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Partially functional Cenpa–GFP fusion protein causes increased chromosome missegregation and apoptosis during mouse embryogenesis

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Abstract

CENP-A is an essential histone H3-like protein that localizes to the centromeric region of eukaryotic chromosomes. Heterozygous and homozygous Cenpa–GFP fusion-protein mouse mutants, generated through targeted insertion of the green fluorescent protein (GFP) gene into the mouse Cenpa gene locus, show specific localized fluorescence at all the centromeres. Heterozygous mice are healthy and fertile. Cenpa–GFP homozygotes (Cenpa g/g) undergo many cell divisions, giving rise to up to one million cells that show relatively accurate differentiation into distinct mouse embryonic tissues until day 10.5 when significant levels of chromosome missegregation, aneuploidy and apoptosis result in death. Cenpa g/g embryos assemble functional kinetochores that bind toa host of centromere-specific structural and mitotic spindle checkpoint proteins (Cenpc, BubR1, Mad2 and Zw10). Examination of the nucleosomal phasing ofcentromeric minor and pericentromeric major satellite sequences indicates that the formation of Cenpa g/g homotypic nucleosomes is not accompanied by any overt alteration to the overall size of the monomeric nucleosomal structure or the spacing of these structures. This study provides the first example of an essential centromeric protein gene variant in which subtle perturbation at the centromeric nucleosomal/chromatin level manifests in a significantly delayed lethality when compared with Cenpa null mice.

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References

  • Belmont AS (2001) Visualizing chromosome dynamics with GFP. Trends Cell Biol 11: 250-257.

    Google Scholar 

  • Blower MD, Karpen GH (2001) The role of Drosophila CID in kinetochore formation, cell-cycle progression and heterochromatin interactions. Nat Cell Biol 3: 730-739.

    Google Scholar 

  • Cahill DP, Lengauer C, Yu J et al. (1998) Mutations of mitotic checkpoint genes in human cancers. Nature 392: 300-303.

    Google Scholar 

  • Chen Y, Baker RE, Keith KC, Harris K, Stoler S, Fitzgerald-Hayes M (2000) The N terminus of the centromere H3-like protein Cse4p performs an essential function distinct from that of the histone fold domain. Mol Cell Biol 20: 7037-7048.

    Google Scholar 

  • Choo KHA (2001) Domain organization at the centromere and neocentromere. Dev Cell 1: 165-177.

    Google Scholar 

  • Cutts SM, Fowler KJ, Kile BT et al. (1999) Defective chromosome segregation, microtubule bundling and nuclear bridging in inner centromere protein gene (Incenp)-disrupted mice. Hum Mol Genet 8: 1145-1155.

    Google Scholar 

  • Dobles M, Liberal V, Scott ML, Benezra R, Sorger PK (2000) Chromosome missegregation and apoptosis in mice lacking the mitotic checkpoint protein Mad2. Cell 101: 635-645.

    Google Scholar 

  • Figueroa J, Saffrich R, Ansorge W, Valdivia M (1998) Microinjection of antibodies to centromere protein CENP-A arrests cells in interphase but does not prevent mitosis. Chromosoma 107: 397-405.

    Google Scholar 

  • Fowler KJ, Hudson DF, Salamonsen LA et al. (2000) Uterine dysfunction and genetic modifiers in centromere protein B-deficient mice. Genome Res 10: 30-41.

    Google Scholar 

  • Fukagawa T, Brown WRA (1997) Efficient conditional mutation of the vertebrate CENP-C gene. Hum Mol Genet 6: 2301-2308.

    Google Scholar 

  • Gavrieli Y, Sherman Y, Ben-Sasson SA (1992) Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J Cell Biol 119: 493-501.

    Google Scholar 

  • Glowczewski L, Yang P, Kalashnikova T, Santisteban MS, Smith MM (2000) Histone-histone interactions and centromere function. Mol Cell Biol 20: 5700-5711.

    Google Scholar 

  • Henikoff S, Ahmad K, Platero JS, van Steensel B (2000) Heterochromatic deposition of centromeric histone H3-like proteins. Proc Natl Acad Sci USA 97: 716-721.

    Google Scholar 

  • Howman EV, Fowler KJ, Newson AJ et al. (2000) Early disruption of centromeric chromatin organization in centromere protein A (Cenpa) null mice. Proc Natl Acad Sci USA 97: 1148-1153.

    Google Scholar 

  • Hudson DF, Fowler KJ, Earle E et al. (1998) Centromere protein B null mice are mitotically and meiotically normal but have lower body and testis weights. J Cell Biol 141: 309-319.

    Google Scholar 

  • Jablonski SA, Chan GK, Cooke CA, Earnshaw WC, Yen TJ (1998) The hBUB1 and hBUBR1 kinases sequentially assemble onto kinetochores during prophase with hBUBR1 concentrating at the kinetochore plates in mitosis. Chromosoma 107: 386-396.

    Google Scholar 

  • Kalitsis P, Fowler KJ, Earle E, Hill J, Choo KHA (1998a) Targeted disruption of mouse centromere protein C gene leads to mitotic disarray and early embryo death. Proc Natl Acad Sci USA 95: 1136-1141.

    Google Scholar 

  • Kalitsis P, MacDonald AC, Newson AJ, Hudson DF, Choo KHA (1998b) Gene structure and sequence analysis of mouse centromere proteins A and C. Genomics 47: 108-114.

    Google Scholar 

  • Kalitsis P, Earle E, Fowler KJ, Choo KHA (2000) Bub3 gene disruption in mice reveals essential mitotic spindle checkpoint function during early embryogenesis. Genes Dev 14: 2277-2282.

