Cell and Tissue Biology

, Volume 9, Issue 6, pp 493–503 | Cite as

The influence of sample preprocessing on in situ identification of 5-methylcytosine in metaphase chromosomes and interphase nuclei

  • N. A. Grudinina
  • L. K. Sasina
  • E. M. Noniashvili
  • E. G. Neronova
  • L. I. Pavlinova
  • I. A. Suchkova
  • G. A. Sofronov
  • E. L. Patkin
Article

Abstract

Qualitative and quantitative analysis of DNA methylation in situ at the level of cells, chromosomes, and the chromosomal domain is extremely important in diagnosis and treatment of various pathologies, as well in studies of aging and the effects of environmental factors. Yet, the questions remain unresolved of whether the detectable in situ methylation patterns correspond to the actual DNA methylation per se and/or reflect the accessibility of DNA to antibodies, which depends on the structural features of chromatin and chromosome condensation. Thus, this phenomenon can result in an incorrect determination of the real DNA methylation pattern. In order to eliminate this disadvantage to the extent possible, we modified the commonly used methodology of in situ detection methylcytosine by means of monoclonal antibodies. In this study, we show that the efficiency of immunofluorescent labeling for 5-methylcytosin in centromeric heterochromatin, chromosome arms and sister chromatids is significantly affected by the conditions of pretreatment of chromosome preparations. We used undifferentiated murine embryonic F9 cells to show that variations in the conditions of storage of chromosome preparations can lead to a sharp reduction of labeling intensity and even disappearance of the fluorescence signal in centromeric heterochromatin. Using the developed method, we discovered asymmetric methylation of sister chromatids in F9 cells and in human peripheral blood lymphocytes. This phenomena can lead to asymmetric cell division and asymmetric transcriptional status in daughter cells. Thus, the modified methodology for detection of 5-methyl cytosine in situ can provide for a more precise assessment of methylation of chromosomes and chromosomal regions.

Keywords

mouse embryonic carcinoma cell line F9 human peripheral blood lymphocytes DNA methylation heterochromatin metaphase chromosomes epigenetic regulation in situ analysis immunocytochemistry monoclonal antibodies methylcytosine asymmetric methylation of sister chromatids 

