bluuebunny/crossref_metadata_2025_split huggingface dataset:
An Inhibitory Region of the DNA-Binding Domain of Thyroid Hormone Receptor Blocks Hormone-Dependent Transactivation
Authors: Liu Ying , Takeshita Akira , Nagaya Takashi , Baniahmad Aria , Chin William W. , Yen Paul M. 
Published: January 2014

AbstractWe have employed a chimeric receptor system in which we cotransfected yeast GAL4 DNA-binding domain/retinoid X receptor β ligand-binding domain chimeric receptor (GAL4RXR), thyroid hormone receptor-β (TRβ), and upstream activating sequence-reporter plasmids into CV-1 cells to study repression, derepression, and transcriptional activation. In the absence of T3, unliganded TR repressed transcription to 20% of basal level, and in the presence of T3, liganded TRβ derepressed transcription to basal level. Using this system and a battery of TRβ mutants, we found that TRβ/RXR heterodimer formation is necessary and sufficient for basal repression and derepression in this system. Additionally, an AF-2 domain mutant (E457A) mediated basal repression but not derepression, suggesting that interaction with a putative coactivator at this site may be critical for derepression. Interestingly, a mutant containing only the TRβ ligand binding domain (LBD) not only mediated derepression, but also stimulated transcriptional activation 10-fold higher than basal level. Studies using deletion and domain swap mutants localized an inhibitory region to the TRβ DNA-binding domain. Titration studies further suggested that allosteric changes promoting interaction with coactivators may account for enhanced transcriptional activity by LBD. In summary, our findings suggest that TR heterodimer formation with RXR is important for repression and derepression, and coactivator interaction with the AF-2 domain may be needed for derepression in this chimeric system. Additionally, there may be an inhibitory region in the DNA-binding domain, which reduces TR interaction with coactivators, and prevents full-length wild-type TRβ from achieving transcriptional activation above basal level in this chimeric receptor system.
Functional Interaction between the Estrogen Receptor and CTF1: Analysis of the Vitellogenin Gene B1 Promoter in Yeast
Authors: Tsai-Pflugfelder Monika , Gasser Susan M. , Wahli Walter 
Published: January 2014

AbstractEukaryotic gene expression depends on a complex interplay between the transcriptional apparatus and chromatin structure. We report here a yeast model system for investigating the functional interaction between the human estrogen receptor (hER) and CTF1, a member of the CTF/NFI transcription factor family. We show that a CTF1-fusion protein and the hER transactivate a synthetic promoter in yeast in a synergistic manner. This interaction requires the proline-rich transactivation domain of CTF1. When the natural estrogen-dependent vitellogenin B1 promoter is tested in yeast, CTF1 and CTF1-fusion proteins are unable to activate transcription, and no synergy is observed between hER, which activates the B1 promoter, and these factors. Chromatin structure analysis on this promoter reveals positioned nucleosomes at −430 to −270 (±20 bp) and at −270 to− 100 (±20 bp) relative to the start site of transcription. The positions of the nucleosomes remain unchanged upon hormone-dependent transcriptional activation of the promoter, and the more proximal nucleosome appears to mask the CTF/NFI site located at −101 to −114. We conclude that a functional interaction of hER with the estrogen response element located upstream of a basal promoter occurs in yeast despite the nucleosomal organization of this promoter, whereas the interaction of CTF1 with its target site is apparently precluded by a nucleosome.
Functional Activities of the A and B Forms of the Human Androgen Receptor in Response to Androgen Receptor Agonists and Antagonists
Authors: Gao Tianshu , McPhaul Michael J. 
Published: January 2014

