Skip to main content
PMC home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1994 Jun;14(6):4225–4232. doi: 10.1128/mcb.14.6.4225

Ubiquitous and tenacious methylation of the CpG site in codon 248 of the p53 gene may explain its frequent appearance as a mutational hot spot in human cancer.

A N Magewu 1, P A Jones 1
PMCID: PMC358788  PMID: 8196660

Abstract

Cytosine methylation at CpG dinucleotides is thought to cause more than one-third of all transition mutations responsible for human genetic diseases and cancer. We investigated the methylation status of the CpG dinucleotide at codon 248 in exon 7 of the p53 gene because this codon is a hot spot for inactivating mutations in the germ line and in most human somatic tissues examined. Codon 248 is contained within an HpaII site (CCGG), and the methylation status of this and flanking CpG sites was analyzed by using the methylation-sensitive enzymes CfoI (GCGC) and HpaII. Codon 248 and the CfoI and HpaII sites in the flanking introns were methylated in every tissue and cell line examined, indicating extensive methylation of this region in the p53 gene. Exhaustive treatment of an osteogenic sarcoma cell line, TE85, with the hypomethylating drug 5-aza-2'-deoxycytidine did not demethylate codon 248 or the CfoI sites in intron 6, although considerable global demethylation of the p53 gene was induced. Constructs containing either exon 7 alone or exon 7 and the flanking introns were transfected into TE85 cells to determine whether de novo methylation would occur. The presence of exon 7 alone caused some de novo methylation to occur at codon 248. More extensive de novo methylation of the CfoI sites in intron 6, which contains an Alu sequence, occurred in cells transfected with a vector containing exon 7 and flanking introns. With longer time in culture, there was increased methylation at the CfoI sites, and de novo methylation of codon 248 and its flanking HpaII sites was observed. These de novo-methylated sites were also resistant to 5-aza-2'-deoxycytidine-induced demethylation. The frequent methylation of codon 248 and adjacent Alu sequence may explain the enhanced mutability of this site as a result of the deamination of the 5-methylcytosine.

Full text

PDF
4227

Images in this article

4227Image
on p.4227
4228Image
on p.4228
4229Image
on p.4229
4229Image
on p.4229
4229Image
on p.4229
4230Image
on p.4230

