Skip to main content
Log in

DNA methylation of the X chromosomes of the human female: an in situ semi-quantitative analysis

  • Original Articles
  • Published:
Chromosoma Aims and scope Submit manuscript

Abstract

We present an in situ semi-quantitative analysis of the global DNA methylation of the X chromosomes of the human female using antibodies raised against 5-methylcytosine. The antibodies were revealed by immunofluorescence. Images were recorded by a CCD camera and the difference in intensity of fluorescence between active (early replicating) and inactive (late-replicating) X chromosomes was measured. Global hypomethylation of the late-replicating X chromosomal DNA was observed in three cases of fibroblast primary cultures that were characterized by numerical and structural aberrations of the X chromosomes [46,X,ter rea(X;X), 48,XXXX and 46,X,t(X;15)]. In these cases, the difference between early and late-replicating X chromosomes was significantly greater than the intrametaphasic variations, measured for a pair of autosomes, that result from experimental procedures. In cells with normal karyotypes, the differneces between the two X chromosomes were in the range of experimental variation. These results demonstrated that late replication and facultative heterochromatinization of the inactive X are two processes that are not related to global hypermethylation of the DNA

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  • AdolphS, hameisterH (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: 132–136

    Google Scholar 

  • BarbinA, MontpellierC, Kokalj-VokacN, GibaudA, NiveleauA, MalfoyB, DutrillauxB, BourgeoisCA (1994) New sites of methylcytosine-rich DNA detected on metaphase chromosomes. Hum Genet 94: 684–692

    Google Scholar 

  • BaylinSB, MakosM, WuJ ChiuYen RW, deBustrosA, VertinoP, NelkinBD (1991) Abnormal patterns of DNa methylation in human neoplasia: potential consequences for tumor progression. Cancer Cells 3: 383–390

    Google Scholar 

  • BiemontMC, LaurentC, CouturierJ, DutrillauxB (1978) Chronologie de la réplication des bandes des chromosomes sexuels dans les lymphocytes de sujets normaux et anormaux. Ann Génét 21: 133–141

    Google Scholar 

  • BourgeoisCA, LaquerriereF, HemonD, HubertJ, BouteilleM (1985) New data on the in situ position of the inactive X chromosome in the interphase nucleus of human fibroblasts. Hum Genet 69: 122–129

    Google Scholar 

  • BoydY, FraserNJ (1990) Methylation patterns at the hypervariable X-chromosome locus DXS255 (M27β): correlation with X-inactivation status. Genomics 7: 182–187

    Google Scholar 

  • BrownCJ, LafrenièreRG, PowersVE, SebastioG, BallabioA, PettigrewA, LedbetterDH, LevyE, CraigIW, WillardHF (1991a) Localization of the X inactivation centre on the human X chromosome in Xq13. Nature 349: 82–84

    Google Scholar 

  • BrownCJ, BallabioA, RupertJL LafrenièreRG, GrompeM, TonlorenziR, WillardHF (1991b) A gene from the region of the human X inactivation centre is expressed exclusively from the inactive X chromosome. Nature 349: 38–44

    Google Scholar 

  • BrownCJ, HendrichBD, RupertJL, LafrenièreRG, XingY, LawrenceJ, WillardHF (1992) The human XIST gene: analysis of a 17 kb inactive X-specific RNA that contains conserved repeats and is highly localized within the nucleus. Cell 71: 527–542

    Google Scholar 

  • DriscollDJ, MigeonBR (1990) Sex difference in methylation of single-copy genes in human meiotic germ cells: implications for X chromosome inactivation, parental imprinting, and origin of CpG mutations. Somatic Cell Mol Genet 16: 267–282

    Google Scholar 

  • DutrillauxB, CouturierJ (1981) La pratique de l'analyse chromosomique. Masson, Paris

    Google Scholar 

  • DyerKA, CanfieldTK, GartlerSM (1989) Molecular cytological differentiation of active from inactive X domains in interphase: implications for X chromosome inactivation. Cytogenet Cell Genet 50: 116–120

    Google Scholar 

  • HeardE, AvnerP (1994) Role play in X-inactivation. Hum Mol Genet 3: 1481–1485

    Google Scholar 

  • JeppesenP, TurnerBM (1993) The inactive X chromosome in female mammals is distinguished by a lack of histone H4 acetylation, a cytogenetic marker for gene expression. Cell 74: 281–289

    Google Scholar 

  • KandaN (1973) A new differential technique for staining the heteropycnotic X-chromosome in female mice. Exp Cell Res 80: 463–467

