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At least 865 records · Page 48Linked to original sources
PubMed1992

Binding of the estrogen receptor DNA-binding domain to the estrogen response element induces DNA bending.

We have used circular permutation analysis to determine whether binding of purified Xenopus laevis estrogen receptor DNA-binding domain (DBD) to a DNA fragment containing an estrogen response element (ERE) causes the DNA to bend. Gel mobility shift assays showed that DBD-DNA complexes formed with fragments containing more centrally located EREs migrated more slowly than complexes formed with fragments containing EREs near the ends of the DNA. DNA bending standards were used to determine that the degree of bending induced by binding of the DBD to an ERE was approximately 34 degrees. A 1.55-fold increase in the degree of bending was observed when two EREs were present in the DNA fragment. These in vitro studies suggest that interaction of nuclear receptors with their hormone response elements in vivo may result in an altered DNA conformation.

Animals↗
PubMed2003

Geldanamycin induction of grp78 requires activation of reactive oxygen species via ER stress responsive elements in 9L rat brain tumour cells.

The molecular mechanism whereby anticancer agent geldanamycin (GA) impacts endoplasmic reticulum (ER) stress pathway is largely unknown. Here, we investigate the effect of GA on the expression of grp78 coding for ER stress protein and the mechanistic relationship of GA signalling to ER stress. GA induces the expression of mRNA and protein of grp78 by Northern blot analysis and metabolic labelling experiment in cultured rat brain tumour 9L cells. The induced grp78 expression is sensitive to antioxidant N-acetylcysteine (NAC) addition, indicating the involvement of reactive oxygen species (ROS) in GA-induced ER stress. Results from direct determination of oxidation status using dichlorodihydrofluorescein diacetate (H(2)DCFDA) showed that accumulation of ROS elicited GA was quenched by addition of NAC. Reporter genes harbouring deletions of transcription elements from grp78 promoter demonstrated that controlling elements of ERSE1, ERSE2 and CRE are required in GA treatment. The critical ROS-dependent elements in grp78 promoter can be confined within ER stress responsive element (ERSE) region, since reporter constructs loss of ERSE elements that lost the susceptibility to be modulated by NAC after GA treatment. Hence, ER stress elements correlate well with ROS-mediated elements in grp78 promoter. Reporter construct loss of ERSE element retains the susceptibility by NAC after GA treatment, indicating that CRE element might represent a ROS-independent, GA-inductive element. Conclusively, we show that ROS is required for GA to launch the transactivation of grp78, and a firm link was established between the ROS signalling pathway to specific promoter elements-ERSE1 and ERSE2 elements in ER stress marker gene grp78 promoter.

Acetylcysteine↗
PubMed1996

High mobility group protein I(Y) is required for function and for c-Rel binding to CD28 response elements within the GM-CSF and IL-2 promoters.

CD28 response elements (CD28REs) within cytokine promoters are variant NF-kappaB-binding sites and are essential for transcription in response to CD28 receptor activation in T cells. We show that the CK-1 element (CD28RE) within the GM-CSF promoter binds the RelA and c-Rel transcription factors in response to CD28 activation. We further show that the high mobility group protein HMG I(Y) can bind to the CD28REs of both GM-CSF and IL-2 and that this binding is critical for c-Rel, but not RelA, binding. A second NF-kappaB site in the GM-CSF promoter that binds p50 and RelA, but neither c-Rel nor HMG I(Y), failed to respond to CD28 activation. Expression of HMG I or c-Rel antisense RNA inhibited CD28 activation of the IL-2 and GM-CSF promoters, implying that HMG I(Y) enhancement of c-Rel binding plays an important role in the activity of the CD28REs.