Selected References

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

  1. Benzer S. ON THE TOPOGRAPHY OF THE GENETIC FINE STRUCTURE. Proc Natl Acad Sci U S A. 1961 Mar;47(3):403–415. doi: 10.1073/pnas.47.3.403. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bestor T. H. Activation of mammalian DNA methyltransferase by cleavage of a Zn binding regulatory domain. EMBO J. 1992 Jul;11(7):2611–2617. doi: 10.1002/j.1460-2075.1992.tb05326.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bird A. P. CpG-rich islands and the function of DNA methylation. Nature. 1986 May 15;321(6067):209–213. doi: 10.1038/321209a0. [DOI] [PubMed] [Google Scholar]
  4. Bolden A. H., Nalin C. M., Ward C. A., Poonian M. S., Weissbach A. Primary DNA sequence determines sites of maintenance and de novo methylation by mammalian DNA methyltransferases. Mol Cell Biol. 1986 Apr;6(4):1135–1140. doi: 10.1128/mcb.6.4.1135. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Boukamp P., Petrussevska R. T., Breitkreutz D., Hornung J., Markham A., Fusenig N. E. Normal keratinization in a spontaneously immortalized aneuploid human keratinocyte cell line. J Cell Biol. 1988 Mar;106(3):761–771. doi: 10.1083/jcb.106.3.761. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Britten R. J., Kohne D. E. Repeated sequences in DNA. Hundreds of thousands of copies of DNA sequences have been incorporated into the genomes of higher organisms. Science. 1968 Aug 9;161(3841):529–540. doi: 10.1126/science.161.3841.529. [DOI] [PubMed] [Google Scholar]
  7. Church G. M., Gilbert W. Genomic sequencing. Proc Natl Acad Sci U S A. 1984 Apr;81(7):1991–1995. doi: 10.1073/pnas.81.7.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cooper D. N., Youssoufian H. The CpG dinucleotide and human genetic disease. Hum Genet. 1988 Feb;78(2):151–155. doi: 10.1007/BF00278187. [DOI] [PubMed] [Google Scholar]
  9. Coulondre C., Miller J. H., Farabaugh P. J., Gilbert W. Molecular basis of base substitution hotspots in Escherichia coli. Nature. 1978 Aug 24;274(5673):775–780. doi: 10.1038/274775a0. [DOI] [PubMed] [Google Scholar]
  10. Doerfler W. Ernst Klenk lecture, November 7, 1988. Patterns of DNA methylation in the mammalian genome. Biol Chem Hoppe Seyler. 1990 Jun;371(6):455–463. [PubMed] [Google Scholar]
  11. Ehrhart J. C., Duthu A., Ullrich S., Appella E., May P. Specific interaction between a subset of the p53 protein family and heat shock proteins hsp72/hsc73 in a human osteosarcoma cell line. Oncogene. 1988 Nov;3(5):595–603. [PubMed] [Google Scholar]
  12. Evans D. R., Irwin R. J., Havre P. A., Bouchard J. G., Kato T., Prout G. R., Jr The activity of the pyrimidine biosynthetic pathway in MGH-U1 transitional carcinoma cells grown in tissue culture. J Urol. 1977 Jun;117(6):712–719. doi: 10.1016/s0022-5347(17)58598-5. [DOI] [PubMed] [Google Scholar]
  13. Fearon E. R., Jones P. A. Progressing toward a molecular description of colorectal cancer development. FASEB J. 1992 Jul;6(10):2783–2790. doi: 10.1096/fasebj.6.10.1321771. [DOI] [PubMed] [Google Scholar]
  14. Feinberg A. P., Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem. 1983 Jul 1;132(1):6–13. doi: 10.1016/0003-2697(83)90418-9. [DOI] [PubMed] [Google Scholar]
  15. Flatau E., Bogenmann E., Jones P. A. Variable 5-methylcytosine levels in human tumor cell lines and fresh pediatric tumor explants. Cancer Res. 1983 Oct;43(10):4901–4905. [PubMed] [Google Scholar]
  16. Gardiner-Garden M., Frommer M. CpG islands in vertebrate genomes. J Mol Biol. 1987 Jul 20;196(2):261–282. doi: 10.1016/0022-2836(87)90689-9. [DOI] [PubMed] [Google Scholar]
  17. Gebara M. M., Drevon C., Harcourt S. A., Steingrimsdottir H., James M. R., Burke J. F., Arlett C. F., Lehmann A. R. Inactivation of a transfected gene in human fibroblasts can occur by deletion, amplification, phenotypic switching, or methylation. Mol Cell Biol. 1987 Apr;7(4):1459–1464. doi: 10.1128/mcb.7.4.1459. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Graham F. L., van der Eb A. J. A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology. 1973 Apr;52(2):456–467. doi: 10.1016/0042-6822(73)90341-3. [DOI] [PubMed] [Google Scholar]
  19. Hasse A., Schulz W. A., Sies H. De novo methylation of transfected CAT gene plasmid constructs in F9 mouse embryonal carcinoma cells. Biochim Biophys Acta. 1992 May 7;1131(1):16–22. doi: 10.1016/0167-4781(92)90092-e. [DOI] [PubMed] [Google Scholar]
  20. Hellmann-Blumberg U., Hintz M. F., Gatewood J. M., Schmid C. W. Developmental differences in methylation of human Alu repeats. Mol Cell Biol. 1993 Aug;13(8):4523–4530. doi: 10.1128/mcb.13.8.4523. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Hoeben R. C., Migchielsen A. A., van der Jagt R. C., van Ormondt H., van der Eb A. J. Inactivation of the Moloney murine leukemia virus long terminal repeat in murine fibroblast cell lines is associated with methylation and dependent on its chromosomal position. J Virol. 1991 Feb;65(2):904–912. doi: 10.1128/jvi.65.2.904-912.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Holliday R., Pugh J. E. DNA modification mechanisms and gene activity during development. Science. 1975 Jan 24;187(4173):226–232. [PubMed] [Google Scholar]
  23. Jones P. A., Taylor S. M. Cellular differentiation, cytidine analogs and DNA methylation. Cell. 1980 May;20(1):85–93. doi: 10.1016/0092-8674(80)90237-8. [DOI] [PubMed] [Google Scholar]
