Advertisement

Chromatin Immunoprecipitation Assays: Analyzing Transcription Factor Binding and Histone Modifications In Vivo

  • Smitha Pillai
  • Piyali Dasgupta
  • Srikumar P. ChellappanEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1288)

Abstract

Studies in the past decade have shown that differential gene expression depends not only on the binding of specific transcription factors to discrete promoter elements but also on the epigenetic modification of the DNA as well as histones associated with the promoter. While techniques like electrophoretic mobility shift assays could detect and characterize the binding of specific transcription factors present in cell lysates to DNA sequences in in vitro binding conditions, they were not effective in assessing the binding in intact cells. Development of chromatin immunoprecipitation technique in the past decade enabled the analysis of the association of regulatory molecules with specific promoters or changes in histone modifications in vivo, without overexpressing any component. ChIP assays can provide a snapshot of how a regulatory transcription factor affects the expression of a single gene, or a variety of genes at the same time. Availability of high quality antibodies that recognizes histones modified in a specific fashion further expanded the use of ChIP assays to analyze even minute changes in histone modification and nucleosomes structure. This chapter outlines the general strategies and protocols used to carry out ChIP assays to study the differential recruitment of transcription factors as well as histone modifications.

Key words

Chromatin immunoprecipitation Histone modification Methylation Acetylation 

Notes

Acknowledgements

Work in the Chellappan lab is supported by the grants CA139612 and CA127725 from the NCI.

