Nuclear Receptor Mobility on Gene Targets

G. L. Hager, D. Walker, J. McNally, G. Rayasam, D. Edwards, M. Becker, T. Johnson, C. Elbi, S. John, A. Nagaich

Laboratory of Receptor Biology & Gene Expression, National Cancer Institute, NIH, Bethesda, MD 20892-5055, U.S.A.

Nuclear receptors exert their physiological effects largely through the modulation of gene expression rates for responsive genes in target tissues. Until recently, the mechanisms of transcription regulation have been modeled in terms of interactions between soluble members of the regulatory apparatus and the DNA template. We now know that a key feature of receptor action lies in the epigenetic modification of the nucleoprotein template. Receptors recruit many activities that change local chromatin structure, and these alterations in turn are central to the resulting activation or suppression of gene activity.

A central concept in endocrine biology has been that hormone bound receptors bind their cognate response element and serve as a platform to recruit the many coactivators and coregulators that participate in modulating transcription rates. Through the use of novel imaging techniques, we observed direct steroid receptor binding to a tandem array of a hormone-responsive promoter in living cells. We unexpectedly found that the glucocorticoid receptor (GR) exchanges rapidly with regulatory elements in the continued presence of ligand [1]. Using fluorescence recovery after photobleaching (FRAP) we have now examined the dynamic behavior of several green fluorescence protein (GFP) tagged transcription factors at the promoter tandem array. The glucocorticoid receptor interacting protein 1 (GRIP1) exhibits a half maximal time for fluorescent recovery of 5 sec., the same rapid exchange as observed for GR. In contrast, the large subunit of RNA polymerase II (RPB1) shows a very slow exchange, requiring thirteen minutes for complete fluorescence recovery.

We have also reconstituted the GR-dependent nucleoprotein transition with chromatin assembled on promoter DNA. The remodeling event is ATP-dependent, and requires either a nuclear extract from HeLa cells or purified human SWI/SNF [2]. Through the use of a direct interaction assay (magnetic bead ”pull-down”), we demonstrate recruitment of human SWI/SNF to promoter chromatin by GR [3]. We find that GR is actively displaced from the chromatin template during the remodeling process. Displacement requires the presence of ATP, can be reversed by the addition of apyrase, and is specific to chromatin templates. The disengagement reaction can be induced with purified human SWI/SNF, but the reaction occurs less efficiently, and is further stimulated by the inclusion of nuclear extract. Binding of the secondary transcription factor, NF-1, to the promoter is facilitated by GR-induced chromatin remodeling.

Using the in vitro template pull down assay, we also demonstrate that progesterone receptor (PR) binding is dynamic on MMTV chromatin. Both PR-A and PR-B liganded with R5020 are actively displaced from MMTV chromatin in the presence of either a HeLa nuclear extract or purified SWI/SNF and ATP. We have examined the effect of different classes of PR antagonists on this displacement reaction. We find that PR liganded to the agonist R5020 and the antagonist RU486 is actively displaced from chromatin during remodeling. However, PR liganded with the antagonist ZK98299 is not displaced from chromatin. We further examined the ability of PR bound to different ligands to recruit BRG1 MMTV chromatin, and our findings indicate that PR ligands exert a differential effect on the ability of the receptor to support dynamic exchange during chromatin remodeling.

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, and is simultaneously lost from the template. These findings suggest a new model for nuclear receptor function [4] in which the receptors reside on response elements in chromatin only for brief periods, then return to the template for successive binding events. We further observed that the template undergoes a cascade of modification events, which are triggered by the initial receptor binding event. We suggest that the receptor may recruit different cofactors during successive ”return-to-template” events, and thus participate in biochemically distinct processes during each binding cycle. This view represents a major departure from classic endocrine models, which envisage the receptor as statically bound to responsive promoters in the continued presence of hormone.

References

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• 2 Becker M, Baumann C T, John S, Walker D, Vigneron M, McNally J G, Hager G L. Dynamic behavior of transcription factors on a natural promoter in living cells.  EMBO Reports. 2002;  3 1188-1194
• 3 Fletcher T M, Ryu B-W, Baumann C T, Warren B S, Fragoso G, John S, Hager G L. Structure and dynamic properties of the glucocorticoid receptor-induced chromatin transition at the MMTV promoter.  Mol Cell Biol. 2000;  20 6466-6475
• 4 Fletcher T M, Xiao N, Mautino G, Baumann C T, Warren B S, Hager G L. ATP-dependent mobilization of the glucocorticoid receptor during chromatin remodeling.  Mol Cell Biol. 2002;  22 3255-3263
• 5 Hager G L, Elbi C, Becker M. Protein dynamics in the nuclear compartment.  Curr Opin Genet Dev. 2002;  12 137-141