Chromosome Research

, 19:457 | Cite as

Genomic size of CENP-A domain is proportional to total alpha satellite array size at human centromeres and expands in cancer cells

  • Lori L. Sullivan
  • Christopher D. Boivin
  • Brankica Mravinac
  • Ihn Young Song
  • Beth A. Sullivan


Human centromeres contain multi-megabase-sized arrays of alpha satellite DNA, a family of satellite DNA repeats based on a tandemly arranged 171 bp monomer. The centromere-specific histone protein CENP-A is assembled on alpha satellite DNA within the primary constriction, but does not extend along its entire length. CENP-A domains have been estimated to extend over 2,500 kb of alpha satellite DNA. However, these estimates do not take into account inter-individual variation in alpha satellite array sizes on homologous chromosomes and among different chromosomes. We defined the genomic distance of CENP-A chromatin on human chromosomes X and Y from different individuals. CENP-A chromatin occupied different genomic intervals on different chromosomes, but despite inter-chromosomal and inter-individual array size variation, the ratio of CENP-A to total alpha satellite DNA size remained consistent. Changes in the ratio of alpha satellite array size to CENP-A domain size were observed when CENP-A was overexpressed and when primary cells were transformed by disrupting interactions between the tumor suppressor protein Rb and chromatin. Our data support a model for centromeric domain organization in which the genomic limits of CENP-A chromatin varies on different human chromosomes, and imply that alpha satellite array size may be a more prominent predictor of CENP-A incorporation than chromosome size. In addition, our results also suggest that cancer transformation and amounts of centromeric heterochromatin have notable effects on the amount of alpha satellite that is associated with CENP-A chromatin.


Chromatin Chromosome Heterochromatin Histone Kinetochore Retinoblastoma 



Centromere protein A


Charged coupled device


Contour-clamped homogenous electric field


Chromatin immunoprecipitation

CSK buffer

Cytoskeleton buffer


Deoxyribonucleic acid


E7 protein subunit of HPV


Fluorescence in situ hybridization


Fluorescein isothiocyanate


Fetal bovine serum




Human papillomavirus


Higher order repeat


Homo sapiens chromosome X


Homo sapiens chromosome Y




Minimal essential medium




Polymerase chain reaction


Polyvinylidene fluoride


Retinoblastoma protein


Roswell Park Memorial Institute medium


Pulsed field gel electrophoresis


Sodium dodecyl sulfate



We thank Cyrus Vaziri (University of North Carolina, Chapel Hill) for providing cell lines DIP3 and DIP3-E7 and Chris Shaw for technical assistance. This work was supported in part by grants from the American Cancer Society (ACS IRG-72-001-29-IRG), March of Dimes Foundation (6-FY06-377 and 6-FY10-294), and NIH NIGMS (R01 GM069514) to BAS.


