title: Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo.
abstract: Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and silencing of genes mediate the response of plants to environmental signals and developmental cues. Therefore, insights into the mechanisms that control plant gene expression are essential to gain a deep understanding of how biological processes are regulated in plants. The chromatin immunoprecipitation (ChIP) technique described here is a procedure to identify the DNA-binding sites of proteins in genes or genomic regions of the model species Arabidopsis thaliana. The interactions with DNA of proteins of interest such as transcription factors, chromatin proteins or posttranslationally modified versions of histones can be efficiently analyzed with the ChIP protocol. This method is based on the fixation of protein-DNA interactions in vivo, random fragmentation of chromatin, immunoprecipitation of protein-DNA complexes with specific antibodies, and quantification of the DNA associated with the protein of interest by PCR techniques. The use of this methodology in Arabidopsis has contributed significantly to unveil transcriptional regulatory mechanisms that control a variety of plant biological processes. This approach allowed the identification of the binding sites of the Arabidopsis chromatin protein EBS to regulatory regions of the master gene of flowering FT. The impact of this protein in the accumulation of particular histone marks in the genomic region of FT was also revealed through ChIP analysis.
authors: Centro de Biotecnología y Genómica de Plantas (CBGP), Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA)-Universidad Politécnica de Madrid.
authors: Dorota N
authors: DN
authors: Komar
authors: Centro de Biotecnología y Genómica de Plantas (CBGP), Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA)-Universidad Politécnica de Madrid.
authors: Alfonso
authors: A
authors: Mouriz
authors: Centro de Biotecnología y Genómica de Plantas (CBGP), Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA)-Universidad Politécnica de Madrid.
authors: José A
authors: JA
authors: Jarillo
authors: Centro de Biotecnología y Genómica de Plantas (CBGP), Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA)-Universidad Politécnica de Madrid; pineiro@inia.es.
authors: Manuel
authors: M
authors: Piñeiro
mesh_terms: D017360:Arabidopsis / Q000235:genetics*; D029681:Arabidopsis Proteins / Q000235:genetics*; D047369:Chromatin Immunoprecipitation / Q000379:methods*; D004247:DNA / Q000378:metabolism; D018398:Homeodomain Proteins / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods; D015533:Transcriptional Activation / Q000235:genetics
chemical_list: D029681:Arabidopsis Proteins; C478974:EBS protein, Arabidopsis; D018398:Homeodomain Proteins; D004247:DNA