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At least 1,279 records · Page 71Linked to original sources
PubMed2003

Interferon regulatory factor-3-mediated activation of the interferon-sensitive response element by Toll-like receptor (TLR) 4 but not TLR3 requires the p65 subunit of NF-kappa.

Interferon regulatory factor (IRF) 3 is a transcription factor that binds the interferon-sensitive response element (ISRE) and is activated by Toll-like receptor 3 (TLR3) and TLR4. We have found that a dominant negative form of I kappa B kinase 2 and a mutant form of I kappa B, which acts as a super-repressor of NF-kappa B, blocked activation of the ISRE by the TLR4 ligand lipopolysaccharide but not the TLR3 ligand poly(I-C). TLR4 failed to activate the ISRE in mouse embryonic fibroblasts bearing a targeted deletion of p65, whereas the response to TLR3 in these cells was normal. The p65 subunit of NF-kappa B was detected in the lipopolysaccharide-activated but not poly(I-C)-activated ISRE-binding complex. Finally, p65 promoted transactivation of gene expression by IRF-3. These results therefore indicate that IRF-3-mediated activation of the ISRE by TLR4 but not TLR3 requires the p65 subunit of NF-kappa B.

Animals↗
PubMed2002

Characterization of a carbohydrate response element regulating the gene for human galactose-1-phosphate uridyltransferase.

Human galactose-1-phosphate uridyltransferase (hGALT) is a central enzyme in the conserved pathway by which galactose is converted to energy, UDP-galactose and UDP-glucose. A natural mutation that deleted -119 to -116 bp (delGTCA) of the promoter decreased hGALT mRNA and enzyme activity and prompted analysis of hGALT gene regulation. Regulatory domains were identified by inspection and confirmed in a reporter system. Previous studies by others were confirmed that HepG2 cells grown in D-glucose increased hGALT enzyme activity and mRNA by 30%. We extended these observations by sequencing the promoter region and identifying a potential carbohydrate response element (ChoRE). The response to glucose rose to 190% when a plasmid construct containing a luciferase reporter and only the -165 bp region as a promoter was transfected into HepG2 and NIH:OVCAR-3. By contrast, fibroblasts transfected with the identical construct failed to respond to glucose. Within the -165 bp region there were two enhancer (E-box) motifs that encompassed the delGTCA mutation. The deletion diminished the positive regulatory response, but an additional GTCA repeat unexpectedly increased the response. Using this postulated ChoRE as a probe in electrophoretic mobility shift assays, multiple nuclear proteins bound and one was identified as a member of the basic/helix-loop-helix/leucine zipper enhancer-binding (b/HLH/LZ) family. Increased binding of proteins correlated with increased hGALT expression when the spacing between E-box motifs was enlarged but the carbohydrate response was dampened. When the 3(')E-box was mutated, b/HLH/LZ binding and gene expression were abolished. We conclude that the hGALT promoter region contains a ChoRE in which the spacing between and the sequence of its E-box motifs are critical. One nuclear protein of the b/HLH/LZ family is necessary, but not sufficient for the carbohydrate response.

Binding Sites↗
PubMedDate not supplied

Identification and characterization of high glucose and glucosamine responsive element in the rat osteopontin promoter.

We have previously reported that high glucose stimulates osteopontin (OPN) expression via a protein kinase C-dependent pathway and a hexosamine pathway in cultured rat aortic smooth muscle cells (SMCs) [Biochem. Biophys. Res. Commun. 258 (1999) 722.]. In the present study, we carried out functional OPN promoter assays using the luciferase expression vector system in cultured rat aortic SMCs to determine a high glucose/glucosamine responsive element. An extensive deletion analysis of the 5'-flanking region of the rat OPN gene revealed that an element involved in high glucose and glucosamine responses was present within a region between -112 and -62 bp of the OPN promoter. This region is highly conserved in the rat, mouse, and human promoters and contains a number of consensus regions, including an E-box and a GC-rich region. Mutation of the E-box or the GC-rich region resulted in a significant loss of both high glucose and glucosamine responses. These results suggest that two cis-acting elements, the E-box and the GC-rich region, are involved at least partly in high glucose/glucosamine-stimulated transcription of the rat OPN gene.

