Genome-Wide Mapping of the Binding Sites of Proteins That Interact with DNA

  • Stephen SpiroEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 881)


The coordinated regulation of the expression of a group of genes by a specific transcription factor frequently lies at the heart of the ability of a bacterium to respond to an environmental signal, or to progress through a developmental program. Thus, in many situations, it is of interest to identify all of the genes that are under the control of a particular regulatory protein. This chapter begins with a brief overview of some of the methods that have been used in attempts to identify some or all of the members of a regulon (i.e., those genes that are the targets for a transcriptional activator or repressor). Thereafter, the chapter will focus on one technique, chromatin immunoprecipitation and microarray analysis (ChIP-chip) and some of its variants. Design considerations and some protocols for ChIP-chip experiments are provided, along with some considerations related to downstream data analysis.

ChIP-chip is a method for the genome-wide localization of protein-binding sites. In a typical ChIP-chip protocol, proteins are cross-linked nonspecifically to DNA in vivo. Chromatin is extracted and sheared, and specific protein–DNA complexes are immunoprecipitated with a suitable antibody. After purification, the DNA is hybridized to a microarray (after an amplification step in some protocols), together with a differentially labeled reference sample. Features on the microarray that show an elevated fluorescence ratio reveal DNA sequences that were enriched by immunoprecipitation. The corresponding genomic locations are those that were enriched, and are therefore close to sites of binding. The use of high-density tiled microarrays allows for binding site localization with quite high resolution. It is likely that ChIP-chip will soon be superseded by ChIP-seq, in which the immunoprecipitated DNA is analyzed directly by next-generation sequencing technologies. ChIP-chip and ChIP-seq applications are not confined to regulatory proteins, since they can be used with any protein that binds to DNA, either directly, or indirectly via an interaction with another protein. Thus, ChIP-chip has been used successfully to map binding sites for nucleoid proteins, and proteins involved in DNA replication.

Key words

Chromatin immunoprecipitation and microarray analysis ChIP-chip DNA-binding proteins 



Research in the author’s laboratory is supported by the National Science Foundation through award MCB-1020470.


