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. 1996 Sep;16(9):5156–5168. doi: 10.1128/mcb.16.9.5156

Surprising deficiency of CENP-B binding sites in African green monkey alpha-satellite DNA: implications for CENP-B function at centromeres.

I G Goldberg 1, H Sawhney 1, A F Pluta 1, P E Warburton 1, W C Earnshaw 1
PMCID: PMC231516  PMID: 8756673

Abstract

Centromeres of mammalian chromosomes are rich in repetitive DNAs that are packaged into specialized nucleoprotein structures called heterochromatin. In humans, the major centromeric repetitive DNA, alpha-satellite DNA, has been extensively sequenced and shown to contain binding sites for CENP-B, an 80-kDa centromeric autoantigen. The present report reveals that African green monkey (AGM) cells, which contain extensive alpha-satellite arrays at centromeres, appear to lack the well-characterized CENP-B binding site (the CENP-B box). We show that AGM cells express a functional CENP-B homolog that binds to the CENP-B box and is recognized by several independent anti-CENP-B antibodies. However, three independent assays fail to reveal CENP-B binding sites in AGM DNA. Methods used include a gel mobility shift competition assay using purified AGM alpha-satellite, a novel kinetic electrophoretic mobility shift assay competition protocol using bulk genomic DNA, and bulk sequencing of 76 AGM alpha-satellite monomers. Immunofluorescence studies reveal the presence of significant levels of CENP-B antigen dispersed diffusely throughout the nuclei of interphase cells. These experiments reveal a paradox. CENP-B is highly conserved among mammals, yet its DNA binding site is conserved in human and mouse genomes but not in the AGM genome. One interpretation of these findings is that the role of CENP-B may be in the maintenance and/or organization of centromeric satellite DNA arrays rather than a more direct involvement in centromere structure.

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Selected References

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  1. Agard D. A., Hiraoka Y., Shaw P., Sedat J. W. Fluorescence microscopy in three dimensions. Methods Cell Biol. 1989;30:353–377. doi: 10.1016/s0091-679x(08)60986-3. [DOI] [PubMed] [Google Scholar]
  2. Alexandrov I. A., Mashkova T. D., Akopian T. A., Medvedev L. I., Kisselev L. L., Mitkevich S. P., Yurov Y. B. Chromosome-specific alpha satellites: two distinct families on human chromosome 18. Genomics. 1991 Sep;11(1):15–23. doi: 10.1016/0888-7543(91)90097-x. [DOI] [PubMed] [Google Scholar]
  3. Alexandrov I. A., Medvedev L. I., Mashkova T. D., Kisselev L. L., Romanova L. Y., Yurov Y. B. Definition of a new alpha satellite suprachromosomal family characterized by monomeric organization. Nucleic Acids Res. 1993 May 11;21(9):2209–2215. doi: 10.1093/nar/21.9.2209. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Blennow E., Telenius H., de Vos D., Larsson C., Henriksson P., Johansson O., Carter N. P., Nordenskjöld M. Tetrasomy 15q: two marker chromosomes with no detectable alpha-satellite DNA. Am J Hum Genet. 1994 May;54(5):877–883. [PMC free article] [PubMed] [Google Scholar]
  5. Broccoli D., Trevor K. T., Miller O. J., Miller D. A. Isolation of a variant family of mouse minor satellite DNA that hybridizes preferentially to chromosome 4. Genomics. 1991 May;10(1):68–74. doi: 10.1016/0888-7543(91)90485-w. [DOI] [PubMed] [Google Scholar]
  6. Cai M., Davis R. W. Yeast centromere binding protein CBF1, of the helix-loop-helix protein family, is required for chromosome stability and methionine prototrophy. Cell. 1990 May 4;61(3):437–446. doi: 10.1016/0092-8674(90)90525-j. [DOI] [PubMed] [Google Scholar]