AbstractThe androgen receptor (AR) is present in many cells in two forms. The B form migrates with an apparent mass of 110 kDa and constitutes more than 80% of the immunoreactive receptor in most cell types. The A form of the AR migrates with an apparent mass of 87 kDa, appears to derive from internal translation initiation at methionine-188 in the AR open-reading frame, and usually constitutes 20% or less of the immunoreactive AR present. Previous experiments designed to examine the functional capacity of the A and B forms of the AR have been hampered by marked differences in the expression levels of the two isoforms, as the nucleotide sequence surrounding the codon encoding methionine-188 causes it to be used inefficiently as a translation initiation site. To circumvent this, we altered the nucleotide sequence surrounding methionine-188 to render it more similar to that surrounding the codon encoding methionine-1. Transfection of a cDNA containing these changes resulted in similar levels of expression of A and B forms of the AR as assessed by immunoblot assays using antibodies directed at an epitope preserved in both. Functional activities of these cDNAs were assessed using cotransfection assays that employed two model androgen-responsive genes (MMTV-luciferase and PRE2-tk-luciferase) in response to mibolerone, a potent androgen agonist, in three different cell lines. These studies demonstrated subtle differences in the activities of the A and B isoforms, which depended on the promoter and cell context. Additional studies failed to reveal any major differences in the responses of the AR-A and AR-B isoforms to a variety of androgen agonists and antagonists, suggesting that the previously reported functional defect of the AR-A is due principally to its level of expression. When assays of AR function are performed under conditions in which levels of expression of the two isoforms are equivalent, the AR-A and AR-B possess similar functional activities.
Structure, Expression, and Function of Human Pituitary Tumor-Transforming Gene (PTTG)
Authors: Zhang Xun , Horwitz Gregory A. , Prezant Toni R. , Valentini Alberto , Nakashima Masahiro , Bronstein Marcello D. , Melmed Shlomo 
Published: January 2014

AbstractDespite advances in characterizing the pathophysiology and genetics of pituitary tumors, molecular mechanisms of their pathogenesis are poorly understood. Recently, we isolated a transforming gene [pituitary tumor-transforming gene (PTTG)] from rat pituitary tumor cells. Here we describe the cloning of human PTTG, which is located on chromosome 5q33 and shares striking sequence homology with its rat counterpart. Northern analysis revealed PTTG expression in normal adult testis, thymus, colon, small intestine, brain, lung, and fetal liver, but most abundant levels of PTTG mRNA were observed in several carcinoma cell lines. Stable transfection of NIH 3T3 cells with human PTTG cDNA caused anchorage-independent transformation in vitro and induced in vivo tumor formation when transfectants were injected into athymic mice. Overexpression of PTTG in transfected NIH 3T3 cells also stimulated expression and secretion of basic fibroblast growth factor, a human pituitary tumor growth-regulating factor. A proline-rich region, which contains two PXXP motifs for the SH3 domain-binding site, was detected in the PTTG protein sequence. When these proline residues were changed by site-directed mutagenesis, PTTG in vitro transforming and in vivo tumor-inducing activity, as well as stimulation of basic fibroblast growth factor, was abrogated. These results indicate that human PTTG, a novel oncogene, may function through SH3-mediated signal transduction pathways and activation of growth factor(s).
Role of the Third Intracellular Loop for the Activation of Gonadotropin Receptors
Authors: Schulz Angela , Schöneberg Torsten , Paschke Ralf , Schultz Günter , Gudermann Thomas 
Published: January 2014

AbstractHyperfunctional endocrine thyroid and testicular disorders can frequently be traced back to gain-of-function mutations in glycoprotein hormone receptor genes. Deletion mutations in the third intracellular (i3) loop of the TSH receptor have recently been identified as a cause of constitutive receptor activity. To examine whether the underlying mechanism of receptor activation applies to all glycoprotein hormone receptors, we created deletion mutations in the LH and FSH receptors. In analogy to the situation with the TSH receptor, a deletion of nine amino acids resulted in constitutive activity irrespective of the location of deletions within the i3 loop of the LH receptor. In contrast, only one (Δ563–566) of four different 4-amino acid deletion mutants displayed agonist-independent activity. Systematic examination of the structural requirements for this effect in the Δ563–566 mutant revealed that only deletions including D564 resulted in constitutive receptor activity. Replacement of D564 by G, K, and N led to agonist-independent cAMP formation while introduction of a negatively charged E silenced constitutive receptor activity, indicating that an anionic amino acid at this position may be required to maintain an inactive receptor conformation. Insertion of A residues up- and downstream of D564 did not perturb receptor quiescence, showing that a certain degree of spatial freedom of the negatively charged amino acid within the context of the i3 loop is well tolerated. In contrast to the results obtained with the LH receptor, deletion of the corresponding D567 from the i3 loop of the FSH receptor did not cause constitutive receptor activation, highlighting significant differences in the activation mechanism of gonadotropin receptors.
Ligand-Activated Retinoic Acid Receptor Inhibits AP-1 Transactivation by Disrupting c-Jun/c-Fos Dimerization
Authors: Zhou Xiao-Feng , Shen Xi-Qiang , Shemshedini Lirim 
Published: January 2014

