All Title Author
Keywords Abstract

PLOS Biology  2007 

Tetrameric Structure of Centromeric Nucleosomes in Interphase Drosophila Cells

DOI: 10.1371/journal.pbio.0050218

Full-Text   Cite this paper   Add to My Lib


Centromeres, the specialized chromatin structures that are responsible for equal segregation of chromosomes at mitosis, are epigenetically maintained by a centromere-specific histone H3 variant (CenH3). However, the mechanistic basis for centromere maintenance is unknown. We investigated biochemical properties of CenH3 nucleosomes from Drosophila melanogaster cells. Cross-linking of CenH3 nucleosomes identifies heterotypic tetramers containing one copy of CenH3, H2A, H2B, and H4 each. Interphase CenH3 particles display a stable association of approximately 120 DNA base pairs. Purified centromeric nucleosomal arrays have typical “beads-on-a-string” appearance by electron microscopy but appear to resist condensation under physiological conditions. Atomic force microscopy reveals that native CenH3-containing nucleosomes are only half as high as canonical octameric nucleosomes are, confirming that the tetrameric structure detected by cross-linking comprises the entire interphase nucleosome particle. This demonstration of stable half-nucleosomes in vivo provides a possible basis for the instability of centromeric nucleosomes that are deposited in euchromatic regions, which might help maintain centromere identity.


