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PubMed1998

Extreme position dependence of a canonical hormone response element.

Hormone response elements (HREs) are considered enhancers, activating transcription in a relatively position- and orientation-independent fashion. Upon binding to an HRE, steroid receptors presumably contact coactivators and/or proteins associated with the transcription initiation complex. As a receptor target site is moved further from a fixed position such as the TATA box, not only will the spatial separation of the receptor with respect to its interaction partners change, so will the orientation due to the rotation of the DNA helix. Additional constraints may be imposed by the assembly of DNA into chromatin. Therefore, we have endeavored to test rigorously the assertion that HRE action is position independent. We have constructed a series of 42 chloramphenicol acetyltransferase expression vectors that contain a single progesterone/glucocorticoid receptor-binding site separated from a TATA box by 4 to 286 bp. The enhancer activity of the HRE was assessed after transient transfection of progesterone receptor-expressing fibroblasts. We find that the position of the HRE has a dramatic influence on induction by progestins. When closely juxtaposed to the TATA box, the HRE was unable to support a hormone response, perhaps due to direct steric hindrance with the transcription initiation complex. Full activity was gained by moving the HRE 10 bp further from the TATA sequence. As the HRE was moved incrementally further, activity remained near maximal over the next 26 bp. HRE activity then declined over the subsequent 26 bp and remained low for another 2.5 helical turns. Surprisingly, a narrow window of HRE activity occurred at an HRE-TATA box separation of 90-100 bp. Little or no hormone-induced transcriptional activity was observed when the HRE was positioned further from the TATA box. The addition of a second HRE or a basal (nuclear factor-1) element failed to relieve this constraint. A similar series of experiments was carried out in a mammary carcinoma cell line that expressed high levels of both glucocorticoid and progestin receptors. Data in these cells indicate that glucocorticoids and progestins supported a similar HRE position-activity profile, but this pattern of HRE activity was quite distinct from that seen in fibroblasts. This may be indicative of cell type-specific interactions between steroid receptors and adapter/coactivator proteins or cell type-specific activities such as acetylases or deacetylases participating in the steroid response.

Base Sequence↗
PubMed2004

Transcription factor NF2d9 (LBP-1a) interacts with the positive regulatory element for the xenobiotic responsive element.

PREX is a positive regulatory element for xenobiotic responsive element (XRE)-mediated gene expression that is located upstream of the XRE in the CYP2A8 gene. Using gel mobility shift assays, we demonstrated that NF2d9 (LBP-1a), a transcription factor related to CP2 (LBP-1c/LSF), bound directly to PREX and also interacts indirectly with XRE. Luciferase-reporter gene assays showed that the overexpression of NF2d9 enhanced PREX and XRE-driven CYP2A8 gene transcriptional induction. These findings suggest that the interaction of NF2d9 with PREX and XRE enhances XRE-driven CYP2A8 gene transcriptional induction.

Animals↗
PubMed1988

Synergistic action of glucocorticoid and estradiol responsive elements.

Modulation of gene expression by steroid hormones is mediated by receptor proteins that associate with regulatory elements of responsive genes upon binding the hormone ligand. The finding that two glucocorticoid responsive elements act cooperatively to stimulate transcription of the tyrosine aminotransferase gene prompted us to explore whether synergistic effects also occur when two different steroid hormone receptors are involved. A region of the chicken vitellogenin II gene that displays homologies to glucocorticoid and estradiol responsive elements was tested for its capability to confer estradiol and glucocorticoid inducibility to a heterologous promoter. When positioned immediately upstream of the thymidine kinase gene promoter, this element enhances expression by either steroid. Combination of both hormones results in a synergistic increase of transcription. Mutational analysis shows that sequences that show similarities of glucocorticoid and estradiol responsive elements are absolutely required for hormone induction. Analysis of the dose dependence of induction by both steroids demonstrates that half-maximal activity is observed at lower hormone concentrations when the other steroid is present in saturating amounts, which suggests that the synergistic induction observed with the combination of hormones is based on a functional interaction of the two hormone receptors.

