Department of Life Science and Research Institute of Natural Sciences, Hanyang University, Seoul 133-791, Republic of Korea.
Anal Biochem. 2013 Oct 15;441(2):147-51. doi: 10.1016/j.ab.2013.07.001. Epub 2013 Jul 16.
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.
为了开始基因转录，几个转录因子必须通过 DNA-蛋白质相互作用结合到特定的 DNA 序列上以形成复合物。我们建立了一种基于 DNA 免疫沉淀（DIP）方法的特异性和灵敏性分析 DNA-蛋白质相互作用的体外方法。我们使用转录因子 CP2c 作为模型，验证了 DIP 测定法在定量测量 DNA-蛋白质结合方面的准确性和效率。通过我们的 DIP 测定法，我们可以检测到 DNA-CP2c 复合物内的特异性相互作用，具有可重复和定量的结合值。此外，我们能够通过添加小分子 FQI1（因子喹啉酮抑制剂 1）有效地测量 DNA-CP2c 结合的变化，该小分子先前被鉴定为这种结合的特异性抑制剂。为了鉴定 DNA-CP2c 结合的新调节剂，我们分析了几个 CP2c 结合肽，发现只有一类肽严重抑制 DNA-CP2c 结合。这些数据表明，我们的 DIP 测定法非常有助于定量检测 DNA-蛋白质复合物的结合动力学。由于在不同的细胞环境中 DNA-蛋白质相互作用非常动态，我们的测定法可以应用于检测活性转录因子，包括正常和疾病条件下的启动子占有率。此外，它可用于开发针对特定 DNA-蛋白质相互作用的靶向调节剂。