Abbreviations

BrdU

5-bromodeoxyuridine

DNMTase

DNA methyltransferase

RT

room temperature

MetCyt

5-methylcytosine

SC

sister chromatid

TelHet

telomeric heterochromatin

CenHet

centromeric heterochromatin

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Alonso, A., Breuer, B., Steuer, B., and Fischer, J., The F9-EC cell line as a model for the analysis of differentiation, Int. J. Dev. Biol., 1991, vol. 35, pp. 389–397.PubMedGoogle Scholar
  2. Baccarelli, A. and Bollati, V., Epigenetics and environmental chemicals, Curr. Opin. Pediatr., 2009, vol. 21, pp. 243–251.PubMedCentralCrossRefPubMedGoogle Scholar
  3. Baranov, V.S. and Kuznetsova, T.V., Tsitogenetika embrional’nogo razvitiya cheloveka (Cytogenetics of Human Embryonal Development), Leningrad: Nauka., 2006.Google Scholar
  4. Barbin, A., Montpellier, C., Kokalj-Vokac, N., Gibaud, A., Niveleau, A., Malfoy, B., Dutrillaux, B., and Bourgeois, C.A., New sites of methylcytosine-rich DNA detected on metaphase chromosomes, Hum. Genet., 1994, vol. 94, pp. 684–692.CrossRefPubMedGoogle Scholar
  5. Bell, C.D., Is mitotic chromatid segregation random?, Histol. Histopathol., 2005, vol. 20, pp. 1313–1320.PubMedGoogle Scholar
  6. Bianchi, N.O., Morgan, W.F., and Cleaver, J.E., Relationship of ultraviolet light-induced DNA-protein cross-linkage to chromatin structure, Exp. Cell Res., 1985, vol. 156, pp. 405–418.CrossRefPubMedGoogle Scholar
  7. Bickmore, WA., Karyotype analysis and chromosome banding, in Encyclopedia of Life Sciences, 2001. Internet references, retrieved from Nature Publishing Group, 1–6. www.els.netGoogle Scholar
  8. Brock, G.J, Charlton, J., and Bird, A., Densely methylated sequences that are preferentially localized at telomereproximal regions of human chromosomes, Gene, 1999, vol. 240, pp. 269–277.CrossRefPubMedGoogle Scholar
  9. Darzynkiewicz, Z., Traganos, F., Sharpless, T., and Melamed, M.R., DNA denaturation in situ. Effect of divalent cations and alcohols, J. Cell Biol., 1976, vol. 68, pp. 1–10.CrossRefPubMedGoogle Scholar
  10. de Capoa, A., Grappelli, C., Febbo, F.R., Spanò, A., Niveleau, A., Cafolla, A., Cordone, I., and Foa, R., Methylation levels of normal and chronic lymphocytic leukemia B lymphocytes: computer-assisted quantitative analysis of anti-5methylcytosine antibody binding to individual nuclei, Cytometry, 1999, vol. 36, pp. 157–159.CrossRefPubMedGoogle Scholar
  11. Fernandez-Peralta, A.M., Navarro, P., Tagarro, I., and Gonzalez-Aguilera, J.J., Digestion of human chromosomes by means of the isoschizomers MspI and HpaII, Genome, 1994, vol. 37, pp. 770–774.CrossRefPubMedGoogle Scholar
  12. Garagna, S., Marziliano, N., Zuccotti, M., Searle, J.B., Capanna, E., and Redi, C.A., Pericentromeric organization at the fusion point of mouse robertsonian translocation chromosomes, Proc. Natl. Acad. Sci. USA, 2001, vol. 98, pp. 171–175.PubMedCentralCrossRefPubMedGoogle Scholar
  13. Gonzalo, S., Jaco, I., Fraga, M.F., Chen, T., Li, E., Esteller, M., and Blasco, M.A., DNA methyltransferases control telomere length and telomere recombination in mammalian cells, Nat. Cell Biol., 2006, vol. 8, pp. 414–426.CrossRefGoogle Scholar
  14. Gupta, R., Nagarajan, A., and Wajapeyee, N., Advances in genome-wide DNA methylation analysis, Biotechniques, 2010, vol. 49, no. 4, pp. iii–xi. doi: 10.2144/000113493PubMedCentralCrossRefPubMedGoogle Scholar
  15. Haaf, T., Methylation dynamics in the early mammalian embryo: implications of genome reprogramming defects for development, Curr. Top Microbiol. Immunol., 2006, vol. 310, pp. 13–22.PubMedGoogle Scholar
  16. Henikoff, S. and Furuyama, T., The unconventional structure of centromeric nucleosomes, Chromosoma, 2012, vol. 121, pp. 341–350.PubMedCentralCrossRefPubMedGoogle Scholar
  17. Jeong, K-S. and Lee, S., Estimating the total mouse DNA methylation according to the B1 repetitive elements, Biochem. Biophys. Res. Commun., 2005, vol. 335, pp. 1211–1216.CrossRefPubMedGoogle Scholar
  18. Jones, P.A. and Baylin, S.B., The fundamental role of epigenetic events in cancer, Nat. Rev. Genet., 2002, vol. 3, pp. 415–428.CrossRefPubMedGoogle Scholar
  19. Joseph, A., Mitchell, A.R., and Miller, O., The organization of the mouse satellite DNA at centromeres, Exp. Cell Res., 1989, vol. 183, pp. 494–500.CrossRefPubMedGoogle Scholar