AbstractIn the presence of retinoic acid (RA), the retinoid receptors, retinoic acid receptor (RAR) and retinoid X receptor (RXR), are able to up-regulate transcription directly by binding to RA-responsive elements on the promoters of responsive genes. Liganded RARs and RXRs are also capable of down-regulating transcription, but, by contrast, this is an indirect effect, mediated by the interaction of these nuclear receptors not with DNA but the transcription factor activating protein-1 (AP-1). AP-1 is a dimeric complex of the protooncoproteins c-Jun and c-Fos and directly regulates transcription of genes important for cellular growth. Previous in vitro results have suggested that RARs can block AP-1 DNA binding. Using a mammalian two-hybrid system, we report here that human RARα (hRARα) can disrupt in a RA-dependent manner the homo- and heterodimerization properties of c-Jun and c-Fos. This inhibition of dimerization is cell specific, occurring only in those cells that exhibit RA-induced repression of AP-1 transcriptional activity. Furthermore, this mechanism appears to be specific for the RARs, since another potent inhibitor of AP-1 activity, the glucocorticoid receptor, does not affect AP-1 dimerization. Our data argue for a novel mechanism by which RARs can repress AP-1 DNA binding, in which liganded RARs are able to interfere with c-Jun/c-Jun homodimerization and c-Jun/c-Fos heterodimerization and, in this way, may prevent the formation of AP-1 complexes capable of DNA binding.
Cell Membrane and Nuclear Estrogen Receptors (ERs) Originate from a Single Transcript: Studies of ERα and ERβ Expressed in Chinese Hamster Ovary Cells
Authors: Razandi Mahnaz , Pedram Ali , Greene Geoffrey L. , Levin Ellis R. 
Published: January 2014

AbstractThe existence of a putative membrane estrogen receptor (ER) has been supported by studies accomplished over the past 20 yr. However, the origin and functions of this receptor are not well defined. To study the membrane receptor, we transiently transfected cDNAs for ERα or ERβ into Chinese hamster ovary (CHO) cells. Transfection of ERα resulted in a single transcript by Northern blot, specific binding of labeled 17β-estradiol (E2), and expression of ER in both nuclear and membrane cell fractions. Competitive binding studies in both compartments revealed near identical dissociation constants (Kds) of 0.283 and 0.287 nm, respectively, but the membrane receptor number was only 3% as great as the nuclear receptor density. Transfection of ERβ also yielded a single transcript and nuclear and membrane receptors with respective Kd values of 1.23 and 1.14 nm; the membrane receptor number was only 2% compared with expressed nuclear receptors. Estradiol binding to CHO-ERα or CHO-ERβ activated Gαq and Gαs proteins in the membrane and rapidly stimulated corresponding inositol phosphate production and adenylate cyclase activity. Binding by 17-β-E2 to either expressed receptor comparably enhanced the nuclear incorporation of thymidine, critically dependent upon the activation of the mitogen-activated protein kinase, ERK (extracellular regulated kinase). In contrast, c-Jun N-terminal kinase activity was stimulated by 17-β-E2 in ERβ-expressing CHO, but was inhibited in CHO-ERα cells. In summary, membrane and nuclear ER can be derived from a single transcript and have near-identical affinities for 17-β-E2, but there are considerably more nuclear than membrane receptors. This is also the first report that cells can express a membrane ERβ. Both membrane ERs activate G proteins, ERK, and cell proliferation, but there is novel differential regulation of c-Jun kinase activity by ERβ and ERα.
Identification of a Placental-Specific Enhancer in the Rat Placental Lactogen II Gene That Contains Binding Sites for Members of the Ets and AP-1 (Activator Protein 1) Families of Transcription Factors
Authors: Sun Yuxiang , Duckworth Mary Lynn 
Published: January 2014

AbstractWe previously identified a 3-kb proximal 5′-flanking region of the rat placental lactogen (rPLII) gene1 that is important for reporter gene transcription in the rat trophoblast cell line, Rcho, and targets expression to the placentas of transgenic mice. In our current studies we have used further deletion analysis and transfection studies in Rcho and GC cells to map more precisely the locations of regulatory elements involved in this placental expression. We show that sequences between −1435 and− 765 are necessary for minimal expression in Rcho cells and that there are negative regulatory elements between −3031 to −2838 and −1729 to− 1435. Most importantly, we have identified a fragment between −1793 to −1729 that is essential for expression levels characteristic of the complete 3-kb 5′-region. When linked to the herpes simplex thymidine kinase minimal promoter, this fragment acts as an enhancing element in Rcho but not GC cells. Deoxyribonuclease I (DNAse I) protection and electrophoretic mobility shift assays with nuclear extracts and in vitro translated proteins identify binding sites for members of the activator protein-1 (AP-1) and Ets families of transcription factors. Site-directed mutagenesis of the individual AP-1- and Ets-binding sites leads to a partial loss of the enhancing activity; a double AP-1/Ets mutation leads to a complete loss of activity, demonstrating the functional importance of these sites. By these criteria, putative GATA-binding sites located within the enhancing fragment are not active. These new data suggest an important role for this enhancing fragment in rPLII placental giant cell expression and are the first to implicate a member of the Ets family in the regulation of this gene family.
Chromatin Recycling of Glucocorticoid Receptors: Implications for Multiple Roles of Heat Shock Protein 90
Authors: Liu Jimin , DeFranco Donald B. 
Published: January 2014