Adenocarcinoma↗
PubMed1994

Delineation of three different thyroid hormone-response elements in promoter of rat sarcoplasmic reticulum Ca2+ATPase gene. Demonstration that retinoid X receptor binds 5' to thyroid hormone receptor in response element 1.

Thyroid hormone (3,5,3'-triiodothyronine) positively regulates transcription of the sarcoplasmic reticulum Ca2+ATPase gene in rat heart, and sequences within 559 nucleotides upstream from the transcription start site confer thyroid hormone responsiveness upon a reporter gene. In the present study, three thyroid hormone-response elements (TREs) are identified between nucleotides -485 and -190. Each TRE is active in transient transfection assays and specifically binds 3,5,3'-triiodothyronine receptors (TRs) alpha 1 and beta 1 alone and in combination with retinoid X receptors (RXRs) alpha and beta. TRE 1 is a direct repeat of two half-sites separated by four nucleotides; TREs 2 and 3 are inverted palindromes of two half-sites separated by four and six nucleotides, respectively. Methylation interference analysis of TRE 1 showed binding of a TR alpha 1 monomer to the 3' half-site, whereas the heterodimer contacts both half-sites. Subsequent studies employed TR beta and RXR alpha mutants in which their P-boxes were replaced with the P-box of the glucocorticoid receptor. Bandshifts of wild type and mutant proteins with either wild type TRE 1 or a mutant version, in which the 5' half-site was converted to a glucocorticoid response element half-site, demonstrated preferential binding of RXR to the 5' half-site and of TR to the 3' half-site of TRE 1.

Animals↗
PubMed1994

The rat vasoactive intestinal polypeptide cyclic AMP response element regulates gene transcriptional responses differently in neonatal and adult rat sensory neurons.

We studied the ability of the rat vasoactive intestinal polypeptide (VIP) cyclic AMP responsive element (CRE) to regulate reporter gene expression through a c-fos promoter in rat sensory neurons transfected in culture by plasmid microinjection. The CRE enhanced the synergistic response of the promoter to combined potassium-evoked depolarisation and forskolin treatment in neonatal but not adult rat neurons. This corresponds to endogenous VIP expression which is induced synergistically by the same stimuli in neonatal but not adult rat neurons. We conclude that VIP expression in sensory neurons, which is induced by axotomy in vivo, could be regulated through the CRE.

Aging↗
PubMed1998

The proximal interferon-stimulated response elements are essential for interferon responsiveness: a promoter analysis of the antiviral MxA gene.

Interferon (IFN)-inducible human MxA protein mediates resistance against influenza and several other RNA viruses. The MxA gene is under the control of type I IFN and, in certain cell types, is also directly activated by viruses. Here we show that in human macrophages, MxA mRNA levels are upregulated by very low doses of IFN-alpha in a dose-dependent manner. A similar, albeit much weaker, dose-dependent induction was seen with IFN-gamma. The induction was rapid and independent of protein synthesis. Interleukin-6 (IL-6) or tumor necrosis factor-alpha (TNF-alpha) did not influence MxA mRNA levels alone or in combination with IFNs, in spite of the presence of putative response elements of these cytokines in the MxA promoter. We show that the promoter of the MxA gene contains two functional IFN-stimulated response elements (ISRE) near the transcription start site and one homologous ISRE-like element, which is apparently nonfunctional, further upstream. The two proximal ISRE sites are essential for IFN-alpha-induced transcription and appear to be binding sites for IFN-stimulated gene factor 3 complex. In addition, EMSA and DNAse I footprinting analysis demonstrated that Spl binds with high affinity to a region encompassing nucleotides -25 and -50 and, thus, may provide means of interaction with the basal transcriptional machinery.

Antiviral Agents↗
PubMed2003

The role of the iron responsive element in the control of ferroportin1/IREG1/MTP1 gene expression.