  24. Jähner D., Jaenisch R. Retrovirus-induced de novo methylation of flanking host sequences correlates with gene inactivity. Nature. 1985 Jun 13;315(6020):594–597. doi: 10.1038/315594a0. [DOI] [PubMed] [Google Scholar]
  25. Kochanek S., Renz D., Doerfler W. DNA methylation in the Alu sequences of diploid and haploid primary human cells. EMBO J. 1993 Mar;12(3):1141–1151. doi: 10.1002/j.1460-2075.1993.tb05755.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Kuhlmann I., Doerfler W. Loss of viral genomes from hamster tumor cells and nonrandom alterations in patterns of methylation of integrated adenovirus type 12 DNA. J Virol. 1983 Sep;47(3):631–636. doi: 10.1128/jvi.47.3.631-636.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Lehrman M. A., Russell D. W., Goldstein J. L., Brown M. S. Alu-Alu recombination deletes splice acceptor sites and produces secreted low density lipoprotein receptor in a subject with familial hypercholesterolemia. J Biol Chem. 1987 Mar 5;262(7):3354–3361. [PubMed] [Google Scholar]
  28. Li E., Bestor T. H., Jaenisch R. Targeted mutation of the DNA methyltransferase gene results in embryonic lethality. Cell. 1992 Jun 12;69(6):915–926. doi: 10.1016/0092-8674(92)90611-f. [DOI] [PubMed] [Google Scholar]
  29. Liu W. M., Schmid C. W. Proposed roles for DNA methylation in Alu transcriptional repression and mutational inactivation. Nucleic Acids Res. 1993 Mar 25;21(6):1351–1359. doi: 10.1093/nar/21.6.1351. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Masters J. R., Hepburn P. J., Walker L., Highman W. J., Trejdosiewicz L. K., Povey S., Parkar M., Hill B. T., Riddle P. R., Franks L. M. Tissue culture model of transitional cell carcinoma: characterization of twenty-two human urothelial cell lines. Cancer Res. 1986 Jul;46(7):3630–3636. [PubMed] [Google Scholar]
  31. McAllister R. M., Gardner M. B., Greene A. E., Bradt C., Nichols W. W., Landing B. H. Cultivation in vitro of cells derived from a human osteosarcoma. Cancer. 1971 Feb;27(2):397–402. doi: 10.1002/1097-0142(197102)27:2<397::aid-cncr2820270224>3.0.co;2-x. [DOI] [PubMed] [Google Scholar]
  32. Michalowsky L. A., Jones P. A. Gene structure and transcription in mouse cells with extensively demethylated DNA. Mol Cell Biol. 1989 Mar;9(3):885–892. doi: 10.1128/mcb.9.3.885. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Mummaneni P., Bishop P. L., Turker M. S. A cis-acting element accounts for a conserved methylation pattern upstream of the mouse adenine phosphoribosyltransferase gene. J Biol Chem. 1993 Jan 5;268(1):552–558. [PubMed] [Google Scholar]
  34. Rideout W. M., 3rd, Coetzee G. A., Olumi A. F., Jones P. A. 5-Methylcytosine as an endogenous mutagen in the human LDL receptor and p53 genes. Science. 1990 Sep 14;249(4974):1288–1290. doi: 10.1126/science.1697983. [DOI] [PubMed] [Google Scholar]
  35. Rigby C. C., Franks L. M. A human tissue culture cell line from a transitional cell tumour of the urinary bladder: growth, chromosone pattern and ultrastructure. Br J Cancer. 1970 Dec;24(4):746–754. doi: 10.1038/bjc.1970.89. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Riggs A. D. X inactivation, differentiation, and DNA methylation. Cytogenet Cell Genet. 1975;14(1):9–25. doi: 10.1159/000130315. [DOI] [PubMed] [Google Scholar]
  37. Selker E. U., Jensen B. C., Richardson G. A. A portable signal causing faithful DNA methylation de novo in Neurospora crassa. Science. 1987 Oct 2;238(4823):48–53. doi: 10.1126/science.2958937. [DOI] [PubMed] [Google Scholar]
  38. Smith S. S., Kan J. L., Baker D. J., Kaplan B. E., Dembek P. Recognition of unusual DNA structures by human DNA (cytosine-5)methyltransferase. J Mol Biol. 1991 Jan 5;217(1):39–51. doi: 10.1016/0022-2836(91)90609-a. [DOI] [PubMed] [Google Scholar]
  39. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  40. Southern P. J., Berg P. Transformation of mammalian cells to antibiotic resistance with a bacterial gene under control of the SV40 early region promoter. J Mol Appl Genet. 1982;1(4):327–341. [PubMed] [Google Scholar]
  41. Spruck C. H., 3rd, Rideout W. M., 3rd, Olumi A. F., Ohneseit P. F., Yang A. S., Tsai Y. C., Nichols P. W., Horn T., Hermann G. G., Steven K. Distinct pattern of p53 mutations in bladder cancer: relationship to tobacco usage. Cancer Res. 1993 Mar 1;53(5):1162–1166. [PubMed] [Google Scholar]
  42. Szyf M., Schimmer B. P., Seidman J. G. Nucleotide-sequence-specific de novo methylation in a somatic murine cell line. Proc Natl Acad Sci U S A. 1989 Sep;86(18):6853–6857. doi: 10.1073/pnas.86.18.6853. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Toth M., Müller U., Doerfler W. Establishment of de novo DNA methylation patterns. Transcription factor binding and deoxycytidine methylation at CpG and non-CpG sequences in an integrated adenovirus promoter. J Mol Biol. 1990 Aug 5;214(3):673–683. doi: 10.1016/0022-2836(90)90285-T. [DOI] [PubMed] [Google Scholar]
  44. 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]
  45. Weiner A. M., Deininger P. L., Efstratiadis A. Nonviral retroposons: genes, pseudogenes, and transposable elements generated by the reverse flow of genetic information. Annu Rev Biochem. 1986;55:631–661. doi: 10.1146/annurev.bi.55.070186.003215. [DOI] [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES