Ligand-Specific Dynamics of the Progesterone Receptor in Living Cells and during Chromatin Remodeling In Vitro

Abstract

Progesterone receptor (PR), a member of the nuclear receptor superfamily, is a key regulator of several processes in reproductive function. We have studied the dynamics of the interaction of PR with a natural target promoter in living cells through the use of fluorescence recovery after photobleaching (FRAP) analysis and also have characterized the dynamics of the interaction of PR with the mouse mammary tumor virus (MMTV) promoter reconstituted into chromatin in vitro. In photobleaching experiments, PR in the presence of the agonist R5020 exhibits rapid exchange with the MMTV promoter in living cells. Two PR antagonists, RU486 and ZK98299, have opposite effects on receptor dynamics in vivo. In the presence of RU486, PR binds to the promoter and is exchanged more slowly than the agonist-activated receptor. In contrast, PR bound to ZK98299 is not localized to the promoter and exhibits higher mobility in the nucleoplasm than the agonist-bound receptor. Significantly, PR bound to R5020 or RU486 can recruit the SWI/SNF chromatin remodeling complex to the promoter, but PR activated with ZK98299 cannot. Furthermore, we found ligand-specific active displacement of PR from the MMTV promoter during chromatin remodeling in vitro and conclude that the interaction of PR with chromatin is highly dynamic both in vivo and in vitro. We propose that factor displacement during chromatin remodeling is an important component of receptor mobility and that ligandspecific interactions with remodeling complexes can strongly influence receptor nuclear dynamics and rates of exchange with chromatin in living cells.

Chromatin Remodeling Complexes Interact Dynamically with a Glucocorticoid Receptor–regulated Promoter

“…In fact, this modulation is one of the key mechanisms essential for the functional role of nuclear hormone receptors (Stenoien et al, 2001;Schaaf and Cidlowski, 2003;Stavreva et al, 2004;Elbi et al, 2004b;Farla et al, 2005;Rayasam et al, 2005). In vivo FRAP has been used to study the dynamic properties of chromatin proteins and that the FRAP recovery kinetics of chromatin proteins are directly related to their chromatin-binding properties (Lefebvre et al, 1991;Fragoso et al, 1998;Lever et al, 2000;Kimura and Cook, 2001;Kimura et al, 2002;Maruvada et al, 2003;Phair et al, 2004;Becker et al, 2005;Chen et al, 2005).…”

“…To further understand the process by which chromatin-remodeling complexes are recruited and regulate target genes to modulate transcription, we have directly visualized the sequence of gene expression events involving the SWI/SNF complex in mouse mammary adenocarcinoma cells that contain a tandem repeat of stably integrated copies of the MMTV-LTR (mouse mammary tumor virus-long terminal repeat). This array which contains 800-1200 binding sites for GR can be visualized by using green fluorescent protein (GFP)-tagged versions of steroid receptors and associated cofactors (Kramer et al, 1999;McNally et al, 2000;Rayasam et al, 2005). We have investigated the molecular basis by which SWI/SNF regulates transcription as well as its influence on the chromatin structure of a steroid hormone-responsive promoter array.…”

“…However, the transfection of GR along with BRG1 or BRM expression plasmids potentiated the hormone response to 33-and 77-fold, respectively ( Figure 1C). ATPase-deficient forms of BRG1 or BRM (BRG1-K-R or BRM-K-R) contain a lysine-to-arginine mutation in the ATP binding pocket (Figure 1, A and B) that abrogates ATP hydrolysis but retains the ability to efficiently incorporate into multisubunit SWI/SNF-like complexes (Rayasam et al, 2005). Consequently, when overexpressed, these mutants function as dominant negatives (de la Serna et al, 2000).…”

“…Cells were fixed in 4% paraformaldehyde and processed for indirect immunofluorescence microscopy as described above. This was followed by a RNA FISH procedure to detect MMTV transcripts as described previously (Parada et al, 2003;Rayasam et al, 2005). All images were acquired with the same exposure times in order to compare across different treatment conditions.…”

Novel arylpyrazole compounds selectively modulate glucocorticoid receptor regulatory activity

“…The eGFP-rGR cell line expressing enhanced GFP-rat GR under control of the Tet-Off synthetic promoter was obtained as a stably transfected derivative of the Tet-Off murine mammary adenocarcinoma cell line 5858 (Rayasam et al 2005). The eGFPrGR construct was cloned into pTRE-tight (Clontech), and the resulting construct was transfected along with a puromycinresistance plasmid into Tet-Off cell line 5858.…”