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. ...

Members of the HMG-I(Y) family of mammalian nonhistone proteins are of importance because they have been demonstrated to bind specifically to the minor groove of A.T-rich sequences both in vitro and in vivo and to function as gene transcriptional regulatory proteins in vivo. Here we report the cloning, sequencing, characterization and chromosomal localization of the human HMG-I(Y) gene. The gene has several potential promoter/enhancer regions, a number of different transcription start sites and numerous alternatively spliced exons making it one of the most complex nonhistone chromatin protein-encoding genes so far reported. The putative promoter/enhancer regions each contain a number of conserved nucleotide sequences for potential binding of inducible regulatory transcription factors. Consistent with the presence of these conserved sequences, we found that transcription of the HMG-I(Y) gene is inducible in human lymphoid cells by factors such as phorbol esters and calcium ionophores. Detailed sequence analysis confirms our earlier suggestion that alternative splicing of precursor mRNAs gives rise to the major HMG-I and HMG-Y isoform proteins found in human cells. Furthermore, the gene's exon-intron arrangement fully accounts for all of the previously cloned human HMG-I(Y) cDNAs (1,2). Also of considerable interest is the fact that each of the three different DNA-binding domain peptides present in an individual HMG-I(Y) protein is coded for by sequences present on separate exons thus potentially allowing for exon 'shuffling' of these functional domains during evolution. And, finally, we localized the gene to the short arm of chromosome 6 (6p) in a region that is known to be involved in rearrangements, translocations and other abnormalities correlated with a number of human cancers.

Nucleosomes associated with transcribing chromatin of mammalian cells have an unfolded structure in which the normally buried cysteinyl-thiol group of histone H3 is exposed. In this study we analyzed transcriptionally active/competent DNA-enriched chromatin fractions from chicken mature and immature erythrocytes for the presence of thiol-reactive nucleosomes using organomercury-agarose column chromatography and hydroxylapatite dissociation chromatography of chromatin fractions labeled with [ 3 H]iodoacetate. In mature and immature erythrocytes, the active DNA-enriched chromatin fractions are associated with histones that are rapidly highly acetylated and rapidly deacetylated. When histone deacetylation was prevented by incubating cells with histone deacetylase inhibitors, sodium butyrate or trichostatin A, thiol-reactive H3 of unfolded nucleosomes was detected in the soluble chromatin and nuclear skeleton-associated chromatin of immature, but not mature, erythrocytes. We did not find thiol-reactive nucleosomes in active DNA-enriched chromatin fractions of untreated immature erythrocytes that had low levels of highly acetylated histones H3 and H4 or in chromatin of immature cells incubated with inhibitors of transcription elongation. This study shows that transcription elongation is required to form, and histone acetylation is needed to maintain, the unfolded structure of transcribing nucleosomes.Acetylation of the core histones (H2A, H2B, H3, and H4) is a dynamic process catalyzed by histone acetyltransferases and histone deacetylases (1, 2). In chicken immature erythrocytes, 4% of the modifiable lysine sites participate in dynamic histone acetylation. These core histones are rapidly acetylated (t1 ⁄2 ϭ 12 min for monoacetylated H4) and rapidly deacetylated (t1 ⁄2 ϭ 5 min for the tetraacetylated isoform of H4) (3, 4). Histones undergoing rapid acetylation and deacetylation are associated with transcriptionally active chromatin (5-7). The recent findings that histone acetyltransferases and deacetylases are transcriptional coactivators and corepressors have increased our understanding of how the process of dynamic histone acetylation is established on transcriptionally active chromatin (2, 8).Transcriptionally active chromatin has a soluble and insoluble nature (9). Transcribed DNA is found in chromatin fragments that are soluble in 0.15 M NaCl and/or 2 mM MgCl 2 and in chromatin fragments associated with the low salt-insoluble residual nuclear material (nuclear skeletons) (for review, see Davie (10)). Chromatin engaged in transcription is thought to be retained by the nuclear skeleton by multiple dynamic attachments between the nuclear matrix and transcribed chromatin; hence rendering the transcribing chromatin insoluble (11, 12). As histone acetyltransferase and deacetylase activities are associated with the nuclear matrix (7, 13), we proposed that these nuclear matrix-bound enzymes may mediate some of the dynamic attachments between active chromatin and nuclear matrix (13,14). Most information on the structure ...

