A highly fluorescent mutant form of the green fluorescent protein (GFP) has been fused to the rat glucocorticoid receptor (GR). When GFP-GR is expressed in living mouse cells, it is competent for normal transactivation of the GR-responsive mouse mammary tumor virus promoter. The unliganded GFP-GR resides in the cytoplasm and translocates to the nucleus in a hormone-dependent manner with ligand specificity similar to that of the native GR receptor. Due to the resistance of the mutant GFP to photobleaching, the translocation process can be studied by time-lapse video microscopy. Confocal laser scanning microscopy showed nuclear accumulation in a discrete series of foci, excluding nucleoli. Complete receptor translocation is induced with RU486 (a ligand with little agonist activity), although concentration into nuclear foci is not observed. This reproducible pattern of transactivation-competent GR reveals a previously undescribed intranuclear architecture of GR target sites.Steroid receptors are hormone-dependent activators of gene expression. It is generally accepted that the unliganded glucocorticoid receptor (GR) resides in the cytoplasm and that hormone activation leads to both nuclear accumulation and gene activation (see refs. 1-6 and references therein). However, the mechanisms involved in nuclear translocation and targeting of steroid receptors to regulatory sites in chromatin are poorly understood. It has been difficult to discriminate between the ability of a given receptor mutant, or a given receptor-ligand combination, to participate in the separate processes of receptor activation, nuclear translocation, sequence-specific DNA binding, and promoter activation.The paucity of information on these issues stems in part from the lack of appropriate technology to study the various stages in nuclear targeting. Because knowledge of these steps is essential for understanding the mechanism of steroid hormone action, we have taken the approach of tagging GR with a chromophore, allowing us to visualize in vivo, with the least perturbation, the changes in receptor subcellular localization upon exposure to activating ligand.Recent characterization of a chromophore, the green fluorescent protein (GFP), provides a general method to label proteins in living cells. Chimeras formed with a highly efficient variant of GFP (7)

Repeating copolymers of (dT-dC)n.(dA-dG)n sequences (TC.AGn) can assume a hinged DNA structure (H-DNA) which is composed of triple-stranded and single-stranded regions. A model for the formation of H-DNA is proposed, based on two-dimensional gel electrophoretic analysis of DNA's with different lengths of (TC.AG)n copolymers. In this model, H-DNA formation is initiated at a small denaturation bubble in the interior of the copolymer, which allows the duplexes on either side to rotate slightly and to fold back, in order to make the first base triplet. This nucleation establishes which of several nonequivalent H-DNA conformations is to be assumed by any DNA molecule, thereby trapping each molecule in one of several metastable conformers that are not freely interconvertible. Subsequently, the acceptor region spools up single-stranded polypyrimidines as they are released by progressive denaturation of the donor region; both the spooling and the denaturation result in relaxation of negative supercoils in the rest of the DNA molecule. From the model, it can be predicted that the levels of supercoiling of the DNA determine which half of the (dT-dC)n repeat is to become the donated third strand.

A naturally occurring (dT-dC)18:(dA-dG)18 repeat in the H conformation of DNA was shown to contain single-stranded nucleotides in the center of the TC18 repeat and on one half of the AG18 repeat. These results support the model that H-DNA is a structure containing both triple-stranded and single-stranded regions. The stability of this structure was affected by both pH and the degree of negative supercoiling: at pH 7.6 to 7.7, a high level of supercoiling was needed to keep about half of the molecules in the H conformation; at pH 6 and pH 5, normal levels of supercoiling supported H-DNA; and at pH 4, no supercoiling was required. At mildly alkaline pH, the TC/AG18 repeat assumed a novel conformation called J-DNA that differed from both the B and H forms. A three-dimensional model for the structure of H-DNA is proposed that accounts both for the single-strandedness of the nucleotides and for the influence of supercoiling on H-DNA formation. This model predicts and evidence is presented that H-DNA introduces a sharp kink in the DNA. Moreover, the angle of this kink appears not to be fixed, so that H-DNA is also a hinged-DNA.

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. ...