BACKGROUND/AIMS: MTP1/Ferroportin1/IREG1, the product of the SLC40A1 gene, is a main iron export protein in mammals. However, the way this gene is regulated by iron is still unclear. The aim of this study was to investigate the functional role of genomic SLC40A1 elements in response to iron. METHODS: Vectors containing either reverse similar 2.6 kb 5' flanking region or deletion constructs, including one devoid of an iron responsive element (SLC40A1-DeltaIRE-Luc), were analyzed by luciferase reporter gene in transfected HepG2, CaCO2 and U937 cells. Expression of iron genes and activity of the iron regulatory protein were also studied. RESULTS: Iron increased and desferrioxamine decreased luciferase activity in all the cell types using both the full-length construct and the promoter deletion constructs, in the absence of changes in SLC40A1 or luciferase mRNA levels. To test the role of the SLC40A1 5' untranslated region, we first demonstrated that wild type and not SLC40A1-DeltaIRE-Luc could bind iron regulatory protein. Then, in cells transfected with SLC40A1-DeltaIRE-Luc, we found that, in spite of iron regulatory protein activation, the response to iron manipulation was lost. CONCLUSIONS: We demonstrate that the iron responsive element in the SLC40A1 gene is functional and that it controls gene expression through the cytoplasmic iron regulatory protein system.

5' Untranslated Regions↗
PubMed2006

Establishing a statistic model for recognition of steroid hormone response elements.

Identification of hormone response elements (HREs) is essential for understanding the mechanism of hormone-regulated gene expression. To date, there has been a lack of effective bioinformatics tools for recognition of specific HRE such as Progesterone Response Elements (PRE). In this paper, a comprehensive survey and comparison of in silico methods is conducted for establishing a more accurate statistic model. Homogeneity of steroid HRE is analyzed and a reliable training dataset is constructed through extensive searching for experimentally validated response elements from more than 150 literature sources. Based on the observation that the verified HREs carry di-nucleotide preservation in comparison with uniform nucleotide distributions, both mono and di-nucleotide Position Weight Matrices are computed to extract the statistic pattern of the positions. It is followed by the sequence transition pattern recognition using a specifically designed profile Hidden Markov Model. Reciprocal combination of the statistic and transition patterns significantly improves the performance of the model in terms of higher sensitivity and specificity. Upon acquisition of the putative response elements in the promoter areas of vertebrate genes, a qualitative scheme is applied to assess the probability for each gene to be a hormone primary target. Using >650 records of experimentally validated steroid hormone response elements, a high sensitivity level of 73% and high specificity level of one prediction per 8.24 kb is reached, allowing this model to be used for further prediction of primary target genes through the analysis of their upstream promoters, for human or other vertebrate genomes of interest. Additional documents, supplementary data and the web-based program developed for response elements prediction are freely available for academic research at . Submission of putative gene promoter regions for recognition of potential regulatory PREs can be as long as 5 kb.

Animals↗
PubMed1994

Isolation of an X-ray-responsive element in the promoter region of tissue-type plasminogen activator: potential uses of X-ray-responsive elements for gene therapy.

Tissue-type plasminogen activator (t-PA) was induced over 50-fold after X irradiation in radioresistant human melanoma cells (Boothman et al., Cancer Res. 51, 5587-5595, 1991). Activities of t-PA were induced 14-fold in ataxia telangiectasia, 9-fold in Bloom's syndrome and 6-fold in Fanconi's anemia cells, compared to normal human fibroblasts (Fukunaga et al., Int. J. Radiat. Oncol. Biol. Phys. 24, 949-957, 1992). X-ray-inducible synthesis of the protease, t-PA, may play a role(s) in damage-inducible repair processes in mammalian cells, similar to the SOS repair systems in lower eukaryotes and prokaryotes. DNA band shift and DNase I footprinting assays were used to determine binding if transcription factors to a previously unknown X-ray-responsive element (XRE) in the t-PA promoter. The major goals of our research with XREs are to understand (a) which transcription factor(s) regulates t-PA induction after X rays, and (b) the role(s) of t-PA in DNA repair, apoptosis or other responses to X rays. The purpose of this paper is to discuss the potential use of an XRE, such as the one in the t-PA promoter, for gene radiotherapy. Several gene therapy strategies are proposed.

Cell Nucleus↗
PubMed1999

5-Hydroxytryptamine2A receptor stimulation induces activator protein-1 and cyclic AMP-responsive element binding with cyclic AMP-responsive element-binding protein and Jun D as common components in cerebellar neurons.