CD28 Antigens↗
PubMed1999

Germ cell-specific cyclic adenosine 3',5'-monophosphate response element modulator expression in rodent and primate testis is maintained despite gonadotropin deficiency.

cAMP response element modulator (CREM) is an important component of the cAMP-mediated signaling pathway and is essential for differentiation of haploid male germ cells. In the rodent, testicular expression of CREM is believed to be controlled by FSH. We studied the expression pattern of CREM and gonadotropic control in the nonhuman primate and rodent testis. Adult cynomolgus monkeys (Macaca fascicularis) received daily either vehicle or the potent GnRH antagonist (ANT) cetrorelix for periods of 25 and 56 days. Rats were also exposed to vehicle or ANT for periods of 14 and 42 days. ANT treatment suppressed pituitary gonadotropin secretion, reduced testis size, and altered spermatogenesis. A rabbit polyclonal antibody raised against recombinant CREM tau and reacting with CREM alpha, -beta, -gamma, -tau1, and -tau2 at similar affinities was used for immunocytochemistry and Western blotting. CREM expression was seen in round spermatids, with highest levels during spermatogenic stages V-VII, but declined with progression of spermatid development in the primate. Similar observations were made for the rat testis. Thus, CREM expression was maximal at the onset of acrosome formation and was low or undetectable upon initiation of spermatid elongation in both species. A weak, but specific, CREM signal was seen in mid- to late pachytene spermatocytes and during meiotic division in both species. After ANT exposure, the germ cell- and stage-specific pattern of CREM expression was quantitatively retained at all time points and in both species. Northern and Western blot analysis confirmed the maintenance of testicular CREM expression despite 25 days of ANT treatment. A retrospective immunocytochemical analysis of rat testes 14 days posthypophysectomy revealed CREM signals in round spermatids. These findings demonstrate that the testicular expression of CREM is not entirely dependent on gonadotropic hormones but, rather, on the maturational stage of haploid round germ cells.

Animals↗
PubMed2003

Brassinolide induces IAA5, IAA19, and DR5, a synthetic auxin response element in Arabidopsis, implying a cross talk point of brassinosteroid and auxin signaling.

Despite numerous physiological studies addressing the interactions between brassinosteroids (BRs) and auxins, little is known about the underlying molecular mechanisms. We studied the expression of IAA5 and IAA19 in response to treatment with indole acetic acid (IAA) or brassinolide (BL), the most active BR. Exogenous IAA induced these genes quickly and transiently, whereas exogenous BL induced them gradually and continuously. We also found that a fusion of DR5, a synthetic auxin response element, with the GUS (beta-glucuronidase) gene was induced with similar kinetics to those of the IAA5 and IAA19 genes in response to both IAA and BL treatment of transgenic plants. These results suggest that the IAA genes are induced by BL, at least in part, via the activation of the auxin response element. Endogenous IAA levels per gram fresh weight did not increase when seedlings of Arabidopsis wild type (WT) or the BR-deficient mutant det2 were treated with BL. Furthermore, the levels of IAA transcripts were lower in the det2 mutant than in the WT, even though endogenous IAA levels per gram fresh weight were higher in the det2 mutant than in the WT. In conclusion, the lack of evidence for auxin-mediated activation of early auxin-inducible genes in response to BL suggests that the BR and auxin signaling pathways independently activate the transcriptional system of the IAA and DR5-GUS genes.

Arabidopsis↗
PubMed2005

Pigment epithelium-derived factor induces pro-survival genes through cyclic AMP-responsive element binding protein and nuclear factor kappa B activation in rat cultured cerebellar granule cells: Implication for its neuroprotective effect.

Pigment epithelium-derived factor (PEDF) protects immature cerebellar granule cell neurons (CGCs) against apoptosis induced by K+ and serum deprivation. However, the precise mechanism of this protection remains unknown. We recently reported that the transcription factor nuclear factor kappa B (NF-kappaB) is activated in PEDF-treated CGCs. Although it is well known that NF-kappaB blocks apoptotic cell death through the induction of pro-survival factors, the effects of PEDF on the expression of these factors are not fully understood. In this study, we employed the use of reverse transcriptase-polymerase chain reaction to analyze the gene expression of certain pro-survival genes and found that genes such as c-IAP1, c-IAP2, FLIPs, A1/Bfl-1 and Mn-SOD were induced in PEDF-treated neurons. On the other hand, no induction was observed of the pro-apoptotic Bcl-2 family members Bax and Bid at any time from 3 to 24 h following PEDF addition. Furthermore, phosphorylation of cyclic AMP-responsive element binding protein (CREB) and increment of nuclear cyclic AMP-response element (CRE)-like DNA binding were observed in PEDF-treated CGCs. The anti-apoptotic effect of PEDF was blocked by overexpression of dominant negative CREB or a mutated form of IkappaBalpha. These results suggested that induction of both CRE- and NF-kappaB-dependent genes is required for the observed neuroprotective effects of PEDF on CGCs.

