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 ligand-specific interactions with remodeling complexes can strongly influence receptor nuclear dynamics and rates of exchange with chromatin in living cells.

ACKNOWLEDGMENTS

We thank G. Crabtree (Stanford University, Stanford, Calif.) and K. Zhao (National Institutes of Health [NIH]) for the generous gift of anti-BRG1 antibody J1. We also thank Carl Wu (NIH) for the Drosophila embryos used for the preparation of chromatin assembly extracts. We acknowledge Anthony Imbalzano (University of Massachusetts Medical School) for the DN-BRG1 construct and the National Cell Culture Center for providing the cell pellets of FL-INI-11 (FLAG-tagged BRG1) and FLAG-tagged DN-BRG1. We thank Jim McNally, Tom Misteli, and Sagar Sengupta for critical reading of the manuscript and discussions. Live-cell microscopy was carried out at the Fluorescence Imaging Facility, Laboratory of Receptor Biology and Gene Expression, National Cancer Institute.