Chromatin Endogenous Cleavage and Psoralen Crosslinking Assays to Analyze rRNA Gene Chromatin In Vivo

  • Joachim Griesenbeck
  • Manuel Wittner
  • Romain Charton
  • Antonio Conconi
Part of the Methods in Molecular Biology book series (MIMB, volume 809)


In eukaryotes, multiple copies of ribosomal RNA (rRNA) genes co-exist in two different chromatin states: actively transcribed (nucleosome depleted) chromatin, and nontranscribed (nucleosomal) chromatin. The presence of two rRNA gene populations compromises the interpretation of analyses obtained by the standard biochemical methods that are used to study chromatin structure (e.g., nuclease digestion and chromatin immunoprecipitation). Here, we provide a protocol to investigate the specific association of proteins with the two rRNA gene chromatin populations in vivo, using Saccharomyces cerevisiae as a model eukaryote.

Key words

Chromatin Chromatin endogenous cleavage Psoralen crosslinking rDNA Ribosomal DNA Ribosomal RNA genes RNA polymerase I 



This work was supported by a mobility program accompanied by the Bavarian Research Alliance GmbH as an assignment from the Bavarian State Chancellery to J.G. and by the Ministère des Relations Internationales du Québec to A.C. M.W. is a recipient of a fellowship from the Elitenetzwerk Bayern and R.C. is supported by the Natural Sciences and Engineering Research Council of Canada (NSERC No. 326873-2009) to A.C.


  1. 1.
    Warner, J. R. (1999) The economics of ribosome biosynthesis in yeast. Trends Biochem. Sci 24, 437–440.Google Scholar
  2. 2.
    Conconi, A., Widmer, R.M., Koller, T., and Sogo, J. M. (1989) Two different chromatin structures coexist in ribosomal RNA genes throughout the cell cycle. Cell 57, 753–61.Google Scholar
  3. 3.
    Merz, K., Hondele, M., Goetze, H., Gmelch, K., Stoeckl, U., and Griesenbeck, J. (2008) Actively transcribed rRNA genes in S. cerevisiae are organized in a specialized chromatin associated with the high-mobility group protein Hmo1 and are largely devoid of histone molecules. Genes Dev 22, 1190–204.Google Scholar
  4. 4.
    Dammann, R., Lucchini, R., Koller, T., and Sogo, J. M. (1993) Chromatin structures and transcription of rDNA in yeast Saccharomyces cerevisiae. Nucleic Acids Res 21, 2331–8.Google Scholar
  5. 5.
    Toussaint, M., Levasseur, G., Tremblay, M., Paquette, M., and Conconi, A. (2005) Psoralen photocrosslinking, a tool to study the chromatin structure of RNA polymerase I--transcribed ribosomal genes. Biochem Cell Biol 83, 449–59.Google Scholar
  6. 6.
    Schmid, M., Durussel, T., and Laemmli, U. K. (2004) ChIC and ChEC; genomic mapping of chromatin proteins. Mol Cell 16, 147–57.Google Scholar
  7. 7.
    Sogo, J. M., Ness, P. J., Widmer, R. M., Parish, R. W., and Koller, T. (1984) Psoralen-crosslinking of DNA as a probe for the structure of active nucleolar chromatin. J. Mol. Biol 178, 897–919.Google Scholar
  8. 8.
    Schmid, M., Arib, G., Laemmli, C., Nishikawa, J., Durussel, T., and Laemmli, U. K. (2006) Nup-PI: the nucleopore-promoter interaction of genes in yeast. Mol. Cell 21, 379–391.Google Scholar
  9. 9.
    Goetze, H., Wittner, M., Hamperl, S., Hondele, M., Merz, K., Stoeckl, U., and Griesenbeck, J. (2010) Alternative Chromatin Structures of the 35S rRNA Genes in Saccharomyces cerevisiae Provide a Molecular Basis for the Selective Recruitment of RNA Polymerases I and II. Mol. Cell. Biol 30, 2028–2045.Google Scholar
  10. 10.
    Sambrook, J., and Russell, D. W. (2006) Southern Blotting: Capillary Transfer of DNA to Membranes. Cold Spring Harb Protoc 2006, pdb.prot4040.Google Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Joachim Griesenbeck
    • 1
  • Manuel Wittner
    • 1
  • Romain Charton
    • 2
  • Antonio Conconi
    • 2
  1. 1.Naturwissenschaftliche Fakultät III, Institut für Biochemie IIIUniversität RegensburgRegensburgGermany
  2. 2.Département de Microbiologie et Infectiologie, Faculté de MédecineUniversité de SherbrookeSherbrookeCanada

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