AbstractUnliganded glucocorticoid receptors (GRs) released from chromatin after hormone withdrawal remain associated with the nucleus within a novel subnuclear compartment that serves as a nuclear export staging area. We set out to examine whether unliganded nuclear receptors cycle between distinct subnuclear compartments or require cytoplasmic transit to regain hormone and chromatin-binding capacity. Hormone-withdrawn rat GrH2 hepatoma cells were permeabilized with digitonin to deplete cytoplasmic factors, and then hormone-binding and chromatin-binding properties of the recycled nuclear GRs were measured. We found that recycled nuclear GRs do not require cytosolic factors or ATP to rebind hormone. Nuclear GRs that rebind hormone in permeabilized cells target to high-affinity chromatin-binding sites at 30 C, but not 0 C, in the presence of ATP. Since geldanamycin, a heat shock protein-90 (hsp90)-binding drug, inhibits hormone binding to recycled nuclear GRs, hsp90 may be required to reassemble the receptor into a form capable of productive interactions with hormone. Geldanamycin also inhibits GR release from chromatin during hormone withdrawal, suggesting that hsp90 chaperone function may play multiple roles to facilitate chromatin recycling of GR.
Transcriptional Activation of E2F1 Gene Expression by 17β-Estradiol in MCF-7 Cells Is Regulated by NF-Y-Sp1/Estrogen Receptor Interactions
Authors: Wang Weili , Dong Lian , Saville Brad , Safe Stephen 
Published: January 2014

Abstract17β-Estradiol (E2) stimulated proliferation and DNA synthesis in MCF-7 human breast cancer cells, and this was accompanied by induction of E2F1 mRNA and protein levels. Analysis of the E2F1 gene promoter showed that the −146 to− 54 region was required for E2-responsiveness in transient transfection assays, and subsequent deletion/mutation analysis showed that a single upstream GC-rich and two downstream CCAAT-binding sites were required for transactivation by E2. Gel mobility shift assays with multiple oligonucleotides and protein antibodies (for supershifts) showed that the −146 to −54 region of the E2F1 gene promoter bound Sp1 and NF-Y proteins in MCF-7 cells. The estrogen receptor (ER) protein enhanced Sp1 interactions with upstream GC-rich sites, and interactions of ER, Sp1, and ER/Sp1 with downstream DNA bound-NF-Y was investigated by kinetic analysis for protein-DNA binding (on- and off-rates), coimmunoprecipitation, and pulldown assays using wild-type and truncated glutathione S-transferase (GST)-Sp1 chimeric proteins. The results showed that Sp1 protein enhanced the Bmax of NF-Y-DNA binding by more than 5-fold (on-rate); in addition, the Sp1-enhanced NF-Y-DNA complex was further stabilized by coincubation with ER and the rate of dissociation (t1/2) was decreased by approximately 50%. Sp1 antibodies immunoprecipitated [35S]NF-YA after coincubation with unlabeled Sp1 protein. Thus, transcriptional activation of E2F1 gene expression in MCF-7 cells by E2 is regulated by multiprotein ER/Sp1-NF-Y interactions at GC-rich and two CCAAT elements in the proximal region of the E2F1 gene promoter. This represents a unique trans-acting protein complex in which ligand-dependent transactivation by the ER is independent of direct ER interactions with promoter elements.
Stat5b Inhibits NFκB-Mediated Signaling
Authors: Luo Guoyang , Yu-Lee Li-yuan 
Published: January 2014