References

  1. 1.
    Bernstein BE, Humphrey EL, Liu CL, Schreiber SL (2004) The use of chromatin immunoprecipitation assays in genome-wide analyses of histone modifications. Methods Enzymol 376:349–360PubMedCrossRefGoogle Scholar
  2. 2.
    Kirmizis A, Farnham PJ (2004) Genomic approaches that aid in the identification of transcription factor target genes. Exp Biol Med (Maywood) 229(8):705–721Google Scholar
  3. 3.
    Johnson KD, Bresnick EH (2002) Dissecting long-range transcriptional mechanisms by chromatin immunoprecipitation. Methods 26(1):27–36PubMedCrossRefGoogle Scholar
  4. 4.
    Umlauf D, Goto Y, Feil R (2004) Site-specific analysis of histone methylation and acetylation. Methods Mol Biol 287:99–120PubMedGoogle Scholar
  5. 5.
    Kouzarides T (2002) Histone methylation in transcriptional control. Curr Opin Genet Dev 12(2):198–209PubMedCrossRefGoogle Scholar
  6. 6.
    Spencer VA, Sun JM, Li L, Davie JR (2003) Chromatin immunoprecipitation: a tool for studying histone acetylation and transcription factor binding. Methods 31(1):67–75PubMedCrossRefGoogle Scholar
  7. 7.
    Stallcup MR (2001) Role of protein methylation in chromatin remodeling and transcriptional regulation. Oncogene 20(24):3014–3020PubMedCrossRefGoogle Scholar
  8. 8.
    Weinmann AS, Farnham PJ (2002) Identification of unknown target genes of human transcription factors using chromatin immunoprecipitation. Methods 26(1):37–47PubMedCrossRefGoogle Scholar
  9. 9.
    Kondo Y, Shen L, Yan PS, Huang TH, Issa JP (2004) Chromatin immunoprecipitation microarrays for identification of genes silenced by histone H3 lysine 9 methylation. Proc Natl Acad Sci U S A 101(19):7398–7403PubMedCentralPubMedCrossRefGoogle Scholar
  10. 10.
    Dasgupta P, Chellappan SP (2005) Chromatin immunoprecipitation assays: molecular analysis of chromatin modification and gene regulation. Humana Press, Totowa, NJGoogle Scholar
  11. 11.
    Pillai S, Dasgupta P, Chellappan SP (2009) Chromatin immunoprecipitation assays: analyzing transcription factor binding and histone modifications in vivo. Methods Mol Biol 523:323–339PubMedCrossRefGoogle Scholar
  12. 12.
    Wells J, Farnham PJ (2002) Characterizing transcription factor binding sites using formaldehyde crosslinking and immunoprecipitation. Methods 26(1):48–56PubMedCrossRefGoogle Scholar
  13. 13.
    Ren B, Dynlacht BD (2004) Use of chromatin immunoprecipitation assays in genome-wide location analysis of mammalian transcription factors. Methods Enzymol 376:304–315PubMedCrossRefGoogle Scholar
  14. 14.
    Wang S, Fusaro G, Padmanabhan J, Chellappan SP (2002) Prohibitin co-localizes with Rb in the nucleus and recruits N-CoR and HDAC1 for transcriptional repression. Oncogene 21(55):8388–8396PubMedCrossRefGoogle Scholar
  15. 15.
    Thorne AW, Myers FA, Hebbes TR (2004) Native chromatin immunoprecipitation. Methods Mol Biol 287:21–44PubMedGoogle Scholar
  16. 16.
    Cosseau C, Grunau C (2011) Native chromatin immunoprecipitation. Methods Mol Biol 791:195–212PubMedCrossRefGoogle Scholar
  17. 17.
    Dasgupta P, Sun J, Wang S, Fusaro G, Betts V, Padmanabhan J, Sebti SM, Chellappan SP (2004) Disruption of the Rb-Raf-1 interaction inhibits tumor growth and angiogenesis. Mol Cell Biol 24(21):9527–9541PubMedCentralPubMedCrossRefGoogle Scholar
  18. 18.
    Dasgupta P, Betts V, Rastogi S, Joshi B, Morris M, Brennan B, Ordonez-Ercan D, Chellappan S (2004) Direct binding of apoptosis signal-regulating kinase 1 to retinoblastoma protein: novel links between apoptotic signaling and cell cycle machinery. J Biol Chem 279(37):38762–38769PubMedCrossRefGoogle Scholar
  19. 19.
    Joshi B, Ordonez-Ercan D, Dasgupta P, Chellappan S (2005) Induction of human metallothionein 1 g promoter by VEGF and heavy metals: differential involvement of E2F and MTF transcription factors. Oncogene 24:2204–2217PubMedCrossRefGoogle Scholar
