bodmer R, Koga H (2002) MBLR, a new RING finger protein resembling mammalian Polycomb gene products, is regulated by cell cycle-dependent phosphorylation. Genes Cell 7:835–850CrossRefGoogle Scholar
  3. Akoulitchev S, Reinberg D (1998) The molecular mechanism of mitotic inhibition of TFIIH is mediated by phosphorylation of CDK7. Genes Dev 12:3541–3550PubMedGoogle Scholar
  4. Atsuta T, Fujimura Y, Moriya H, Vidal M, Akasaka T, Koseki H (2001) Production of monoclonal antibodies against mammalian Ring1B proteins. Hybridoma 20:43–46CrossRefPubMedGoogle Scholar
  5. Bostock CJ, Prescott DM, Kirkpatrick JB (1971) An evaluation of the double thymidine block for synchronizing mammalian cells at the G1-S border. Exp Cell Res 68:163–168PubMedGoogle Scholar
  6. Bouniol-Baly C, Hamraoui L, Guibert J, Beaujean N, Szollosi MS, Debey P (1999) Differential transcriptional activity associated with chromatin configuration in fully grown mouse germinal vesicle oocytes. Biol Reprod 60:580–587PubMedGoogle Scholar
  7. Core N, Bel S, Gaunt SJ, Aurrand-Lions M, Pearce J, Fisher A, Djabali M (1997) Altered cellular proliferation and mesoderm patterning in Polycomb-M33-deficient mice. Development 124:721–729PubMedGoogle Scholar
  8. del Mar Lorente M, Marcos-Gutierrez C, Perez C, Schoorlemmer J, Ramirez A, Magin T, Vidal M (2000) Loss- and gain-function mutations show a Polycomb group function for Ring1A in mice. Development 127:5093–5100PubMedGoogle Scholar
  9. Franke A, DeCamillis M, Zink D, Cheng N, Brock HW, Paro R (1992) Polycomb and polyhomeotic are constituents of a multimeric protein complex in chromatin of Drosophila melanogaster. EMBO J 11:2941–2950PubMedGoogle Scholar
  10. Gebara MM, Sayre MH, Corden JL (1997) Phosphorylation of the carboxy-terminal repeat domain in RNA polymerase II by cyclin-dependent kinases is sufficient to inhibit transcription. J Cell Biochem 64:390–402CrossRefPubMedGoogle Scholar
  11. Gebuhr TC, Bultman SJ, Magnuson T (2000) Pc-G/trx-G and the SWI/SNF connection: developmental gene regulation through chromatin remodeling. Genesis 26:189–197CrossRefPubMedGoogle Scholar
  12. Gunster MJ, Satijn DP, Hamer KM, den Baauwen JL, de Bruijn D, Alkema MJ, van Lohuizen M, van Driel R, Otte AP (1997) Identification and characterization of interactions between the vertebrate Polycomb-group protein Bmi1 and human homologs of polyhomeotic. Mol Cell Biol 17:2326–2335PubMedGoogle Scholar
  13. Hemenway CS, Halligan BW, Levy LS (1998) The Bmi-1 oncoprotein interacts with dinG and Mph2: the role of RING finger domains. Oncogene 16:2541–2547Google Scholar
  14. Hemenway CS, Halligan BW, Gould GC, Levy LS (2000) Identification and analysis of a third mouse Polycomb gene, MPc3. Gene 242:31–40CrossRefPubMedGoogle Scholar
  15. Itahara K, Zou Y, Itahara Y, Martinez JL, Beausejour C, Jacobs JJ, Van Lohuizen M, Band V, Campisi J, Dimri GP (2003) Control of the replicative life span of human fibroblasts by p16 and the Polycomb protein Bmi-1. Mol Cell Biol 23:389–401CrossRefPubMedGoogle Scholar
  16. Kamijo T, Zindy F, Roussel MF, Quelle DE, Downing JR, Ashmun RA, Grosveld G, Sherr CJ (1997) Tumor suppression at the mouse INK4a locus mediated by the alternative reading frame product p19ARF. Cell 91:649–659PubMedGoogle Scholar
  17. King IF, Francis NJ, Kingston RE (2002) Native and recombinant Polycomb group complexes establish a selective block to template accessibility to repress transcription in vitro. Mol Cell Biol 22:7919–7928CrossRefPubMedGoogle Scholar
  18. Laible G, Wolf A, Dorn R, Reuter G, Nislow C, Lebersorger A, Popkin D, Pillus L, Jenuwein T (1997) Mammalian homologues of the Polycomb-group gene Enhancer of zeste mediate gene silencing in Drosophila heterochromatin and S. cerevisiae telomeres. EMBO J 16:3219–3232CrossRefPubMedGoogle Scholar
  19. Leresche A, Wolf VJ, Gottesfeld JM (1996) Repression of RNA polymerase II and III transcription during M phase of the cell cycle. Exp Cell Res 229:282–288CrossRefPubMedGoogle Scholar