[1]  Amor DJ, Kalitsis P, Sumer H, Choo KH (2004) Building the centromere: From foundation proteins to 3D organization. Trends Cell Biol 14: 359–368.
[2]  Malik HS, Henikoff S (2003) Phylogenomics of the nucleosome. Nat Struct Biol 10: 882–891.
[3]  Tomonaga T, Matsushita K, Yamaguchi S, Oohashi T, Shimada H, et al. (2003) Overexpression and mistargeting of centromere protein-A in human primary colorectal cancer. Cancer Res 63: 3511–3516.
[4]  Ahmad K, Henikoff S (2002) Histone H3 variants specify modes of chromatin assembly. Proc Natl Acad Sci U S A 99(Suppl 4): 16477–16484.
[5]  Heun P, Erhardt S, Blower MD, Weiss S, Skora AD, et al. (2006) Mislocalization of the Drosophila centromere-specific histone CID promotes formation of functional ectopic kinetochores. Dev Cell 10: 303–315.
[6]  Moreno-Moreno O, Torras-Llort M, Azorin F (2006) Proteolysis restricts localization of CID, the centromere-specific histone H3 variant of Drosophila, to centromeres. Nucleic Acids Res 34: 6247–6255.
[7]  Collins KA, Furuyama S, Biggins S (2004) Proteolysis contributes to the exclusive centromere localization of the yeast Cse4/CENP-A histone H3 variant. Curr Biol 14: 1968–1972.
[8]  Pidoux AL, Allshire RC (2004) Kinetochore and heterochromatin domains of the fission yeast centromere. Chromosome Res 12: 521–534.
[9]  Jansen LE, Black BE, Foltz DR, Cleveland DW (2007) Propagation of centromeric chromatin requires exit from mitosis. J Cell Biol 176: 795–805.
[10]  Shelby RD, Monier K, Sullivan KF (2000) Chromatin assembly at kinetochores is uncoupled from DNA replication. J Cell Biol 151: 1113–1118.
[11]  Ahmad K, Henikoff S (2001) Centromeres are specialized replication domains in heterochromatin. J Cell Biol 153: 101–110.
[12]  Schuh M, Lehner CF, Heidmann S (2007) Incorporation of Drosophila CID/CENP-A and CENP-C into centromeres during early embryonic anaphase. Curr Biol 17: 237–243.
[13]  Palmer DK, O'Day K, Wener MH, Andrews BS, Margolis RL (1987) A 17-kD centromere protein (CENP-A) copurifies with nucleosome core particles and with histones. J Cell Biol 104: 805–815.
[14]  Palmer DK, O'Day K, Trong HL, Charbonneau H, Margolis RL (1991) Purification of the centromere-specific protein CENP-A and demonstration that it is a distinctive histone. Proc Natl Acad Sci U S A 88: 3734–3738.
[15]  Sullivan KF, Hechenberger M, Masri K (1994) Human CENP-A contains a histone H3 related histone fold that is required for targeting to the centromere. J Cell Biol 127: 581–592.
[16]  Yoda K, Ando S, Morishita S, Houmura K, Hashimoto K, et al. (2000) Human centromere protein A (CENP-A) can replace histone 3 in nucleosome reconstitution in vitro. Proc Natl Acad Sci U S A 97: 7266–7271.
[17]  Furuyama T, Dalal Y, Henikoff S (2006) Chaperone-mediated assembly of centromeric chromatin in vitro. Proc Natl Acad Sci U S A 103: 6172–6177.
[18]  Bloom KS, Amaya E, Carbon J, Clarke L, Hill A, et al. (1984) Chromatin conformation of yeast centromeres. J Cell Biol 99: 1559–1568.
[19]  Polizzi C, Clarke L (1991) The chromatin structure of centromeres from fission yeast: Differentiation of the central core that correlates with function. J Cell Biol 112: 191–201.
[20]  Takahashi K, Murakami S, Chikashige Y, Funabiki H, Niwa O, et al. (1992) A low copy number central sequence with strict symmetry and unusual chromatin structure in fission yeast centromere. Mol Biol Cell 3: 819–835.
[21]  Thomas JO, Kornberg RD (1975) An octamer of histones in chromatin and free in solution. Proc Natl Acad Sci U S A 72: 2626–2630.
[22]  Kornberg RD, Thomas JO (1974) Chromatin structure; oligomers of the histones. Science 184: 865–868.
[23]  Blower MD, Sullivan BA, Karpen GH (2002) Conserved organization of centromeric chromatin in flies and humans. Developmental Cell 2: 319–330.
[24]  Westermann S, Cheeseman IM, Anderson S, Yates JR, Drubin DG, et al. (2003) Architecture of the budding yeast kinetochore reveals a conserved molecular core. J Cell Biol 163: 215–222.
[25]  Henikoff S, Ahmad K, Platero JS, van Steensel B (2000) Heterochromatic deposition of centromeric histone H3-like proteins. Proc Natl Acad Sci U S A 97: 716–721.
[26]  Stein A, Page D (1980) Core histone associations in solutions of high salt. An osmotic pressure study. J Biol Chem 255: 3629–3637.
[27]  Polizzi C, Clarke L (1991) The chromatin structure of centromeres from fission yeast: Differentiation of the central core that correlates with function. J Cell Biol 112: 191–201.
