Human Genetics

, Volume 94, Issue 6, pp 684–692 | Cite as

New sites of methylcytosine-rich DNA detected on metaphase chromosomes

  • Agnès Barbin
  • Claire Montpellier
  • Nadja Kokalj-Vokac
  • Anne Gibaud
  • Alain Niveleau
  • Bernard Malfoy
  • Bernard Dutrillaux
  • Claire A. Bourgeois
Original Investigation

Abstract

In situ immunofluorescence detection of antibodies against 5-methylcytosine on metaphase chromosomes prepared by a new procedure allows the display of new 5-methylcytosine-rich sites as compared to previously published methods. In short-term culture lymphocytes, the immunofluorescent signals give a recurrent pattern in which four types of binding sites can be distinguished. Type I sites are the secondary constrictions and a few juxtacentromeric regions, type II sites correspond to T-bands. Both types I and II sites emit a strong fluorescence. Type III sites form an R-band pattern and emit a weaker fluorescence. Type IV sites are the short arms of acrocentrics, they emit strong but polymorphic signals. The results obtained from control experiments suggest that the pattern observed is rather the expression of an uneven distribution of 5-methylcytosine-rich sites than a consequence of the various treatments used. In a lymphoblastoid cell line known to have a reduced 5-methylcytosine content, it was possible to demonstrate a heterogeneous hypomethylation among chromosome structures, principally involving type I sites. The method opens the possibility of studying in situ on chromosomes, regional variations of methylation in pathological conditions.

