Purification of the MeCP2/Histone Deacetylase Complex from Xenopus laevis

  • Peter L. Jones
  • Paul A. Wade
  • Alan P. Wolffe
Part of the Methods in Molecular Biology™ book series (MIMB, volume 181)

Abstract

DNA methylation has long been associated with stable transcriptional silencing and a repressive chromatin structure (reviewed in refs. 1,2). Differential methylation is associated with imprinting, carcinogenesis, silencing of repetitive DNA, and allows for differentiating cells to efficiently shut off unnecessary genes. In vertebrates, where 60-90% of genomic CpG dinucleotides are methylated, methylation-dependent repression is vital for proper embryonic development (3). Microinjection experiments using methylated DNA templates implicate chromatin structure as an underlying mechanism of methylation-dependent silencing (4,5). Methyl-specific transcriptional repression requires chromatin assembly, and can be partially relieved by the histone deacetylase inhibitor Trichostatin A. In addition, several proteins have been identified that specifically bind to methylated DNA (6-8). Two of these methyl-DNA binding proteins, MeCP1 and MeCP2, have been shown to mediate transcriptional repression (6,7). MeCP1 is a relatively uncharacterized complex that requires at least 12 symmetrical methyl-CpGs for DNA binding (6). MeCP2 is a single polypeptide containing a methyl-binding domain capable of binding a single methyl-CpG, and a transcriptional repression domain (9). Recently MeCP2 was shown to interact with the Sin3 corepressor and histone deacetylase (10,11). Changes in the acetylation state of the core histone tails correlates with changes in transcription (reviewed in refs. 12,13), and several transcriptional repression complexes containing histone deacetylases have recently been described (10,14,15). These data provide a direct link between methyl-dependent transcriptional repression and the modification of chromatin structure. Here, we describe techniques for purifying the MeCP2-contining histone deacetylase complex from Xenopus laevis oocytes.