  7. Choo K. H., Vissel B., Nagy A., Earle E., Kalitsis P. A survey of the genomic distribution of alpha satellite DNA on all the human chromosomes, and derivation of a new consensus sequence. Nucleic Acids Res. 1991 Mar 25;19(6):1179–1182. doi: 10.1093/nar/19.6.1179. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Clarke L., Carbon J. Isolation of a yeast centromere and construction of functional small circular chromosomes. Nature. 1980 Oct 9;287(5782):504–509. doi: 10.1038/287504a0. [DOI] [PubMed] [Google Scholar]
  9. Cooke C. A., Bazett-Jones D. P., Earnshaw W. C., Rattner J. B. Mapping DNA within the mammalian kinetochore. J Cell Biol. 1993 Mar;120(5):1083–1091. doi: 10.1083/jcb.120.5.1083. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Cooke C. A., Bernat R. L., Earnshaw W. C. CENP-B: a major human centromere protein located beneath the kinetochore. J Cell Biol. 1990 May;110(5):1475–1488. doi: 10.1083/jcb.110.5.1475. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Cooke C. A., Heck M. M., Earnshaw W. C. The inner centromere protein (INCENP) antigens: movement from inner centromere to midbody during mitosis. J Cell Biol. 1987 Nov;105(5):2053–2067. doi: 10.1083/jcb.105.5.2053. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Doak T. G., Doerder F. P., Jahn C. L., Herrick G. A proposed superfamily of transposase genes: transposon-like elements in ciliated protozoa and a common "D35E" motif. Proc Natl Acad Sci U S A. 1994 Feb 1;91(3):942–946. doi: 10.1073/pnas.91.3.942. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Doheny K. F., Sorger P. K., Hyman A. A., Tugendreich S., Spencer F., Hieter P. Identification of essential components of the S. cerevisiae kinetochore. Cell. 1993 May 21;73(4):761–774. doi: 10.1016/0092-8674(93)90255-O. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Earnshaw W. C., Bernat R. L., Cooke C. A., Rothfield N. F. Role of the centromere/kinetochore in cell cycle control. Cold Spring Harb Symp Quant Biol. 1991;56:675–685. doi: 10.1101/sqb.1991.056.01.076. [DOI] [PubMed] [Google Scholar]
  15. Earnshaw W. C., Ratrie H., 3rd, Stetten G. Visualization of centromere proteins CENP-B and CENP-C on a stable dicentric chromosome in cytological spreads. Chromosoma. 1989 Jun;98(1):1–12. doi: 10.1007/BF00293329. [DOI] [PubMed] [Google Scholar]
  16. Earnshaw W. C., Rothfield N. Identification of a family of human centromere proteins using autoimmune sera from patients with scleroderma. Chromosoma. 1985;91(3-4):313–321. doi: 10.1007/BF00328227. [DOI] [PubMed] [Google Scholar]
  17. Earnshaw W. C., Sullivan K. F., Machlin P. S., Cooke C. A., Kaiser D. A., Pollard T. D., Rothfield N. F., Cleveland D. W. Molecular cloning of cDNA for CENP-B, the major human centromere autoantigen. J Cell Biol. 1987 Apr;104(4):817–829. doi: 10.1083/jcb.104.4.817. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Ellis L., Clauser E., Morgan D. O., Edery M., Roth R. A., Rutter W. J. Replacement of insulin receptor tyrosine residues 1162 and 1163 compromises insulin-stimulated kinase activity and uptake of 2-deoxyglucose. Cell. 1986 Jun 6;45(5):721–732. doi: 10.1016/0092-8674(86)90786-5. [DOI] [PubMed] [Google Scholar]
  19. Ferns M. J., Hall Z. W. How many agrins does it take to make a synapse? Cell. 1992 Jul 10;70(1):1–3. doi: 10.1016/0092-8674(92)90525-h. [DOI] [PubMed] [Google Scholar]
  20. Fitzgerald D. J., Dryden G. L., Bronson E. C., Williams J. S., Anderson J. N. Conserved patterns of bending in satellite and nucleosome positioning DNA. J Biol Chem. 1994 Aug 19;269(33):21303–21314. [PubMed] [Google Scholar]