Animals↗
PubMed2005

Identification of novel steroid target genes through the combination of bioinformatics and functional analysis of hormone response elements.

Steroid hormone receptors including androgen receptor (AR), glucocorticoid receptor (GR), progesterone receptor (PR), and mineralocorticoid receptor (MR) recognize and bind to identical consensus hormone response elements (HREs), which consist of two hexameric half-sites (5'-AGAACA-3') arranged as inverted repeats with a 3-bp spacer. Although only a few near-consensus HRE sequences have been identified in the transcriptional regulatory regions of known steroid target genes, it has been unclear whether the exact consensus sequences function as bona fide HREs in vivo. A genome-wide in silico screening of palindromic HREs identified 565 exact consensus sequences in human genome (NCBI 35 assembly). In this study, of 565 exact consensus elements, functional in vivo receptor binding was evaluated regarding 26 sequences located within 10 kb upstream to the 5' end of annotated genes through chromatin immunoprecipitation (ChIP) assay using cells endogenously expressing steroid hormone receptors. Hormone responsiveness of proximal gene expression was examined through quantitative RT-PCR. As far as performing ChIP assay for AR, GR, and PR, 14 of 26 elements significantly recruited at least one of the receptors by hormone treatment (>2-fold enrichment versus vehicle). In terms of gene expression in the vicinity of the above 14 functional perfect HREs, four genes were upregulated by >2-fold with hormone treatment. The present data suggest that the combination of bioinformatics analysis and quantitative experimental evaluation is useful to identify novel functional HREs that may contribute to the transcriptional regulation of steroid target genes.

Cell Line, Tumor↗
PubMed2005

JunD activates transcription of the human ferritin H gene through an antioxidant response element during oxidative stress.

Ferritin is the major intracellular iron storage protein that sequesters excess free iron to minimize generation of iron-catalysed reactive oxygen species. We previously demonstrated that expression of ferritin heavy chain (ferritin H) was induced by pro-oxidants, which is a part of cellular antioxidant response to protect cells from oxidative damage. In this study, we have identified that the antioxidant/electrophile response element (ARE) located 4.5 kb upstream to the human ferritin H transcription initiation site is responsible for the oxidant response. The human ferritin H ARE comprises two copies of bidirectional AP1 motifs. Mutations in each AP1 motif significantly impaired protein binding and the function of the ARE, indicating that both of the AP1 motifs are required for pro-oxidant-mediated activation of the ferritin H gene. We identified that JunD, an AP1 family basic-leucine zipper (bZip) transcription factor, is one of the ferritin H ARE binding proteins and activates ferritin H transcription in HepG2 hepatocarcinoma cells. Gel retardation assay demonstrated that H2O2 (hydrogen peroxide) or t-BHQ (tert-butylhydroquinone) treatment increased total protein binding as well as JunD binding to the ferritin H ARE. Chromatin immunoprecipitation assay showed that H2O2 treatment induced JunD binding to the ferritin H ARE. Both H2O2 and t-BHQ induced phosphorylation of JunD at Ser-100, an activated form of JunD. Furthermore, overexpression of JunD induced endogenous ferritin H protein synthesis. Since JunD has recently been demonstrated to protect cells from several stress stimuli including oxidative stress, these results suggest that, in addition to NFE2-related factor 2 (Nrf2) as a major ARE regulatory protein, JunD is another ARE regulatory protein for transcriptional activation of the human ferritin H gene and probably other antioxidant genes containing the conserved ARE sequences by which JunD may confer cytoprotection during oxidative stress.