  1. 1.
    Kenyon CJ, Walker GC (1980) DNA-damaging agents stimulate gene expression at specific loci in Escherichia coli. Proc Natl Acad Sci U S A 77:2819–2823PubMedCrossRefGoogle Scholar
  2. 2.
    Stojiljkovic I, Baumler A, Hantke K (1994) Fur regulon in gram-negative bacteria. Identification and characterization of new iron-regulated Escherichia coli genes by a Fur titration assay. J Mol Biol 236:531–545PubMedCrossRefGoogle Scholar
  3. 3.
    Mathiopoulos C, Sonenshein AL (1989) Identification of Bacillus subtilis genes expressed early during sporulation. Mol Microbiol 3:1071–1081PubMedCrossRefGoogle Scholar
  4. 4.
    O'Farrell PH (2008) The pre-omics era: the early days of two-dimensional gels. Proteomics 8:4842–4852PubMedCrossRefGoogle Scholar
  5. 5.
    Hoskisson PA, Hobbs G (2005) Continuous culture—making a comeback? Microbiology 151:3153–3159PubMedCrossRefGoogle Scholar
  6. 6.
    Nonaka G, Blankschien M, Herman C, Gross CA, Rhodius VA (2006) Regulon and promoter analysis of the E. coli heat-shock factor, σ32, reveals a multifaceted cellular response to heat stress. Genes Dev 20:1776–1789PubMedCrossRefGoogle Scholar
  7. 7.
    MacLellan SR, Eiamphungporn W, Helmann JD (2009) ROMA: an in vitro approach to defining target genes for transcription regulators. Methods 47:73–77PubMedCrossRefGoogle Scholar
  8. 8.
    Cao M, Kobel PA, Morshedi MM, Wu MFW, Peddon C, Helmann JD (2002) Defining the Bacillus subtilis σW regulon: a comparative analysis of promoter consensus search, run-off transcription/macroarray analysis (ROMA), and transcriptional profiling approaches. J Mol Biol 316:443–457PubMedCrossRefGoogle Scholar
  9. 9.
    Zheng D, Constantinidou C, Hobman JL, Minchin SD (2004) Identification of the CRP regulon using in vitro and in vivo transcriptional profiling. Nucleic Acids Res 32:5874–5893PubMedCrossRefGoogle Scholar
  10. 10.
    Petrova OE, Sauer K (2010) The novel two-component regulatory system BfiSR regulates biofilm development by controlling the small RNA rsmZ through CafA. J Bacteriol 192:5275–5288PubMedCrossRefGoogle Scholar
  11. 11.
    Liu X, Noll DM, Lieb JD, Clarke ND (2005) DIP-chip: rapid and accurate determination of DNA-binding specificity. Genome Res 15:421–427PubMedCrossRefGoogle Scholar
  12. 12.
    Butala M, Busby SJW, Lee DJ (2009) DNA sampling: a method for probing protein binding at specific loci on bacterial chromosomes. Nucleic Acids Res 37:e37PubMedCrossRefGoogle Scholar
  13. 13.
    Vora T, Hottes AK, Tavazoie S (2009) Protein occupancy landscape of a bacterial genome. Mol Cell 35:247–253PubMedCrossRefGoogle Scholar
  14. 14.
    Munch R, Hiller K, Grote A, Scheer M, Klein J, Schobert M, Jahn D (2005) Virtual Footprint and PRODORIC: an integrative framework for regulon prediction in prokaryotes. Bioinformatics 21:4187–4189PubMedCrossRefGoogle Scholar
  15. 15.
    Rodionov DA (2007) Comparative genomic reconstruction of transcriptional regulatory networks in bacteria. Chem Rev 107:3467–3497PubMedCrossRefGoogle Scholar
  16. 16.
    Rodionov DA, Dubchak IL, Arkin AP, Alm EJ, Gelfand MS (2005) Dissimilatory metabolism of nitrogen oxides in bacteria: comparative reconstruction of transcriptional networks. PLoS Comput Biol 1:e55PubMedCrossRefGoogle Scholar
  17. 17.
    Bodenmiller DM, Spiro S (2006) The yjeB (nsrR) gene of Escherichia coli encodes a nitric oxide sensitive transcriptional regulator. J Bacteriol 188:874–881PubMedCrossRefGoogle Scholar
  18. 18.
    Pavesi G, Mereghetti P, Mauri G, Pesole G (2004) Weeder Web: discovery of transcription factor binding sites in a set of sequences from co-regulated genes. Nucleic Acids Res 32:W199–W203PubMedCrossRefGoogle Scholar
