DNA-binding profiling of human hormone nuclear receptors via fluorescence correlation spectroscopy in a cell-free system.
The nuclear hormone receptors (NHRs), a family of transcription factors, bind directly to the hormone response elements (HREs) to regulate gene expression. In this study, we describe a comprehensive NHR-HRE profiling analysis with a new high-throughput DNA binding assay system utilizing wheat germ cell-free protein production and fluorescence correlation spectroscopy (FCS). This approach revealed NHR binding to natural response elements and new heterodimeric NHR-HRE bindings. We analyzed 408 possible binding combinations between 34 human NHRs and 12 different HREs, and identified 205 NHR-HRE binding combinations, 124 of which have not been previously reported. Thus, this study provides a novel biochemical classification of the human NHRs, as well as describing a novel approach to the large-scale analysis of DNA-protein interactions.
Kobayashi
Tamiyo
T
Cell-Free Science and Technology Research Center, and The Venture Business Laboratory, Ehime University, 3 Bunkyo-cho, Matsuyama, Ehime 790-8577, Japan.
Kodani
Yoshiko
Y
Nozawa
Akira
A
Endo
Yaeta
Y
Sawasaki
Tatsuya
T
D002474:Cell-Free System; D004247:DNA / Q000378:metabolism*; D006801:Humans; D040081:Protein Array Analysis; D011485:Protein Binding; D018160:Receptors, Cytoplasmic and Nuclear / Q000145:classification* / Q000235:genetics / Q000378:metabolism*; D020218:Response Elements; D047491:Retinoid X Receptor gamma / Q000145:classification / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D014157:Transcription Factors / Q000145:classification* / Q000235:genetics / Q000378:metabolism*
D018160:Receptors, Cytoplasmic and Nuclear; D047491:Retinoid X Receptor gamma; D014157:Transcription Factors; D004247:DNA