  20. 20.
    Hebbes TR, Clayton AL, Thorne AW, Crane-Robinson C (1994) Core histone hyperacetylation co-maps with generalized DNase I sensitivity in the chicken beta-globin chromosomal domain. EMBO J 13(8):1823–1830PubMedCentralPubMedGoogle Scholar
  21. 21.
    Litt MD, Simpson M, Recillas-Targa F, Prioleau MN, Felsenfeld G (2001) Transitions in histone acetylation reveal boundaries of three separately regulated neighboring loci. EMBO J 20(9):2224–2235PubMedCentralPubMedCrossRefGoogle Scholar
  22. 22.
    Buck MJ, Lieb JD (2004) ChIP-chip: considerations for the design, analysis, and application of genome-wide chromatin immunoprecipitation experiments. Genomics 83(3):349–360PubMedCrossRefGoogle Scholar
  23. 23.
    Robyr D, Grunstein M (2003) Genomewide histone acetylation microarrays. Methods 31(1):83–89PubMedCrossRefGoogle Scholar
  24. 24.
    Roh TY, Ngau WC, Cui K, Landsman D, Zhao K (2004) High-resolution genome-wide mapping of histone modifications. Nat Biotechnol 22(8):1013–1016PubMedCrossRefGoogle Scholar
  25. 25.
    Rodriguez BA, Huang TH (2005) Tilling the chromatin landscape: emerging methods for the discovery and profiling of protein-DNA interactions. Biochem Cell Biol 83(4):525–534PubMedCrossRefGoogle Scholar
  26. 26.
    Oberley MJ, Tsao J, Yau P, Farnham PJ (2004) High-throughput screening of chromatin immunoprecipitates using CpG-island microarrays. Methods Enzymol 376:315–334PubMedCrossRefGoogle Scholar
  27. 27.
    Bieda M, Xu X, Singer MA, Green R, Farnham PJ (2006) Unbiased location analysis of E2F1-binding sites suggests a widespread role for E2F1 in the human genome. Genome Res 16(5):595–605PubMedCentralPubMedCrossRefGoogle Scholar
  28. 28.
    Jin VX, Rabinovich A, Squazzo SL, Green R, Farnham PJ (2006) A computational genomics approach to identify cis-regulatory modules from chromatin immunoprecipitation microarray data–a case study using E2F1. Genome Res 16(12):1585–1595PubMedCentralPubMedCrossRefGoogle Scholar
  29. 29.
    Fusaro G, Dasgupta P, Rastogi S, Joshi B, Chellappan SP (2003) Prohibitin induces the transcriptional activity of p53 and is exported from the nucleus upon apoptotic signaling. J Biol Chem 278:47853–47861PubMedCrossRefGoogle Scholar
  30. 30.
    Strahl-Bolsinger S, Hecht A, Luo K, Grunstein M (1997) SIR2 and SIR4 interactions differ in core and extended telomeric heterochromatin in yeast. Genes Dev 11(1):83–93PubMedCrossRefGoogle Scholar
  31. 31.
    Boyd KE, Wells J, Gutman J, Bartley SM, Farnham PJ (1998) c-Myc target gene specificity is determined by a post-DNAbinding mechanism. Proc Natl Acad Sci U S A 95(23):13887–13892PubMedCentralPubMedCrossRefGoogle Scholar
  32. 32.
    Boyd KE, Farnham PJ (1999) Coexamination of site-specific transcription factor binding and promoter activity in living cells. Mol Cell Biol 19(12):8393–8399PubMedCentralPubMedGoogle Scholar
  33. 33.
    Dahl JA, Collas P (2007) Q2ChIP, a quick and quantitative chromatin immunoprecipitation assay, unravels epigenetic dynamics of developmentally regulated genes in human carcinoma cells. Stem Cells (Dayton, Ohio) 25(4):1037–1046CrossRefGoogle Scholar
  34. 34.
    Farnham PJ (2002) In vivo assays to examine transcription factor localization and target gene specificity. Methods 26(1):1–2PubMedCrossRefGoogle Scholar
  35. 35.
    Wells J, Graveel CR, Bartley SM, Madore SJ, Farnham PJ (2002) The identification of E2F1-specific target genes. Proc Natl Acad Sci U S A 99(6):3890–3895PubMedCentralPubMedCrossRefGoogle Scholar
  36. 36.
    Kirmizis A, Bartley SM, Kuzmichev A, Margueron R, Reinberg D, Green R, Farnham PJ (2004) Silencing of human polycomb target genes is associated with methylation of histone H3 Lys 27. Genes Dev 18(13):1592–1605PubMedCentralPubMedCrossRefGoogle Scholar
