The glucocorticoid receptor is an inducible transcription factor which plays important roles in many physiological processes. Upon activation, GR interacts with regulatory elements and modulates the expression of genes. Although GR is widely expressed in multiple tissues, its binding sites within chromatin and the genes it regulates are tissue specific. Many accessory proteins and cofactors are thought to play a role in dictating GR’s function; however, mechanisms involved in targeting GR to specific sites in the genome are not well understood. Here we describe a high-throughput fluorescence-based method to identify factors involved in GR loading at response elements. This screen utilizes a genetically engineered cell line that contains 200 repeats of a glucocorticoid response promoter and expresses GFP-tagged GR. Upon treatment with corticosteroids, GFP–GR forms a steady-state distribution at the promoter array, and its concentration at this focal point can be quantitatively determined. This system provides a novel approach to identify activities important for GR loading at its response element using siRNA libraries to target factors that enhance or inhibit receptor localization.
Wiench M, Miranda TB, Hager GL (2011) Control of nuclear receptor function by local chromatin structure. FEBS J 278:2211–2230PubMedCrossRefGoogle Scholar
Kramer P, Fragoso G, Pennie WD et al (1999) Transcriptional state of the mouse mammary tumor virus promoter can effect topological domain size in vivo. J Biol Chem 274:28590–28597PubMedCrossRefGoogle Scholar
Walker D, Htun H, Hager GL (1999) Using inducible vectors to study intracellular trafficking of GFP-tagged steroid/nuclear receptors in living cells. Methods 19:386–393PubMedCrossRefGoogle Scholar
Voss TC, Schiltz RL, Sung MH et al (2011) Dynamic exchange at regulatory elements during chromatin remodeling underlies assisted loading mechanism. Cell 146:544–554PubMedCrossRefGoogle Scholar
Voss TC, John S, Hager GL (2006) Single cell analysis of glucocorticoid receptor action reveals that stochastic post-chromatin association mechanisms regulate ligand-specific transcription. Mol Endocrinol 20:2641–2655PubMedCrossRefGoogle Scholar
Schulz M, Eggert M, Baniahmad A et al (2002) RU486-induced glucocorticoid receptor agonism is controlled by the receptor N terminus and by corepressor binding. J Biol Chem 277:26238–26243PubMedCrossRefGoogle Scholar
Szapary D, Huang Y, Simons SS Jr (1999) Opposing effects of corepressor and coactivators in determining the dose–response curve of agonists, and residual agonist activity of antagonists, for glucocorticoid receptor-regulated gene expression. Mol Endocrinol 13:2108–2121PubMedCrossRefGoogle Scholar