title: High-throughput fluorescence-based screen to identify factors involved in nuclear receptor recruitment to response elements.
abstract: 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.
authors: Laboratory of Receptor Biology and Gene Expression, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA.
authors: Tina B
authors: TB
authors: Miranda
authors: Ty C
authors: TC
authors: Voss
authors: Gordon L
authors: GL
authors: Hager
mesh_terms: D000305:Adrenal Cortex Hormones / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D002843:Chromatin / Q000378:metabolism; D003907:Dexamethasone / Q000494:pharmacology; D024581:Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating) / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics; D057166:High-Throughput Screening Assays / Q000379:methods*; D051379:Mice; D015735:Mifepristone / Q000494:pharmacology; D011401:Promoter Regions, Genetic; D034622:RNA Interference; D034741:RNA, Small Interfering; D011965:Receptors, Glucocorticoid / Q000378:metabolism*; D020218:Response Elements / Q000235:genetics*
chemical_list: D000305:Adrenal Cortex Hormones; D002843:Chromatin; D034741:RNA, Small Interfering; D011965:Receptors, Glucocorticoid; D049452:Green Fluorescent Proteins; D015735:Mifepristone; D003907:Dexamethasone; D024581:Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating)
references: FEBS J. 2011 Jul;278(13):2211-30
references: 21501385
references: Mol Endocrinol. 1999 Dec;13(12):2108-21
references: 10598585
references: Cell. 2011 Aug 19;146(4):544-54
references: 21835447
references: Methods. 1999 Nov;19(3):386-93
references: 10579933
references: J Biol Chem. 2002 Jul 19;277(29):26238-43
references: 12011091
references: Mol Endocrinol. 2006 Nov;20(11):2641-55
references: 16873444
references: Stress. 2009 May;12(3):193-205
references: 19051126
references: J Biol Chem. 1999 Oct 1;274(40):28590-7
references: 10497225