  37. 37.
    Savic D, Gertz J, Jain P, Cooper GM, Myers RM (2013) Mapping genome-wide transcription factor binding sites in frozen tissues. Epigenetics Chromatin 6(1):30PubMedCentralPubMedCrossRefGoogle Scholar
  38. 38.
    Visel A, Blow MJ, Li Z, Zhang T, Akiyama JA, Holt A, Plajzer-Frick I, Shoukry M, Wright C, Chen F et al (2009) ChIP-seq accurately predicts tissue-specific activity of enhancers. Nature 457(7231):854–858PubMedCentralPubMedCrossRefGoogle Scholar
  39. 39.
    Ross-Innes CS, Stark R, Teschendorff AE, Holmes KA, Ali HR, Dunning MJ, Brown GD, Gojis O, Ellis IO, Green AR et al (2012) Differential oestrogen receptor binding is associated with clinical outcome in breast cancer. Nature 481(7381):389–393PubMedCentralPubMedGoogle Scholar
  40. 40.
    Chaya D, Zaret KS (2004) Sequential chromatin immunoprecipitation from animal tissues. Methods Enzymol 376:361–372PubMedCrossRefGoogle Scholar
  41. 41.
    Forsberg EC, Downs KM, Bresnick EH (2000) Direct interaction of NF-E2 with hypersensitive site 2 of the beta-globin locus control region in living cells. Blood 96(1):334–339PubMedGoogle Scholar
  42. 42.
    Luo RX, Dean DC (1999) Chromatin remodeling and transcriptional regulation. J Natl Cancer Inst 91(15):1288–1294PubMedCrossRefGoogle Scholar
  43. 43.
    Im H, Grass JA, Johnson KD, Boyer ME, Wu J, Bresnick EH (2004) Measurement of protein-DNA interactions in vivo by chromatin immunoprecipitation. Methods Mol Biol 284:129–146PubMedGoogle Scholar
  44. 44.
    Blais A, Dynlacht BD (2004) Hitting their targets: an emerging picture of E2F and cell cycle control. Curr Opin Genet Dev 14(5):527–532PubMedCrossRefGoogle Scholar
  45. 45.
    Harbour JW, Dean DC (2000) Chromatin remodeling and Rb activity. Curr Opin Cell Biol 12(6):685–689PubMedCrossRefGoogle Scholar
  46. 46.
    Harbour JW, Dean DC (2001) Corepressors and retinoblastoma protein function. Curr Top Microbiol Immunol 254:137–144PubMedGoogle Scholar
  47. 47.
    Skowronska-Krawczyk D, Ballivet M, Dynlacht BD, Matter JM (2004) Highly specific interactions between bHLH transcription factors and chromatin during retina development. Development 131(18):4447–4454PubMedCrossRefGoogle Scholar
  48. 48.
    Elefant F, Cooke NE, Liebhaber SA (2000) Targeted recruitment of histone acetyltransferase activity to a locus control region. J Biol Chem 275(18):13827–13834PubMedCrossRefGoogle Scholar
  49. 49.
    Johnson KD, Christensen HM, Zhao B, Bresnick EH (2001) Distinct mechanisms control RNA polymerase II recruitment to a tissue-specific locus control region and a downstream promoter. Mol Cell 8(2):465–471PubMedCrossRefGoogle Scholar
  50. 50.
    Nielsen SJ, Schneider R, Bauer UM, Bannister AJ, Morrison A, O’Carroll D, Firestein R, Cleary M, Jenuwein T, Herrera RE et al (2001) Rb targets histone H3 methylation and HP1 to promoters. Nature 412(6846):561–565PubMedCrossRefGoogle Scholar
  51. 51.
    Ren B, Cam H, Takahashi Y, Volkert T, Terragni J, Young RA, Dynlacht BD (2002) E2F integrates cell cycle progression with DNA repair, replication, and G(2)/M checkpoints. Genes Dev 16(2):245–256PubMedCentralPubMedCrossRefGoogle Scholar
  52. 52.
    Dahl JA, Collas P (2008) MicroChIP–a rapid micro chromatin immunoprecipitation assay for small cell samples and biopsies. Nucleic Acids Res 36(3):e15PubMedCentralPubMedCrossRefGoogle Scholar
  53. 53.
    Dahl JA, Collas P (2008) A rapid micro chromatin immunoprecipitation assay (microChIP). Nat Protoc 3(6):1032–1045PubMedCrossRefGoogle Scholar
  54. 54.
    Collas P (2011) A chromatin immunoprecipitation protocol for small cell numbers. Methods Mol Biol 791:179–193PubMedCrossRefGoogle Scholar
  55. 55.