AbstractSignal transducers and activators of transcription (Stat) are latent transcription factors that participate in cytokine signaling by regulating the expression of early response genes. Our previous studies showed that Stat5 functions not only as a transcriptional activator but also as a transcriptional inhibitor, depending on the target promoter. This report further investigates the mechanism of Stat5b-mediated inhibition and demonstrates that PRL-inducible Stat5b inhibits nuclear factorκB (NFκB) signaling to both the interferon regulatory factor-1 promoter and to the thymidine kinase promoter containing multimerized NFκB elements (NFκB-TK). Further, PRL-inducible Stat5b inhibits tumor necrosis factor-α signaling presumably by inhibiting endogenous NFκB. This Stat5b-mediated inhibitory effect on NFκB signaling is independent of Stat5b-DNA interactions but requires the carboxyl terminus of Stat5b as well as Stat5b nuclear translocation and/or accumulation, suggesting that Stat5b is competing for a nuclear factor(s) necessary for NFκB-mediated activation of target promoters. Increasing concentrations of the coactivator p300/CBP reverses Stat5b inhibition at both the interferon-regulatory factor-1 and NFκB-TK promoters, suggesting that Stat5b may be squelching limiting coactivators via protein-protein interactions as one mechanism of promoter inhibition. These results further substantiate our observation that Stat factors can function as transcriptional inhibitors. Our studies reveal cross-talk between the Stat5b and NFκB signal transduction pathways and suggest that Stat5b-mediated inhibition of target promoters occurs at the level of protein-protein interactions and involves competition for limiting coactivators.
Endocrine Disrupting Chemicals, Phthalic Acid and Nonylphenol, Activate Pregnane X Receptor-Mediated Transcription
Authors: Masuyama Hisashi , Hiramatsu Yuji , Kunitomi Mamoru , Kudo Takafumi , MacDonald Paul N. 
Published: January 2014

Abstract
Recently, Pregnane X receptor (PXR), a new member of the nuclear receptor superfamily, was shown to mediate the effects of several steroid hormones, such as progesterone, glucocorticoid, pregnenolone, and xenobiotics on cytochrome P450 3A genes (CYP3A) through the specific DNA sequence for CYP3A, suggesting that PXR may play a role in steroid hormone metabolism. In this paper, we demonstrated that phthalic acid and nonylphenol, endocrine-disrupting chemicals (EDCs), stimulated PXR-mediated transcription at concentrations comparable to those at which they activate estrogen receptor-mediated transcription using a transient reporter gene expression assay in COS-7 cells. However, bisphenol A, another EDC, had no effect on PXR-mediated transcription, although this chemical significantly enhanced ER-mediated transcription. In the yeast two-hybrid protein interaction assay, PXR interacted with two nuclear receptor coactivator proteins, steroid hormone receptor coactivator-1 and receptor interacting protein 140, in the presence of phthalic acid or nonylphenol. Thus, EDC-occupied PXR may regulate its specific gene expression through the receptor-coactivator interaction. In contrast, these EDCs had no effect on the interaction between PXR and suppressor for gal 1, a component of proteasome. Finally, the expression of CYP3A1 mRNA in the liver of rats exposed to phthalic acid or nonylphenol markedly increased compared with that in rats treated with estradiol, bisphenol A, or ethanol as assessed by competitive RT-PCR. These data suggest that EDCs may affect endocrine functions by altering steroid hormone metabolism through PXR.
Molecular Cloning, Expression, and Characterization of Rat Homolog of Human AP-2α That Stimulates Neuropeptide Y Transcription Activity in Response to Nerve Growth Factor
Authors: Li Bing-Sheng , Kramer Phillip R. , Zhao Weiqin , Ma Wu , Stenger David A. , Zhang Lei 
Published: January 2014

AbstractNeuropeptide Y (NPY) plays an important role in the central regulation of neuronal activity, endocrine and sexual behavior, and food intake. Although transcription activity of the NPY gene in PC12 cells is regulated by a number of agents such as nerve growth factor (NGF), the mechanism responsible for the NGF-elicited increase in the transcription of the NPY gene remains to be explored. In this study, we isolated and characterized a nuclear protein that is bound to NGF-response elements (NGFRE) that lie between nucleotide −87 and −33 of the rat NPY promoter gene. This nuclear protein is identical to the rat homolog of human transcription factor AP-2α. We further demonstrated that rat AP-2α promotes efficient NPY transcription activity in response to NGF. Finally, we provide direct evidence that the mice lacking transcription factor AP-2α exhibit reduced expression of NPY mRNA compared with wild-type mice, further supporting the hypothesis that AP-2α is an important transcription factor in regulating NPY transcription activity.