We studied the interaction of the androgen receptor with the MMTV promoter in living cells, and found a pronounced ligand effect on mobility of the receptor. We found that AR antagonists induce a high rate of exchange for the receptor with response elements, while agonist liganded receptors have a lower mobility. These results indicate that the longer residence time of the receptor is assoicated with a producitve promoter interation. Action of the glucocorticoid receptor (GR) was studied at the single cell level through the use of mammary carcinoma cell line containing a tandem array of MMTV promoter-reporter gene cassettes integrated at a single genomic locus. Direct binding of a GFP-GR fusion protein to the MMTV regulatory elements can be observed in living cells. After ligand treatment, MMTV-dependent transcription in individual cells was detected by RNA fluorescence in situ hybridization (FISH). High-resolution fluorescence images were acquired from large numbers of randomly selected cells. Images were analyzed with a novel automated computer algorithm, measuring the RNA FISH signal and the relative GFP-GR fluorescence intensity at the MMTV array for each cell. A pronounced cell-to-cell variability was observed in RNA FISH signal and GR-MMTV association within treatment groups. A nonlinear relationship was observed between GR-MMTV association and RNA FISH in individual cells, indicating that differences in GR-MMTV interaction account for some, but not all, of the transcriptional heterogeneity between individual cells. In selected cell subpopulations with equal levels of GR-MMTV association, there was a decrease in RNA FISH signal with RU486 treatment compared with dexamethasone treatment. These results indicate that stochastic events occurring after GR-promoter association, such as the actions of chromatin remodeling complexes or other cofactors, change in a ligand-dependent manner and regulate heterogeneous transcription in individual cells. We found that cochaperone p23-dependent disruption of GR-driven transcription depended on the ligand binding domain (LBD), and examined the importance of the LBD and of ligand dissociation in GR-GRE dissociation in living cells. We showed in fluorescence recovery after photobleaching studies that dissociation of GR from GREs is faster in the absence of the LBD. Furthermore, GR interaction with a target promoter revealed ligand-specific exchange rates. However, using covalently binding ligands, we demonstrated that ligand dissociation is not required for receptor dissociation from GREs. Thus, activities impinging on the LBD regulate GR exchange with GREs, but the dissociation of GR from GREs is independent from ligand dissociation.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Intramural Research (Z01)
Project #
1Z01BC010308-09
Application #
7592658
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
9
Fiscal Year
2007
Total Cost
$415,385
Indirect Cost
Name
National Cancer Institute Division of Basic Sciences
Department
Type
DUNS #
City
State
Country
United States
Zip Code
Stavreva, Diana A; Wiench, Malgorzata; John, Sam et al. (2009) Ultradian hormone stimulation induces glucocorticoid receptor-mediated pulses of gene transcription. Nat Cell Biol 11:1093-102
Meijsing, Sebastiaan H; Elbi, Cem; Luecke, Hans F et al. (2007) The ligand binding domain controls glucocorticoid receptor dynamics independent of ligand release. Mol Cell Biol 27:2442-51
Huh, Jung-Im; Qiu, Ting Hu; Chandramouli, Gadisetti V R et al. (2007) 2-methoxyestradiol induces mammary gland differentiation through amphiregulin-epithelial growth factor receptor-mediated signaling: molecular distinctions from the mammary gland of pregnant mice. Endocrinology 148:1266-77
Klokk, Tove I; Kurys, Piotr; Elbi, Cem et al. (2007) Ligand-specific dynamics of the androgen receptor at its response element in living cells. Mol Cell Biol 27:1823-43
Voss, Ty C; John, Sam; Hager, Gordon L (2006) Single-cell analysis of glucocorticoid receptor action reveals that stochastic post-chromatin association mechanisms regulate ligand-specific transcription. Mol Endocrinol 20:2641-55
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Rayasam, Geetha V; Elbi, Cem; Walker, Dawn A et al. (2005) Ligand-specific dynamics of the progesterone receptor in living cells and during chromatin remodeling in vitro. Mol Cell Biol 25:2406-18
Becker, Matthias; Becker, Antje; Miyara, Faical et al. (2005) Differential in vivo binding dynamics of somatic and oocyte-specific linker histones in oocytes and during ES cell nuclear transfer. Mol Biol Cell 16:3887-95
Dohoney, Kathleen M; Guillerm, Claire; Whiteford, Craig et al. (2004) Phosphorylation of p53 at serine 37 is important for transcriptional activity and regulation in response to DNA damage. Oncogene 23:49-57
Elbi, Cem; Walker, Dawn A; Lewis, Marcia et al. (2004) A novel in situ assay for the identification and characterization of soluble nuclear mobility factors. Sci STKE 2004:pl10

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