  1. Abruzzo MA, Griffin DK, Millie EA, Sheean LA, Hassold TJ (1996) The effect of Y-chromosome alpha-satellite array length on the rate of sex chromosome disomy in human sperm. Hum Genet 97:819–823PubMedCrossRefGoogle Scholar
  2. Alonso A, Mahmood R, Li S, Cheung F, Yoda K, Warburton PE (2003) Genomic microarray analysis reveals distinct locations for the CENP-A binding domains in three human chromosome 13q32 neocentromeres. Hum Mol Genet 12:2711–2721PubMedCrossRefGoogle Scholar
  3. Alonso A, Fritz B, Hasson D, Abrusan G, Cheung F, Yoda K, Radlwimmer B, Ladurner AG, Warburton PE (2007) Co-localization of CENP-C and CENP-H to discontinuous domains of CENP-A chromatin at human neocentromeres. Genome Biol 8:R148PubMedCrossRefGoogle Scholar
  4. Alonso A, Hasson D, Cheung F, Warburton PE (2010) A paucity of heterochromatin at functional human neocentromeres. Epigenetics Chromatin 3:6PubMedCrossRefGoogle Scholar
  5. Amato A, Schillaci T, Lentini L, Di Leonardo A (2009) CENPA overexpression promotes genome instability in pRb-depleted human cells. Mol Cancer 8:119PubMedCrossRefGoogle Scholar
  6. Bashamboo A, Rahman MM, Prasad A, Chandy SP, Ahmad J, Ali S (2005) Fate of SRY, PABY, DYS1, DYZ3 and DYZ1 loci in Indian patients harbouring sex chromosomal anomalies. Mol Hum Reprod 11:117–127PubMedCrossRefGoogle Scholar
  7. Bergmann JH, Rodriguez MG, Martins NM, Kimura H, Kelly DA, Masumoto H, Larionov V, Jansen LE, Earnshaw WC (2011) Epigenetic engineering shows H3K4me2 is required for HJURP targeting and CENP-A assembly on a synthetic human kinetochore. EMBO J 30:328–340PubMedCrossRefGoogle Scholar
  8. Black BE, Jansen LE, Maddox PS, Foltz DR, Desai AB, Shah JV, Cleveland DW (2007) Centromere identity maintained by nucleosomes assembled with histone H3 containing the CENP-A targeting domain. Mol Cell 25:309–322PubMedCrossRefGoogle Scholar
  9. Blower MD, Sullivan BA, Karpen GH (2002) Conserved organization of centromeric chromatin in flies and humans. Dev Cell 3:1–11CrossRefGoogle Scholar
  10. Brinkley BR, Ouspenski I, Zinkowski RP (1992) Structure and molecular organization of the centromere–kinetochore complex. Trends Cell Biol 2:15–21PubMedCrossRefGoogle Scholar
  11. Cherry LM, Faulkner AJ, Grossberg LA, Balczon R (1989) Kinetochore size variation in mammalian chromosomes: an image analysis study with evolutionary implications. J Cell Sci 92(Pt 2):281–289PubMedGoogle Scholar
  12. Chueh AC, Wong LH, Wong N, Choo KH (2005) Variable and hierarchical size distribution of L1-retroelement-enriched CENP-A clusters within a functional human neocentromere. Hum Mol Genet 14:85–93PubMedCrossRefGoogle Scholar
  13. Dyson N, Howley PM, Munger K, Harlow E (1989) The human papilloma virus-16 E7 oncoprotein is able to bind to the retinoblastoma gene product. Science 243:934–937PubMedCrossRefGoogle Scholar
  14. Farr CJ, Stevanovic M, Thomson EJ, Goodfellow PN, Cooke HJ (1992) Telomere-associated chromosome fragmentation: applications in genome manipulation and analysis. Nat Genet 2:275–282PubMedCrossRefGoogle Scholar
  15. Floridia G, Zatterale A, Zuffardi O, Tyler-Smith C (2000) Mapping of a human centromere onto the DNA by topoisomerase II cleavage. EMBO Rep 1:489–493PubMedGoogle Scholar
  16. Gonzalo S, Blasco MA (2005) Role of Rb family in the epigenetic definition of chromatin. Cell Cycle 4:752–755PubMedCrossRefGoogle Scholar
  17. Gonzalo S, Garcia-Cao M, Fraga MF, Schotta G, Peters AH, Cotter SE, Eguia R, Dean DC, Esteller M, Jenuwein T, Blasco MA (2005) Role of the RB1 family in stabilizing histone methylation at constitutive heterochromatin. Nat Cell Biol 7:420–428PubMedCrossRefGoogle Scholar
  18. Grimes BR, Rhoades AA, Willard HF (2002) Alpha-satellite DNA and vector composition influence rates of human artificial chromosome formation. Mol Ther 5:798–805PubMedCrossRefGoogle Scholar