[28]  Sun X, Le HD, Wahlstrom JM, Karpen GH (2003) Sequence analysis of a functional Drosophila centromere. Genome Res 13: 182–194.
[29]  Hewish DR, Burgoyne LA (1973) Chromatin sub-structure. The digestion of chromatin DNA at regularly spaced sites by a nuclear deoxyribonuclease. Biochem Biophys Res Commun 52: 504–510.
[30]  Noll M (1974) Subunit structure of chromatin. Nature 251: 249–251.
[31]  Annunziato AT (2005) Split decision: What happens to nucleosomes during DNA replication? J Biol Chem 280: 12065–12068.
[32]  Olins AL, Senior MB, Olins DE (1976) Ultrastructural features of chromatin nu bodies. J Cell Biol 68: 787–793.
[33]  Woodcock CL, Frado LL, Rattner JB (1984) The higher-order structure of chromatin: Evidence for a helical ribbon arrangement. J Cell Biol 99: 42–52.
[34]  Lohr D, Bash R, Wang H, Yodh J, Lindsay S (2007) Using atomic force microscopy to study chromatin structure and nucleosome remodeling. Methods 41: 333–341.
[35]  Tomschik M, Karymov MA, Zlatanova J, Leuba SH (2001) The archaeal histone-fold protein HMf organizes DNA into bona fide chromatin fibers. Structure 9: 1201–1211.
[36]  Tagami H, Ray-Gallet D, Almouzni G, Nakatani Y (2004) Histone H3.1 and H3.3 complexes mediate nucleosome assembly pathways dependent or independent of DNA synthesis. Cell 116: 51–61.
[37]  Weintraub H, Worcel A, Alberts B (1976) A model for chromatin based upon two symmetrically paired half-nucleosomes. Cell 9: 409–417.
[38]  Henikoff S, Furuyama T, Ahmad A (2004) Histone variants, nucleosome assembly and epigenetic inheritance. Trends Genet 20: 320–326.
[39]  McEwen BF, Hsieh CE, Mattheyses AL, Rieder CL (1998) A new look at kinetochore structure in vertebrate somatic cells using high- pressure freezing and freeze substitution. Chromosoma 107: 366–375.
[40]  Maiato H, Hergert PJ, Moutinho-Pereira S, Dong Y, Vandenbeldt KJ, et al. (2006) The ultrastructure of the kinetochore and kinetochore fiber in Drosophila somatic cells. Chromosoma 115: 469–480.
[41]  Talbert PB, Masuelli R, Tyagi AP, Comai L, Henikoff S (2002) Centromeric localization and adaptive evolution of an Arabidopsis histone H3 variant. Plant Cell 14: 1053–1066.
[42]  Maddox PS, Hyndman F, Monen J, Oegema K, Desai A (2007) Functional genomics identifies a Myb domain-containing protein family required for assembly of CENP-A chromatin. J Cell Biol 176: 757–763.
[43]  Li B, Carey M, Workman JL (2007) The role of chromatin during transcription. Cell 128: 707–719.
[44]  Dion MF, Kaplan T, Kim M, Buratowski S, Friedman N, et al. (2007) Dynamics of replication-independent histone turnover in budding yeast. Science 315: 1405–1408.
[45]  Henikoff S, Ahmad K (2005) Assembly of variant histones into chromatin. Ann Rev Cell Dev Biol 21: 133–153.
[46]  Wieland G, Orthaus S, Ohndorf S, Diekmann S, Hemmerich P (2004) Functional Complementation of Human Centromere Protein A (CENP-A) by Cse4p from . Mol Cell Biol 24: 6620–6630.
[47]  Mellone BG, Allshire RC (2003) Stretching it: Putting the CEN(P-A) in centromere. Curr Opin Genet Dev 13: 191–198.
[48]  Henikoff S, Dalal Y (2005) Centromeric chromatin: What makes it unique? Curr Opin Genet Dev 15: 177–184.
[49]  Yan H, Jin W, Nagaki K, Tian S, Ouyang S, et al. (2005) Transcription and histone modifications in the recombination-free region spanning a rice centromere. Plant Cell 17: 3227–3238.
[50]  Sullivan BA, Karpen GH (2004) Centromeric chromatin exhibits a histone modification pattern that is distinct from both euchromatin and heterochromatin. Nat Struct Mol Biol 11: 1076–1083.
[51]  Nakashima H, Nakano M, Ohnishi R, Hiraoka Y, Kaneda Y, et al. (2005) Assembly of additional heterochromatin distinct from centromere-kinetochore chromatin is required for de novo formation of human artificial chromosome. J Cell Sci 118: 5885–5898.
[52]  Echalier G (1997) Drosophila cells in culture. New York: Academic Press. 702 p.
[53]  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.
[54]  Mito Y, Henikoff J, Henikoff S (2005) Genome-scale profiling of histone H3.3 replacement patterns. Nat Genet 37: 1090–1097.
[55]  Wang H, Bash R, Yodh JG, Hager GL, Lohr D, et al. (2002) Glutaraldehyde modified mica: a new surface for atomic force microscopy of chromatin. Biophys J 83: 3619–3625.


comments powered by Disqus