Animals↗
PubMed2003

Herpesvirus quiescence in neuronal cells. V: forskolin-responsiveness of the herpes simplex virus type 1 alpha0 promoter and contribution of the putative cAMP response element.

The herpes simplex virus (HSV)-1 alpha0 promoter contains a putative cAMP response element (CRE) located at positions -68 to -60 with respect to the initiation of transcription. In this report, the authors examined the functionality of this element using (1) luciferase reporter gene assays in nerve growth factor-differentiated (ND)-PC12 cells and (2) virus-induced activation from quiescently infected (QIF)-PC12 cells. The putative alpha0 CRE was completely eliminated by digestion with the restriction enzyme Tsp45I followed by mung bean nuclease treatment. The mutated region was verified by DNA sequencing and was inserted into the alpha0-luciferase reporter plasmid (pRDalpha0-LUC) creating (pRDalpha0deltaCRE-LUC), and into the HSV-1 genome of strain 17(+)(alpha0deltaCRE). Insertion into both copies of the alpha0 promoter was verified by Southern blot analysis. ND-PC12 cells transfected with pRDalpha0-LUC and pRDalpha0deltaCRE-LUC plasmids responded similarly to forskolin (50 microM), with approximately 250% increases in luciferase activity compared to mock-treated cultures as measured 3 days following treatment. When QIF-PC12 cultures established with HSV-1 strain 17(+) and alpha0deltaCRE were treated with forskolin (50 microM) 17 days post infection, virus was detected in 9/24 (37.5%) and 13/24 (54.2%) of induced cultures by day 8 post treatment, respectively. In contrast, virus was detected in 0/23 and 1/24 (4.2%) of mock-treated cultures by day 8 post treatment for wild-type and mutant viruses, respectively. These findings indicate that the alpha0 promoter is forskolin responsive, the purported CRE of the alpha0 promoter does not confer forskolin responsiveness in ND-PC12 cells, and this element is not required for reactivation of HSV-1 from QIF-PC12 cells.

Animals↗
PubMed2000

Transcriptional regulation of the antioxidant response element. Activation by Nrf2 and repression by MafK.

The antioxidant response element (ARE) mediates the transcriptional activation of many genes encoding phase II drug-metabolizing enzymes in response to oxidative stress. Recent studies using knockout mice suggest that NF-E2-related factor 2 (Nrf2), along with small Maf proteins, binds and activates the ARE. In this study, using in vitro binding assays, Nrf2/MafK heterodimers were found to interact with high affinity to the ARE. However, distinct differences were observed when this interaction was compared with that formed with nuclear proteins from H4II EC3 or HepG2 cells. Overexpression of Nrf2 activated ARE-mediated transcription in HepG2 cells, and this activation was further increased by tert-butylhydroquinone. In HeLa cells, overexpression of Nrf2 resulted in activation of the ARE, but this activation was no longer induced by tert-butylhydroquinone. Using ARE constructs with point mutations in the core sequence, we found that only mutations at the T or G nucleotides within the core (TGAC) render the ARE unresponsive to Nrf2. Overexpression of MafK led to dose-dependent repression of ARE activity. Activation of the ARE by Nrf2 was similarly antagonized by MafK. These data suggest that Nrf2 plays an important role mediating basal activity of the ARE but that small Maf proteins are repressors and not activators of ARE-mediated transcription.

Animals↗
PubMed2004

Human glutamate cysteine ligase gene regulation through the electrophile response element.

Glutathione (GSH) is the primary nonprotein thiol in the cell. It has many important roles in cell function, including regulating redox-dependent signal transduction pathways. The content of GSH within the cell varies with stress. In many cases, a process involving GSH synthesis results in adaptation to subsequent stressors. Sustained increases in GSH content are controlled primarily through induction of two genes, Gclc and Gclm, leading to the synthesis of the rate-limiting enzyme for GSH synthesis, glutamate cysteine ligase. Each of these genes in humans has a number of putative enhancer elements in their promoters. Overall, the most important element in both Gclc and Gclm expression is the electrophile response element. We review the evidence that has led to this conclusion and the implications for the redox-dependent regulation of this critical intracellular antioxidant.