Antioxidants↗
PubMed2004

Protein kinase A contributes to sciatic ligation-associated early activation of cyclic AMP response element binding protein in the rat spinal dorsal horn.

We examined whether early injury-associated activation of cyclic AMP response element binding protein (CREB) in the spinal dorsal horn was mediated by the cyclic AMP-dependent protein kinase A (PKA) pathway. Significant increases in the levels of phosphorylated CREB (pCREB), phosphorylated PKAIIalpha regulatory subunit (pPKA), and PKAalpha catalytic subunit (PKAalpha cat) were elicited 2 h after loose ligation of the sciatic nerve. These injury-elicited increases were significantly reduced by dorsal horn application of the cell-permeable PKA inhibitor Rp-8-Br-cAMPS. The cell-permeable PKA activator Sp-8-Br-cAMPS significantly increased the levels of pCREB, pPKA and PKAalpha cat 2 h after application onto the dorsal horn of control, uninjured animals. Our data lent further support to the notion that activation of PKA may play an important role in the early stages of nerve injury-elicited plasticity in the dorsal horn.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1992

Presence of nuclear factors bound to both cAMP-responsive element and AP1 factor binding site in the porcine anterior pituitary.

Factors binding to consensus sequences of the cAMP-responsive element (CRE) and the AP1 factor binding site (AP1) were investigated using porcine anterior pituitary nuclear extracts. Each element showed specific gel mobility shifts. By reciprocal competition for the AP1 and CRE binding, CRE prevented AP1 binding completely. On the other hand, AP1 decreased the CRE binding considerably to 20%, suggesting that approximately 80% of the total CRE binding is due to factors which bind to a common site shared by both CRE and AP1, whereas proteins binding to AP1 alone are absent. Relative binding affinities of AP1 against CRE estimated from the reciprocal competition data were 0.17 for CRE binding and 0.56 for AP1 binding. UV cross-linking experiments showed that CRE and AP1 gave different patterns consisting of different molecular size. Inconsistency of the relative binding affinities and the multiple molecular size of binding factors, cannot be explained simply by the presence of two types of binding factor, common CRE/AP1-binding and specific CRE-binding factors. A more likely explanation is that the CRE/AP1-binding factors alter the dimer form by changing each respective partner to bind CRE and/or AP1.

Animals↗
PubMed1989

Hyperinducible gene expression from a metallothionein promoter containing additional metal-responsive elements.

We describe the development of metallothionein-based vectors with low basal levels of expression that are hyperinducible upon treatment with heavy metals. Vectors were constructed by substituting a region in the hMTIIA promoter (bp -70 to -129) containing an element (BLE) involved in basal level expression with multiple metal responsive elements (MREs). In expression studies utilizing cat as a reporter gene, heavy metal inducibility was examined in both transiently transfected and permanently transformed Chinese hamster ovary (CHO) cells. Our results demonstrate that, within the same promoter structure, inducibility can be increased by altering the ratio of MREs to BLEs. Optimal induction of expression in permanently transformed CHO cells was achieved by exposure to heavy metals for 48 h prior to cell harvest, with an additional boost 12 h before harvest. These vectors have the potential to be used for production of proteins in cultured mammalian cells and in gene expression in transgenic animals.

Animals↗
PubMed1991

Structural analysis of the interaction between the human immunodeficiency virus Rev protein and the Rev response element.

The specific interaction between a defined structural element of the human immunodeficiency virus mRNA (RRE, the Rev response element) and the virus-encoded protein Rev has been implicated in the regulation of the export of unspliced or singly spliced mRNA from the nucleus to the cytoplasm. Rev protein was expressed and purified from insect cells using the baculovirus expression system. Chemical and RNase probes were used to analyze the structure of the RRE and the regions involved in Rev binding. Increased reactivity to single-strand-specific probes of nucleotides in two helical domains indicates that Rev binding induces conformational changes in the RRE. Binding of Rev to the RRE primarily protects helical segments and adjacent nucleotides in domain II. A Rev unit binding site is proposed that consists of a six-base-pair helical segment and three adjacent nucleotides. The data also suggest that multiple Rev proteins bind to repeated structural elements of the RRE.

