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. 1982 Jul;79(13):3949–3953. doi: 10.1073/pnas.79.13.3949

Variability of DNA methylation patterns during serial passage of human diploid fibroblasts

Robert J Shmookler Reis 1,2, Samuel Goldstein 1,2
PMCID: PMC346553  PMID: 6287455

Abstract

We have examined DNA methylation in diploid human fibroblasts, early and late in their replicative life-span. The extent of methylation of -C-C-G-G- was measured by comparison of fragment sizes after digestion with methylation-specific restriction enzyme Hpa II or Msp I, or both. Methylation of -C-C-G-G- sites in total DNA, occurring predominantly at internal (3′) cytosines, increased from 59% to 64% of sites in one cell strain at late passage, remained constant in another, and decreased in four other strains (54% to 48%, 58.5% to 48%, 55% to 51.5%, and 52% to 44.5%). Base composition analysis confirmed a substantial loss of total DNA 5-methylcytosine (mC) in one strain. Seven clonal isolates, examined at middle to late passage, ranged from 33% to 51% methylation of 3′ cytosines in -C-C-G-G- sites. Three discrete classes of highly repetitive DNA were found which contained Msp I sites at intervals of 45, 110, and 175 base pairs. These repeat families consistently had 70-80% of sites methylated at 3′ cytosines, in all clones and in all strains examined both at early and at late passage. Thus, altered methylation of repetitive sequences is unlikely to account for the variable -C-C-G-G- methylation observed in total DNA. When DNA from one fibroblast strain and from eight pure clones isolated from that parental culture was digested with Msp I or Hpa II followed by EcoRI and probed for γ-globin gene sequences, considerable interclonal and intraclonal heterogeneity was observed for methylation at four -C-C-G-G- sites in the γ-globin coding region of DNA. Therefore, the pattern of methylation in endogenous gene regions appears to undergo random drift during replication of diploid fibroblasts.

Keywords: 5-methylcytosine, repetitive (satellite) DNA, γ-globin genes, clonal heterogeneity, cellular aging

