Protocol

Plant Signal Transduction

Volume 1363 of the series Methods in Molecular Biology pp 25-35

DNA-Binding Factor Target Identification by Chromatin Immunoprecipitation (ChIP) in Plants

  • David PoséAffiliated withDepartamento de Biología Molecular y Bioquímica, Facultad de Ciencias, Instituto de Hortofruticultura Subtropical y Mediterránea, Universidad de Málaga-Consejo Superior de Investigaciones Científicas Email author 
  • , Levi YantAffiliated withDepartment of Cell and Development Biology, John Innes Centre, Norwich Research park

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Abstract

Chromatin immunoprecipitation (ChIP) allows the precise identification of genomic loci that physically interact with a protein of interest, whether that protein is a transcription factor, a core polymerase, a histone, or other chromatin-associated protein. In short, tissue is first cross-linked to freeze a population of DNA-protein interactions at a stage of interest. Chromatin is then extracted, fragmented, and incubated with a specific antibody against the protein of interest. Next, the resultant DNA-protein complexes are immunoprecipitated and captured using beads that bind to the antibody constant region. Samples are finally reverse cross-linked to separate the bound fragments and the DNA is purified. This DNA is analyzed by quantitative PCR for enrichment of genomic regions expected to be bound by the protein under study. The protocol detailed in this chapter has been successfully applied in the identification of target genes for seven transcriptional regulators of diverse classes involved in Arabidopsis thaliana floral transition.

Key words

Chromatin immunoprecipitation ChIP ChIP-seq ChIP-chip Transcription factor Antibody Direct target