Several lines of our research show that the organizational status of regulatory elements in the eukaryotic nucleus can impact strongly on their utilization. We have developed a reagent that will be very useful in testing models related to the domain structure of chromatin and intranuclear organization. We have constructed chimeras between the green fluorescent protein (GFP) and several of the major nuclear receptors, including the glucocorticoid receptor, progesterone receptor, estrogen receptor, AhR receptor, PPAR receptor, RAR receptor, and the thyroid receptor. All of the receptor chimeras have been shown to be efficiently expressed in cultured cells, and to be functional in terms of promoter transactivation and ligand binding. GFP-GR has been characterized in particular detail. Using a cell line, 3134, that harbors a 200-copy, head-to-tail tandem repeat of an MMTV LTR-ras-BPV fusion, we have been able to demonstrate direct binding of the receptor to its cognate response element in living cells. Using these cells, a bright ribbon of GFP-GR localization is observed that represents binding to the tandem array. An antagonist, RU486, gives complete translocation of the receptor into the nucleus, but fails to target GR to the tandem array. This technology allows us, for the first time, to characterize the interactions of nuclear receptors with regulatory elements in living cells in real time.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Intramural Research (Z01)
Project #
1Z01BC010027-02
Application #
6161119
Study Section
Special Emphasis Panel (LRBG)
Project Start
Project End
Budget Start
Budget End
Support Year
2
Fiscal Year
1997
Total Cost
Indirect Cost
Name
National Cancer Institute Division of Basic Sciences
Department
Type
DUNS #
City
State
Country
United States
Zip Code
Voss, Ty C; Schiltz, R Louis; Sung, Myong-Hee et al. (2009) Combinatorial probabilistic chromatin interactions produce transcriptional heterogeneity. J Cell Sci 122:345-56
Martinez, Elisabeth D; Rayasam, Geetha V; Dull, Angie B et al. (2005) An estrogen receptor chimera senses ligands by nuclear translocation. J Steroid Biochem Mol Biol 97:307-21
Cremazy, Frederic G E; Manders, Erik M M; Bastiaens, Philippe I H et al. (2005) Imaging in situ protein-DNA interactions in the cell nucleus using FRET-FLIM. Exp Cell Res 309:390-6
Hager, G L; Fletcher, T M; Xiao, N et al. (2000) Dynamics of gene targeting and chromatin remodelling by nuclear receptors. Biochem Soc Trans 28:405-10
Hager, G L; Lim, C S; Elbi, C et al. (2000) Trafficking of nuclear receptors in living cells. J Steroid Biochem Mol Biol 74:249-54
McNally, J G; Muller, W G; Walker, D et al. (2000) The glucocorticoid receptor: rapid exchange with regulatory sites in living cells. Science 287:1262-5
Kramer, P R; Fragoso, G; Pennie, W et al. (1999) Transcriptional state of the mouse mammary tumor virus promoter can affect topological domain size in vivo. J Biol Chem 274:28590-7
Baumann, C T; Lim, C S; Hager, G L (1999) Intracellular localization and trafficking of steroid receptors. Cell Biochem Biophys 31:119-27
Htun, H; Holth, L T; Walker, D et al. (1999) Direct visualization of the human estrogen receptor alpha reveals a role for ligand in the nuclear distribution of the receptor. Mol Biol Cell 10:471-86
Walker, D; Htun, H; Hager, G L (1999) Using inducible vectors to study intracellular trafficking of GFP-tagged steroid/nuclear receptors in living cells. Methods 19:386-93