  19. Harrington JJ, Van Bokkelen G, Mays RW, Gustashaw K, Willard HF (1997) Formation of de novo centromeres and construction of first-generation human artificial microchromosomes. Nat Genet 15:345–355PubMedCrossRefGoogle Scholar
  20. Heun P, Erhardt S, Blower MD, Weiss S, Skora AD, Karpen GH (2006) Mislocalization of the Drosophila centromere-specific histone CID promotes formation of functional ectopic kinetochores. Dev Cell 10:303–315PubMedCrossRefGoogle Scholar
  21. Irvine DV, Amor DJ, Perry J, Sirvent N, Pedeutour F, Choo KH, Saffery R (2004) Chromosome size and origin as determinants of the level of CENP-A incorporation into human centromeres. Chromosome Res 12:805–815PubMedCrossRefGoogle Scholar
  22. Joglekar AP, Bouck D, Finley K, Liu X, Wan Y, Berman J, He X, Salmon ED, Bloom KS (2008) Molecular architecture of the kinetochore-microtubule attachment site is conserved between point and regional centromeres. J Cell Biol 181:587–594PubMedCrossRefGoogle Scholar
  23. Lam AL, Boivin CD, Bonney CF, Rudd MK, Sullivan BA (2006) Human centromeric chromatin is a dynamic chromosomal domain that can spread over noncentromeric DNA. Proc Natl Acad Sci USA 103:4186–4191PubMedCrossRefGoogle Scholar
  24. Lo AW, Liao GC, Rocchi M, Choo KH (1999) Extreme reduction of chromosome-specific alpha-satellite array is unusually common in human chromosome 21. Genome Res 9:895–908PubMedCrossRefGoogle Scholar
  25. Lo AWI, Magliano DJ, Sibson MC, Kalitsis P, Craig JM, Choo KHA (2001) A novel chromatin immunoprecipitation and array (CIA) analysis identifies a 460-kb CENP-A-binding neocentromere DNA. Genome Res 11:448–457PubMedCrossRefGoogle Scholar
  26. Maggert KA, Karpen GH (2001) The activation of a neocentromere in Drosophila requires proximity to an endogenous centromere. Genetics 158:1615–1628PubMedGoogle Scholar
  27. Mahtani MM, Willard HF (1990) Pulsed-field gel analysis of alpha-satellite DNA at the human X chromosome centromere: high-frequency polymorphisms and array size estimate. Genomics 7:607–613PubMedCrossRefGoogle Scholar
  28. Mahtani MM, Willard HF (1998) Physical and genetic mapping of the human X chromosome centromere: repression of recombination. Genome Res 8:100–110PubMedGoogle Scholar
  29. Manning AL, Longworth MS, Dyson NJ (2010) Loss of pRB causes centromere dysfunction and chromosomal instability. Genes Dev 24:1364–1376PubMedCrossRefGoogle Scholar
  30. Mravinac B, Sullivan LL, Reeves JW, Yan CM, Kopf KS, Farr CJ, Schueler MG, Sullivan BA (2009) Histone modifications within the human X centromere region. PLoS ONE 4:e6602PubMedCrossRefGoogle Scholar
  31. Munger K, Phelps WC, Bubb V, Howley PM, Schlegel R (1989) The E6 and E7 genes of the human papillomavirus type 16 together are necessary and sufficient for transformation of primary human keratinocytes. J Virol 63:4417–4421PubMedGoogle Scholar
  32. Nakano M, Cardinale S, Noskov VN, Gassmann R, Vagnarelli P, Kandels-Lewis S, Larionov V, Earnshaw WC, Masumoto H (2008) Inactivation of a human kinetochore by specific targeting of chromatin modifiers. Dev Cell 14:507–522PubMedCrossRefGoogle Scholar
  33. Oakey R, Tyler-Smith C (1990) Y chromosome DNA haplotyping suggests that most European and Asian men are descended from one to two males. Genomics 7:325–330PubMedCrossRefGoogle Scholar
  34. Rudd MK, Willard HF (2004) Analysis of the centromeric regions of the human genome assembly. Trends Genet 20:529–533PubMedCrossRefGoogle Scholar
  35. Schueler MG, Higgins AW, Rudd MK, Gustashaw K, Willard HF (2001) Genomic and genetic definition of a functional human centromere. Science 294:109–115PubMedCrossRefGoogle Scholar
