Eukaryotic regulatory elements lurking in plasmid DNA: the activator protein-1 site in pUC.

Kushner, Peter J.; Baxter, John D.; Duncan, Keith G.; Lopez, Gabriela N.; Schaufele, Fred; Uht, Rosalie Maire; Webb, Paul; West, Brian L.

doi:10.1210/mend.8.4.8052261

Cited by 30 publications

(29 citation statements)

References 6 publications

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“…Mutation of the AP-1 or the CREB2 site diminished the response to AP-1, and the lowest activities were seen with simultaneous mutation of the AP-1 and CREB2 motifs. The residual stimulation seen with constructs in which the AP-1 site is mutated was also observed with the minimal promoter construct PGDH-80/luc3 and is probably due to a cryptic AP-1 motif in the plasmid backbone, as reported by other investigators (Kushner et al 1994). Mutation of the CREB1 site was without effect on basal or AP-1-stimulated promoter activity (Fig.…”

Section: Transcriptional Regulation Of the Human Pgdh Genesupporting

confidence: 83%

Genomic structure and transcriptional regulation of the human NAD+-dependent 15-hydroxyprostaglandin dehydrogenase gene

Nandy¹,

Jenatschke²,

Hartung³

et al. 2003

Journal of Molecular Endocrinology

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The NAD + -dependent 15-hydroxyprostaglandin dehydrogenase (PGDH) is a catabolic enzyme that controls the biological activities of prostaglandins by converting them into inactive keto-metabolites. Here we report the genomic organisation of the complete human PGDH gene and characterise its transcriptional regulation. The PGDH gene spans about 31 kb on chromosome 4 and contains 7 exons. Within 2·4 kb of the 5′-flanking sequence we identified two regions with clustered putative transcription factor binding sites. The distal promoter element PGDH-DE (positions−2152/−1944 relative to the start codon) contains binding sites for Ets and activating protein-1 (AP-1) flanked by two cAMP-responsive element-binding protein binding sites (CREB1, CREB2), whereas the proximal element PGDH-PE (−235/−153) includes an Ets and an AP-1 binding sequence. By electrophoretic mobility shift assay, no high affinity binding of Ets or AP-1 factors was observed with PGDH-PE, whereas we confirmed interaction of members of the Ets, AP-1 and CREB families of transcription factors with PGDH-DE. Transcriptional control of the PGDH promoter was assessed by transiently transfecting JEG-3 choriocarcinoma cells. A luciferase reporter gene construct containing the PGDH-PE was not induced by c-jun/c-fos in the absence or presence of co-expressed Ets-1. A construct carrying the PGDH-DE in front of the minimal homologous promoter was activated by co-transfection of expression vectors for AP-1 proteins. Mutation of the AP-1 or CREB2 site reduced the response to c-jun/c-fos, whereas mutation of the Ets site of the distal element reduced basal promoter activity. CREB activated the PGDH-DE construct through the CREB1 site. These results defined the distal element as an integrator of transcriptional regulation by AP-1, Ets and CREB proteins.

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Section: Transcriptional Regulation Of the Human Pgdh Genesupporting

confidence: 83%

Genomic structure and transcriptional regulation of the human NAD+-dependent 15-hydroxyprostaglandin dehydrogenase gene

Nandy¹,

Jenatschke²,

Hartung³

et al. 2003

Journal of Molecular Endocrinology

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“…Assays of GST-ER fusions were carried out in a 100-l volume that contained 40 l of bead suspension (equivalent to 10 l of compact bead volume) and 1 to 2 l of 35 S-labeled in vitro-translated TBP (prepared with plasmid GPP 26 [48]) in IPAB-80-2.5% nonfat milk and incubated for 1.5 h at 4ЊC. Beads were washed five to six times with IPAB-80 containing 0.05% Nonidet P-40.…”

Section: Methodsmentioning

confidence: 99%

“…For assays of GST-TBP fusions, appropriate ER and UL80 cDNAs were transcribed in vitro, translated, and labeled with 35 S, using the TNT7-coupled rabbit reticulocyte lysate system as described by the manufacturer (Promega Corp., Madison, Wis.). The TBP-GST fusion protein was prepared and purified as previously described (20).…”

Section: Methodsmentioning

confidence: 99%

“…To test whether the functional interaction of ER and TBP might reflect biochemical interactions, we examined the abilities of various ER derivatives fused to GST and attached to glutathione beads to pellet in vitro-translated, 35 S-radiolabeled TBP. We tested full-length human ER and the isolated AF-1 and AF-2 domains.…”

Section: Each Of the Er Activation Domains Binds Tbpmentioning

confidence: 99%

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Transcriptional Activators Differ in Their Responses to Overexpression of TATA-Box-Binding Protein

