Chromatin Immunoprecipitation Assay as a Tool for Analyzing Transcription Factor Activity

  • Padmaja Gade
  • Dhan V. KalvakolanuEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 809)


Differential gene expression is facilitated by transcriptional regulatory mechanisms and chromatin modifications through DNA–protein interactions. One of the widely used assays to study this is chromatin immunoprecipitation (ChIP) assay, which enables analysis of association of regulatory molecules to specific promoters and histone modifications in vivo. This is of immense value as ChIP assays can provide glimpse of the regulatory mechanisms involved in gene expression in vivo. This article outlines the general strategies and protocols to study ChIP assays in differential recruitment of transcriptional factors (TFs) and also global analysis of transcription factor recruitment is discussed. Further, the applications of ChIP assays for discovering novel genes that are dependent on specific transcription factors were addressed.

Key words

ChIP dapk1 IFN-γ C/EBP-β Transcription factor activity Regulation of gene expression DNA–protein interactions Transcription factor recruitment 



This work is supported by NIH grants CA78282 and CA105005 to D.V.K.


  1. 1.
    Fried, M.G. (1989) Measurement of protein-DNA interaction parameters by electrophoresis mobility shift assay. Electrophoresis 10: 366–76.PubMedCrossRefGoogle Scholar
  2. 2.
    Christy, R.J., et al. (1991) CCAAT/enhancer binding protein gene promoter: binding of nuclear factors during differentiation of 3T3-L1 preadipocytes. Proc Natl Acad Sci USA 88: 2593–7.PubMedCrossRefGoogle Scholar
  3. 3.
    Pillai, S., P. Dasgupta, and S.P. Chellappan (2009) Chromatin immunoprecipitation assays: analyzing transcription factor binding and histone modifications in vivo. Methods Mol Biol 523: 323–39.PubMedCrossRefGoogle Scholar
  4. 4.
    Yan, Y., H. Chen, and M. Costa (2004) Chromatin immunoprecipitation assays. Methods Mol Biol 287: 9–19.PubMedGoogle Scholar
  5. 5.
    Nowak, D.E., B. Tian, and A.R. Brasier (2005) Two-step cross-linking method for identification of NF-kappaB gene network by chromatin immunoprecipitation. Biotechniques 39: 715–25.PubMedCrossRefGoogle Scholar
  6. 6.
    Dasgupta, P. and S.P. Chellappan (2007) Chromatin immunoprecipitation assays: molecular analysis of chromatin modification and gene regulation. Methods Mol Biol 383: 135–52.PubMedCrossRefGoogle Scholar
  7. 7.
    Kurdistani, S.K. and M. Grunstein (2003) In vivo protein-protein and protein-DNA crosslinking for genomewide binding microarray. Methods 31: 90–5.PubMedCrossRefGoogle Scholar
  8. 8.
    Breiling, A., et al. (2001) General transcription factors bind promoters repressed by Polycomb group proteins. Nature 412: 651–5.PubMedCrossRefGoogle Scholar
  9. 9.
    Dedon, P.C., et al. (1991) A simplified formaldehyde fixation and immunoprecipitation technique for studying protein-DNA interactions. Anal Biochem 197: 83–90.PubMedCrossRefGoogle Scholar
  10. 10.
    Mukhopadhyay, A., et al. (2008) Chromatin immunoprecipitation (ChIP) coupled to detection by quantitative real-time PCR to study transcription factor binding to DNA in Caenorhabditis elegans. Nat Prot 3: 698–709.CrossRefGoogle Scholar
  11. 11.
    Hassan, M.Q., et al. (2004) Dlx3 transcriptional regulation of osteoblast differentiation: temporal recruitment of Msx2, Dlx3, and Dlx5 homeodomain proteins to chromatin of the osteocalcin gene. Mol Cell Biol 24: 9248–61.PubMedCrossRefGoogle Scholar
  12. 12.
    Botquin, V., et al. (1998) New POU dimer configuration mediates antagonistic control of an osteopontin preimplantation enhancer by Oct-4 and Sox-2. Genes Dev 12: 2073–90.PubMedCrossRefGoogle Scholar
  13. 13.
    Weinmann, A.S. and P.J. Farnham (2002) Identification of unknown target genes of human transcription factors using chromatin immunoprecipitation. Methods 26: 37–47.PubMedCrossRefGoogle Scholar
  14. 14.
    Weinmann, A.S., et al. (2001) Use of chromatin immunoprecipitation to clone novel E2F target promoters. Mol Cell Biol 21: 6820–32.PubMedCrossRefGoogle Scholar
  15. 15.
    Martone, R., et al. (2003) Distribution of NF-kappaB-binding sites across human chromosome 22. Proc Natl Acad Sci USA 100: 12247–52.PubMedCrossRefGoogle Scholar
  16. 16.
    Heckman, C.A. and L.M. Boxer (2002) Allele-specific analysis of transcription factors binding to promoter regions. Methods 26: 19–26.PubMedCrossRefGoogle Scholar
  17. 17.
    Johnson, K.D. and E.H. Bresnick (2002) Dissecting long-range transcriptional mechanisms by chromatin immunoprecipitation. Methods 26: 27–36.PubMedCrossRefGoogle Scholar
  18. 18.
