title: A DNA immunoprecipitation assay used in quantitative detection of in vitro DNA-protein complex binding.
abstract: To begin gene transcription, several transcription factors must bind to specific DNA sequences to form a complex via DNA-protein interactions. We established an in vitro method for specific and sensitive analyses of DNA-protein interactions based on a DNA immunoprecipitation (DIP) method. We verified the accuracy and efficiency of the DIP assay in quantitatively measuring DNA-protein binding using transcription factor CP2c as a model. With our DIP assay, we could detect specific interactions within a DNA-CP2c complex, with reproducible and quantitative binding values. In addition, we were able to effectively measure the changes in DNA-CP2c binding by the addition of a small molecule, FQI1 (factor quinolinone inhibitor 1), previously identified as a specific inhibitor of this binding. To identify a new regulator of DNA-CP2c binding, we analyzed several CP2c binding peptides and found that only one class of peptide severely inhibits DNA-CP2c binding. These data show that our DIP assay is very useful in quantitatively detecting the binding dynamics of DNA-protein complex. Because DNA-protein interaction is very dynamic in different cellular environments, our assay can be applied to the detection of active transcription factors, including promoter occupancy in normal and disease conditions. Moreover, it may be used to develop a targeted regulator of specific DNA-protein interaction.
authors.affiliation: Department of Life Science and Research Institute of Natural Sciences, Hanyang University, Seoul 133-791, Republic of Korea.
authors.forename: Min Young
authors.initials: MY
authors.lastname: Kim
authors.forename: Ji Hyung
authors.initials: JH
authors.lastname: Chae
authors.forename: Chang-Ho
authors.initials: CH
authors.lastname: Oh
authors.forename: Chul Geun
authors.initials: CG
authors.lastname: Kim
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D045744:Cell Line, Tumor; D004247:DNA / Q000032:analysis / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000032:analysis / Q000378:metabolism*; D006801:Humans; D047468:Immunoprecipitation / Q000379:methods*; D051379:Mice; D011485:Protein Binding; D014157:Transcription Factors / Q000032:analysis / Q000378:metabolism*
chemical_list: D004268:DNA-Binding Proteins; C501906:TFCP2 protein, human; C501905:Tfcp2 protein, mouse; D014157:Transcription Factors; D004247:DNA
keywords: DNA immunoprecipitation; DNA–protein binding dynamics; DNA–protein interaction; Transcription factor CP2c