  19. 19.
    Bailey TL, Williams N, Misleh C, Li WW (2006) MEME: discovering and analyzing DNA and protein sequence motifs. Nucleic Acids Res 34:W369–W373PubMedCrossRefGoogle Scholar
  20. 20.
    Molle V, Fujita M, Jensen ST, Eichenberger P, Gonzalez-Pastor JE, Liu JS, Losick R (2003) The Spo0A regulon of Bacillus subtilis. Mol Microbiol 50:1683–1701PubMedCrossRefGoogle Scholar
  21. 21.
    Dufour YS, Kiley PJ, Donohue TJ (2010) Reconstruction of the core and extended regulons of global transcription factors. PLoS Genet 6:e1001027PubMedCrossRefGoogle Scholar
  22. 22.
    Navarre WW, Porwollik S, Wang Y, McClelland M, Rosen H, Libby SJ, Fang FC (2006) Selective silencing of foreign DNA with low GC content by the H-NS protein in Salmonella. Science 313:236–238PubMedCrossRefGoogle Scholar
  23. 23.
    Lucchini S, Rowley G, Goldberg MD, Hurd D, Harrison M, Hinton JCD (2006) H-NS mediates the silencing of laterally acquired genes in bacteria. PLoS Pathog 2:e81PubMedCrossRefGoogle Scholar
  24. 24.
    Grainger DC, Hurd D, Goldberg MD, Busby SJW (2006) Association of nucleoid proteins with coding and non-coding segments of the Escherichia coli genome. Nucleic Acids Res 34:4642–4652PubMedCrossRefGoogle Scholar
  25. 25.
    Breier AM, Grossman AD (2007) Whole-genome analysis of the chromosome partitioning and sporulation protein Spo0J (ParB) reveals spreading and origin-distal sites on the Bacillus subtilis chromosome. Mol Microbiol 64:703–718PubMedCrossRefGoogle Scholar
  26. 26.
    Breier AM, Grossman AD (2009) Dynamic association of the replication initiator and transcription factor DnaA with the Bacillus subtilis chromosome during replication stress. J Bacteriol 191:486–493PubMedCrossRefGoogle Scholar
  27. 27.
    Grainger DC, Hurd D, Harrison M, Holdstock J, Busby SJW (2005) Studies of the distribution of Escherichia coli cAMP-receptor protein and RNA polymerase along the E. coli chromosome. Proc Natl Acad Sci U S A 102:17693–17698PubMedCrossRefGoogle Scholar
  28. 28.
    Dufour YS, Wesenberg GE, Tritt AJ, Glasner JD, Perna NT, Mitchell JC, Donohue TJ (2010) chipD: a web tool to design oligonucleotide probes for high-density tiling arrays. Nucleic Acids Res 38:W321–W325PubMedCrossRefGoogle Scholar
  29. 29.
    Liu ET, Pott S, Huss M (2010) Q&A: ChIP-seq technologies and the study of gene regulation. BMC Biol 8:56PubMedCrossRefGoogle Scholar
  30. 30.
    Park PJ (2009) ChIP-seq: advantages and challenges of a maturing technology. Nat Rev Genet 10:669–680PubMedCrossRefGoogle Scholar
  31. 31.
    Raha D, Hong M, Snyder M (2010) ChIP-Seq: a method for global identification of regulatory elements in the genome. Curr Protoc Mol Biol Chapter 21:Unit 21.19.1-14Google Scholar
  32. 32.
    Datsenko K, Wanner B (2000) One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci U S A 97:6640–6645PubMedCrossRefGoogle Scholar
  33. 33.
    Uzzau S, Figueroa-Bossi N, Rubino S, Bossi L (2001) Epitope tagging of chromosomal genes in Salmonella. Proc Natl Acad Sci U S A 98:15264–15269PubMedCrossRefGoogle Scholar
  34. 34.
    Vine CE, Justino MC, Saraiva LM, Cole J (2010) Detection by whole genome microarrays of a spontaneous 126-gene deletion during construction of a ytfE mutant: confirmation that a ytfE mutation results in loss of repair of iron-sulfur centres in proteins damaged by oxidative or nitrosative stress. J Microbiol Methods 81:77–79PubMedCrossRefGoogle Scholar
  35. 35.
    Lee DJ, Bingle LEH, Heurlier K, Pallen MJ, Penn CW, Busby SJW, Hobman JL (2009) Gene doctoring: a method for recombineering in laboratory and pathogenic Escherichia coli strains. BMC Microbiol 9:252PubMedCrossRefGoogle Scholar
