Genomewide Identification of Protein Binding Locations Using Chromatin Immunoprecipitation Coupled with Microarray
Interactions between cis-acting elements and proteins play a key role in transcriptional regulation of all known organisms. To better understand these interactions, researchers developed a method that couples chromatin immunoprecipitation with microarrays (also known as ChIP-chip), which is capable of providing a whole-genome map of protein-DNA interactions. This versatile and high-throughput strategy is initiated by formaldehyde-mediated cross-linking of DNA and proteins, followed by cell lysis, DNA fragmentation, and immunopurification. The immunoprecipitated DNA fragments are then purified from the proteins by reverse-cross-linking followed by amplification, labeling, and hybridization to a whole-genome tiling microarray against a reference sample. The enriched signals obtained from the microarray then are normalized by the reference sample and used to generate the whole-genome map of protein-DNA interactions. The protocol described here has been used for discovering the genomewide distribution of RNA polymerase and several transcription factors of Escherichia coli.
KeywordsChIP-chip chromatin immunoprecipitation microarray RNA polymerase transcription factor transcription factor binding
The protocol described here was based on previous work by many other research groups in this field. The pioneers in this field are Dr. Young's group at MIT, Dr. Lieb's group at the University of North Carolina, Dr. Grunstein's group at Yale University, Dr. Ren's group at UCSD, and others. We thank anyone whose work was not referenced in here. This work is supported by NIH research grant no. GM62791.
- 4.4. Lee TI, Rinaldi NJ, Robert F, Odom DT, Bar-Joseph Z, Gerber GK, Hannett NM, Harbison CT, Thompson CM, Simon I, Zeitlinger J, Jennings EG, Murray HL, Gordon DB, Ren B, Wyrick JJ, Tagne J, Volkert TL, Fraenkel E, Gifford DK, Young RA (2002) Transcriptional regulatory networks in Saccharomyces cerevisiae. Science 298:799–804PubMedCrossRefGoogle Scholar
- 13.Wade JT, Roa DC, Grainger DC, Hurd D, Busby JW, Struhl K, Nudler E (2006) Extensive functional overlap between σ factors in Escherichia coli. Nat Struct Mol Biol. doi: 10.1038/ nsmb1130Google Scholar