The full-length cDNA encoding a novel human intracellular serine proteinase inhibitor has been sequenced and found to encode a 376 amino acid protein (M(r) approximately 42.5K) that we designate as cytoplasmic antiproteinase. Analysis of the primary structure revealed that the cytoplasmic antiproteinase has the majority of structural motifs conserved among the greater superfamily of serine proteinase inhibitors, or serpins. On the basis of several criteria such as amino acid identity and the absence of a classical N-terminal signal peptide, the cytoplasmic antiproteinase represents a new member of the intracellular serpin family. Further inspection of the cytoplasmic antiproteinase amino acid sequence identified three potential N-glycosylation sites and Arg341-Cys342 as the reactive site P1-P1' residues, respectively. We have also employed the slow binding kinetic approach to detail the mechanism of bovine trypsin and human factor Xa inhibition by the novel cytoplasmic antiproteinase. Inhibition of trypsin by the cytoplasmic antiproteinase was preceded by a two-step mechanism corresponding to the formation of an initial loose complex, followed by an isomerization step to a more stable, tight complex. The binding of the cytoplasmic antiproteinase to trypsin occurred with a second-order association rate constant of 2.8 x 10(6) M-1 s-1 and an overall equilibrium constant of 22.5 pM, demonstrating that the factor is a potent inhibitor of this proteinase. Under the appropriate conditions, the tight complex between trypsin and the cytoplasmic inhibitor was reversible, indicated by an exponential regeneration of proteinase amidolytic activity from the preformed complex. Therefore, the tight complex appears to be stabilized predominantly by reversible bonds that form between trypsin and the cytoplasmic inhibitor. In contrast to the inhibition of trypsin, the inhibition of factor Xa amidolytic activity by the cytoplasmic antiproteinase followed a single-step binding mechanism. The apparent first-order rate constant for factor Xa inhibition was found to increase as a linear function of the inhibitor concentration range studied. Formation of the inhibitory complex between factor Xa and the cytoplasmic antiproteinase occurred with a second-order association rate constant of approximately 1.3 x 10(5) M-1 s-1 and a equilibrium constant of 3.7 nM. These findings suggests that the cytoplasmic inhibitor may initially encounter significant energy barriers for proper alignment with the substrate binding cleft of factor Xa. However, once aligned, the reaction proceeds rapidly to a tight factor Xa.inhibitor complex that dissociates at a slow rate.

Tagging hormone receptors with the green fluorescent protein (GFP) has increased our knowledge of ligand dependent sub-cellular trafficking of hormone receptors. However, the effect of the tagged hormone receptor expression on the corresponding wild type hormone receptor and endogenous gene expression has not been investigated. In this study, we constructed a MCF-7 cell line stably expressing GFP-tagged human estrogen receptor-alpha (ER) under control of the tetracycline-on system to determine the effect of GFP-ER expression on cell proliferation and expression of endogenous ER and hormone-responsive genes. Further, the inducible system was applied to determine the ligand dependent turnover rates of GFP-ER protein and mRNA. Our results demonstrate that GFP-ER expression did not affect cell cycling. Independent of ligand, GFP-ER markedly reduced the level of endogenous ER mRNA and protein, suggesting that ER negatively autoregulates its expression. Cisplatin cross-linking studies showed that GFP-ER is associated with nuclear DNA in situ, suggesting that GFP-ER is partially replacing ER at estrogen response elements. Furthermore, GFP-ER expression did not affect the estradiol induced temporal expression of hormone responsive genes c-myc and pS2.