  20. Kadauke, S. and Blobel, G.A., Mitotic bookmarking by transcription factors, Epigenetics Chromatin, 2013, vol. 6, p. 6.PubMedCentralCrossRefPubMedGoogle Scholar
  21. Kim, J., Kim, J.-Y., and Issa, J.P., Aging and DNA methylation, Curr. Chem. Biol., 2009, vol. 3, pp. 321–329.CrossRefGoogle Scholar
  22. Klenov, M.S. and Gvozdev, V.A., Heterochromatin formation: role of short RNAs and DNA methylation, Biochemistry (Moscow), 2005, vol. 70, no. 11, pp. 1187–1198.CrossRefGoogle Scholar
  23. Komissarov, A.S., Gavrilova, E.V., Demin, S.J., Ishov, A.M., and Podgornaya, O.I., Tandemly repeated DNA families in the mouse genome, BMC Genomics, 2011, vol. 12, pp. 531–552.PubMedCentralCrossRefPubMedGoogle Scholar
  24. Lansdorp, P.M., Falconer, E., Tao, J., Brind’Amour, J., and Naumann, U., Epigenetic differences between sister chromatids?, Ann. N.Y. Acad. Sci., 2012, vol. 1266, pp. 1–6.PubMedCentralCrossRefPubMedGoogle Scholar
  25. Lewis, J.D., Meeham, R.R., Henzel, W.J., Maurer-Fogy, I., Jeppesen, P., Klein, F., and Bird, A., Purification, sequence, and cellular localization of a novel chromosome protein that binds to methylated DNA, Cell, 1992, vol. 69, pp. 905–914.CrossRefPubMedGoogle Scholar
  26. Lister, R., Pelizzola, M., Dowen, R.H., Hawkins, R.D., Hon, G., Tonti-Filippini, J., Nery, J.R., Lee, L., Ye, Z., Ngo, Q.M., Edsall, L., Antosiewicz-Bourget, J., Stewart, R., Ruotti, V., Millar, A.H., Thomson, J.A., Ren, B., and Ecker, J.R., Human DNA methylomes at base resolution show widespread epigenomic differences, Nature, 2009, vol. 462, pp. 315–322.PubMedCentralCrossRefPubMedGoogle Scholar
  27. Mascetti, G., Carrara, S., and Vergani, L., Relationship between chromatin compactness and dye uptake for in situ chromatin stained with DAPI, Cytometry, 2001, vol. 44, pp. 113–119.CrossRefPubMedGoogle Scholar
  28. Metivier, R., Gallais, R., Tiffoche, C., Le Péron, C., Jurkowska, R.Z., Carmouche, R.P., Ibberson, D., Barath, P., Demay, F., Reid, G., Benes, V., Jeltsch, A., Gannon, F., and Salbert, G., Cyclical DNA methylation of a transcriptionally active promoter, Nature, 2008, vol. 452, pp. 45–52.CrossRefPubMedGoogle Scholar
  29. Mezzanotte, R., Vanni, R., Flore, O., Ferrucci, L., and Sumner, AT., Ageing of fixed cytological preparations produces gradation of chromosomal DNA, Cytogenet. Cell Genet., 1988, vol. 48, pp. 60–62.CrossRefPubMedGoogle Scholar
  30. Miniou, P., Jeanpierre, M., Bourchis, D., Coutinho, Barbosa, A.C., Blanquet, V., and Viegas-Péquignot, E., Alphasatellite DNA methylation in normal individuals and in ICF patients: heterogeneous methylation of constitutive heterochromatin in adult and fetal samples, Hum. Genet., 1997, vol. 99, pp. 738–745.CrossRefPubMedGoogle Scholar
  31. Patkin, E.L., Asymmetry of sister chromatids methylation of preimplantation mouse embryo chromosomes as revealed by nick translation in situ, Cytogenet. Cell Genet., 1997, vol. 77, pp. 82.Google Scholar
  32. Patkin, E.L., Epigenetic mechanisms for primary differentiation in mammalian embryos, Int. Rev. Cytol., 2002, vol. 216, pp. 81–129.CrossRefPubMedGoogle Scholar
  33. Patkin, E.L., Epigeneticheskie mekhanizmy rasprostranennykh zabolevanii cheloveka Epigenetic Mechanisms of the Common Human Diseases), St. Petersburg: NestorIstoriya, 2008.Google Scholar
  34. Patkin, E.L. and Sorokin, A.V., The level of chromosomal DNA methylation in mice in the early stages of embryogenesis studied by the action of restriction endonucleases on the chromosomes, Tsitologiia, 1992, vol. 34, no. 1, pp. 65–69.PubMedGoogle Scholar
  35. Patkin, E.L.and Quinn, J., Epigenetical mechanisms of susceptibility to complex human diseases, Russ. J. Genet.: Appl. Res., 2011, vol. 1, pp. 436–447.CrossRefGoogle Scholar
  36. Patkin, E.L., Kustova, M.E., and Dyban, A.P., Spontaneous sister chromatid differentiation (SCD) and sister chromatid exchange (SCE) in mouse blastocyst chromosomes, Cytogenet. Cell Genet., 1994, vol. 66, pp. 31–32.CrossRefPubMedGoogle Scholar
  37. Pendina, A.A., Efimova, O.A., Fedorova, I.D., Leont’eva, O.A., Shilnikova, E.M., Lezhnina, J.G., Kuznetzova, T.V., and Baranov, V.S., DNA methylation patterns of metaphase chromosomes in human preimplantation embryos, Cytogenet. Genome Res., 2011, vol. 132, pp. 1–7.CrossRefPubMedGoogle Scholar
