Skip to main content
SpringerLink
Log in

Enhanced Gene Targeting Frequency in ES Cells with Low Genomic Methylation Levels

  • Published:
Transgenic Research Aims and scope Submit manuscript

Abstract

Increased methylation in promoter/enhancer regions typically results in transcriptional downregulation. The direct correlation between gene expression and homologous recombination (HR) is also widely acknowledged, and suggests that actively transcribed, hypomethylated targets may be more accessible to the HR machinery. Consistent with this hypothesis, we report that DNA methyltransferase 1 (Dnmt1)-knockout ES cells show a 2-fold increase in gene targeting frequency. However, the use of hypomethylated targeting vectors or the ectopic expression of a putative DNA demethylase did not enhance targeting frequency. These observations are discussed in the context of devising more efficient targeting protocols by transiently modifying genomic methylation levels.

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

Access this article

Log in via an institution

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

  • Alt FW, Blackwell TK, DePinho RA, Reth MG and Yancopoulos GD (1986) Regulation of genome rearrangement events during lymphocyte differentiation. Immunol Rev 89: 5–30.

    Google Scholar 

  • Assaad FF and Signer ER (1992) Somatic and germinal recombination of a direct repeat in Arabidopsis. Genetics 132: 553–566.

    Google Scholar 

  • Bhattacharya SK, Ramchandani S, Cervoni N and Szyf M (1999) A mammalian protein with specific demethylase activity for mCpG DNA. Nature 397: 579–583.

    Google Scholar 

  • Bird A (1992) The essentials of DNA methylation. Cell 70: 5–8.

    Google Scholar 

  • Blackwell TK, Moore MW, Yancopoulos GD, Suh H, Lutzker S, Selsing E et al. (1986) Recombination between immunoglobulin variable region gene segments is enhanced by transcription. Nature 324: 585–589.

    Google Scholar 

  • Boyes J and Bird A (1991) Repression of genes by DNA methylation depends on CpG density and promoter strength: evidence for involvement of a methyl-CpG binding protein. EMBO J 11: 327–333.

    Google Scholar 

  • Charollais RH, Buquet C and Mester J (1990) Butyrate blocks the accumulation of CDC2 mRNA in late G1 phase but inhibits both the early and late G1 progression in chemically transformed mouse fibroblasts BP-A31.J Cell Physiol 145: 46–52.

    Google Scholar 

  • Chen RZ, Pettersson U, Beard C, Jackson-Grusby L and Jaenisch R (1998) DNA hypomethylation leads to elevated mutation rates.Nature 395: 89–93.

    Google Scholar 

  • Counts JL and Goodman JI (1995) Alterations in DNA methylation may play a variety of roles in carcinogenesis. Cell 83: 13–15.

    Google Scholar 

  • Detich N, Theberge J and Szyf M (2002) Promoter-specific activation and demethylation by MBD2/demethylase. J Biol Chem 277: 35791–35794.

    Google Scholar 

  • Dion LD, Goldsmith KT, Tang DC, Engler JA, Yoshida M and Garver RI Jr. (1997) Amplification of recombinant adenoviral transgene products occurs by inhibition of histone deacetylase. Virology 231: 201–209.

    Google Scholar 

  • Domínguez-Bendala J, Priddle H, Clarke A and McWhir J (2003) Elevated expression of exogenous Rad51 leads to identical increases in gene targeting frequency in murine embryonic stem (ES) cells with both functional and dysfunctional p53 genes. Exp Cell Res 286: 298–307.

    Google Scholar 

  • Eden S and Cedar H (1994) Role of DNA methylation in the regulation of transcription. Curr Opin Genet Dev 4: 255–259.

    Google Scholar 

  • Fuks F, Burgers WA, Brehm A, Hughes-Davies L and Kouzarides T (2000) DNA methyltransferase Dnmt1 associates with histone deacetylase activity. Nat Genet 24: 88–91.

    Google Scholar 

  • García-Villalba P, Jiménez-Lara AM, Castillo AI and Aranda A (1997) Histone acetylation influences thyroid hormone and retinoic acid-mediated gene expression. DNA Cell Biol 16: 421–431.

    Google Scholar 

  • Garrison PM, Rogers JM, Brackney WR and Denison MS (2000) Effects of histone deacetylase inhibitors on the Ah receptor gene promoter. Arch Biochem Biophys 374: 161–171.

