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.
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 examined the dynamic behavior of several green fluorescence protein (GFP)-tagged transcription factors at the promoter tandem array. The GR interacting protein 1 (GRIP 1) exhibits a half maximal time of 5 seconds for fluorescent recovery, the same rapid exchange as observed for GR. In contrast, the large subunit of RNA polymerase II (RPB1) shows a very slow exchange, requiring 13 minutes for complete fluorescence recovery.
We 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/Snf1(2). Using a direct interaction assay (magnetic bead "pull-down"), we demonstrated recruitment of human Swi/Snf to promoter chromatin by GR(3). We found 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. 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.
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