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Adolph S, Hameister H (1990) In situ nick translation of human metaphase chromosomes with the restriction enzymes MspI and HpaII reveals an R-band pattern. Cytogenet Cell Genet 54: 132–136Google Scholar
  2. Almeida A, Kokalj-Vokac N, Lefrançois D, Viegas-Péquignot E, Jeanpierre M, Dutrillaux B, Malfoy B (1993) Hypomethylation of classical satellite DNA and chromosome instability in lymphoblastoid cell lines. Hum Genet 91: 538–546Google Scholar
  3. Baylin SB, Makos M, Wu J, Chiu Yen RW, de Bustros A, Vertino P, Nelkin BD (1991) Abnormal patterns of DNA methylation in human neoplasia: potential consequence for tumor progression. Cancer Cell 3: 383–390Google Scholar
  4. Bianchi NO, Vidal-Rioja L, Cleaver JE (1986) Direct visualization of the sites of DNA methylation in human, and mosquito chromosomes. Chromosoma 94: 362–366Google Scholar
  5. Bird A (1992) The essentials of DNA methylation. Cell 70: 5–8Google Scholar
  6. Bloom SE, Goodpasture C (1976) An improved technique for silver staining of nucleolar organizer regions in human chromosomes. Hum Genet 34: 199–206PubMedGoogle Scholar
  7. Bobrow M, Madan K (1973) A comparison of chimpanzee and human chromosomes using the Giemsa-11 and other chromosome banding techniques. Cytogenet Cell Genet 12: 107–116Google Scholar
  8. Bobrow M, Madan K, Pearson PL (1972) Staining of some specific regions of human chromosomes, particularly the secondary constriction of no. 9. Nature 238: 122–124Google Scholar
  9. Caspersson T, Zech L, Johansson C, Modeste J (1970) Identification of human chromosomes by DNA-binding fluorescent agents. Chromosoma 30: 215–227Google Scholar
  10. Catania J, Fairweather DS (1991) DNA methylation and cellular ageing. Mutat Res 256: 283–293Google Scholar
  11. Dante R, Percy ME, Baldini A, Markovic VD, Miller DA, Rocchi M, Niveleau A, Miller OJ (1992) Methylation of the 5′ flanking sequences of the ribosomal DNA in human cell lines and in a human-hamster hybrid cell line. J Cell Biochem 50: 357–362Google Scholar
  12. Dutrillaux B (1973) Nouveau système de marquage chromosomique: les bandes T. Chromosoma 41: 395–402Google Scholar
  13. Dutrillaux B, Couturier J (1981) La pratique de l'analyse chromosomique. Masson, ParisGoogle Scholar
  14. Evans HJ, Buckland RA, Pardue ML (1974) Location of the genes coding for 18S and 28S ribosomal RNA in the human genome. Chromosoma 45: 405–426Google Scholar
  15. Feinberg AP (1993) Genomic imprinting and gene activation in cancer. Nature Genet 4: 110–113Google Scholar
  16. Ferrucci L, Mezzanotte R, Vanni R, Stuppia R, Guanciali-Franchi P, Calabrese G, Palka G, Bianchi U, Summer AT (1988) Effect of HpaII and MspI restriction endonucleases on chronic myelogenous leukemia chromosomes. Cancer Genet Cytogenet 34: 251–256Google Scholar
  17. Foss HM, Roberts CJ, Claeys KM, Selker EU (1993) Abnormal chromosome behavior in Neurospora mutants defective in DNA methylation. Science 262: 1737–1741Google Scholar
  18. Henderson AS, Warburton D, Atwood KC (1972) Location of ribosomal DNA in the human chromosome complement. Proc Natl Acad Sci USA 69: 3215–3219Google Scholar
  19. Holmquist GP (1992) Chromosome bands, their chromatin flavors, and their functional features. Am J Hum Genet 51: 17–37Google Scholar
  20. Jackson MS, Mole SE, Ponder BAJ (1992) Characterisation of a boundary between satellite III and alphoid sequences on human chromosome 10. Nucleic Acids Res 20: 4781–4787Google Scholar
  21. Jeanpierre M, Turleau C, Aurias A, Prieur M, Ledeist F, Fischer A, Viegas-Péquignot E (1993) An embryonic-like methylation pattern of classical satellite DNA is observed in ICF syndrome. Hum Mol Genet 2: 731–735PubMedGoogle Scholar
  22. Jones PA, Buckley JD (1990) The role of DNA methylation in cancer. Adv Cancer Res 54: 1–23Google Scholar
  23. Jones KW, Corneo G (1971) Location of satellite and homogeneous DNA sequences on human chromosomes. Nature New Biol 233: 268–271Google Scholar
  24. Kokalj-Vokac N, Almeida A, Viegas-Péquignot E, Jeanpierre M, Malfoy B, Dutrillaux B (1993) Specific induction of uncoiling and recombination by azacytidine in classical satellite-containing constitutive heterochromatin. Cytogenet Cell Genet 63: 11–15PubMedGoogle Scholar
  25. Lange T de, Shiue L, Myers RM, Cox DR, Naylor SL, Killery AM, Varmus HE (1990) Structure and variability of human chromosome ends. Mol Cell Biol 10: 518–527Google Scholar
  26. Lefrançois D, Kokalj N, Viegas-Péquignot E, Montagnier L, Dutrillaux B (1991) High recurrence of rearrangements involving chromosome 14 in ataxia telangiectasia lymphoblastoid cell line and in its mutagen-treated derivatives. Hum Genet 86: 475–480Google Scholar
  27. Lemieux N, Dutrillaux B, Viegas-Péquignot E (1992) A simple method for simultaneous R- or G-banding and fluorescence in situ hybridization of small single-copy genes. Cytogenet Cell Genet 59: 311–312Google Scholar
  28. Miller OJ, Schnedl W, Allen J, Erlanger BF (1974) 5-Methylcytosine localised in mammalian constitutive heterochromatin. Nature 251: 636–637Google Scholar