  38. Plohl, M., Luchetti, A., Meštrovi, N., and Mantovani, B., Satellite DNAs between selfishness and functionality: structure, genomics and evolution of tandem repeats in centromeric (hetero) chromatin, Gene, 2008, vol. 409, pp. 72–82.CrossRefPubMedGoogle Scholar
  39. Pogribny, I., Raiche, J., Slovack, M., and Kovalchuk, O., Dose-dependence, sexand tissue-specificity, and persistence of radiation-induced genomic DNA methylation changes, Biochem. Biophys. Res. Commun., 2004, vol. 320, pp. 1253–1261.CrossRefPubMedGoogle Scholar
  40. Rougier, N., Bourchis, D., Gomes, D.M., Niveleau, A., Plachot, M., Pàldi, A., and Viegas-Péquignot, E., Chromosome methylation patterns during mammalian preimplantation development, Genes Devel., 1998, vol. 12, pp. 2108–2113.PubMedCentralCrossRefPubMedGoogle Scholar
  41. Salozhin, S.V., Prokhorchuk, E.B., and Georgiev, G.P., Methylation of DNA—one of the major epigenetic markers, Biochemistry (Moscow), 2005, vol. 70, no. 5, pp. 525–532.CrossRefGoogle Scholar
  42. Santos, F. and Dean, W., Using immunofluorescence to observe methylation changes in mammalian preimplantation embryos, Methods Mol. Biol., 2006, vol. 325, pp. 129–137.PubMedGoogle Scholar
  43. Sasai, N., and Defossez, P.A., Many paths to one goal? The proteins that recognize methylated DNA in eukaryotes, Int. J. Dev. Biol., 2009, vol. 53, pp. 323–334.CrossRefPubMedGoogle Scholar
  44. Sasina, L.K., Fedorova, E.M., Grudinina, N.A., Belotserkovskaya, E.V., Solovyov, K.V., Suchkova, I.O., and Patkin, E.L., Modulation of reporter EGFP gene expression by a disease-associated human intra-intronic minisatellite upon transient and stable transfection, Int. J. Biol. Engin., 2013, vol. 3, pp. 1–10.Google Scholar
  45. Schneider, L., and d’Adda di Fagagna, F., Neural stem cells exposed to BrdU lose their global DNA methylation and undergo astrocytic differentiation, Nucl. Acids Res., 2012, vol. 40, pp. 5332–5342.PubMedCentralCrossRefPubMedGoogle Scholar
  46. Suchkova, I.O., Baranova, T.V., Kustova, M.E., Kisljakova, T.V., Vassiliev, V.B., Slominskaja, N.A., Alenina, N.V., and Patkin, E.L., Bovine satellite DNA induces heterochromatinization of host chromosomal DNA in cells of transsatellite mouse embryonal carcinoma, Tsitologiia, 2004, vol. 46, pp. 53–61.PubMedGoogle Scholar
  47. Tajbakhsh, S., Rocheteau, P., and Le Roux, I., Asymmetric cell divisions and asymmetric cell fates, Annu. Rev. Cell Dev. Biol., 2009, vol. 25, pp. 671–699.CrossRefPubMedGoogle Scholar
  48. Teubner, B. and Schulz, W.A., Exemption of satellite DNA from demethylation in immortalized differentiated derivatives of F9 mouse embryonal carcinoma cells, Exp. Cell Res., 1994, vol. 210, pp. 192–200.CrossRefPubMedGoogle Scholar
  49. Tran, V., Feng, L., and Chen, X., Asymmetric distribution of histones during Drosophila male germline stem cell asymmetric divisions, Chromosome Res., 2013, vol. 21, pp. 255–269.PubMedCentralCrossRefPubMedGoogle Scholar
  50. Vanyushin, B.F., DNA methylation and epigenetics, Russ. J. Genet., 2006, vol. 42, no. 9, pp. 1186–1199.CrossRefGoogle Scholar
  51. Xu, N., Azziz, R., and Goodarzi, M.O., Epigenetics in polycystic ovary syndrome: a pilot study of global DNA methylation, Fert. Steril., 2010, vol. 94, pp. 781–783.CrossRefGoogle Scholar
  52. Yates, P.A., Robert, W. Burman, R.W., Mummaneni, P. Krussel, S., and Turker, M.S., Turker tandem B1 elements located in a mouse methylation center provide a target for de novo DNA methylation, J. Biol. Chem., 1999, vol. 274, pp. 36357–36361.CrossRefPubMedGoogle Scholar
  53. Zaitseva, I., Zaitsev, S., Alenina, N., Bader, M., and Krivokharchenko, A., Dynamics of DNA-demethylation in early mouse and rat embryos developed in vivo and in vitro, Mol. Reprod. Dev., 2007, vol. 74, pp. 1255–1261.CrossRefPubMedGoogle Scholar

Copyright information

© Pleiades Publishing, Ltd. 2015

Authors and Affiliations

  • N. A. Grudinina
    • 1
  • L. K. Sasina
    • 1
  • E. M. Noniashvili
    • 1
  • E. G. Neronova
    • 3
  • L. I. Pavlinova
    • 1
    • 2
  • I. A. Suchkova
    • 1
  • G. A. Sofronov
    • 1
  • E. L. Patkin
    • 1
  1. 1.Institute of Experimental Medicine of the Russian Academy of SciencesSt. PetersburgRussia
  2. 2.Pavlov Institute of PhysiologyRussian Academy of SciencesSt. PetersburgRussia
  3. 3.All-Russia Center of Emergency and Radiation MedicineMinistry of Emergency SituationsNikiforov, St. PetersburgRussia

Personalised recommendations