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Goldstein S., Moerman E. J., Soeldner J. S., Gleason R. E., Barnett D. M. Diabetes mellitus and genetic prediabetes. Decreased replicative capacity of cultured skin fibroblasts. J Clin Invest. 1979 Mar;63(3):358–370. doi: 10.1172/JCI109311. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Groudine M., Eisenman R., Weintraub H. Chromatin structure of endogenous retroviral genes and activation by an inhibitor of DNA methylation. Nature. 1981 Jul 23;292(5821):311–317. doi: 10.1038/292311a0. [DOI] [PubMed] [Google Scholar]
  3. Harbers K., Harbers B., Spencer J. H. Nucleotide clusters in deoxyribonucleic acids. XII. The distribution of 5-methylcytosine in pyrimidine oligonucleotides of mouse L-cell satellite DNA and main band DNA. Biochem Biophys Res Commun. 1975 Sep 16;66(2):738–746. doi: 10.1016/0006-291x(75)90572-0. [DOI] [PubMed] [Google Scholar]
  4. Harley C. B., Goldstein S. Cultured human fibroblasts: distribution of cell generations and a critical limit. J Cell Physiol. 1978 Dec;97(3 Pt 2 Suppl 1):509–516. doi: 10.1002/jcp.1040970326. [DOI] [PubMed] [Google Scholar]
  5. Jeffreys A. J. DNA sequence variants in the G gamma-, A gamma-, delta- and beta-globin genes of man. Cell. 1979 Sep;18(1):1–10. doi: 10.1016/0092-8674(79)90348-9. [DOI] [PubMed] [Google Scholar]
  6. Little P. F., Flavell R. A., Kooter J. M., Annison G., Williamson R. Structure of the human fetal globin gene locus. Nature. 1979 Mar 15;278(5701):227–231. doi: 10.1038/278227a0. [DOI] [PubMed] [Google Scholar]
  7. Mandel J. L., Chambon P. DNA methylation: organ specific variations in the methylation pattern within and around ovalbumin and other chicken genes. Nucleic Acids Res. 1979 Dec 20;7(8):2081–2103. doi: 10.1093/nar/7.8.2081. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Maniatis T., Jeffrey A., Kleid D. G. Nucleotide sequence of the rightward operator of phage lambda. Proc Natl Acad Sci U S A. 1975 Mar;72(3):1184–1188. doi: 10.1073/pnas.72.3.1184. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Mann M. B., Smith H. O. Specificity of Hpa II and Hae III DNA methylases. Nucleic Acids Res. 1977 Dec;4(12):4211–4221. doi: 10.1093/nar/4.12.4211. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. McGhee J. D., Ginder G. D. Specific DNA methylation sites in the vicinity of the chicken beta-globin genes. Nature. 1979 Aug 2;280(5721):419–420. doi: 10.1038/280419a0. [DOI] [PubMed] [Google Scholar]
  11. Pollack Y., Stein R., Razin A., Cedar H. Methylation of foreign DNA sequences in eukaryotic cells. Proc Natl Acad Sci U S A. 1980 Nov;77(11):6463–6467. doi: 10.1073/pnas.77.11.6463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Razin A., Riggs A. D. DNA methylation and gene function. Science. 1980 Nov 7;210(4470):604–610. doi: 10.1126/science.6254144. [DOI] [PubMed] [Google Scholar]
  13. Rosen S. W., Weintraub B. D., Aaronson S. A. Nonrandom ectopic protein production by malignant cells: direct evidence in vitro. J Clin Endocrinol Metab. 1980 May;50(5):834–841. doi: 10.1210/jcem-50-5-834. [DOI] [PubMed] [Google Scholar]
  14. Shen C. K., Maniatis T. Tissue-specific DNA methylation in a cluster of rabbit beta-like globin genes. Proc Natl Acad Sci U S A. 1980 Nov;77(11):6634–6638. doi: 10.1073/pnas.77.11.6634. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Shmookler Reis R. J., Goldstein S. Loss of reiterated DNA sequences during serial passage of human diploid fibroblasts. Cell. 1980 Oct;21(3):739–749. doi: 10.1016/0092-8674(80)90437-7. [DOI] [PubMed] [Google Scholar]
  16. Shmookler Reis R., Timmis J. N., Ingle J. Divergence, differential methylation and interspersion of melon satellite DNA sequences. Biochem J. 1981 Jun 1;195(3):723–734. doi: 10.1042/bj1950723. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Singal D. P., Goldstein S. Absence of detectable HL-A antigens on cultured fibroblasts in progeria. J Clin Invest. 1973 Sep;52(9):2259–2263. doi: 10.1172/JCI107412. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Singer J., Roberts-Ems J., Riggs A. D. Methylation of mouse liver DNA studied by means of the restriction enzymes msp I and hpa II. Science. 1979 Mar 9;203(4384):1019–1021. doi: 10.1126/science.424726. [DOI] [PubMed] [Google Scholar]
  19. Singer J., Stellwagen R. H., Roberts-Ems J., Riggs A. D. 5-Methylcytosine content of rat hepatoma DNA substituted with bromodeoxyuridine. J Biol Chem. 1977 Aug 10;252(15):5509–5513. [PubMed] [Google Scholar]
  20. Slightom J. L., Blechl A. E., Smithies O. Human fetal G gamma- and A gamma-globin genes: complete nucleotide sequences suggest that DNA can be exchanged between these duplicated genes. Cell. 1980 Oct;21(3):627–638. doi: 10.1016/0092-8674(80)90426-2. [DOI] [PubMed] [Google Scholar]
  21. Waalwijk C., Flavell R. A. DNA methylation at a CCGG sequence in the large intron of the rabbit beta-globin gene: tissue-specific variations. Nucleic Acids Res. 1978 Dec;5(12):4631–4634. doi: 10.1093/nar/5.12.4631. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Waalwijk C., Flavell R. A. MspI, an isoschizomer of hpaII which cleaves both unmethylated and methylated hpaII sites. Nucleic Acids Res. 1978 Sep;5(9):3231–3236. doi: 10.1093/nar/5.9.3231. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Wahl G. M., Stern M., Stark G. R. Efficient transfer of large DNA fragments from agarose gels to diazobenzyloxymethyl-paper and rapid hybridization by using dextran sulfate. Proc Natl Acad Sci U S A. 1979 Aug;76(8):3683–3687. doi: 10.1073/pnas.76.8.3683. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Wigler M., Levy D., Perucho M. The somatic replication of DNA methylation. Cell. 1981 Apr;24(1):33–40. doi: 10.1016/0092-8674(81)90498-0. [DOI] [PubMed] [Google Scholar]
  25. Wolf S. F., Migeon B. R. Studies of X chromosome DNA methylation in normal human cells. Nature. 1982 Feb 25;295(5851):667–671. doi: 10.1038/295667a0. [DOI] [PubMed] [Google Scholar]
  26. Yagi M., Koshland M. E. Expression of the J chain gene during B cell differentiation is inversely correlated with DNA methylation. Proc Natl Acad Sci U S A. 1981 Aug;78(8):4907–4911. doi: 10.1073/pnas.78.8.4907. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. van der Ploeg L. H., Flavell R. A. DNA methylation in the human gamma delta beta-globin locus in erythroid and nonerythroid tissues. Cell. 1980 Apr;19(4):947–958. doi: 10.1016/0092-8674(80)90086-0. [DOI] [PubMed] [Google Scholar]

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