  21. Gruss P., Sauer G. Repetitive primate DNA containing the recognition sequences for two restriction endonucleases which generate cohesive ends. FEBS Lett. 1975 Dec 1;60(1):85–88. doi: 10.1016/0014-5793(75)80424-8. [DOI] [PubMed] [Google Scholar]
  22. Guldner H. H., Lakomek H. J., Bautz F. A. Human anti-centromere sera recognise a 19.5 kD non-histone chromosomal protein from HeLa cells. Clin Exp Immunol. 1984 Oct;58(1):13–20. [PMC free article] [PubMed] [Google Scholar]
  23. Haaf T., Mater A. G., Wienberg J., Ward D. C. Presence and abundance of CENP-B box sequences in great ape subsets of primate-specific alpha-satellite DNA. J Mol Evol. 1995 Oct;41(4):487–491. doi: 10.1007/BF00160320. [DOI] [PubMed] [Google Scholar]
  24. Hayashi T., Ohtsuka H., Kuwabara K., Mafune Y., Miyashita N., Moriwaki K., Takahashi Y., Kominami R. A variant family of mouse minor satellite located on the centromeric region of chromosome 2. Genomics. 1993 Aug;17(2):490–492. doi: 10.1006/geno.1993.1352. [DOI] [PubMed] [Google Scholar]
  25. Hyman A. A., Middleton K., Centola M., Mitchison T. J., Carbon J. Microtubule-motor activity of a yeast centromere-binding protein complex. Nature. 1992 Oct 8;359(6395):533–536. doi: 10.1038/359533a0. [DOI] [PubMed] [Google Scholar]
  26. Ikeno M., Masumoto H., Okazaki T. Distribution of CENP-B boxes reflected in CREST centromere antigenic sites on long-range alpha-satellite DNA arrays of human chromosome 21. Hum Mol Genet. 1994 Aug;3(8):1245–1257. doi: 10.1093/hmg/3.8.1245. [DOI] [PubMed] [Google Scholar]
  27. Jiang W., Lechner J., Carbon J. Isolation and characterization of a gene (CBF2) specifying a protein component of the budding yeast kinetochore. J Cell Biol. 1993 May;121(3):513–519. doi: 10.1083/jcb.121.3.513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Jiang W., Middleton K., Yoon H. J., Fouquet C., Carbon J. An essential yeast protein, CBF5p, binds in vitro to centromeres and microtubules. Mol Cell Biol. 1993 Aug;13(8):4884–4893. doi: 10.1128/mcb.13.8.4884. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Kipling D., Mitchell A. R., Masumoto H., Wilson H. E., Nicol L., Cooke H. J. CENP-B binds a novel centromeric sequence in the Asian mouse Mus caroli. Mol Cell Biol. 1995 Aug;15(8):4009–4020. doi: 10.1128/mcb.15.8.4009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Kipling D., Wilson H. E., Mitchell A. R., Taylor B. A., Cooke H. J. Mouse centromere mapping using oligonucleotide probes that detect variants of the minor satellite. Chromosoma. 1994 Mar;103(1):46–55. doi: 10.1007/BF00364725. [DOI] [PubMed] [Google Scholar]
  31. Kurnit D. M., Maio J. J. Subnuclear redistribution of DNA species in confluent and growing mammalian cells. Chromosoma. 1973 May 14;42(1):23–36. doi: 10.1007/BF00326328. [DOI] [PubMed] [Google Scholar]
  32. Lechner J., Carbon J. A 240 kd multisubunit protein complex, CBF3, is a major component of the budding yeast centromere. Cell. 1991 Feb 22;64(4):717–725. doi: 10.1016/0092-8674(91)90501-o. [DOI] [PubMed] [Google Scholar]
  33. Machamer C. E., Rose J. K. A specific transmembrane domain of a coronavirus E1 glycoprotein is required for its retention in the Golgi region. J Cell Biol. 1987 Sep;105(3):1205–1214. doi: 10.1083/jcb.105.3.1205. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Masumoto H., Masukata H., Muro Y., Nozaki N., Okazaki T. A human centromere antigen (CENP-B) interacts with a short specific sequence in alphoid DNA, a human centromeric satellite. J Cell Biol. 1989 Nov;109(5):1963–1973. doi: 10.1083/jcb.109.5.1963. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Matera A. G., Ward D. C. Oligonucleotide probes for the analysis of specific repetitive DNA sequences by fluorescence in situ hybridization. Hum Mol Genet. 1992 Oct;1(7):535–539. doi: 10.1093/hmg/1.7.535. [DOI] [PubMed] [Google Scholar]
