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An in vitro assay to study the recruitment and substrate specificity of chromatin modifying enzymes

Biological Procedures Online volume 6, pages 157–162 (2004)Cite this article

Abstract

Post-translational modifications of core histones play an important role in regulating fundamental biological processes such as DNA repair, transcription and replication. In this paper, we describe a novel assay that allows sequential targeting of distinct histone modifying enzymes to immobilized nucleosomal templates using recombinant chimeric targeting molecules. The assay can be used to study the histone substrate specificity of chromatin modifying enzymes as well as whether and how certain enzymes affect each other’s histone modifying activities. As such the assay can help to understand how a certain histone code is established and interpreted.

References

  1. Becker PB, Horz W. ATP-dependent nucleosome remodeling. Annual Review of Biochemistry 2002; 71:247–273.

    Article  PubMed  CAS  Google Scholar 

  2. Fischle W, Wang Y, Allis CD. Histone and chromatin crosstalk. Current Opinion in Cell Biology 2003; 15:172–183.

    Article  PubMed  CAS  Google Scholar 

  3. Jenuwein T, Allis CD. Translating the histone code. Science 2001; 293:1074–1080.

    Article  PubMed  CAS  Google Scholar 

  4. Kundu TK, Palhan VB, Wang Z, An W, Cole PA, Roeder RG. Activator-dependent transcription from chromatin in vitro involving targeted histone acetylation by p300. Molecular Cell 2000; 6:551–561.

    Article  PubMed  CAS  Google Scholar 

  5. Utley RT, Ikeda K, Grant PA, Cote J, Steger DJ, Eberharter A, John S, Workman JL. Transcriptional activators direct histone acetyltransferase complexes to nucleosomes. Nature 1998; 394:498–502.

    Article  PubMed  CAS  Google Scholar 

  6. Valcarcel R, Stunnenberg HG. Retinoid-dependent in vitro transcription. Methods in Enzymology 1996; 274:149–161.

    Article  PubMed  CAS  Google Scholar 

  7. Vermeulen M, Carroza MJ, Lasonder E, Workman JL, Logie C, Stunnenberg HG. In vitro targeting reveals histone tail specificity of the Sin3/Histone Deacetylase and NCoR/SMRT co-repressor complexes. Molecular and Cellular Biology 2004; 24:2364–2372.

    Article  PubMed  CAS  Google Scholar 

  8. Kolle D, Brosch G, Lechner T, Lusser A, Loidl P. Biochemical methods for analysis of histone deacetylases. Methods 1998; 15:323–331.

    Article  PubMed  CAS  Google Scholar 

  9. Eberharter A, John S, Grant PA, Utley RT, Workman JL. Identification and analysis of yeast nucleosomal histone acetyltransferase complexes. Methods 1998; 15:315–321.

    Article  PubMed  CAS  Google Scholar 

  10. Carrozza MJ, John S, Sil AK, Hopper JE, Workman JL. Gal80 confers specificity on HAT complex interactions with activators. Journal of Biological Chemistry 2002; 277:24648–24652.

    Article  PubMed  CAS  Google Scholar 

  11. Steger DJ, Workman JL. Transcriptional analysis of purified histone acetyltransferase complexes. Methods 1999; 19:410–416.

    Article  PubMed  CAS  Google Scholar 

  12. Logie C, Peterson CL. Purification and biochemical properties of yeast SWI/SNF complex. Methods in Enzymology 1999; 304:726–741.

    Article  PubMed  CAS  Google Scholar 

  13. Luger K, Mader AW, Richmond RK, Sargent DF, Richmond TJ. Crystal structure of the nucleosome core particle at 2.8 A resolution. Nature 1997; 389:251–260.

    Article  PubMed  CAS  Google Scholar 

  14. Glass CK, Rosenfeld MG. The coregulator exchange in transcriptional functions of nuclear receptors. Genes & Development 1997; 14:121–141.

    Google Scholar 

  15. Spronk CA, Tessari M, Kaan AM, Jansen JF, Vermeulen M, Stunnenberg HG, Vuister GW. The Mad1-Sin3B interaction involves a novel helical fold. Nature Structural Biology 2000; 7:1100–1104.

    Article  PubMed  CAS  Google Scholar 

  16. Brubaker K, Cowley SM, Huang K, Loo L, Yochum GS, Ayer DE, Eisenman RN, Radhakrishnan I. Solution structure of the interacting domains of the Mad-Sin3 complex: implications for recruitment of a chromatin-modifying complex. Cell 2000; 103:655–665.

    Article  PubMed  CAS  Google Scholar 

  17. Mohana-Borges R, Pacheco AB, Sousa FJ, Foguel D, Almeida DF, Silva JL. LexA repressor forms stable dimers in solution. The role of specific dna in tightening protein-protein interactions. Journal of Biological Chemistry 2000; 275:4708–4712.

    Article  PubMed  CAS  Google Scholar 

  18. Deckert J, Struhl K. Targeted recruitment of Rpd3 histone deacetylase represses transcription by inhibiting recruitment of Swi/Snf, SAGA, and TATA binding protein. Molecular and Cellular Biology 2002; 22:6458–6470.

    Article  PubMed  CAS  Google Scholar 

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Authors and Affiliations

  1. Department of Molecular Biology, NCMLS 191, University of Nijmegen, P.O. Box 9101, 6500 HB, Nijmegen, The Netherlands

    Michiel Vermeulen & Hendrik G. Stunnenberg

Authors
  1. Michiel Vermeulen
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  2. Hendrik G. Stunnenberg
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Correspondence to Hendrik G. Stunnenberg.

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Published: July 27, 2004.

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Vermeulen, M., Stunnenberg, H.G. An in vitro assay to study the recruitment and substrate specificity of chromatin modifying enzymes. Biol. Proced. Online 6, 157–162 (2004). https://doi.org/10.1251/bpo85

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  • DOI: https://doi.org/10.1251/bpo85

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Biological Procedures Online

ISSN: 1480-9222

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