Curcumin↗
PubMed2000

Cloning of cDNA encoding ethylene-responsive element binding protein-5 in the cultured cells of Nicotiana tabacum.

We have isolated a full-length cDNA clone from the cultured cells of Nicotiana tabacum, of which the deduced 225 amino acid sequence showed significant homology to the known ethylene-responsive element binding proteins (EREBPs) from tobacco and Arabidopsis. Although the clone shared substantial homology with an Arabidopsis ethylene-responsive element binding factor-3 (AtERF-3) throughout the full length, such a clone had not been found in N. tabacum cells yet; the clone was found to be 5th homologue of EREBPs on N. tabacum.

Amino Acid Sequence↗
PubMed1999

Characterization of the genomic structure of the murine Spam1 gene and its promoter: evidence for transcriptional regulation by a cAMP-responsive element.

The Sperm Adhesion Molecule 1 (SPAM1), also known as PH-20, is a sperm membrane-bound protein that has been shown to have bifunctional roles in fertilization. It is encoded by a gene that is widely conserved in mammalian species, underscoring its importance in the fertilization process. Here we determined the genomic structure of the murine homologue, Spam1, using PCR analysis, and studied its transcriptional regulation. The gene covers approximately 10.5 kb of genomic DNA, is encoded by four exons, and the splice site consensus sequence is maintained in all intron-exon junctions, similar to that reported for the human homologue. With primer extension analysis, two transcription initiation sites were detected. One was assigned to the residue C and the other (a minor site) to the residue G, at positions 1 and -56, respectively. These are at 313 and 369 nucleotides upstream of the translation initiation codon, ATG. In about 770 bp upstream region of Spam1 that has been cloned and sequenced, multiple transcription factor binding sites including a CRE (cAMP-responsive element) were found. We specifically studied the function of the eight nucleotide CRE sequence (TGATGTCA) at -57 (or -62 depending on the strain of mice) of the promoter region. It can bind to the transcription factor CREM (cAMP-responsive element modulator) in gel mobility shift assays using mouse testis nuclear extract, and the binding can be inhibited by a 28 bp oligonucleotide containing the CRE sequence. Similar binding and inhibition assays using rat nuclear extract suggest the existence of a rat CRE sequence and the involvement of CREM in rat Spam1 expression. In vitro transcription assays suggest that CRE is necessary for the transcriptional activity of the murine promoter, and Northern analysis shows that Spam1 transcripts are absent in CREM-knockout mice. The results strongly suggest that the murine Spam1 expression is under the control of CREM, and that this transcriptional regulator for Spam1 might be conserved in other mammals, at least in the rat.

Animals↗
PubMed1994

The human J kappa recombination signal sequence binding protein (RBP-J kappa) targets the Epstein-Barr virus EBNA2 protein to its DNA responsive elements.

The Epstein-Barr virus (EBV) protein EBNA2, which is essential for the immortalization of human primary B cells by EBV, acts as a transcriptional activator of cellular and viral genes. Specific responsive elements have been characterized in several of the promoters activated by EBNA2. They all share the core sequence GTGGGAA. EBNA2 does not, however, bind to these sequences directly, but appears to be targeted to them by a cellular protein. A similar core sequence has recently been identified as a high-affinity binding site for the human recombination signal sequence binding protein RBP-J kappa. Here we provide evidence that RBP-J kappa binds to specific sequences in EBNA2-responsive elements. Our results also demonstrate that RBP-J kappa makes direct physical contact with EBNA2 in solution and recruits EBNA2 to its cognate DNA sequences, suggesting that RBP-J kappa may mediate EBNA2 transactivation of both cellular and viral genes.

Antigens, Viral↗
PubMed1998

Cyclic adenosine 3',5'-monophosphate-responsive element modulator gene expression in germ cells of normo- and oligoazoospermic men.