  36. Middleton K., Carbon J. KAR3-encoded kinesin is a minus-end-directed motor that functions with centromere binding proteins (CBF3) on an in vitro yeast kinetochore. Proc Natl Acad Sci U S A. 1994 Jul 19;91(15):7212–7216. doi: 10.1073/pnas.91.15.7212. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Muro Y., Masumoto H., Okazaki T., Ohashi M. Purification of a human centromere antigen (CENP-B) and application of DNA immunoprecipitation to quantitative assay for anti-CENP-B antibodies. J Invest Dermatol. 1991 Aug;97(2):378–380. doi: 10.1111/1523-1747.ep12480836. [DOI] [PubMed] [Google Scholar]
  38. Muro Y., Masumoto H., Yoda K., Nozaki N., Ohashi M., Okazaki T. Centromere protein B assembles human centromeric alpha-satellite DNA at the 17-bp sequence, CENP-B box. J Cell Biol. 1992 Feb;116(3):585–596. doi: 10.1083/jcb.116.3.585. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Nicol L., Jeppesen P. Human autoimmune sera recognize a conserved 26 kD protein associated with mammalian heterochromatin that is homologous to heterochromatin protein 1 of Drosophila. Chromosome Res. 1994 May;2(3):245–253. doi: 10.1007/BF01553325. [DOI] [PubMed] [Google Scholar]
  40. Ohashi H., Wakui K., Ogawa K., Okano T., Niikawa N., Fukushima Y. A stable acentric marker chromosome: possible existence of an intercalary ancient centromere at distal 8p. Am J Hum Genet. 1994 Dec;55(6):1202–1208. [PMC free article] [PubMed] [Google Scholar]
  41. Palmer D. K., Margolis R. L. Kinetochore components recognized by human autoantibodies are present on mononucleosomes. Mol Cell Biol. 1985 Jan;5(1):173–186. doi: 10.1128/mcb.5.1.173. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Pankov R., Lemieux M., Hancock R. An antigen located in the kinetochore region in metaphase and on polar microtubule ends in the midbody region in anaphase, characterised using a monoclonal antibody. Chromosoma. 1990 Apr;99(2):95–101. doi: 10.1007/BF01735324. [DOI] [PubMed] [Google Scholar]
  43. Pfarr C. M., Coue M., Grissom P. M., Hays T. S., Porter M. E., McIntosh J. R. Cytoplasmic dynein is localized to kinetochores during mitosis. Nature. 1990 May 17;345(6272):263–265. doi: 10.1038/345263a0. [DOI] [PubMed] [Google Scholar]
  44. Pluta A. F., Saitoh N., Goldberg I., Earnshaw W. C. Identification of a subdomain of CENP-B that is necessary and sufficient for localization to the human centromere. J Cell Biol. 1992 Mar;116(5):1081–1093. doi: 10.1083/jcb.116.5.1081. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Rosenberg H., Singer M., Rosenberg M. Highly reiterated sequences of SIMIANSIMIANSIMIANSIMIANSIMIAN. Science. 1978 Apr 28;200(4340):394–402. doi: 10.1126/science.205944. [DOI] [PubMed] [Google Scholar]
  46. Saitoh H., Tomkiel J., Cooke C. A., Ratrie H., 3rd, Maurer M., Rothfield N. F., Earnshaw W. C. CENP-C, an autoantigen in scleroderma, is a component of the human inner kinetochore plate. Cell. 1992 Jul 10;70(1):115–125. doi: 10.1016/0092-8674(92)90538-n. [DOI] [PubMed] [Google Scholar]
  47. Segal S., Garner M., Singer M. F., Rosenberg M. In situ hybridization of repetitive monkey genome sequences isolated from defective simian virus 40 DNA. Cell. 1976 Oct;9(2):247–257. doi: 10.1016/0092-8674(76)90116-1. [DOI] [PubMed] [Google Scholar]
  48. Singer D. S. Arrangement of a highly repeated DNA sequence in the genome and chromatin of the African green monkey. J Biol Chem. 1979 Jun 25;254(12):5506–5514. [PubMed] [Google Scholar]