    Cosma, M.P., T. Tanaka, and K. Nasmyth (1999) Ordered recruitment of transcription and chromatin remodeling factors to a cell cycle- and developmentally regulated promoter. Cell 97: 299–311.PubMedCrossRefGoogle Scholar
  19. 19.
    Metivier, R., et al. (2004) Transcriptional complexes engaged by apo-estrogen receptor-alpha isoforms have divergent outcomes. EMBO J 23: 3653–66.PubMedCrossRefGoogle Scholar
  20. 20.
    Flanagin, S., et al. (2008) Microplate-based chromatin immunoprecipitation method, Matrix ChIP: a platform to study signaling of complex genomic events. Nucleic Acids Res 36: e17.PubMedCrossRefGoogle Scholar
  21. 21.
    Dahl, J.A. and P. Collas (2007) Q2ChIP, a quick and quantitative chromatin immunoprecipitation assay, unravels epigenetic dynamics of developmentally regulated genes in human carcinoma cells. Stem Cells 25: 1037–46.PubMedCrossRefGoogle Scholar
  22. 22.
    Gade, P., et al. (2008) Critical role for transcription factor C/EBP-beta in regulating the expression of death-associated protein kinase 1. Mol Cell Biol 28: 2528–48.PubMedCrossRefGoogle Scholar
  23. 23.
    Li, H., et al. (2008) The Med1 subunit of transcriptional mediator plays a central role in regulating CCAAT/enhancer-binding protein-beta-driven transcription in response to interferon-gamma. J Biol Chem, 283: 13077–86.PubMedCrossRefGoogle Scholar
  24. 24.
    Gade, P., et al. (2009) Down-regulation of the transcriptional mediator subunit Med1 contributes to the loss of expression of metastasis-associated dapk1 in human cancers and cancer cells. Int J Cancer 125: 1566–74.PubMedCrossRefGoogle Scholar
  25. 25.
    Orlando, V. (2000) Mapping chromosomal proteins in vivo by formaldehyde-crosslinked-chromatin immunoprecipitation. Trends Biochem Sci 25: 99–104.PubMedCrossRefGoogle Scholar
  26. 26.
    Nguyen, J., et al. (2008) Assessment of sera for chromatin-immunoprecipitation. Biotechniques 44: 66–68.PubMedCrossRefGoogle Scholar
  27. 27.
    Wells, J. and P.J. Farnham (2002) Characterizing transcription factor binding sites using formaldehyde crosslinking and immunoprecipitation. Methods 26: 48–56.PubMedCrossRefGoogle Scholar
  28. 28.
    Nelson, J.D., et al. (2006) Fast chromatin immunoprecipitation assay. Nucleic Acids Res 34: p. e2.Google Scholar
  29. 29.
    Hug, B.A., et al. (2004) A chromatin immunoprecipitation screen reveals protein kinase Cbeta as a direct RUNX1 target gene. J Biol Chem, 279: 825–30.PubMedCrossRefGoogle Scholar
  30. 30.
    Hartman, S.E., et al. (2005) Global changes in STAT target selection and transcription regulation upon interferon treatments. Genes Dev 19: 2953–68.PubMedCrossRefGoogle Scholar
  31. 31.
    Odom, D.T., et al. (2004) Control of pancreas and liver gene expression by HNF transcription factors. Science 303: 1378–81.PubMedCrossRefGoogle Scholar
  32. 32.
    Euskirchen, G., et al. (2004) CREB binds to multiple loci on human chromosome 22. Mol Cell Biol 24: 3804–14.PubMedCrossRefGoogle Scholar
  33. 33.
    Cawley, S., et al. (2004) Unbiased mapping of transcription factor binding sites along human chromosomes 21 and 22 points to widespread regulation of noncoding RNAs. Cell 116: 499–509.PubMedCrossRefGoogle Scholar
  34. 34.
    Kim, J., et al. (2005) Mapping DNA-protein interactions in large genomes by sequence tag analysis of genomic enrichment. Nat Methods, 2: 47–53.PubMedCrossRefGoogle Scholar
  35. 35.
    Impey, S., et al. (2004) Defining the CREB regulon: a genome-wide analysis of transcription factor regulatory regions. Cell 119: 1041–54.PubMedGoogle Scholar
  36. 36.
    Wei, C.L., et al. (2006) A global map of p53 transcription-factor binding sites in the human genome. Cell, 124: 207–19.PubMedCrossRefGoogle Scholar
  37. 37.
    Loh, Y.H., et al. (2006) The Oct4 and Nanog tran-scription network regulates pluripotency in mouse embryonic stem cells. Nat Genet 38: 431–40.PubMedCrossRefGoogle Scholar
  38. 38.
    Bonifacino, J.S. and E.C. Dell’Angelica (2001) Immunoprecipitation. Curr Protoc Cell Biol, Chapter 7: p. Unit 7 2.Google Scholar
  39. 39.
    O’Neill, L.P. and B.M. Turner (2003) Immunoprecipitation of native chromatin: NChIP. Methods 31: 76–82.PubMedCrossRefGoogle Scholar
  40. 40.
    Thorne, A.W., F.A. Myers, and T.R. Hebbes (2004) Native chromatin immunoprecipitation. Methods Mol Biol 287: 21–44.PubMedGoogle Scholar
  41. 41.
    Hebbes, T.R., et al. (1994) Core histone hyperacetylation co-maps with generalized DNase I sensitivity in the chicken beta-globin chromosomal domain. EMBO J, 13: 1823–30.PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  1. 1.Department of Microbiology & Immunology, Greenebaum Cancer CenterUniversity of Maryland School of MedicineBaltimoreUSA

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