    Google Scholar 

  • Gross DS and Garrard WT (1988) Nuclease hypersensitive sites in chromatin. Ann Rev Biochem 57: 159–197.

    Google Scholar 

  • Hooper M, Hardy K, Hanyside A, Hunter S and Monk M (1987) HPRT-deficient (Lesch-Nyhan) mouse embryos derived from germline colonization by cultured cells. Nature 326: 292–295.

    Google Scholar 

  • Ikeda H and Matsumoto T (1979) Transcription promotes recAindependent recombination mediated by DNA-dependent RNA polymerase in Escherichia coli. Proc Natl Acad Sci USA 76: 4571–4575.

    Google Scholar 

  • Jones PA (1999) The DNA methylation paradox. Trends Genet 1999 15: 34–37.

    Google Scholar 

  • Kass SU, Pruss D and Wolffe AP (1997) How does DNA methylation repress transcription? Trends Genet 13: 444–449.

    Google Scholar 

  • Kassavetis GA and Geiduschek EP (1993) RNA polymerase marching backward. Science 259: 944–945.

    Google Scholar 

  • Kuehn MR, Bradley A, Robertson E and Evans MJ (1987) A potential animal model for Lesch-Nyhan syndrome through introduction of mutations into mice. Nature 326: 295–298.

    Google Scholar 

  • Latham KE (1996) X chromosome imprinting and inactivation in the early mammalian embryo. Trends Genet 12: 134–138.

    Google Scholar 

  • Liang F and Jasin M (1995) Studies on the influence of cytosine methylation on DNA recombination and end-joining in mammalian cells. J Biol Chem 270: 23838–23844.

    Google Scholar 

  • Maloisel L and Rossignol JL (1998) Suppression of crossingover by DNA methylation in Ascobolus. Genes Dev 12: 1381–1389.

    Google Scholar 

  • Meehan RR, Lewis JD, McKay S, Kleiner EL and Bird AP (1989) Identification of a mammalian protein that binds specifically to DNA containing methylated CpGs. Cell 58: 499–507.

    Google Scholar 

  • Nan X, Cross S and Bird A (1998) Gene silencing by methyl-CpGbinding proteins. Novartis Found Symp 214: 6–16; discussion 16–21, 46–50.

    Google Scholar 

  • Ng HH, Zhang Y, Hendrich B, Johnson CA, Turner BM, Erdjument-Bromage H, Tempst P, Reinberg D and Bird A (1999) MBD2 is a transcriptional repressor belonging to the MeCP1 histone deacetylase complex. Nat Genet 23: 58–61.

    Google Scholar 

  • Nickoloff JA and Reynolds RJ (1990) Transcription stimulates homologous recombination in mammalian cells. Mol Cell Biol 10: 4837–4845.

    Google Scholar 

  • Niki T, Rombouts K, De Bleser P, De Smet K, Rogiers V, Schuppan D, Yoshida M, Gabbiani G and Geerts A (1999) A histone deacetylase inhibitor, trichostatin A, suppresses myofibroblastic differentiation of rat hepatic stellate cells in primary culture. Hepatology 29: 858–867.

    Google Scholar 

  • Nudelman A, Ruse M, Aviram A, Rabizadeh E, Shaklai M, Zimrah Y and Rephaeli A (1992) Novel anticancer prodrugs of butyric acid. 2. J Med Chem 35: 687–694.

    Google Scholar 

  • Paldi A, Gyapay G and Jami J (1995) Imprinted chromosomal regions of the human genome display sex-specific meiotic recombination frequencies. Curr Biol 5: 1030–1035.

    Google Scholar 

  • Prado F, Piruat JI and Aguilera A (1997) Recombination between DNA repeats in yeast hpr1delta cells is linked to transcription elongation. EMBO J 16: 2826–2835.

    Google Scholar 

  • Qu GZ and Ehrlich M (1999) Demethylation and expression of methylated plasmid DNA stably transfected into HeLa cells. Nucl Acids Res 27: 2332–2338.

    Google Scholar 

  • Razin A and Riggs AD (1980) DNA methylation and gene function. Science 210: 604–610.

    Google Scholar 

  • Razin A and Shemer R (1995) DNA methylation in early development. Hum Mol Genet 4: 1751–1755.

