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.

Keywords::

• GR
• GRE
• polymorphism
• rSNP
• yeast one-hybrid assay

Acknowledgments

We would like to thank Dr. Marc Vidal for gifts of the plasmid pJH3, Dr. Xiao-Dong Gao and Akiko Nishikawa for yeast strains and technical advice. We also thank Dr. A. Fukamizu, for their helpful discussion and encouragements. We are also grateful to Miss Miko Shigeno, Miss Kanako Yamaguchi, Miss Miki Maeda, Mr Naoki Takahashi, Mr Munemitsu Yuasa, and Miss Mariko Takahashi for excellent technical assistance. This work was supported by the Research Grant for the Genome Network Project from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT) of the Japanese Government, the Grant-in-Aid for Young Scientists (A) No. 19681021 from MEXT of Japan, and the faculty of science special grant for promoting scientific research at Toho University.

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.