Previous studies from our laboratory have demonstrated that stimulation of 5-hydroxytryptamine2A receptors in rat cerebellar granule cells produces an increase in the levels of 5-hydroxytryptamine2A receptor messenger RNA and binding sites, and that this up-regulation requires de novo RNA and protein synthesis. Here we showed that up-regulation of 5-hydroxytryptamine2A receptor binding sites induced by stimulation with the 5-hydroxytryptamine2A/2C receptor agonist, (+/-)-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI), is associated with an increase in the 5-hydroxytryptamine2A receptor transcription rate. To examine the possible role of transcriptional activation in DOI-induced 5-hydroxytryptamine2A receptor up-regulation, we studied the effects of DOI on transcription factor binding to activator protein-1 and cyclic AMP-responsive element (CRE) DNA consensus sequences. We found that DOI induces a time-dependent increase in activator protein-1 and CRE transcription factor binding activity, which is blocked by 5-hydroxytryptamine2A receptor antagonists. Similar to 5-hydroxytryptamine2A receptor up-regulation, DOI-induced activator protein-1 binding is suppressed by inhibitors of calmodulin and Ca2+/calmodulin-dependent kinases. The increased activator protein-1 binding is effectively competed by excessive activator protein-1 and CRE sequences as well as endogenous activator protein-1-like sequences present in the rat 5-hydroxytryptamine2A receptor gene. Supershift assays revealed that cAMP-responsive element-binding protein (CREB) and Jun D are common components of both activator protein-1 and CRE binding complexes. DOI also increased the level of phospho-CREB in a time-dependent manner. The binding of phospho-CREB transcription factor to the activator protein-1 site suggests that CREB may modulate the transcription of genes that contain activator protein-1 but lack CRE site in their promoters, through interaction with the activator protein-1 site. The rat 5-hydroxytryptamine2A receptor up-regulation may involve such a mechanism.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1999

Cellular response to cancer chemopreventive agents: contribution of the antioxidant responsive element to the adaptive response to oxidative and chemical stress.

Cancer chemopreventive agents can act by inhibiting either the acquisition of mutations or the neoplastic processes that occur subsequent to mutagenesis. Compounds that reduce the rate at which mutations arise, referred to as blocking agents, exert their effects largely through their ability to induce the expression of antioxidant and detoxification proteins. This is achieved by the transcriptional activation of a small number of genes that are co-regulated through the presence of an antioxidant responsive element (ARE) in their promoters. Blocking agents can cause gene induction by producing oxidative and/or chemical stress within the cell and, as the inducible proteins act to ameliorate the metabolic insult, the process represents a form of adaptive response. The transcription factors which mediate this response through the ARE are members of the basic leucine zipper superfamily. The mechanism whereby cells sense and respond to the chemical signal(s) generated by chemopreventive blocking agents is discussed.

Adaptation, Physiological↗
PubMed2004

Protein hydrolysates stimulate proglucagon gene transcription in intestinal endocrine cells via two elements related to cyclic AMP response element.

AIMS/HYPOTHESIS: Protein hydrolysates (peptones) increase not only glucagon-like peptide-1 (GLP-1) secretion but also transcription of the proglucagon ( PG) gene in the intestine. The critical physiological roles of gut-derived GLPs raised hope for their therapeutic use in several disorders, especially GLP-1 in diabetes. We aimed to investigate the molecular mechanisms involved in this nutrient- PG gene interaction. METHODS: Wild-type and mutated PG promoter fragments fused to the luciferase reporter gene were transfected into enteroendocrine STC-1 cells, which were then either treated or not with peptones. Co-transfection with expression vectors of dominant-negative forms of cAMP response element binding protein (CREB) and protein kinase A (PKA) proteins were performed, as well as electrophoresis mobility shift assays. RESULTS: Deletion analysis showed that the promoter region spanning between -350 and -292 bp was crucial for the transcriptional stimulation induced by peptones. Site-directed mutagenesis of the canonical cAMP response element (CRE(PG)) and of the adjacent putative CRE site (CRE-like1) led to a dramatic inhibition of the promoter responsiveness to peptones. Over expression of a dominant-negative mutant of CREB or of PKA produced a comparable and selective inhibitory effect on the activity of transfected promoter fragment containing the -350/-292 sequence. EMSA showed that CREB and fra2 transcription factors bound to CRE(PG) and CRE-like1 elements respectively, independently of peptone treatment. CONCLUSIONS/INTERPRETATION: Our report identified cis- and trans-regulatory elements implicated in the transcriptional control of PG gene by nutrients in enteroendocrine cells. It highlights the role of a previously unsuspected CRE-like1 element, and emphasises the importance of CRE-related sequences in the regulation of PG gene transcription in the intestine.