    Google Scholar 

  • Reik W and Walter J (1998) Imprinting mechanisms in mammals. Curr Opin Genet Dev 8: 154–164.

    Google Scholar 

  • Rossignol JL and Faugeron G (1994) Gene inactivation triggered by recognition between DNA repeats. Experientia 50: 307–317.

    Google Scholar 

  • Ruh MF, Tian S, Cox LK and Ruh TS (1999) The effects of histone acetylation on estrogen responsiveness in MCF-7 cells. Endocrine 11: 157–164.

    Google Scholar 

  • Sambrook J, Fritsch EF and Maniatis T (1989) Molecular Cloning. A Laboratory Manual (2nd edition). Cold Spring Harbor Laboratory Press, USA.

    Google Scholar 

  • Siegfried Z, Eden S, Mendelsohn M, Feng X, Tsuberi BZ and Cedar H (1999) DNA methylation represses transcription in vivo. Nat Genet 22: 203–206.

    Google Scholar 

  • Takahashi I, Miyaji H, Yoshida T, Sato S and Mizukami T (1996) Selective inhibition of IL-2 gene expression by trichostatin A, a potent inhibitor of mammalian histone deacetylase. J Antibiot (Tokyo) 49: 453–457.

    Google Scholar 

  • Tang DC and Taylor MW (1990) Transcriptional activation of the adenine phosphoribosyltransferase promoter by an upstream butyrate-induced Moloney murine sarcoma virus enhancerpromoter element.J Virol 64: 2907–2911.

    Google Scholar 

  • Thomas BJ and Rothstein R (1989) Elevated recombination rates in transcriptionally active DNA. Cell 56: 619–630.

    Google Scholar 

  • Thyagarajan B, Johnson BL and Campbell C (1995) The effect of target site transcription on gene targeting in human cells in vitro.Nucl Acids Res 23: 2784–2790.

    Google Scholar 

  • Whitkus R, Doebley J and Lee M (1992) Comparative genome mapping of Sorghum and maize. Genetics 132: 1119–1130.

    Google Scholar 

  • Xu L, Ruh TS and Ruh MF (1997) Effect of the histone deacetylase inhibitor trichostatin A on the responsiveness of rat hepatocytes to dioxin. Biochem Pharmacol 53: 951–957.

    Google Scholar 

  • Yisraeli J, Frank D, Razin A and Cedar H (1988) Effect of in vitro DNA methylation on beta-globin gene expression. Proc Natl Acad Sci USA 85: 4638–4642.

    Google Scholar 

  • Yoshida M, Kijima M, Akita M and Beppu T (1990) Potent and specific inhibition of mammalian histone deacetylase both in vivo and in vitro by trichostatin A. J Biol Chem 265: 17174–17179.

    Google Scholar 

  • Zhang ZX, Kumar V, Rivera RT, Pasion SG, Chisholm J and Biswas DK (1989) Suppression of prolactin gene expression in GH cells correlates with site-specific DNA methylation. DNA 8: 605–613.

    Google Scholar 

  • Zhang Y, Shields T, Crenshaw T, Hao Y, Moulton T and Tycko B (1993) Imprinting of human H19: allele-specific CpG methylation, loss of the active allele in Wilms tumor, and potential for somatic allele switching. Am J Hum Genet 53: 113–124.

    Google Scholar 

Download references

Author information

Author notes

  1. Juan Domínguez-Bendala

    Present address: Diabetes Research Institute, University of Miami School of Medicine, 1450 NW, 10th Ave Miami, FL, 33136, USA

Authors and Affiliations

  1. Roslin Institute, Roslin, Midlothian, Scotland, EH25 9PS, UK

    Juan Domínguez-Bendala & Jim McWhir

Authors
  1. Juan Domínguez-Bendala
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jim McWhir
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Jim McWhir.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Domínguez-Bendala, J., McWhir, J. Enhanced Gene Targeting Frequency in ES Cells with Low Genomic Methylation Levels. Transgenic Res 13, 69–74 (2004). https://doi.org/10.1023/B:TRAG.0000017176.77847.80

Download citation

  • Issue Date:

  • DOI: https://doi.org/10.1023/B:TRAG.0000017176.77847.80

Search

Navigation