  36. Scott KC, Merrett SL, Willard HF (2006) A heterochromatin barrier partitions the fission yeast centromere into discrete chromatin domains. Curr Biol 16:119–129PubMedCrossRefGoogle Scholar
  37. Siddiqui H, Fox SR, Gunawardena RW, Knudsen ES (2007) Loss of RB compromises specific heterochromatin modifications and modulates HP1alpha dynamics. J Cell Physiol 211:131–137PubMedCrossRefGoogle Scholar
  38. Sims JK, Houston SI, Magazinnik T, Rice JC (2006) A trans-tail histone code defined by monomethylated H4 Lys-20 and H3 Lys-9 demarcates distinct regions of silent chromatin. J Biol Chem 281:12760–12766PubMedCrossRefGoogle Scholar
  39. Song IY, Palle K, Gurkar A, Tateishi S, Kupfer GM, Vaziri C (2010) Rad18-mediated translesion synthesis of bulky DNA adducts is coupled to activation of the Fanconi anemia DNA repair pathway. J Biol Chem 285:31525–31536PubMedCrossRefGoogle Scholar
  40. Spence JM, Critcher R, Ebersole TA, Valdivia MM, Earnshaw WC, Fukagawa T, Farr CJ (2002) Co-localization of centromere activity, proteins and topoisomerase II within a subdomain of the major human X alpha-satellite array. EMBO J 21:5269–5280PubMedCrossRefGoogle Scholar
  41. Sullivan B, Warburton P (1999) Studying the progression of vertebrate chromosomes through mitosis by immunofluorescence and FISH. In: Bickmore W (ed) Chromosome structural analysis: a practical approach. IRL Press, Oxford, pp 81–101Google Scholar
  42. Tomkiel J, Cooke CA, Saitoh H, Bernat RL, Earnshaw WC (1994) CENP-C is required for maintaining proper kinetochore size and for a timely transition to anaphase. J Cell Biol 125:531–545PubMedCrossRefGoogle Scholar
  43. Tomonaga T, Matsushita K, Yamaguchi S, Oohashi T, Shimada H, Ochiai T, Yoda K, Nomura F (2003) Overexpression and mistargeting of centromere protein-A in human primary colorectal cancer. Cancer Res 63:3511–3516PubMedGoogle Scholar
  44. Van Hooser AA, Ouspenski II, Gregson HC, Starr DA, Yen TJ, Goldberg ML, Yokomori K, Earnshaw WC, Sullivan KF, Brinkley BR (2001) Specification of kinetochore-forming chromatin by the histone H3 variant CENP-A. J Cell Sci 114:3529–3542PubMedGoogle Scholar
  45. Warburton PE, Greig GM, Haaf T, Willard HF (1991) PCR amplification of chromosome-specific alpha satellite DNA: definition of centromeric STS markers and polymorphic analysis. Genomics 11:324–333PubMedCrossRefGoogle Scholar
  46. Waye JS, Willard HF (1987) Nucleotide sequence heterogeneity of alpha satellite repetitive DNA: a survey of alphoid sequences from different human chromosomes. Nucleic Acids Res 15:7549–7569PubMedCrossRefGoogle Scholar
  47. Wevrick R, Willard HF (1989) Long-range organization of tandem arrays of alpha satellite DNA at the centromeres of human chromosomes: high-frequency array-length polymorphism and meiotic stability. Proc Natl Acad Sci USA 86:9394–9398PubMedCrossRefGoogle Scholar
  48. Willard HF, Waye JS (1987) Hierarchical order in chromosome-specific human alpha satellite DNA. Trends Genet 3:192–198CrossRefGoogle Scholar
  49. Zeng K, de las Heras JI, Ross A, Yang J, Cooke H, Shen MH (2004) Localisation of centromeric proteins to a fraction of mouse minor satellite DNA on a mini-chromosome in human, mouse and chicken cells. Chromosoma 113:84–91PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2011

Authors and Affiliations

  • Lori L. Sullivan
    • 1
  • Christopher D. Boivin
    • 2
  • Brankica Mravinac
    • 1
    • 4
  • Ihn Young Song
    • 2
  • Beth A. Sullivan
    • 1
    • 3
  1. 1.Duke Institute for Genome Sciences & PolicyDuke UniversityDurhamUSA
  2. 2.Department of Genetics and GenomicsBoston University School of MedicineBostonUSA
  3. 3.Department of Molecular Genetics and MicrobiologyDuke University Medical CenterDurhamUSA
  4. 4.Division of Molecular BiologyRuder Boskovic InstituteZagrebCroatia

Personalised recommendations