title: DNA-Binding Factor Target Identification by Chromatin Immunoprecipitation (ChIP) in Plants.
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.
authors.affiliation: Departamento de Biología Molecular y Bioquímica, Facultad de Ciencias, Instituto de Hortofruticultura Subtropical y Mediterránea, Universidad de Málaga-Consejo Superior de Investigaciones Científicas, 29071, Málaga, Spain. dpose@uma.es.
authors.forename: David
authors.initials: D
authors.lastname: Posé
authors.affiliation: Department of Cell and Development Biology, John Innes Centre, Norwich Research park, Norwich, NR4 7UH, UK.
authors.forename: Levi
authors.initials: L
authors.lastname: Yant
mesh_terms: D001665:Binding Sites; D002843:Chromatin / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D047369:Chromatin Immunoprecipitation / Q000379:methods*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D010944:Plants / Q000235:genetics* / Q000378:metabolism*
chemical_list: D002843:Chromatin; D004268:DNA-Binding Proteins
keywords: Antibody; ChIP; ChIP-chip; ChIP-seq; Chromatin immunoprecipitation; Direct target; Transcription factor