    Google Scholar 

  • KaslowDC, MigeonBR (1987) DNA methylation stabilizes X chromosome inactivation in eutherians but not in marsupials: evidence for multistep maintenance of mammalian X dosage compensation. Proc Natl Acad Sci USA 84: 6210–6214

    Google Scholar 

  • LindsayS, MonkM, HollidayR, HuschtschaL, DaviesKE, RiggsAD, FlavellRA (1985) Differences in methylation on the active and inactive human X chromosomes. Ann Hum Genet 49: 115–127

    Google Scholar 

  • LyonMF (1961) Gene action in the X-chromosome of the mouse (Mus musculus L.). Nature 190: 372–373

    Google Scholar 

  • LyonMF (1993) Epigenetic inheritance in mammals. Trends Genet 9: 123–128

    Google Scholar 

  • MigeonBR (1990) Insights into X chromosome inactivation from studies of species variation, DNA methylation and replication, and vice versa. Genet Res 56: 91–98

    Google Scholar 

  • MigeonBR (1994) X-chromosome inactivation: molecular mechanisms and genetic consequences. Trends Genet 10: 230–235

    Google Scholar 

  • MigeonBR, HollandMM, DriscollDJ, RobinsonJC (1991) Programmed demethylation in CpG islands during human fetal development. Somatic Cell Mol Genet 17: 159–168

    Google Scholar 

  • MillerDA, OkamotoE, ErlangerBF, MillerOJ (1982) Is DNA methylation responsible for mammalian X chromosome inactivation? Cytogenet Cell Genet 33: 345–349

    Google Scholar 

  • MiniouP, JeanpierreM, BlanquetV, SibellaV, BonneauD, HerbelinC, FischerA, NiveleauA, Viegas-PéquignotE (1994) Abnormal methylation pattern in constitutive and facultative (X inactive chromosome) heterochromatin of ICF patients. Hum Mol Genet 3: 2093–2102

    Google Scholar 

  • MondelloC, GoodfellowPJ, GoodfellowPN (1988) Analysis of methylation of a human X located gene which escapes X inactivation. Nucleic Acids Res 16: 6813–6824

    Google Scholar 

  • MontpelierC, BourgeoisCA, Kokalj-VokacN, MulerisM, NiveleauA, ReynaudC, GibaudA, MalfoyB, DutrillauxB (1994) Detection of methylcytosine-rich heterochromatin on banded chromosomes. Application to cells with various status of DNA methylation. Cancer Genet Cytogenet 78: 87–93

    Google Scholar 

  • PranteraG, FerraroM (1990) Analysis of methylation and distribution of CpG sequences on human active X chromosomes by in situ nick translation. Chromosoma 99: 18–23

    Google Scholar 

  • RazinA, CedarH (1991) DNA methylation and gene expression. Microbiol Rev 55: 451–458

    Google Scholar 

  • ReynaudC, BrunoC, BoullangerP, Grange, BarbestiS, NiveleauA (1991) Monitoring of urinary excretion of modified nucleosides in cancer patients using a set of six monoclonal antibodies. Cancer Lett 61: 255–262

    Google Scholar 

  • ShafferCD, WallrathLL, ElginSCR (1993) Regulating genes by packaging domains: bits of heterochomatin in euchromatin? Trends Genet 9: 35–37

    Google Scholar 

  • Singer-SamJ, RiggsAD (1993) X chromosome inactivation and DNA methylation. In: JostJP, SaluzHP (eds) DNA methylation: molecular biology and biological significance. Saluz, Birkhäuser Basel, pp 358–384

    Google Scholar 

  • VeilleuxC, bernardinoJ, GibaudA, NiveleauA, MalfoyB, DutrillauxB, BourgeoisCA (1995) Changes in methylation in tumor cells: a new in situ quantitative approach on interphase nuclei and chromosomes. Bull Cancer 82: 939–945

    Google Scholar 

  • Viegas-PequignotE, DutrillauxB, ThomasG (1988) Inactive X chromosome has the highest concentration of unmethylated Hha I sites. Proc Natl Acad Sci USA 85: 7657–7660

    Google Scholar 

  • WolfSF, JollyDJ, LunnenKD, FriedmannT, MigeonBR (1984) Methylation of the hypoxanthine phosphoribosyltransferase locus on the human X chromosome: implications for X-chromosome inactivation. Proc Natl Acad Sci USA 81: 2806–2810

    Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Rights and permissions

Reprints and permissions

About this article

Cite this article

Bernardino, J., Lamoliatte, E., Lombard, M. et al. DNA methylation of the X chromosomes of the human female: an in situ semi-quantitative analysis. Chromosoma 104, 528–535 (1996). https://doi.org/10.1007/BF00352117

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00352117

Keywords

Navigation