    Google Scholar 

  • Kapoor M, Montes de Oca Luna R, Liu G et al. (1998) The cenpB gene is not essential in mice. Chromosoma 107: 570-576.

    Google Scholar 

  • Lo AWI, Craig JM, Saffery R et al. (2001) A 330 kb CENP-A binding domain and altered replication timing at a human neocentromere. Embo J 20: 2087-2096.

    Google Scholar 

  • Luger K, Mader AW, Richmond RK, Sargent DF, Richmond TJ (1997) Crystal structure of the nucleosome core particle at 2.8 A resolution. Nature 389: 251-260.

    Google Scholar 

  • Michel LS, Liberal V, Chatterjee A et al. (2001) MAD2 haplo-insufficiency causes premature anaphase and chromosome instability in mammalian cells. Nature 409: 355-359.

    Google Scholar 

  • Nishihashi A, Haraguchi T, Hiraoka Y et al. (2002) CENP-I is essential for centromere function in vertebrate cells. Dev Cell 2: 463-476.

    Google Scholar 

  • Oegema K, Desai A, Rybina S, Kirkham M, Hyman AA (2001) Functional analysis of kinetochore assembly in Caenorhabditis elegans. J Cell Biol 153: 1209-1226.

    Google Scholar 

  • Perez-Castro AV, Shamanski FL, Meneses JJ et al. (1998) Centromeric protein B null mice are viable with no apparent abnormalities. Dev Biol 201: 135-143.

    Google Scholar 

  • Putkey F, Cramer T, Morphew M et al. (2002) Unstable kinetochore-microtubule capture and chromosomal instability following deletion of CENP-E. Dev Cell 3: 351.

    Google Scholar 

  • Saffery R, Earle E, Irvine DV, Kalitsis P, Choo KHA (1999) Conservation of centromere protein in vertebrates. Chromosome Res 7: 261-265.

    Google Scholar 

  • Saitoh H, Tomkiel J, Cooke CA et al. (1992) CENP-C, an auto-antigen in scleroderma, is a component of the human inner kinetochore plate. Cell 70: 115-125.

    Google Scholar 

  • Starr DA, Williams BC, Li Z, Etemad-Moghadam B, Dawe RK, Goldberg ML (1997) Conservation of the centromere/ kinetochore protein ZW10. J Cell Biol 138: 1289-1301.

    Google Scholar 

  • Stoler S, Keith KC, Curnick KE, Fitzgerald-Hayes M(1995) A mutation in CSE4, an essential gene encoding a novel chromatin-associated protein in yeast, causes chromosome nondisjunction and cell cycle arrest at mitosis. Genes Dev. 9: 573-586.

    Google Scholar 

  • Sugata N, Li S, Earnshaw WC et al. (2000) Human CENP-H multimers colocalize with CENP-A and CENP-C at active centromere-kinetochore complexes. Hum Mol Genet 9: 2919-2926.

    Google Scholar 

  • Sullivan KF (2001) A solid foundation: functional specialization of centromeric chromatin. Curr Opin Genet Dev 11: 182-188.

    Google Scholar 

  • Sullivan BA, Blower MD, Karpen GH (2001) Determining centromere identity: cyclical stories and forking paths. Nat Rev Genet 2: 584-596.

    Google Scholar 

  • Takahashi K, Chen ES, Yanagida M (2000) Requirement of Mis6 centromere connector for localizing a CENP-A-like protein in fission yeast. Science 288: 2215-2219.

    Google Scholar 

  • Tsien RY (1998) The green fluorescent protein. Annu Rev Biochem 67: 509-544.

    Google Scholar 

  • Uren AG, Wong L, Pakusch M et al. (2000) Survivin and the inner centromere protein INCENP show similar cell-cycle localization and gene knockout phenotype. Curr Biol 10: 1319-1328.

    Google Scholar 

  • Van Hooser AA, Ouspenski II, Gregson HC et al. (2001) Specification of kinetochore-forming chromatin by the histone H3 variant CENP-A. J Cell Sci 114: 3529-3542.

    Google Scholar 

  • Waters JC, Chen RH, Murray AW, Salmon ED (1998) Localization of Mad2 to kinetochores depends on microtubule attachment, not tension. J Cell Biol 141: 1181-1191.

    Google Scholar 

  • Wong AK, Rattner JB (1988) Sequence organization and cytological localization of the minor satellite of mouse. Nucleic Acids Res 16: 11645-11661.

    Google Scholar 

  • Yoda K, Ando S, Morishita S et al. (2000) Human centromere protein A (CENP-A) can replace histone H3 in nucleosome reconstitution in vitro. Proc Natl Acad Sci USA 97: 7266-7271.

    Google Scholar 

  • Zeitlin SG, Barber CM, Allis CD, Sullivan KF (2001) Differential regulation of CENP-A and histone H3 phosphorylation in G2/M. J Cell Sci 114: 653-661.

    Google Scholar 

  • Zhang Y, Reinberg D (2001) Transcription regulation by histone methylation: interplay between different covalent modifications of the core histone tails. Genes Dev 15: 2343-2360.

    Google Scholar 

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Correspondence to K. H. Andy Choo.

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Kalitsis, P., Fowler, K.J., Earle, E. et al. Partially functional Cenpa–GFP fusion protein causes increased chromosome missegregation and apoptosis during mouse embryogenesis. Chromosome Res 11, 345–357 (2003). https://doi.org/10.1023/A:1024044008009

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