  29. Montpellier C, Bourgeois CA, Kokalj-Vokac N, Muleris M, Niveleau A, Reynaud C, Gibaud A, Malfoy B, Dutrillaux B Detection of methylcytosine rich heterochromatin on banded chromosomes. Application to cells with various status of DNA methylation. Cancer Genet Cytogenet (in press)Google Scholar
  30. Okamoto E, Miller DA, Erlanger BF, Miller OJ (1981) Polymorphism of 5-methylcytosine-rich DNA in human acrocentric chromosomes. Hum Genet 58: 255–259Google Scholar
  31. Prantera G, Ferraro M (1990) Analysis of methylation and distribution of CpG sequences on human active and inactive X chromosomes by in situ nick translation. Chromosoma 99: 18–23Google Scholar
  32. Razin A, Cedar H (1991) DNA methylation and gene expression. Microbiol Rev 55: 451–458Google Scholar
  33. Razin A, Cedar H (1993) DNA methylation and embryogenesis. In: Jost JB, Saluz HP (eds) DNA methylation: molecular biology and biological significance. Birkhäuser Verlag, Basel, pp 343–357Google Scholar
  34. Reynaud C, Bruno C, Boullanger P, Grange J, Barbesti S, Niveleau A (1991) Monitoring of urinary excretion of modified nucleosides in cancer patients using a set of six monoclonal anti-bodies. Cancer Lett 61: 255–262Google Scholar
  35. Saccone S, De Sario A, Delia Valle G, Bernardi G (1992) The highest gene concentrations in the human genome are in telomeric bands of metaphase chromosomes. Proc Natl Acad Sci USA 89: 4913–4917Google Scholar
  36. de Sario A, Aïssani B, Bernardi G (1991) Compositional properties of telomeric regions from human chromosomes. FEBS Lett 295: 22–26Google Scholar
  37. Sasaki H, Allen ND, Surani MA (1993) DNA methylation and genomic imprinting in mammals. In: Jost JP, Saluz HP (eds) DNA methylation: molecular biology and biological significance. Birkhäuser Verlag, Basel, pp 469–486Google Scholar
  38. Schnedl W, Dev VG, Tantravahi R, Miller DA, Erlanger BF, Miller OJ (1975) 5-Methylcytosine in heterochromatic regions of chromosomes: chimpanzee and gorilla compared to the human. Chromosoma 52: 59–66Google Scholar
  39. Schnedl W, Erlanger BF, Miller OJ (1976) 5-Methylcytosine in heterochromatic regions of chromosomes in bovidae. Hum Genet 31: 21–26Google Scholar
  40. Schutte B, Reynders MMJ, Bosman FT, Blijham GH (1987) Effect of tissue fixation on anti-bromodeoxyuridine immunohistochemistry. J Histochem Cytochem 35: 1343–1345Google Scholar
  41. Schwarzacher-Robinson T, Cram LS, Meyne J, Moyzis RK (1988) Characterization of human heterochromatin by in situ hybridization with satellite DNA clones. Cytogenet Cell Genet 47: 192–196Google Scholar
  42. Schweizer D, Ambros P, Andrle M (1978) Modification of DAPI banding on human chromosomes by prestaining with a DNA-binding oligopeptide antibiotic, distamycin A. Exp Cell Res 111: 327–332Google Scholar
  43. Sentís C, Ludeña P, Fernández-Piqueras J (1993) Non-uniform distribution of methylatable CCGG sequences on human chromosomes as shown by in situ methylation. Chromosoma 102: 267–271Google Scholar
  44. Spruck CH III, Rideout WM III, Jones PA (1993) DNA methylation and cancer. In: Jost JP, Saluz HP (eds) DNA methylation: molecular biology and biological significance. Birkhäuser Verlag, Basel, pp 487–509Google Scholar
  45. Summer AT, de la Torre J, Stuppia L (1993) The distribution of genes on chromosomes: a cytological approach. J Mol Evol 37: 117–122Google Scholar
  46. Tantravahi U, Guntaka RV, Erlanger BF, Miller OJ (1981) Amplified ribosomal RNA genes in a rat hepatoma cell line are enriched in 5-methylcytosine. Proc Natl Acad Sci USA 78: 489–493Google Scholar
  47. de la Torre J, Sumner AT, Gosalvez J, Stuppia L (1992) The distribution of genes on human chromosomes as studied by in situ nick translation. Genome 35: 890–894Google Scholar
  48. Trowell HE, Nagy A, Vissel B, Andy Choo KH (1993) Longrange analyses of the centromeric regions of human chromosomes 13, 14 and 21: identification of a narrow domain containing two key centromeric DNA elements. Hum Mol Genet 2: 1639–1649Google Scholar
  49. Vertino PM, Spillare EA, Harris CC, Baylin SB (1993) Altered chromosomal methylation patterns accompany oncogene-induced transformation of human bronchial epithelial cells. Cancer Res 53: 1684–1689Google Scholar
  50. Viegas-Péquignot E, Dutrillaux B, Thomas G (1988) Inactive X chromosome has the highest concentration of unmethylated HhaI sites. Proc Natl Acad Sci USA 85: 7657–7660Google Scholar
  51. Vogt P (1990) Potential genetic functions of tandem repeated DNA sequences blocks in the human genome are based on a highly conserved “chromatin folding code”. Hum Genet 84: 301–336Google Scholar
  52. Yeivin A, Razin A (1993) Gene methylation patterns and expression. In: Jost JP, Saluz HP (eds) DNA methylation: molecular biology and biological significance. Birkhäuser Verlag, Basel, pp523–568Google Scholar

Copyright information

© Springer-Verlag 1994

Authors and Affiliations

  • Agnès Barbin
    • 1
  • Claire Montpellier
    • 2
  • Nadja Kokalj-Vokac
    • 3
  • Anne Gibaud
    • 2
  • Alain Niveleau
    • 4
  • Bernard Malfoy
    • 2
  • Bernard Dutrillaux
    • 2
  • Claire A. Bourgeois
    • 2
  1. 1.CNRS URA 147, Institut Gustave RoussyVillejuif CedexFrance
  2. 2.CNRS URA 620, Institut CurieParis CedexFrance
  3. 3.Cytogenetic LaboratoryMariborSlovenie
  4. 4.CNRS URA 1459. Institut PasteurLyon CedexFrance

Personalised recommendations