Several lines of our research show that the organizational status of regulatory elements in the eukaryotic nucleus can impact strongly on their utilization. We have developed a reagent that will be very useful in testing models related to the domain structure of chromatin and intranuclear organization. We have constructed chimeras between the green fluorescent protein (GFP) and several of the major nuclear receptors, including the glucocorticoid receptor, progesterone receptor, estrogen receptor, AhR receptor, PPAR receptor, RAR receptor, and the thyroid receptor. All of the receptor chimeras have been shown to be efficiently expressed in cultured cells, and to be functional in terms of promoter transactivation and ligand binding. GFP-GR has been characterized in particular detail. Using a cell line, 3134, that harbors a 200-copy, head-to-tail tandem repeat of an MMTV LTR-ras-BPV fusion, we have been able to demonstrate direct binding of the receptor to its cognate response element in living cells. Using these cells, a bright region of GFP-GR localization is observed that represents binding to the tandem array. This technology allows us, for the first time, to characterize the interactions of nuclear receptors with regulatory elements in living cells in real time. Using GFP fusions with the estrogen receptor, we showed that the receptor is nuclear localized in living cells in the absence of ligand, but moves to new sites within the nucleus in response to ligand stimulation. Thus the mechansim of intranuclear localization of this nuclear receptor is more complex than simple binding to chromatin. We have also studied subcellular trafficking for the A and B forms of the progesterone receptor (PR), the thyroid receptor, and the RAR receptor, and find that the subcellular localization for these nuclear receptors is based not only on so-called nuclear localization signals, or NLS's, but more directly on the involvement of the receptors in large multiprotein complexes, both in the nucleus and the cytoplasm, and the effect of ligand stimulation on the interaction of the various receptors with these complexes.