Animals↗
PubMed1999

Involvement of the cyclic AMP-responsive element binding protein gene transcription factor in genetic preference for alcohol drinking behavior.

BACKGROUND: Cyclic adenosine 3',5'-monophosphate (cAMP)-responsive element binding (CREB) protein is a gene transcription factor that can integrate the signals mediated via the cAMP second messenger cascade at the gene expression level, which then controls neuronal functions. METHODS: To examine if the protein kinase A --> CREB signaling cascade is involved in genetic alcohol drinking preference, different measures of CREB were determined in various brain structures of alcohol-preferring (P) and alcohol-nonpreferring (NP) rats. RESULTS: We show here that CRE-DNA binding activity is significantly decreased in the amygdala but not in the cortex, hippocampus, or striatum of P rats compared with NP rats. The levels of total CREB and phosphorylated CREB protein in the amygdala are significantly lower in P rats compared with NP rats. On the other hand, levels of the alpha-isoform of the catalytic subunit of protein kinase A protein, and basal as well as cAMP-stimulated protein kinase A activity are similar in the amygdala of both P and NP rats. CONCLUSIONS: Because P and NP rats are genetically bred for high and low alcohol drinking behavior, respectively, these results suggest the possibility that decreased expression of CREB protein in the amygdala may be associated with the high alcohol drinking behavior of P rats.

Activating Transcription Factor 2↗
PubMed2002

Analysis of two distinct retinoic acid response elements in the homeobox gene Hoxb1 in transgenic mice.

Expression of vertebrate Hox genes is regulated by retinoids such as retinoic acid (RA) in cell culture and in early embryonic development. Retinoic acid response elements (RAREs) have been identified in Hox gene regulatory regions, suggesting that endogenous retinoids may be involved in the direct control of Hox gene patterning functions. Previously, two RAREs located 3' of the murine Hoxb1 gene, a DR(2) RARE and a DR(5) RARE, have been shown to regulate Hoxb1 mRNA expression in the neural epithelium and the foregut region, respectively; the foregut develops into the esophagus, liver, pancreas, lungs, and stomach. We have now examined the functional roles of these two types of 3' RAREs in regulating Hoxb1 expression at different stages of gestation, from embryonic day 7.5 to 13.5, in transgenic mice carrying specific RARE mutations. We demonstrate that the DR(5) RARE is required for the regulation of Hoxb-1 transgene region-specific expression in the gut and extraembryonic tissues, as well as for the RA-induced anteriorization of Hoxb-1 transgene expression in the gut. In contrast, expression of the Hoxb1 transgene in the neural epithelium requires only the DR(2) RARE. By in situ hybridization, we have identified a new site of Hoxb1 expression in the developing forelimbs at approximately day 12.5, and we show that, in transgenic embryos, expression in the forelimb buds requires that either the DR(2) or the DR(5) RARE is functional. Attainment of a high level of Hoxb1 transgene expression in other regions, such as in rhombomere 4 (r4) and in the somites, requires that both the DR(2) and DR(5) RAREs are functional. In addition, our transgenic data indicate that the Hoxb1 gene is expressed in other tissues such as the hernia gut, genital eminence, and lung. Our analysis shows that endogenous retinoids act through individual DR(2) and DR(5) RAREs to regulate Hoxb1 expression in different regions of the embryo and that functional redundancy between these DR(2) and DR(5) RAREs does not exist with respect to neural epithelium and the gut Hoxb1 expression.

Animals↗
PubMed1988

Two functional estrogen response elements are located upstream of the major chicken vitellogenin gene.