In about one third of infertile men the cause of impaired spermatogenesis is not known. Spermatogenesis appears to be mediated at least in part by the pituitary gonadotropins, which activate the cAMP-dependent signaling pathway. The end point of this pathway is the activation of nuclear transcription factors, such as cAMP-responsive element-binding protein and cAMP-responsive element modulator (CREM). These factors, upon binding to gene sequences identified as cAMP response elements, modulate the expression of germ cell-specific genes that, in turn, promote the completion of spermatogenesis. The expressions of the cAMP-responsive element-binding protein and CREM genes create different isoforms, which can be divided into two groups: activators or repressors of gene regulation. Only CREM repressors are expressed in premeiotic germ cells in mice, whereas a switch to the expression of the CREM activator tau is observed from postmeiotic germ cells onward. Completion of germ cell maturation appears to be dependent on this phenomenon. Recently, mice lacking CREM gene expression have been generated. These animals were infertile and presented a developmental arrest of germ cell maturation at the stage of early spermatid. In this report we demonstrate that CREM gene expression also occurs in human germ cells. In particular, we determined by RT-PCR that a switch from the expression of CREM repressors to CREM activators is present in postmeiotic germ cells in normospermic men. Conversely, in oligoazoospermic patients only the expression of CREM repressors was detected. These data were confirmed by in situ hybridization studies in which transcripts for CREM activators were detected in postmeiotic germ cells in testis specimens showing conserved spermatogenesis, but not in specimens showing maturation arrest at the spermatid stage. Thus, our results indicate that the lack of a switch in the expression of CREM gene isoforms may be related to impaired spermatogenesis in humans.

Cyclic AMP Response Element Modulator↗
PubMed1994

Definition of a lipopolysaccharide-responsive element in the 5'-flanking regions of MuRantes and crg-2.

Macrophages are stimulated by lipopolysaccharide (LPS) of gram-negative organisms. The changes in LPS-stimulated macrophages include transcriptional activation of multiple immediate-early genes, which may contribute to the natural immunity to microorganisms. We have defined by deletion and mutational analysis LPS-responsive elements (LREs) in two chemokine genes, MuRantes and crg-2, which are activated in an immediate-early manner. LRE consists of two motifs, TCAYR, which is an AP-1 half site with two flanking bases, and (A/T) (G/C)NTTYC(A/T)NTTY, which resembles in part the interferon-stimulated responsive element (ISRE). The orientation of these two motifs relative to each other in MuRantes differed from that in crg-2. These two motifs are separated by 10 and 6 nonconsensus nucleotides in the MuRantes and crg-2 LREs, respectively. Stimulation of macrophage-like RAW 264.7 cells with alpha/beta interferon did not activate MuRantes, indicating that the ISRE-like motif in MuRantes does not have ISRE activity. Upon stimulation of RAW 264.7 cells with LPS, proteins capable of binding to LRE accumulate in the nuclei as measured by electrophoretic mobility shift assay. These LRE-binding proteins include c-Jun and CREB.

Animals↗
PubMed1989

Specific protein-DNA interactions at a xenobiotic-responsive element: copurification of dioxin receptor and DNA-binding activity.

Upon binding of 2,3,7,8-tetrachlorodibenzo-p-dioxin (called dioxin or TCDD), the dioxin receptor exhibits increased affinity for the cell nucleus in vivo and for DNA in vitro. To define the recognition sequence of the dioxin receptor and its relationship with that of the glucocorticoid receptor, oligonucleotides derived from dioxin-responsive elements of the rat cytochrome P-450c gene were tested for their ability to form specific protein-DNA complexes in a gel retardation assay. We found that a previously defined sequence motif that is similar to the glucocorticoid-responsive element and exhibits strong enhancer activity in response to dioxin receptor ligands bound a dioxin-inducible factor with high specificity but was not recognized by the DNA-binding domain of the glucocorticoid receptor. Binding to this element was only observed in nuclear extracts of wild-type mouse hepatoma cells in a time- and dose-dependent manner and not in nuclear extracts from a nonresponsive mutant cell line deficient in DNA binding of the dioxin receptor. The specific DNA-binding activity in wild-type nuclear extracts comigrated in a Superose size-exclusion column and cosedimented on sucrose gradients with the in vivo labeled dioxin receptor. These experiments strongly suggest that the dioxin receptor is a sequence-specific DNA-binding protein and is not only biochemically but also functionally similar to the steroid receptor family.

Animals↗
PubMed1991

Identification of a novel mammary cell line-specific enhancer element in the long terminal repeat of mouse mammary tumor virus, which interacts with its hormone-responsive element.