Keywords

Glycerol EDTA Fluoride Glycine Heparin 

References

  1. 1.
    Kass, S. U., Pruss, D., and Wolffe, A. P. (1997) How does DNA methylation repress transcription?Trends Genet. 13, 444–449.PubMedCrossRefGoogle Scholar
  2. 2.
    Razin, A. (1998)CpG methylation, chromatin structure and gene silencing-a three-way connection EMBO J. 17, 4905–4908.PubMedCrossRefGoogle Scholar
  3. 3.
    Tate, P., Skarnes, W., and Bird, A. P. (1996) The methyl-CpG binding protein MeCP2 is essential for embryonic development in the mouse. Nature Genet. 12, 205–208.PubMedCrossRefGoogle Scholar
  4. 4.
    Buschhausen, G., Wittig, B., Graessmann, M., and Graessmann, A. (1987) Chromatin structure is required to block transcription of the methylated herpes simplex virus thymidine kinase gene. Proc. Natl. Acad. Sci. USA 84 1177–1181.PubMedCrossRefGoogle Scholar
  5. 5.
    Kass, S. U., Landsberger, N., and Wollfe, A. P. (1997) DNA methylation directs a time-dependent repression of transcription initiation Curr. Biol. 7 157–165.PubMedCrossRefGoogle Scholar
  6. 6.
    Meehan, R. R., Lewis, J. D., McKay, S., Kleiner, E. L., and Bird, A. P. (1989)Identification of a mammalian protein that binds specifically to DNA containing methylated CpGs Cell 58, 499–507.PubMedCrossRefGoogle Scholar
  7. 7.
    Lewis, J. D., Meehan, R. R., Henzel, W. J., Maurer-Fogey, I., Jeppesen, P., Klein, F., and Bird, A. P. (1989) Purification, sequence, and cellular localization of a novel chromosomal protein that binds to methylated DNA. Cell 69 905–914.CrossRefGoogle Scholar
  8. 8.
    Hendrich, B.and Bird, A. (1998)Identification and characterization of a family of mammalian methyl-CpG binding proteins. Mol. Cell Biol. 18, 6538–6547.PubMedGoogle Scholar
  9. 9.
    Nan, X., Campoy, F. J., and Bird, A. P.(1997) MeCP2 is a transcriptional repressor with abundant binding sites in genomic chromatin. Cell 88, 471–481.PubMedCrossRefGoogle Scholar
  10. 10.
    Jones, P. L., Veenstra, G. J. C., Wade, P. A., Vermaak, D., Kass, S. U., Landsberger, N., Strouboulis, J., and Wolffe, A. P. (1998) Methylated DNA and MeCP2 recruit histone deacetylase to repress transcription. Nature Genet. 19, 187–190.PubMedCrossRefGoogle Scholar
  11. 11.
    Nan, X., Ng, H., Johnson, C. A., Laherty, C. D., Turner, B. M., Eisenman, R. N., and Bird, A. P. (1998) Transcriptional repression by the methyl-CpG-binding protein MeCP2 involves a histone deacetylase complex. Nature 393, 386–389.PubMedCrossRefGoogle Scholar
  12. 12.
    Struhl, K. (1998) Histone acetylation and transcriptional regulatory mechanisms. Genes Dev. 12 599–606.PubMedCrossRefGoogle Scholar
  13. 13.
    Grunstein, M. (1997) Histone acetylation in chromatin structure and transcription Nature 389, 349–352.PubMedCrossRefGoogle Scholar
  14. 14.
    Wade, P. A., Jones, P. L., Vermaak, D., and Wolffe, A. P. (1998) A multiple subunit Mi-2 histone deacetylase from Xenopus laevis cofractionates with an associated Snf2 superfamily ATPase. Curr. Biol.8, 843–846.PubMedCrossRefGoogle Scholar
  15. 15.
    Lavinsky, R. M., Mullen, T. M., Soderstrom, M., Laherty, C. D., Torchia, J., et al., (1997) A complex containing N-CoR, mSin3 and histone deacetylase mediates transcriptional repression. Nature 387, 43–48.PubMedCrossRefGoogle Scholar
  16. 16.
    Miskimins, W. K., Roberts, M. P., McClelland, A., and Ruddle, F. H. (1985) Use of a protein-blotting procedure and a specific DNA probe to identify nuclear proteins that recognize the promoter region of the transferrin receptor gene. Proc. Natl. Acad. Sci. USA 32, 6741–6744.CrossRefGoogle Scholar
  17. 17.
    Vinson, C., LaMarco, K., Johnson, P., Landschulz, W., and McKnight, S. (1988) In situ detection of sequence-specific DNA binding activity specified by a recombinant bacteriophage. Genes Dev. 2, 801–806.PubMedCrossRefGoogle Scholar
  18. 18.
    Shimamura, A.and Worcel, A. (1989) The assembly of regularly spaced nucleosomes in the Xenopus oocyte S-150 extract is accompanied by deacetylation of histone H4. J. Biol. Chem. 264, 14,524–14,530.PubMedGoogle Scholar
  19. 19.
    Chandler, S. P.and Wolffe, A. P. (1998) Analysis of linker histone binding to mono-and dinucleosomes, in Methods in Molecular Biology: Chromatin Protocols (Becker, P. B., ed.), Humana, London.Google Scholar
  20. 20.
    Parthun, M. R., Widom, J., and Gottschling, D. E. (1996) The major cytoplasmic histone acetyltransferase in yeast: links to chromatin replication and histone metabolism. Cell 87, 85–94.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 2002

Authors and Affiliations

  • Peter L. Jones
    • 1
  • Paul A. Wade
    • 2
  • Alan P. Wolffe
    • 3
  1. 1.Department of Cell and Structural BiologyUniversity of Illinois, Urbana-ChampaignUrbana
  2. 2.Laboratory of Molecular EmbryologyNational Institute of Child Health and Human Development, National Institutes of HealthBethesda
  3. 3.Sangamo BioSciences Inc.Richmond

Personalised recommendations