    Morimoto RI (2002) Dynamic remodeling of transcription complexes by molecular chaperones. Cell 110(3):281–284PubMedCrossRefGoogle Scholar
  56. 56.
    Christova R (2013) Detecting DNA-protein interactions in living cells-ChIP approach. Adv Protein Chem Struct Biol 91:101–133PubMedCrossRefGoogle Scholar
  57. 57.
    Shang Y, Hu X, DiRenzo J, Lazar MA, Brown M (2000) Cofactor dynamics and sufficiency in estrogen receptor-regulated transcription. Cell 103(6):843–852PubMedCrossRefGoogle Scholar
  58. 58.
    Tse C, Sera T, Wolffe AP, Hansen JC (1998) Disruption of higher-order folding by core histone acetylation dramatically enhances transcription of nucleosomal arrays by RNA polymerase III. Mol Cell Biol 18(8):4629–4638PubMedCentralPubMedGoogle Scholar
  59. 59.
    Nelson JD, Denisenko O, Bomsztyk K (2006) Protocol for the fast chromatin immunoprecipitation (ChIP) method. Nat Protoc 1(1):179–185PubMedCrossRefGoogle Scholar
  60. 60.
    Zhang Q, Lei X, Lu H (2014) Alterations of epigenetic signatures in hepatocyte nuclear factor 4alpha deficient mouse liver determined by improved ChIP-qPCR and (h)MeDIP-qPCR assays. PLoS One 9(1):e84925PubMedCentralPubMedCrossRefGoogle Scholar
  61. 61.
    Noma K, Allis CD, Grewal SI (2001) Transitions in distinct histone H3 methylation patterns at the heterochromatin domain boundaries. Science 293(5532):1150–1155PubMedCrossRefGoogle Scholar
  62. 62.
    Gregory RI, Randall TE, Johnson CA, Khosla S, Hatada I, O'Neill LP, Turner BM, Feil R (2001) DNA methylation is linked to deacetylation of histone H3, but not H4, on the imprinted genes Snrpn and U2af1-rs1. Mol Cell Biol 21(16):5426–5436PubMedCentralPubMedCrossRefGoogle Scholar
  63. 63.
    Gregory RI, Feil R (1999) Analysis of chromatin in limited numbers of cells: a PCR-SSCP based assay of allele-specific nuclease sensitivity. Nucleic Acids Res 27(22):e32PubMedCentralPubMedCrossRefGoogle Scholar
  64. 64.
    Orita M, Iwahana H, Kanazawa H, Hayashi K, Sekiya T (1989) Detection of polymorphisms of human DNA by gel electrophoresis as single-strand conformation polymorphisms. Proc Natl Acad Sci U S A 86(8):2766–2770PubMedCentralPubMedCrossRefGoogle Scholar
  65. 65.
    Uejima H, Lee MP, Cui H, Feinberg AP (2000) Hot-stop PCR: a simple and general assay for linear quantitation of allele ratios. Nat Genet 25(4):375–376PubMedCrossRefGoogle Scholar
  66. 66.
    Weinmann AS, Bartley SM, Zhang T, Zhang MQ, Farnham PJ (2001) Use of chromatin immunoprecipitation to clone novel E2F target promoters. Mol Cell Biol 21(20):6820–6832PubMedCentralPubMedCrossRefGoogle Scholar
  67. 67.
    Furlan-Magaril M, Rincon-Arano H, Recillas-Targa F (2009) Sequential chromatin immunoprecipitation protocol: ChIP-reChIP. Methods Mol Biol 543:253–266PubMedCrossRefGoogle Scholar
  68. 68.
    Brookes E, de Santiago I, Hebenstreit D, Morris KJ, Carroll T, Xie SQ, Stock JK, Heidemann M, Eick D, Nozaki N et al (2012) Polycomb associates genome-wide with a specific RNA polymerase II variant, and regulates metabolic genes in ESCs. Cell Stem Cell 10(2):157–170PubMedCentralPubMedCrossRefGoogle Scholar
  69. 69.
    de Medeiros RB (2011) Sequential chromatin immunoprecipitation assay and analysis. Methods Mol Biol 791:225–237PubMedCrossRefGoogle Scholar
  70. 70.
    Pillai S, Kovacs M, Chellappan S (2010) Regulation of vascular endothelial growth factor receptors by Rb and E2F1: role of acetylation. Cancer Res 70(12):4931–4940PubMedCentralPubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Smitha Pillai
    • 1
  • Piyali Dasgupta
    • 1
    • 2
  • Srikumar P. Chellappan
    • 1
    Email author
  1. 1.Department of Tumor BiologyH. Lee Moffitt Cancer Center and Research InstituteTampaUSA
  2. 2.Department of Pharmacology, Physiology, and ToxicologyMarshall UniversityHuntingtonUSA

Personalised recommendations