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Methylation analysis by genomic sequencing of 5′ region of mousePgk-1 gene and a cautionary note concerning the method

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Somatic Cell and Molecular Genetics

Abstract

We have used genomic sequencing aided by ligation-mediated PCR (LMPCR) to assay for 5-methylcytosine in the CpG-rich promoter region of the mouse X-linked phosphoglycerate kinase gene (Pgk-1). Earlier studies showed that there was very heavy methylation of CpG dinucleotides in the CpG-rich promoter of the humanPGK1 gene on the inactive X chromosome (the Xi), but that these same sites were completely unmethylated on the active X chromosome (the Xa). For mousePgk-1, previous restriction enzyme analysis had shown apparently complete methylation of only one cytosine in the promoter region on the Xi, at HpaII site H7, which is located in the untranslated region, 28 nucleotides upstream of the translation start site. We analyzed this potentially critical region by combining the use of HpaII with LMPCR, and find that the CpG dinucleotides near H7 are either unmethylated or only partially methylated on the Xi. LMPCR analysis of male and female DNA over a 490-bp sequence including the promoter and enhancer extend the finding of relative hypomethylation on the mouse Xi to include all CpG dinucleotides in this region. These results are relevant to the role of DNA methylation in stabilizing the inactive state of chromatin. In addition, we find that caution must be exercised in using LMPCR for methylation analysis of some sequences. A DNA concentration-dependent band-suppression artifact can incorrectly suggest methylation of both CpG and nonCpG dinucleotides.

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Literature cited

  1. Riggs, A.D., and Pfeifer, G.P. (1992).Trends Genet. 8169–174.

    Google Scholar 

  2. Hall, J.G. (1990).Am. J. Hum. Genet. 46857–873.

    Google Scholar 

  3. Reik, W. (1992).Nature 359362–363.

    Google Scholar 

  4. Sutcliffe, J.S., Nelson, D.L., Zhang, F., Pieretti, M., Caskey, C.T., Saxe, D., and Warren, S.T. (1992).Hum. Mol. Genet. 1397–400.

    Google Scholar 

  5. Li, E., Bestor, T.H., and Jaenisch, R. (1992).Cell 69915–926.

    Google Scholar 

  6. Grant, S.G., and Chapman, V.M. (1988).Annu. Rev. Genet. 22199–233.

    Google Scholar 

  7. Riggs, A.D. (1990).Aust. J. Zool. 37419–441.

    Google Scholar 

  8. Singer-Sam, J., and Riggs, A.D. (1993). InDNA Methylation: Molecular Biology and Biological Significance (ed.) Jost, J.P., Saluz, H.P. (Birkhäuser Verlag, Berlin), pp. 358–384.

    Google Scholar 

  9. Brockdorff, N., Ashworth, A., Kay, G.F., Cooper, P., Smith, S., McCabe, V.M., Norris, D.P., Penny, G.D., Patel, D., and Rastan, S. (1991).Nature 351329–331.

    Google Scholar 

  10. Brown, C.J., Lafreniere, R.G., Powers, V.E., Sebastio, G., Ballabio, A., Pettigrew, A.L., Ledbetter, D.H., Levy, E., Craig, I.W., and Willard, H.F. (1991).Nature 34982–84.

    Google Scholar 

  11. Adra, C.N., Boer, P.H., and McBurney, M.W. (1987).Gene 6065–74.

    Google Scholar 

  12. Singer-Sam, J., Keith, D.H., Tani, K., Simmer, R.L., Shively, L., Lindsay, S., Yoshida, A., and Riggs, A.D. (1984).Gene 32409–417.

    Google Scholar 

  13. Antequera, F., and Bird, A. (1993). InDNA Methylation: Molecular Biology and Biological Significance (eds.) Jost, J.P., Saluz, H.P. (Birkhäuser Verlag, Berlin), pp. 169–185.

    Google Scholar 

  14. Pfeifer, G.P., Tanguay, R.L., Steigerwald, S.D., and Riggs, A.D. (1990).Genes Dev. 41277–1287.

    Google Scholar 

  15. Yeivin, A., and Razin, A. (1993). InDNA Methylation: Molecular Biology and Biological Significance (eds.) Jost, J.P., Saluz, H.P. (Birkhäuser Verlag, Berlin), pp. 523–568.

    Google Scholar 

  16. Norris, D.P., Brockdorff, N., and Rastan, S. (1991).Mammal. Genome 178–83.

    Google Scholar 

  17. Pfeifer, G.P., and Riggs, A.D. (1991).Genes Dev. 51102–1113.

    Google Scholar 

  18. Yang, T.P., Singer-Sam, J., Flores, J.C., and Riggs, A.D. (1988).Somat. Cell Mol. Genet. 14461–472.

    Google Scholar 

  19. Bartlett, M.H., Adra, C.N., Park, J., Chapman, V.M., and McBurney, M.W. (1991).Somat. Cell Mol. Genet. 1735–47.

    Google Scholar 

  20. Singer-Sam, J., Yang, T.P., Mori, N., Tanguay, R.L., LeBon, J.M., Flores, J.C., and Riggs, A.D. (1990). InNucleic Acid Methylation, Vol. 128, (eds.) Clawson, G., Willis, D., Weissbach, A., and Jones, P. (Alan R. Liss, New York), 285–298.

    Google Scholar 

  21. Maniatis, T., Fritsch, E.F., and Sambrook, J. (1982).Molecular Cloning: A Laboratory Manual, (Cold Spring Harbor Laboratory, New York).

