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.