We used a transient-expression assay to identify two estrogen response elements (EREs) associated with the major chicken vitellogenin gene (VTGII). Each element was characterized by its ability to confer estrogen responsiveness when cloned in either orientation next to a chimeric reporter gene consisting of the herpes simplex virus thymidine kinase promoter and the chloramphenicol acetyl transferase-coding region. Deletion analyses indicated that sequences necessary for the distal ERE resided within the region from -626 to -613 (nucleotide positions relative to the VTGII start site) whereas those necessary for the proximal ERE were within the region from -358 to -335. These distal and proximal elements contain, respectively, a perfect copy and an imperfect copy of the 13-base-pair sequence that is an essential feature of the EREs associated with two frog vitellogenin genes. These chicken VTGII EREs mapped near regions that were restructured at the chromatin level when the endogenous VTGII gene was expressed in the liver in response to estradiol. These data suggest a model for the tissue-specific expression of this estrogen-responsive gene.

Acetyltransferases↗
PubMed2001

Binding of dimeric aminoglycosides to the HIV-1 rev responsive element (RRE) RNA construct.

Through a series of elegant fluorescence measurements, particularly through stopped-flow kinetic measurements, it was recently demonstrated that aminoglycoside antibiotics are able to bind to the HIV-1 Rev responsive element (RRE) RNA construct in more than a 1:1 stoichiometry (Lacourciere, K. A.; Stivers, J. T.; Marino, J. P. Biocheminstry 2000, 39, 5630). Here, we present the binding study results of dimeric neomycin ligands through fluorescence anisotropy studies, to the HIV-1 RRE RNA construct. The dimeric neomycin molecules are observed to be able to bind the HIV-1 RRE RNA construct approximately 17-fold higher when compared to the monomeric neomycin, lending evidence that there are indeed two or more neomycin binding sites within the HIV-1 RRE construct.

Anti-Bacterial Agents↗
PubMed1994

Insulin and other growth factors induce binding of the ternary complex and a novel protein complex to the c-fos serum response element.

Rapid, transient induction of c-fos transcription follows treatment of cells with insulin and other growth factors. This is mediated through the serum response element (SRE), which binds the serum response factor (SRF), as well as accessory factors such as p62 ternary complex factor. Using a gel shift assay we found that formation of the ternary complex increased transiently within 2 min of insulin or phorbol ester treatment of several insulin-sensitive cell lines. However, mutations that prevented formation of this ternary complex did not inhibit insulin- or phorbol ester-stimulated induction of c-fos transcription in these cell lines. We also identified a novel SRF-containing multiprotein complex that forms on the SRE within 2 min following insulin, phorbol ester, or other growth factor treatment. Formation of this novel complex, called band 3, occurred rapidly and transiently, with a time course parallel to the induction of c-fos transcription. Band 3 also formed with gamma-actin and zif268/3 SRE probes. Methylation and carboxy ethylation interference analysis, as well as extensive SRE mutagenesis, suggest that only the SRF directly contacts the SRE in forming band 3. Formation of this novel complex appears to involve protein-protein interactions between SRF and other nuclear protein(s) that may play a role in growth factor stimulation of c-fos transcription.

3T3 Cells↗
PubMed1996

Translational regulation in vivo of the Drosophila melanogaster mRNA encoding succinate dehydrogenase iron protein via iron responsive elements.

Some mRNA encoding proteins related to iron metabolism contain a specific stem-loop structure--iron responsive element (IRE)-in the 5' UTR. Binding of the iron regulatory protein (IRP) to the IRE, in response to decreases in cellular iron levels, leads to a block in translation of these mRNAs. We here describe the drosophila melanogaster succinate dehydrogenase iron protein (SDH-IP) as a fourth example of an mRNA species being translationally regulated by an IRE, based on iron dependent regulation of SDH-IP translation in vivo by immunoprecipitations and northern blotting in drosophila cell lines. Addition of hemin to the drosophila cells lead to fragmentation of the SDH-IP, which might suggest additional mode to specifically down-regulate the expression of this protein and Krebs cycle function.

