Steroid receptors bind to site-specific response elements in chromatin and modulate gene expression in a hormone-dependent fashion. With the use of a tandem array of mouse mammary tumor virus reporter elements and a form of glucocorticoid receptor labeled with green fluorescent protein, targeting of the receptor to response elements in live mouse cells was observed. Photobleaching experiments provide direct evidence that the hormone-occupied receptor undergoes rapid exchange between chromatin and the nucleoplasmic compartment. Thus, the interaction of regulatory proteins with target sites in chromatin is a more dynamic process than previously believed.

An ultrafast UV laser crosslinking assay has provided novel insights into the progression of the SWI/SNF-mediated chromatin-remodeling reaction and transcription factor binding in real time. We demonstrate site-specific crosslinking between the glucocorticoid receptor (GR), the hSWI/SNF chromatin-remodeling complex, and the mouse mammary tumor virus (MMTV) promoter assembled in an array of correctly positioned nucleosomes. GR first demonstrates rapid binding to the promoter and then is actively displaced from the template during the remodeling reaction. This displacement reaction requires the hSWI/SNF complex and ATP, is specific to the nucleoprotein template, and is accompanied by a core histone rearrangement. The hSWI/SNF complex associates with random positions on the chromatin template in the absence of GR but is recruited specifically to the B/C region when GR is included. These results indicate that enhancement of hSWI/SNF-mediated factor accessibility, a hallmark of chromatin remodeling, is in some cases transient, reversible, and periodic.

The human estrogen receptor ␣ (ER ␣) has been tagged at its amino terminus with the S65T variant of the green fluorescent protein (GFP), allowing subcellular trafficking and localization to be observed in living cells by fluorescence microscopy. The tagged receptor, GFP-ER, is functional as a ligand-dependent transcription factor, responds to both agonist and antagonist ligands, and can associate with the nuclear matrix. Its cellular localization was analyzed in four human breast cancer epithelial cell lines, two ERϩ (MCF7 and T47D) and two ERϪ (MDA-MB-231 and MDA-MB-435A), under a variety of ligand conditions. In all cell lines, GFP-ER is observed only in the nucleus in the absence of ligand. Upon the addition of agonist or antagonist ligand, a dramatic redistribution of GFP-ER from a reticular to punctate pattern occurs within the nucleus. In addition, the full antagonist ICI 182780 alters the nucleocytoplasmic compartmentalization of the receptor and causes partial accumulation in the cytoplasm in a process requiring continued protein synthesis. GFP-ER localization varies between cells, despite being cultured and treated in a similar manner. Analysis of the nuclear fluorescence intensity for variation in its frequency distribution helped establish localization patterns characteristic of cell line and ligand. During the course of this study, localization of GFP-ER to the nucleolar region is observed for ERϪ but not ERϩ human breast cancer epithelial cell lines. Finally, our work provides a visual description of the "unoccupied" and ligand-bound receptor and is discussed in the context of the role of ligand in modulating receptor activity. INTRODUCTIONSteroid hormones elicit diverse biological responses, important during growth, differentiation, inflammation, pregnancy, and homeostasis among many other processes. The genomic actions of steroid hormones are mediated by steroid receptors, members of the nuclear receptor superfamily of ligand-dependent transcription factors. In the absence of hormone, steroid receptors exist in a complex with chaperone proteins capable of high-affinity binding to steroid hormones. Hormone binding leads to a conformational change in the receptor that results in its dissociation from chaperone proteins and ultimately in the binding of the receptor as a homodimer to cognate sites in steroid-responsive genes (reviewed in Tsai and O'Malley, 1994;Mangelsdorf et al., 1995;Beato et al., 1996).Immunohistochemistry and biochemical fractionation show the unoccupied steroid receptors to reside ‡ These authors contributed equally to this work. Corresponding author. E-mail address: hagerg@exchange.nih.gov. predominantly in the cytoplasm, the nucleus, or both compartments, depending on the receptor, in a complex with chaperone proteins (Jensen, 1991;DeFranco et al., 1995;Beato et al., 1996;Pratt and Toft, 1997). For the predominantly nuclear receptors, such as the estrogen receptor (ER), the unoccupied receptor exists in the nucleus either bound or not bound to its cognate site in target genes. ...