Animals↗
PubMed1989

Regulation of interaction of the iron-responsive element binding protein with iron-responsive RNA elements.

The 5' untranslated region of the ferritin heavy-chain mRNA contains a stem-loop structure called an iron-responsive element (IRE), that is solely responsible for the iron-mediated control of ferritin translation. A 90-kilodalton protein, called the IRE binding protein (IRE-BP), binds to the IRE and acts as a translational repressor. IREs also explain the iron-dependent control of the degradation of the mRNA encoding the transferrin receptor. Scatchard analysis reveals that the IRE-BP exists in two states, each of which is able to specifically interact with the IRE. The higher-affinity state has a Kd of 10 to 30 pM, and the lower affinity state has a Kd of 2 to 5 nM. The reversible oxidation or reduction of a sulfhydryl is critical to this switching, and the reduced form is of the higher affinity while the oxidized form is of lower affinity. The in vivo rate of ferritin synthesis is correlated with the abundance of the high-affinity form of the IRE-BP. In lysates of cells treated with iron chelators, which decrease ferritin biosynthesis, a four- to fivefold increase in the binding activity is seen and this increase is entirely caused by an increase in high-affinity binding sites. In desferrioxamine-treated cells, the high-affinity form makes up about 50% of the total IRE-BP, whereas in hemin-treated cells, the high-affinity form makes up less than 1%. The total amount of IRE-BP in the cytosol of cells is the same regardless of the prior iron treatment of the cell. Furthermore, a mutated IRE is not able to interact with the IRE-BP in a high-affinity form but only at a single lower affinity Kd of 0.7 nM. Its interaction with the IRE-BP is insensitive to the sulfhydryl status of the protein.

Base Sequence↗
PubMed2003

Thioredoxin-dependent redox regulation of the antioxidant responsive element (ARE) in electrophile response.

Thioredoxin is a redox-regulating protein, the expression of which is induced by various forms of oxidative stress. Thioredoxin controls the interactions of various transcription factors through redox regulation. In K562 cells, we have previously reported that hemin induces activation of the thioredoxin gene by regulating NF-E2-related factor (Nrf2) through the antioxidant responsive element (ARE). We showed here that tert-butylhydroquinone (tBHQ), an electrophile stressor, activates the thioredoxin gene through the ARE. In an electrophoretic mobility shift assay, a specific Nrf2/small Maf binding complex was induced by tBHQ and bound to the ARE. Overexpression of Nrf2 increased the tBHQ-induced thioredoxin gene activation through the ARE, whereas that of Jun and Fos suppressed the activation. The tBHQ-induced ARE binding activity was completely abrogated by an oxidizing agent, diamide, whereas 2-mercaptoethanol (2-ME) reversibly recovered the inhibitory effects of diamide, suggesting that ARE binding activity is redox-dependent. Moreover, overexpression of thioredoxin enhanced the ARE-mediated thioredoxin gene activation by tBHQ. Therefore, ARE-mediated induction of thioredoxin expression is a mechanism of enhancing signal transduction through the ARE in electrophile-induced stress responses.

Antioxidants↗
PubMed2001

Functional characterization of transcription regulators that interact with the electrophile response element.

