title: Identification and characterization of glucocorticoid receptor-binding sites in the human genome.
abstract: Glucocorticoids regulate gene expression via binding of the ligand-activated glucocorticoid receptor (GR) to glucocorticoid-responsive elements (GRE). To identify GR-binding sites, we developed a modified yeast one-hybrid system which enables rapid and efficient identification of genomic targets for DNA-binding proteins. The human GR expression vector was transformed into yeast cells containing a library of human genomic fragments cloned upstream of the reporter gene URA3. The genomic fragments with GR-binding sites were identified by growth of yeast clones in media lacking uracil but containing dexamethasone. DNA fragments were recovered by colony-direct PCR and GRE sequences were predicted by in silico analysis. Using electrophoretic mobility shift assay and fluorescence correlation spectroscopy, we demonstrated that 314 predicted GREs could directly interact with recombinant human GR proteins. In addition, when the genomic fragments were inserted in front of the heterologous SV40 promoter, at least 150 fragments could function as GREs in HEK293 cells. Furthermore, we identified four functional regulatory polymorphisms which may influence individual variation in sensitivity to glucocorticoids. These results provide insights into the molecular mechanisms underlying the physiological and pathological actions of glucocorticoid.
authors.affiliation: Department of Gene Diagnostics and Therapeutics, Research Institute, International Medical Center of Japan, Shinjuku, Tokyo, Japan.
authors.forename: Keiko
authors.initials: K
authors.lastname: Taniguchi-Yanai
authors.forename: Yoshiko
authors.initials: Y
authors.lastname: Koike
authors.forename: Takashi
authors.initials: T
authors.lastname: Hasegawa
authors.forename: Yuichi
authors.initials: Y
authors.lastname: Furuta
authors.forename: Masakuni
authors.initials: M
authors.lastname: Serizawa
authors.forename: Noriko
authors.initials: N
authors.lastname: Ohshima
authors.forename: Norihiro
authors.initials: N
authors.lastname: Kato
authors.forename: Kazuyuki
authors.initials: K
authors.lastname: Yanai
mesh_terms: D001665:Binding Sites; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D003907:Dexamethasone / Q000494:pharmacology*; D024202:Electrophoretic Mobility Shift Assay; D015972:Gene Expression Regulation, Neoplastic; D017930:Genes, Reporter; D015894:Genome, Human; D006367:HeLa Cells; D006801:Humans; D020641:Polymorphism, Single Nucleotide / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011485:Protein Binding; D011965:Receptors, Glucocorticoid / Q000235:genetics / Q000378:metabolism*; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics*; D020798:Two-Hybrid System Techniques
chemical_list: D004268:DNA-Binding Proteins; D011965:Receptors, Glucocorticoid; D003907:Dexamethasone