  20. Levine SS, Weiss A, Erdjument-Bromage H, Shao Z, Tempst P, Kingston RE (2002) The core of the Polycomb repressive complex is compositionally and functionally conserved in flies and humans. Mol Cell Biol 22:6261–6271CrossRefPubMedGoogle Scholar
  21. Nomura M, Takihara Y, Shimada K (1994) Isolation and characterization of retinoic acid-inducible cDNA clones in F9 cells: one of the early inducible clones encodes a novel protein sharing several highly homologous regions with a Drosophila polyhomeotic protein. Differentiation 57:39–50CrossRefPubMedGoogle Scholar
  22. Nusslein-Volhard C, Kluding H, Jurgens G (1985) Genes affecting the segmental subdivision of the Drosophila embryo. Cold Spring Harb Symp Quant Biol 50:145–154PubMedGoogle Scholar
  23. Ogawa H, Ishiguro K, Gaubatz S, Livingston DM, Nakatani Y (2002) A complex with chromatin modifiers that occupies E2F- and Myc-responsive genes in G0 cells. Science 296:1132–1136CrossRefPubMedGoogle Scholar
  24. Paro R (1995) Propagating memory of transcription status. Trends Genet 8:295–297CrossRefGoogle Scholar
  25. Parsons GG, Spencer CA (1997) Mitotic repression of RNA polymerase II transcription is accompanied by release of transcription elongation complexes. Mol Cell Biol 17:5791–5802PubMedGoogle Scholar
  26. Pearce J, Singh PB, Gaunt SJ (1992) The mouse has a Polycomb-like chromobox gene. Development 114:921–929PubMedGoogle Scholar
  27. Pirrotta V (1997) Chromatin-silencing mechanisms in Drosophila maintain patterns of gene expression. Trends Genet 8:314–318CrossRefGoogle Scholar
  28. Satijn DP, Gunster MJ, van der Vlag J, Hamer KM, Schul W, Alkema MJ, Saurin AJ, Freemont PS, van Driel R, Otte AP (1997a) RING1 is associated with the Polycomb group protein complex and act as a transcriptional repressor. Mol Cell Biol 17:4105–4113PubMedGoogle Scholar
  29. Satijn DP, Olson DJ, van del Vlag, Hamer KM, Lambrechts C, Masselink H, Gunster MJ, Sewalt RG, van der Driel R, Otte AP (1997b) Interference with the expression of a novel human Polycomb protein, hPC2, results in cellular transformation and apoptosis. Mol Cell Biol 17:6076–6086PubMedGoogle Scholar
  30. Saurin AJ, Shiels C, Williamson J, Otte AP, Freemont PS (1998) The human Polycomb group complex associates with pericentromeric heterochromatin to form a novel nuclear domain. J Cell Biol 142:887–898CrossRefPubMedGoogle Scholar
  31. Schoorlemmer J, Marcosa-Gutierrez C, Were F, Martinez R, Garcia E, Satijn DP, Otte AP, Vidal M (1997) Ring1A is a transcriptional repressor that interacts with the Polycomb-M33 protein and is expressed at rhombomere boundaries in the mouse hindbrain. EMBO J 16:5930–5942CrossRefPubMedGoogle Scholar
  32. Schumacher A, Faust C, Magnuson T (1996) Positional cloning of a global regulator of anterior-posterior patterning in mice. Nature 383:250–253PubMedGoogle Scholar
  33. Segil N, Guermah M, Hoffmann A, Roeder RG, Heintz N (1996) Mitotic regulation of TFIID: inhibition of activator-dependent transcription and changes in subcellular localization. Genes Dev 10:2389–2400PubMedGoogle Scholar
  34. Sewalt RG, van der Vlag J, Gunster MJ, Hamer KM, den Blaauwen JL, Satijn DP, Hendrix T, van Driel R, Otte AP (1998) Characterization of interactions between the mammalian Polycomb-group proteins Enx1/EZH2 and EED suggests the existence of different mammalian Polycomb-group protein complexes. Mol Cell Biol 18:3572–3579PubMedGoogle Scholar
  35. Shao Z, Raible F, Mollaaghababa R, Guyon JR, Wu CT, Bender W, Kingston RE (1999) Stabilization of chromatin structure by PRC1, a Polycomb complex. Cell 98:37–46PubMedGoogle Scholar
  36. Storre J, Elsasser HP, Fuchs M, Ullmann D, Livingston DM, Gaubatz S (2002) Homeotic transformations of the axial skeleton that accompany a targeted deletion of E2f6. EMBO Rep 3:695–700CrossRefPubMedGoogle Scholar