The electrophile response element (EpRE), also referred to as the antioxidant responsive element (ARE), is found in the 5'-regulatory region of a number of genes encoding phase II, drug-metabolizing enzymes. Gene knockout studies have demonstrated the primary regulatory role that an Nrf2:Maf dimer plays by binding to nucleotides within the EpRE consensus sequence. Current models of transcription regulation have also shown the involvement of higher-order transcriptional coactivators, proteins that nucleate around DNA sequence-specific transcription factors, enhancing transcription of the target gene by interacting with components of the basal transcriptional apparatus and by enabling chromatin remodeling. Here, we hypothesized that multiple transcriptional regulators, including: (i) a primary Nrf2-Maf heterodimer, (ii) a proposed secondary, EpRE-specific, p160 family coactivator, ARE-binding protein-1, and (iii) a tertiary coactivator, CBP/p300, nucleate to form a complex at the EpRE that regulates transcription of the dependent gene. To test this hypothesis, we constructed a HepG2 cell line which contains a stably integrated green fluorescent protein (GFP) gene; its inducible expression is regulated by a synthetic TK promoter containing a linked EpRE. To identify the involvement of specific, primary and higher-order transcriptional regulators in the EpRE-mediated regulation of the GFP reporter gene, we microinjected antibodies directed against specific transcription factors into the HepG2/GFP cells and determined their effect upon tBHQ-induced expression of the GFP gene. The results demonstrate that microinjected antibodies directed against Nrf2, MafK, CBP and p300 could each, individually, significantly inhibit tBHQ-induced GFP expression. This directly demonstrates the role that the tertiary regulators, CBP or p300, play in mediating EpRE activation of phase II genes, and also implicates the involvement of secondary, p160 family coactivators. Moreover, we found that the same anti-MafK antibody that blocked induction of the EpRE-regulated GFP gene completely ablated the gel-shift complex that we hypothesize contains an Nrf2:Maf dimer, ARE-binding protein-1, and CBP or p300.

Base Sequence↗
PubMed1996

Identification of the human insulin negative regulatory element as a negative glucocorticoid response element.

Insulin gene transcription in adults is restricted to pancreatic beta cells. Studies with both transgenic mice and islet cell lines have demonstrated that beta cell specific expression is conferred by the 5' flanking region of the insulin gene. Transfection analysis has shown that cell specific expression involved an interaction between both positive and negative promoter cis elements. An upstream region (between -258 and -279) of the human insulin promoter served as a site of negative regulation. Transfection analysis in the pancreatic cell line HIT T-15 M 2.2.2 revealed that a DNA fragment containing this region causes a 45% reduction in promoter activity when linked to the native insulin promoter and a 72% reduction when linked to a heterologous tk promoter. Electrophoretic mobility shift analysis of this negative regulatory region (NRE) reveals a complex pattern of binding, wherein two major and several minor complexes are observed. Competition experiments demonstrated that formation of the fastest mobility complex is completely inhibited with excess cold glucocorticoid responsive element (GRE) consensus oligonucleotide. Purified glucocorticoid receptor binding domain (T7X556) demonstrated binding to the NRE oligonucleotide. Functional studies showed that dexamethasone treatment of HIT T-15 M 2.2.2 cells containing an NRE-tk CAT plasmid decreased CAT gene expression by 48%. Analysis of the NRE revealed 73% homology with the negative GRE consensus sequence. These data show that the human insulin NRE is a negative glucocorticoid response element.

Animals↗
PubMed1994

Muscle homeodomain protein MHox inhibits ternary complex formation at the c-fos serum response element.

The Serum Response Element in the c-fos promoter is the target of growth factor-regulated signal transduction pathways. The ternary complex of Serum Response Element-binding protein Serum Response Factor and its accessory protein Ternary Complex Factor are important for transcriptional stimulation of the c-fos promoter in fibroblasts. However, this promoter is repressed in differentiating muscle cells. We discovered that MHox, a muscle homeodomain protein, was capable of inhibiting formation of the ternary complex by direct physical interaction between MHox and Serum Response Factor accessory protein-1 (SAP-1), one member of the Ternary Complex Factor family of proteins. Furthermore, exogenous MHox protein inhibited serum-inducibility of a Serum Response Element-dependent reporter gene in permeabilized fibroblasts. Taken together, these results imply that MHox is involved in blocking mitogenic signals during myogenesis.

3T3 Cells↗