  49. Smit A. F., Riggs A. D. Tiggers and DNA transposon fossils in the human genome. Proc Natl Acad Sci U S A. 1996 Feb 20;93(4):1443–1448. doi: 10.1073/pnas.93.4.1443. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Steuer E. R., Wordeman L., Schroer T. A., Sheetz M. P. Localization of cytoplasmic dynein to mitotic spindles and kinetochores. Nature. 1990 May 17;345(6272):266–268. doi: 10.1038/345266a0. [DOI] [PubMed] [Google Scholar]
  51. Sullivan K. F., Glass C. A. CENP-B is a highly conserved mammalian centromere protein with homology to the helix-loop-helix family of proteins. Chromosoma. 1991 Jul;100(6):360–370. doi: 10.1007/BF00337514. [DOI] [PubMed] [Google Scholar]
  52. Tyler-Smith C., Brown W. R. Structure of the major block of alphoid satellite DNA on the human Y chromosome. J Mol Biol. 1987 Jun 5;195(3):457–470. doi: 10.1016/0022-2836(87)90175-6. [DOI] [PubMed] [Google Scholar]
  53. Voullaire L. E., Slater H. R., Petrovic V., Choo K. H. A functional marker centromere with no detectable alpha-satellite, satellite III, or CENP-B protein: activation of a latent centromere? Am J Hum Genet. 1993 Jun;52(6):1153–1163. [PMC free article] [PubMed] [Google Scholar]
  54. Warburton P. E., Waye J. S., Willard H. F. Nonrandom localization of recombination events in human alpha satellite repeat unit variants: implications for higher-order structural characteristics within centromeric heterochromatin. Mol Cell Biol. 1993 Oct;13(10):6520–6529. doi: 10.1128/mcb.13.10.6520. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Waye J. S., Willard H. F. Structure, organization, and sequence of alpha satellite DNA from human chromosome 17: evidence for evolution by unequal crossing-over and an ancestral pentamer repeat shared with the human X chromosome. Mol Cell Biol. 1986 Sep;6(9):3156–3165. doi: 10.1128/mcb.6.9.3156. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Willard H. F. Centromeres of mammalian chromosomes. Trends Genet. 1990 Dec;6(12):410–416. doi: 10.1016/0168-9525(90)90302-m. [DOI] [PubMed] [Google Scholar]
  57. Wong A. K., Rattner J. B. Sequence organization and cytological localization of the minor satellite of mouse. Nucleic Acids Res. 1988 Dec 23;16(24):11645–11661. doi: 10.1093/nar/16.24.11645. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Wordeman L., Mitchison T. J. Identification and partial characterization of mitotic centromere-associated kinesin, a kinesin-related protein that associates with centromeres during mitosis. J Cell Biol. 1995 Jan;128(1-2):95–104. doi: 10.1083/jcb.128.1.95. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Yen T. J., Compton D. A., Wise D., Zinkowski R. P., Brinkley B. R., Earnshaw W. C., Cleveland D. W. CENP-E, a novel human centromere-associated protein required for progression from metaphase to anaphase. EMBO J. 1991 May;10(5):1245–1254. doi: 10.1002/j.1460-2075.1991.tb08066.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Yoda K., Kitagawa K., Masumoto H., Muro Y., Okazaki T. A human centromere protein, CENP-B, has a DNA binding domain containing four potential alpha helices at the NH2 terminus, which is separable from dimerizing activity. J Cell Biol. 1992 Dec;119(6):1413–1427. doi: 10.1083/jcb.119.6.1413. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Yoda K., Nakamura T., Masumoto H., Suzuki N., Kitagawa K., Nakano M., Shinjo A., Okazaki T. Centromere protein B of African green monkey cells: gene structure, cellular expression, and centromeric localization. Mol Cell Biol. 1996 Sep;16(9):5169–5177. doi: 10.1128/mcb.16.9.5169. [DOI] [PMC free article] [PubMed] [Google Scholar]

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