Animals↗
PubMed1993

An insulin-responsive element in the pancreatic enhancer of the amylase gene.

We have identified a distinct insulin-responsive element (IRE) located within the pancreatic enhancer of the mouse amylase gene Amy-2.2. A 30-base pair (bp) fragment was previously shown to be sufficient to transfer insulin response to a heterologous promoter. The 30-bp fragment overlaps the tissue-specific pancreatic enhancer that binds the putative transcriptional activator PTF1. To determine whether enhancer and IRE activities could be separated, we introduced three 10-base pair substitutions into the 30-bp region. The mutated regulatory regions were cloned upstream of a heterologous promoter and transferred to transgenic mice. Mutants 1 and 2 retained PTF1 binding activity and insulin response. Mutant 3 retained PTF1 binding, but was defective in insulin response. The results indicate that the IRE is functionally distinct from the PTF1-binding site, although the two overlap physically. In contrast to the wild-type gene, mutant 3 is characterized by constitutive expression in diabetic animals, suggesting that a binding site for a repressor has been destroyed. A nuclear protein with affinity for the IRE was detected in normal and diabetic pancreas. This protein does not bind mutant 3, suggesting that it may be involved in negative regulation of amylase in diabetic pancreas.

Amylases↗
PubMedDate not supplied

Characterization of the CDP-like/CTAS-1 binding site in the okadaic acid response element (OARE) of the human CDK1(p34cdc2) promoter.

BACKGROUND: Transcription of CDK1 is induced at the G0/G1-phase of the cell cycle and after okadaic acid treatment and we identified the Site I okadaic acid response element (OARE -944 to -763nt) enhancer in the human CDK1 promoter. MATERIALS AND METHODS: The OARE region of the CDK1 promoter was characterized for enhancer/repressor activities. RESULTS: Transient transfection of upper and lower Site I subregions suggested enhanced transcription activity was divided between both while mobility shift assays demonstrated sequence-specific protein binding to Site IA. Site IA also formed shift complexes following serum starvation/refeeding and putative transcription factor binding sites clustered in Site IA. Oligonucleotides encoding a consensus CDP transcription factor binding site effectively competed against authentic Site IA in mobility shift assays. Mutation of the CDP-like binding sequence substantially reduced competition. DNaseI footprinting revealed binding at this site. CONCLUSION: The CDP-like recognition sequence appears to comprise the OARE binding authentic CDP and/or related factors. We termed this site the CDK1 transcription activation sequence-1 (CTAS-1) enhancer of the human CDK1 promoter.

Base Sequence↗
PubMed1999

Determinants of DNA sequence specificity of the androgen, progesterone, and glucocorticoid receptors: evidence for differential steroid receptor response elements.

While androgen, progesterone, and glucocorticoid receptors perform distinct physiological functions by regulating unique sets of genes, in vitro they can transactivate a common high-affinity DNA-binding target. Naturally occurring steroid response elements display nucleotide divergence that lowers binding affinity in comparison to the optimal binding element, but enhances receptor-type specificity. We investigated the role of nucleotide deviations within the DNA-binding site for contribution to steroid receptor specificity. We hypothesized that receptor specificity drives the evolution of binding site sequence, rather than strictly receptor-binding affinity. Receptor-selective targets can evolve by some nucleotides selected on the basis of additional bond energy, and others may be selected by differential tolerance to discourage binding from inappropriate receptors. To identify receptor-specific binding sites, we mimicked these dual selection pressures in a receptor-competitive environment in which DNA binding sites for the androgen or progesterone receptors were selected in the presence of the glucocorticoid receptor. These analyses also demonstrated that steroid receptors strongly select nucleotides in the spacer and flanking regions of the half-site and do so in an asymmetric fashion, indicating that steroid receptors interact with DNA in an allosteric manner that affects the transcriptional activation potential.

Amino Acid Sequence↗