  37. Suzuki M, Mizutani-Koseki Y, Fujimura Y, Miyagishima H, Kaneko T, Takada Y, Akasaka T, Tanzawa H, Takihara Y, Nakano M, Masumoto H, Vidal M, Koseki H (2002) Involvement of the Polycomb-group gene Ring1B in the specification of the anterior-posterior axis in mice. Development 129:4171–4183PubMedGoogle Scholar
  38. Tachibana M, Sugimoto K, Fukushima T, Shinkai Y (2001) Set domain-containing protein, G9a, is a novel lysine-preferring mammalian histone methyltransferase with hyperactivity and specific selectivity to lysines 9 and 27 of histone H3. J Biol Chem 276:25309–25317CrossRefPubMedGoogle Scholar
  39. Tagawa M, Sakamoto T, Shigemoto K, Matsubara H, Tamura Y, Ito T, Nakamura I, Okitsu A, Imai K, Taniguchi M (1990) Expression of novel DNA-binding protein with zinc finger structure in various tumor cells. J Biol Chem 265:20021–20026PubMedGoogle Scholar
  40. Takihara Y, Tomotsune D, Shirai M, Katoh-Fukui Y, Nishii K, Motaleb MA, Nomura M, Tsuchiya R, Fujita Y, Shibata Y, Higashinakagawa T, Shimada T (1997) Targeted disruption of the mouse homlogue of the Drosophila polyhomeotic gene leads to altered anteroposterior patterning and neural crest defects. Development 124:3673–3682PubMedGoogle Scholar
  41. Trimarchi JM, Fairchild B, Wen J, Lees JA (2001) The E2F6 transcription factor is a component of the mammalian Bmi1-containing Polycomb complex. Proc Natl Acad Sci U S A 98:1519–1524CrossRefPubMedGoogle Scholar
  42. van der Lugt NM, Domen J, Linders K, van Roon M, Robanus-Maandag E, te Riele H, van der Valk M, Deschamps J, Sofroniew M, van Lohuizen M, et al (1994) Posterior transformation, neurological abnormalities, and severe hematopoetic defects in mice with a targeted deletion of the bmi-1 proto-oncogene. Genes Dev 8:757–769PubMedGoogle Scholar
  43. van der Vlag J, den Blaauwen JL, Sewalt RG, van Driel R, Otte AP (1999) Transcriptional repression mediated by Polycomb group proteins and other chromatin-associated repressors is selectively blocked by insulators. J Biol Chem 275:697–704Google Scholar
  44. van Lohuizen M, Verbeek S, Scheijen B, Wientjens E, van der Gulden H, Berns A (1991) Identification of cooperating oncogenes in E mu-myc transgenic mice by provirus tagging. Cell 65:737–752PubMedGoogle Scholar
  45. van Lohuizen M, Tijms M, Voncken JW, Schumacher A, Wientjens E (1998) Interaction of mouse Polycomb-group (Pc-G) proteins Enx1 and Enx2 with Eed: indication for separate Pc-G complexes. Mol Cell Biol 18:3572–3579PubMedGoogle Scholar
  46. Voncken JW, Schweizer D, Aagaard L, Sattler L, JantschMF, van Lohuizen M (1999) Chromatin-association of the Polycomb group protein BMI1 is cell cycle-regulated and correlates with its phosphorylation status. J Cell Sci 112:4627–4639PubMedGoogle Scholar
  47. Wang G, Horsley D, Ma A, Otte AP, Hutchings A, Butcher GW, Singh PB (1997) M33, a mammalian homologue of Drosophila Polycomb localizes to euchromatin within interphase nuclei but is enriched within the centromeric heterochromatin of metaphase chromosomes. Cytogenet Cell Genet 78:50–55PubMedGoogle Scholar

Copyright information

© Springer-Verlag 2003

Authors and Affiliations

  • Hiro Miyagishima
    • 1
    • 2
  • Kyoichi Isono
    • 1
  • Yuichi Fujimura
    • 1
  • Masaomi Iyo
    • 2
  • Yoshihiro Takihara
    • 3
  • Hiroshi Masumoto
    • 4
  • Miguel Vidal
    • 5
  • Haruhiko Koseki
    • 1
    • 6
  1. 1.Department of Molecular Embryology, Graduate School of MedicineChiba UniversityChibaJapan
  2. 2.Department of Psychiatry, Graduate School of MedicineChiba UniversityChibaJapan
  3. 3.Department of Developmental Biology and MedicineOsaka Medical Center for Cancer and Cardiovascular DiseasesOsakaJapan
  4. 4.Division of Biological Science, Graduate School of ScienceNagoya UniversityNagoyaJapan
  5. 5.Centro de Investigaciones BiologicasDepartment of Development and Cell BiologyMadridSpain
  6. 6.RIKEN Research Center for Allergy and ImmunologyYokohamaJapan

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