  36. 36.
    Nobile CJ, Nett JE, Hernday AD, Homann OR, Deneault J-S, Nantel A, Andes DR, Mitchell AP (2009) Biofilm matrix regulation by Candida albicans Zap1. PLoS Biol 7:e1000133PubMedCrossRefGoogle Scholar
  37. 37.
    Buck MJ, Lieb JD (2004) ChIP-chip: considerations for the design, analysis, and application of genome-wide chromatin immunoprecipitation experiments. Genomics 83:349–360PubMedCrossRefGoogle Scholar
  38. 38.
    Waldminghaus T, Skarstad K (2010) ChIP on Chip: surprising results are often artifacts. BMC Genomics 11:414PubMedCrossRefGoogle Scholar
  39. 39.
    Partridge JD, Bodenmiller DM, Humphrys MS, Spiro S (2009) NsrR targets in the Escherichia coli genome: new insights into DNA sequence requirements for binding and a role for NsrR in the regulation of motility. Mol Microbiol 73:680–694PubMedCrossRefGoogle Scholar
  40. 40.
    Efromovich S, Grainger D, Bodenmiller D, Spiro S (2008) Genome-wide identification of binding sites for the nitric oxide-sensitive transcriptional regulator NsrR. Methods Enzymol 437:211–233PubMedCrossRefGoogle Scholar
  41. 41.
    Grainger DC, Aiba H, Hurd D, Browning DF, Busby SJW (2006) Transcription factor distribution in Escherichia coli: studies with FNR protein. Nucl Acids Res 35:269–278PubMedCrossRefGoogle Scholar
  42. 42.
    Wade JT, Roa DC, Grainger DC, Hurd D, Busby SJW, Struhl K, Nudler E (2006) Extensive functional overlap between σ factors in Escherichia coli. Nat Struct Mol Biol 13:806–814PubMedCrossRefGoogle Scholar
  43. 43.
    Buck MJ, Nobel AB, Lieb JD (2005) ChIPOTle: a user-friendly tool for the analysis of ChIP-chip data. Genome Biol 6:R97PubMedCrossRefGoogle Scholar
  44. 44.
    Bieda M, Xu X, Singer M, Green R, Farnham PJ (2006) Unbiased location analysis of E2F1-binding sites suggests a widespread role for E2F1 in the human genome. Genome Res 16:595–605PubMedCrossRefGoogle Scholar
  45. 45.
    Wade JT, Struhl K, Busby SJW, Grainger DC (2007) Genomic analysis of protein-DNA interactions in bacteria: insights into transcription and chromosome organization. Mol Microbiol 65(1):21–26PubMedCrossRefGoogle Scholar
  46. 46.
    Tompa M, Li N, Bailey TL, Church GM, De Moor B, Eskin E, Favorov AV, Frith MC, Fu Y, Kent WJ, Makeev VJ, Mironov AA, Noble WS, Pavesi G, Pesole G, Regnier M, Simonis M, Sinha S, Thiis G, Van Helden J, Vandenbogaert M, Weng Z, Workman C, Ye C, Zhu Z (2005) Assessing computational tools for the discovery of transcription factor binding sites. Nat Biotechnol 23:137–144PubMedCrossRefGoogle Scholar
  47. 47.
    D'Haeseleer P (2006) How does DNA sequence motif discovery work? Nat Biotechnol 24:959–961PubMedCrossRefGoogle Scholar
  48. 48.
    Hu J, Li B, Kihara D (2005) Limitations and potentials of current motif discovery algorithms. Nucleic Acids Res 33:4899–4913PubMedCrossRefGoogle Scholar
  49. 49.
    Dufour YS, Landick R, Donohue TJ (2008) Organization and evolution of the biological response to singlet oxygen stress. J Mol Biol 383:713–730PubMedCrossRefGoogle Scholar
  50. 50.
    Pepke S, Wold B, Mortazavi A (2009) Computation for ChIP-seq and RNA-seq studies. Nat Methods 6:S22–S32PubMedCrossRefGoogle Scholar
  51. 51.
    Wilbanks EG, Facciotti MT (2010) Evaluation of algorithm performance in ChIP-seq peak detection. PLoS One 5:e11471PubMedCrossRefGoogle Scholar
  52. 52.
    Rhodius VA, Wade JT (2009) Technical considerations in using DNA microarrays to define regulons. Methods 47:63–72PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  1. 1.Department of Molecular and Cell BiologyUniversity of Texas at DallasRichardsonUSA

Personalised recommendations