Sedimentation and Immunoprecipitation Assays for Analyzing Complexes that Repress Transcription

  • Ping Lu
  • Bruce S. Hostager
  • Paul B. Rothman
  • John D. Colgan
Part of the Methods in Molecular Biology book series (MIMB, volume 977)


Co-repressor proteins function as platforms for the assembly of multi-subunit complexes that mediate transcriptional repression. Common components of such complexes are histone deacetylases, which catalyze the removal of acetyl groups from the tails of histones within nucleosomes, resulting in chromatin compaction and gene repression. In addition, co-repressor complexes generally interact with sequence-specific DNA-binding proteins that direct association with regulatory elements in the genome. Thus, identifying proteins that stably associate with co-repressors can provide insights regarding the biochemical function and target gene specificity of these molecules. Here, we describe a density gradient fractionation method for determining whether a co-repressor is incorporated into high-molecular complexes within cells and for identifying potential constituents of these complexes. We also describe a co-immunoprecipitation assay for confirming and further studying interactions between co-repressors and other proteins that may represent functional partners.

Key words

Sucrose gradient sedimentation Co-immunoprecipitation Immunoblots Co-repressor proteins DNA-binding proteins Histone deacetylases 


  1. 1.
    Laherty CD, Yang WM, Sun JM, Davie JR, Seto E, Eisenman RN (1997) Histone deacetylases associated with the mSin3 corepressor mediate mad transcriptional repression. Cell 89:349–356PubMedCrossRefGoogle Scholar
  2. 2.
    Zhang Y, Iratni R, Erdjument-Bromage H, Tempst P, Reinberg D (1997) Histone deacetylases and SAP18, a novel polypeptide, are components of a human Sin3 complex. Cell 89:357–364PubMedCrossRefGoogle Scholar
  3. 3.
    Lutterbach B, Westendorf JJ, Linggi B, Patten A, Moniwa M, Davie JR, Huynh KD, Bardwell VJ, Lavinsky RM, Rosenfeld MG, Glass C, Seto E, Hiebert SW (1998) ETO, a target of t(8;21) in acute leukemia, interacts with the N-CoR and mSin3 corepressors. Mol Cell Biol 18:7176–7184PubMedGoogle Scholar
  4. 4.
    Fleischer TC, Yun UJ, Ayer DE (2003) Identification and characterization of three new components of the mSin3A corepressor complex. Mol Cell Biol 23:3456–3467PubMedCrossRefGoogle Scholar
  5. 5.
    Cunliffe VT (2008) Eloquent silence: developmental functions of Class I histone deacetylases. Curr Opin Genet Dev 18:404–410PubMedCrossRefGoogle Scholar
  6. 6.
    Grzenda A, Lomberk G, Zhang JS, Urrutia R (2009) Sin3: master scaffold and transcriptional corepressor. Biochim Biophys Acta 1789:443–450PubMedCrossRefGoogle Scholar
  7. 7.
    Perissi V, Jepsen K, Glass CK, Rosenfeld MG (2010) Deconstructing repression: evolving models of co-repressor action. Nat Rev Genet 11:109–123PubMedCrossRefGoogle Scholar
  8. 8.
    Lu P, Hankel IL, Knisz J, Marquardt A, Chiang MY, Grosse J, Constien R, Meyer T, Schroeder A, Zeitlmann L, Al-Alem U, Friedman AD, Elliott EI, Meyerholz DK, Waldschmidt TJ, Rothman PB, Colgan JD (2010) The justy mutation identifies Gon4-like as a gene that is essential for B lymphopoiesis. J Exp Med 207:1359–1367PubMedCrossRefGoogle Scholar
  9. 9.
    Lu P, Hankel IL, Hostager BS, Swartzendruber JA, Friedman AD, Brenton JL, Rothman PB, Colgan JD (2011) The developmental regulator protein Gon4l associates with protein YY1, co-repressor Sin3a, and histone deacetylase 1 and mediates transcriptional repression. J Biol Chem 286:18311–18319PubMedCrossRefGoogle Scholar
  10. 10.
    Völkl A (2010) Ultracentrifugation. In: Finazzi Agrò A (ed) Encyclopedia of life sciences (ELS). Wiley, Chichester. doi:  10.1002/9780470015902.a0002969.pub2
  11. 11.
    Hamano T, Kim KJ, Leiserson WM, Asofsky R (1982) Establishment of B cell hybridomas with B cell surface antigens. J Immunol 129:1403–1406PubMedGoogle Scholar
  12. 12.
    Dignam JD, Lebovitz RM, Roeder RG (1983) Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res 11: 1475–1489PubMedCrossRefGoogle Scholar
  13. 13.
    Sambrook J, Russell DW (2001) Molecular clonining: a laboratory manual, 3rd edn. Cold Spring Harbor Press, Cold Spring HarborGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2013

Authors and Affiliations

  • Ping Lu
    • 1
  • Bruce S. Hostager
    • 1
  • Paul B. Rothman
    • 1
  • John D. Colgan
    • 1
  1. 1.Department of Internal Medicine, Roy J. and Lucille A. Carver College of MedicineUniversity of IowaIowa CityUSA

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