Ligand-Specific Dynamics of the Androgen Receptor at Its Response Element in Living Cells

Department of Molecular Biosciences, University of Oslo, Postboks 1041 Blindern, 0316 Oslo, Norway.
Molecular and Cellular Biology (Impact Factor: 4.78). 04/2007; 27(5):1823-43. DOI: 10.1128/MCB.01297-06
Source: PubMed


Androgens have key roles in normal physiology and in male sexual differentiation as well as in pathological conditions such as prostate cancer. Androgens act through the androgen receptor (AR), which is a ligand-modulated transcription factor. Antiandrogens block AR function and are widely used in disease states, but little is known about their mechanism of action in vivo. Here, we describe a rapid differential interaction of AR with target genomic sites in living cells in the presence of agonists which coincides with the recruitment of BRM ATPase complex and chromatin remodeling, resulting in transcriptional activation. In contrast, the interaction of antagonist-bound or mutant AR with its target was found to be kinetically different: it was dramatically faster, occurred without chromatin remodeling, and resulted in the lack of transcriptional inhibition. Fluorescent resonance energy transfer analysis of wild-type AR and a transcriptionally compromised mutant at the hormone response element showed that intramolecular interactions between the N and C termini of AR play a key functional role in vivo compared to intermolecular interactions between two neighboring ARs. These data provide a kinetic and mechanistic basis for regulation of gene expression by androgens and antiandrogens in living cells.

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    • "Stable cell lines expressing GFP-AR (3108) and GFP-GR (3617) under the control of the Tet-Off inducible system were previously described [14], [15]. The cells were grown at 37°C with 5% CO2 in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 5 mg/ml penicillin/streptomycin (Life Technologies, Inc.) and 10 µg/ml tetracycline (Sigma) (to suppress GFP-AR and GFP-GR expression). "
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    ABSTRACT: Closely related transcription factors (TFs) can bind to the same response elements (REs) with similar affinities and activate transcription. However, it is unknown whether transcription is similarly orchestrated by different TFs bound at the same RE. Here we have compared the recovery half time (t1/2), binding site occupancy and the resulting temporal changes in transcription upon binding of two closely related steroid receptors, the androgen and glucocorticoid receptors (AR and GR), to their common hormone REs (HREs). We show that there are significant differences at all of these levels between AR and GR at the MMTV HRE when activated by their ligands. These data show that two TFs bound at the same RE can have significantly different modes of action that can affect their responses to environmental cues.
    Full-text · Article · Aug 2014 · PLoS ONE
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    • "DNA FISH was performed on NCI-H716 cells treated with colcemid (0.02 µg/ml for 3 hours) and on microarray tissue cores as described previously [19], [20] using bacterial artificial chromosome clones RP11-62L18 for FGFR2 probes. This FISH probe contains the genomic sequence of Chr10: 123,224,100–123,398,498, which encompasses the entirety of the FGFR2 gene at Chr10: 123,237,844–123,353,481. Probes were labeled directly using Spectrum Orange dUTP and Spectrum Green dUTP (Abbott Molecular Inc., Des Plaines, IL). "
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    ABSTRACT: Aberrant kinase activation resulting from mutation, amplification, or translocation can drive growth and survival in a subset of human cancer. FGFR2 is amplified in breast and gastric cancer, and we report here the first characterization of FGFR2 gene amplification in colorectal cancer in the NCI-H716 colorectal cancer cell line. FGFR2 is highly expressed and activated in NCI-H716 cells, and FGFR selective small molecule inhibitors or FGFR2 shRNA strongly inhibited cell viability in vitro, indicating "addiction" of NCI-H716 cells to FGFR2. NCI-H716 growth in a xenograft model was also inhibited by an FGFR small molecule inhibitor. FGFR2 was required for activation of multiple downstream signaling proteins including AKT, ERK, S6RP and NFKB. Inhibition of downstream kinases such as AKT or ERK alone had modest effects on proliferation, whereas combined inhibition of AKT and ERK signaling resulted in a loss of viability similar to FGFR2 inhibition. We identified elevated FGFR2 expression in a small subset of primary colorectal cancer, however FGFR2 amplification was not observed. Although FGFR2 amplification is not common in primary colon cancer or lymph node and liver metastases, other subsets of colorectal cancer such as ascites, from which the NCI-H716 cell line was derived, have yet to be tested. These results suggest that emerging FGFR inhibitor therapeutics may have efficacy in a subset of colon cancer driven by FGFR2 amplification.
    Full-text · Article · Jun 2014 · PLoS ONE
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    • "In the presence of agonists, AR adopts a conformational change allowing communication between AF-1 and AF-2 and subsequent transcriptional activation (Kemppainen et al., 1999; Langley et al., 1995; Schaufele et al., 2005). To test our hypothesis regarding inhibition of AR activity, we used a fluorescence resonance energy transfer (FRET)-based assay (Jones et al., 2009; Klokk et al., 2007; Schaufele et al., 2005) to determine the effect of differentiation cocktail on the intramolecular interaction between CFP-AF-1 and YFP-AF-2 domains in full-length AR. In this assay, agonist binding induces a conformational change that brings the cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) into close proximity, enabling energy transfer, which precedes nuclear translocation and transcriptional activity. "
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    ABSTRACT: We compared transcriptomes of terminally differentiated mouse 3T3-L1 and human adipocytes to identify cell-specific differences. Gene expression and high content analysis (HCA) data identified the androgen receptor (AR) as both expressed and functional, exclusively during early human adipocyte differentiation. The AR agonist dihydrotestosterone (DHT) inhibited human adipocyte maturation by downregu-lation of adipocyte marker genes, but not in 3T3-L1. It is interesting that AR induction corresponded with dexamethasone activation of the glucocorticoid receptor (GR); however, when exposed to the differentiation cocktail required for adipocyte maturation, AR adopted an antagonist conformation and was transcriptionally repressed. To further explore effectors within the cocktail, we applied an image-based support vector machine (SVM) classification scheme to show that adipocyte differentiation components inhibit AR action. The results demonstrate human adipocyte differentiation, via GR activation, upregu-lates AR but also inhibits AR transcriptional activity.
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