We analyzed the long terminal repeat (LTR) of mouse mammary tumor virus for sequences that influence its promoter activity by using the chloramphenicol acetyltransferase assay. A series of LTR deletion mutants and recombinants between LTR and simian virus 40 regulatory sequences were used for these studies. Through transfection experiments in three different human cell lines (T47D, MCF-7, and HeLa), we identified a novel mammary cell line-specific enhancer element on a 98-bp BanII fragment (from position -1075 to -978 upstream of the start site of transcription) which interacts with the hormone-responsive element of LTR. We also identified nuclear factors that specifically interacted with this BanII fragment in the nuclear extract from the mammary tumor cell line, T47D, but not from the HeLa cell line.

Breast Neoplasms↗
PubMed2000

A response element for the homeodomain transcription factor Ptx3 in the tyrosine hydroxylase gene promoter.

Tyrosine hydroxylase (TH) is the rate-limiting enzyme in the biosynthesis of catecholamines, which takes place in different types of neuronal systems and nonneuronal tissues. The transcriptional regulation of the TH gene, which is complex and highly variable among different tissues, reflects this heterogeneity. We recently isolated a homeodomain transcription factor, named Ptx3, that is uniquely expressed in the dopaminergic neurons of the substantia nigra pars compacta and ventral tegmental area, which together form the mesencephalic dopaminergic system. This strict localization and its coinciding induction of expression with the TH gene during development suggested a possible role for this transcription factor in the control of the TH gene. We report here the presence of a responsive element for Ptx3 located at position -50 to -45 of the rat TH promoter. Transient transfections using TH promoter constructs and electrophoretic mobility shift assays using Ptx3-containing nuclear extracts demonstrated that this region binds Ptx3 protein and confers a transcriptional effect on the TH gene. Depending on the cell type, the effect of Ptx3 was an eight- to 12-fold enhancement of TH promoter activity in Neuro2A neuroblastoma cells, or a 60-80% repression in nonneuronal human embryonic kidney 293 cells. Despite the close association of the Ptx3-binding site and the major cyclic AMP-response element in the TH gene, no interplay was found between Ptx3 and cyclic AMP-modulating agents. In combination with the orphan nuclear receptor Nurr1, which is required for the induction of the TH gene in mesencephalic dopaminergic neurons, the TH promoter activity to Ptx3 was enhanced in Neuro2A cells. Nurr1 alone displayed only very weak activity on the TH promoter in this cell type. The results demonstrate that the homeodomain protein Ptx3 has the potential to act on the promoter of the TH gene in a markedly cell type-dependent fashion. This suggests that Ptx3 contributes to the regulation of TH expression in mesencephalic dopaminergic neurons.

Animals↗
PubMed1998

cAMP-response-element-binding-protein-binding protein (CBP) and p300 are transcriptional co-activators of early growth response factor-1 (Egr-1).

Egr-1 (early-growth response factor-1) is a sequence-specific transcription factor that plays a regulatory role in the expression of many genes important for cell growth, development and the pathogenesis of disease. The transcriptional co-activators CBP (cAMP-response-element-binding-protein-binding protein) and p300 interact with sequence-specific transcription factors as well as components of the basal transcription machinery to facilitate RNA polymerase II recruitment and transcriptional initiation. Here we demonstrate a unique way in which Egr-1 physically and functionally interacts with CBP/p300 to modulate gene transcription. CBP/p300 potentiated Egr-1 mediated expression of 5-lipoxygenase (5-LO) promoter-reporter constructs, and the degree of trans-activation was proportional to the number of Egr-1 consensus binding sites present in wild-type and naturally occurring mutants of the 5-LO promoter. The N- and C-terminal domains of CBP interact with the transcriptional activation domain of Egr-1, as demonstrated by a mammalian two-hybrid assay. Direct protein-protein interactions between CBP/p300 and Egr-1 were demonstrated by glutathione S-transferase fusion-protein binding and co-immunoprecipitation/Western-blot studies. These data suggest that CBP and p300 act as transcriptional co-activators for Egr-1-mediated gene expression and that variations between individuals in such co-activation could serve as a genetic basis for variability in gene expression.

Animals↗