ATP-Dependent Mobilization of the Glucocorticoid Receptor during Chromatin Remodeling

Abstract

Chromatin remodeling by the glucocorticoid receptor (GR) is associated with activation of transcription at the mouse mammary tumor virus (MMTV) promoter. We reconstituted this nucleoprotein transition with chromatin assembled on MMTV DNA. The remodeling event was ATP dependent and required either a nuclear extract from HeLa cells or purified human Swi/Snf. Through the use of a direct interaction assay (magnetic bead pull-down), we demonstrated recruitment of human Swi/Snf to MMTV chromatin by GR. Unexpectedly, we found that GR is actively displaced from the chromatin template during the remodeling process. ATP-dependent GR displacement was reversed by the addition of apyrase and was specific to chromatin templates. The disengagement reaction could also be induced with purified human Swi/Snf. Although GR apparently dissociated during chromatin remodeling by Swi/Snf, it participated in binding of the secondary transcription factor, nuclear factor 1. These results are paralleled by a recent discovery that the hormone-occupied receptor undergoes rapid exchange between chromatin and the nucleoplasmic compartment in living cells. Both the in vitro and in vivo results are consistent with a dynamic model (hit and run) in which GR first binds to chromatin after ligand activation, recruits a remodeling activity, facilitates transcription factor binding, and is simultaneously lost from the template.

T.M.F. and N.X. contributed equally to this work.

We thank Michael Bustin for generously providing HMG-1. We are also grateful for the assistance of Carl Wu, Raphael Sandaltopoulus, and Ju-Gyeong Kang (NIH) for providing the Drosophila embryos for chromatin assembly extract preparation. We thank Catharine Smith and Denise Thurber (NIH) for the MMTV construct containing the mutant NF-1 site, Robert Kingston (Harvard Medical School) for permission to use the FL-INI1-11 cell line, and the National Cell Culture Center for providing cell pellets. The anti-BRG1 antibodies were generously provided by Keji Zhou and Gerald Crabtree (Stanford University), and anti-Snf2 h was provided by Robert Roeder (Rockefeller University). We thank Matthias Becker and Sam John for critical reading of the manuscript.