    Google Scholar 

  22. Pfeifer, G.P., Singer-Sam, J., and Riggs, A.D. (1993).Methods Enzymol. 225567–583.

    Google Scholar 

  23. Holmquist, G.P., and Caston, L.A. (1986).Biochim. Biophys. Acta 868164–177.

    Google Scholar 

  24. Maxam, A.M., and Gilbert, W. (1980).Methods Enzymol. 65499–560.

    Google Scholar 

  25. Mueller, P.R., and Wold, B. (1989).Science 246780–786.

    Google Scholar 

  26. Pfeifer, G.P., Steigerwald, S.D., Mueller, P.R., Wold, B., and Riggs, A.D. (1989).Science 246810–813.

    Google Scholar 

  27. Garrity, P.A., and Wold, B.J. (1992).Proc. Natl. Acad. Sci. U.S.A. 891021–1025.

    Google Scholar 

  28. Bednarczuk, T.A., Wiggins, R.C., and Konat, G.W. (1991).Biotechniques 10478.

    Google Scholar 

  29. McBurney, M.W., Sutherland, L.C., Adra, C.N., Leclair, B., Rudnicki, M.A., and Jardine, K. (1991).Nucleic Acids Res. 195755–5761.

    Google Scholar 

  30. Tamaru, M., Nagao, Y., Taira, M., Masamune, Y., and Nakanishi, Y. (1990).Biochim. Biophys. Acta. 1049331–338.

    Google Scholar 

  31. Hansen, R.S., and Gartler, S.M. (1990).Proc. Natl. Acad. Sci. U.S.A. 874174–4178.

    Google Scholar 

  32. Pfeifer, G.P., Steigerwald, S.D., Hansen, R.S., Gartler, S.M., and Riggs, A.D. (1990).Proc. Natl. Acad. Sci. U.S.A. 878252–8256.

    Google Scholar 

  33. Singer-Sam, J., Grant, M., LeBon, J.M., Okuyama, K., Chapman, V., Monk, M., and Riggs, A.D. (1990).Mol. Cell. Biol. 104987–4989.

    Google Scholar 

  34. Monk, M. (1986).BioEssays 4204–208.

    Google Scholar 

  35. Ehrlich, M., and Ehrlich, K.C. (1993). InDNA Methylation: Molecular Biology and Biological Significance, (eds.) Jost, J.P., Saluz, H.P. (Birkhäuser Verlag, Berlin), pp. 145–168.

    Google Scholar 

  36. Jost, J.-P., and Hofsteenge, J. (1992).Proc. Natl. Acad. Sci. U.S.A. 899499–9503.

    Google Scholar 

  37. Lewis, J., and Bird, A. (1991).FEBS Lett. 285155–159.

    Google Scholar 

  38. Lewis, J.D., Meehan, R.R., Henzel, W.J., Maurer-Fogy, I., Jeppesen, P., Klein, F., and Bird, A. (1992).Cell 69905–914.

    Google Scholar 

  39. Hansen, R.S., Ellis, N., and Gartler, S.M. (1988).Mol. Cell. Biol. 84692–4699.

    Google Scholar 

  40. Brown, S.D.M. (1991).BioEssays 13607–611.

    Google Scholar 

  41. Lock, L.F., Takagi, N., and Martin, G.R. (1987).Cell 4839–46.

    Google Scholar 

  42. Singer-Sam, J., LeBon, J.M., Tanguay, R.L., and Riggs, A.D. (1990).Nucleic Acids Res. 18687.

    Google Scholar 

  43. Grant, M., Zuccotti, M., and Monk, M. (1992).Nature Genet. 2161–166.

    Google Scholar 

  44. Singer-Sam, J., Goldstein, L., Dai, A., Gartler, S.M., and Riggs, A.D. (1992).Proc. Natl. Acad. Sci. U.S.A. 891413–1417.

    Google Scholar 

  45. Adams, R.L.P., Lindsay, H., Reale, A., Seivwright, C., Kass, S., Cummings, M., and Houlston, C. (1993). InDNA Methylation: Molecular Biology and Biological Significance, (eds.) Jost, J.P., Saluz, H.P. (Birkhäuser Verlag, Berlin), pp. 120–144.

    Google Scholar 

  46. Woodcock, D.M., Crowther, P.J., and Diver, W.P. (1987).Biochem. Biophys. Res. Commun. 145888–894.

    Google Scholar 

  47. Holliday, R., and Pugh, J.E. (1975).Science 187226–232.

    Google Scholar 

  48. Riggs, A.D. (1975).Cytogenet. Cell Genet. 149–25.

    Google Scholar 

  49. Toth, M., Müller, U., and Doerfler, W. (1990).J. Mol. Biol. 214673–683.

    Google Scholar 

  50. Frommer, M., McDonald, L.E., Millar, D.S., Collis, C.M., Watt, F., Grigg, G.W., Molloy, P.L., and Paul, C.L. (1992).Proc. Natl. Acad. Sci. U.S.A. 891827–1831.

    Google Scholar 

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Authors and Affiliations

  1. Biology Department, Beckman Research Institute of the City of Hope, 91010, Duarte, California

    Stella Tommasi, Jeanne M. LeBon, Arthur D. Riggs & Judith Singer-Sam

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  1. Stella Tommasi
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  2. Jeanne M. LeBon
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  3. Arthur D. Riggs
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  4. Judith Singer-Sam
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Tommasi, S., LeBon, J.M., Riggs, A.D. et al. Methylation analysis by genomic sequencing of 5′ region of mousePgk-1 gene and a cautionary note concerning the method. Somat Cell Mol Genet 19, 529–541 (1993). https://doi.org/10.1007/BF01233380

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  • DOI: https://doi.org/10.1007/BF01233380

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