Sadovsky

Webb

Lopez

et al. 1995

Molecular and Cellular Biology

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152

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We investigated how overexpression of human TATA-box-binding protein (TBP) affects the action of estrogen receptor (ER) and compared the response with that of other activators. When ER activates a simple promoter, consisting of a response element and either the collagenase or tk TATA box, TBP overexpression potentiates transcription. TBP potentiates only estrogen-induced and not basal transcription and does so independent of spacing between response element and TATA box. TBP overexpression also reduces autoinhibition by overexpressed ER, suggesting that one target of the autoinhibition may be TBP itself. Both AF-1 and AF-2 domains of ER are potentiated by TBP, and each domain binds TBP in vitro. Like ER, chimeric GAL4/VP16 and GAL4/Tat activators are also potentiated by TBP, as is the synergistic activation by ER and GAL4/VP16 on a complex promoter. Unlike ER, GAL4/Sp1 and GAL4/NF-I become less potent when TBP is overexpressed. Furthermore, synergy between ER and Sp1 or between ER and NF-I, whether these are supplied by transfected GAL4 fusions or by the endogenous genes, is inhibited by TBP overexpression. Thus, ER resembles VP16 in response to TBP overexpression and is different from Sp1 and NF-I, which predominate over ER in setting the response on complex promoters.The estrogen receptor (ER) is an upstream activator protein that binds an estrogen response element (ERE) on DNA and enhances transcription from nearby promoters. Two ER activation domains contribute to this process: AF-1 in the amino terminus, which is constitutive, and AF-2 in the C-terminal ligand-binding domain (LBD), which is active only when hormone is bound (13, 57). Neither of the ER domains is marked by an abundance of glutamine, proline, or acidic amino acids as has been noted for many other transcriptional activation domains.The mechanism whereby the ER domains contribute to transcriptional activation is unknown, but by analogy with betterunderstood viral activators, it is thought that interactions with target proteins within the transcriptional apparatus are important (for a review, see reference 55; see also references 11 and 27 and references therein). The TATA-box-binding protein (TBP) is one candidate target. TBP binding to the promoter is a pivotal event leading to unwinding of the DNA at the TATA box and widening of the minor groove (31,32,47). TBP binding is needed for the subsequent recruitment of TFIIB and RNA polymerase II (reviewed in reference 60). Several activators bind TBP in vitro. These include the acidic activator VP16 (26, 52), E1a (3, 24, 37), c-Rel (30, 59), and Tax 1 (7). For several of these activator mutations that decrease binding to TBP decrease transcriptional activation, suggesting that the binding is of functional significance. In higher eukaryotes, TBP is tightly associated with a class of coactivators called TBPassociated factors (TAFs). This complex, but not isolated TBP, mediates transcriptional enhancement by activator proteins in vitro (10, 14, 53, 61; reviewed in reference 55). TAFs and activators al...

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“…These results indicate that cAMP regulation of 1273 ACE is selective and that CREs in the ACE promoter have the same inducibility of 4 tandem repeats of canonical CREs. One could argue that cAMP responsiveness is nonspecific because of cryptic CREs in the vector backbone 34 and that SV40ϩE resistance to cAMP induction is only apparent because the SV40 enhancer is already maximally activated in the basal state. We find this hypothesis untenable because a very low activity promoter such as the enhancertrap adenovirus E1b tata box was not activated by cAMP when fused to the luciferase backbone (from 0.05Ϯ0.02 to 0.045Ϯ0.017 ␤-galactosidase-normalized luciferase units).…”

Section: Xavier-neto Et Almentioning

confidence: 99%

Rat Angiotensin-Converting Enzyme Promoter Regulation by β-Adrenergics and cAMP in Endothelium

et al. 1999

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Abstract-To shed light on mechanisms of angiotensin-converting enzyme (ACE) upregulation, we used a rabbit endothelial cell model to characterize intracellular pathways of ␤-adrenergic stimulation. In these cells, ACE activity is increased by isoproterenol (ISO). The stably transfected 1273-bp ACE promoter is stimulated by ISO in the presence of isobutyl methylxanthine. This effect is abolished by propranolol. Promoter stimulation is mimicked by cholera toxin, forskolin, and 8BrcAMP, but not by 8BrcGMP. Promoter stimulation by ISO and isobutyl methylxanthine is blocked by protein kinase A inhibitors, indicating that ␤-adrenergic stimulation of the ACE gene depends on phosphorylation of protein kinase A targets. Activation by cAMP, resistance to phorbol ester, and lack of synergism between cAMP and phorbol ester suggest that promoter regulation is due to cAMP responsive element rather than to activating protein-2 sequences. Okadaic acid potentiation of 8BrcAMP induction indicated that promoter activation by cAMP is regulated by phosphatases controlling activation of typical cAMP responsive element regulated genes. In summary, ␤-adrenergic activation of rat ACE promoter is specific; uses G s proteins, adenylyl cyclase, protein kinase A; and probably includes cAMP responsive element-like sequences. (Hypertension. 1999;34:31-38.)Key Words: angiotensin-converting enzyme Ⅲ endothelium Ⅲ receptors, adrenergic, beta Ⅲ cyclic AMP Ⅲ luciferase A ngiotensin-converting enzyme (ACE) is the most important enzyme controlling the activation of angiotensin peptides and the inactivation of kinins. 1 Tissue ACE distribution is wide, 1 which suggests that control of ACE gene expression depends on the particular cell type and environment or, alternatively, that ACE gene expression is controlled by a restricted array of non-cell-specific mechanisms. In cultured cells, expression of the endothelial ACE variant is modulated by hormones, 2-4 ionophores, 5 second messengers, 5 and growth factors. 6 Cardiac hypertrophy is one of the most likely pathophysiological conditions in which tissuespecific regulation of endothelial ACE might play an important role. 7-9 Strong evidence implicates the renin-angiotensin system, and ACE in particular, in the development of hypertrophy. 10 It is known that angiotensin II acts either as a hypertrophic or growth factor agent 9 ; that ACE and ACE mRNA (as well as angiotensinogen and its mRNA) are increased in early cardiac hypertrophy 7,8 ; and that ACE inhibitors prevent the development, ameliorate the course, and reduce the mortality of ventricular hypertrophy. 10 Recently, a histochemical study suggested that endothelial cells are the main cell type expressing ACE in the heart. 11 However, only a few studies have been done to understand the molecular basis of renin-angiotensin system activation and, in particular, of ACE upregulation in endothelial cells. 2,4,12,13 Surprisingly, the molecular mechanisms underlying cAMP induction of ACE activity have not yet been explored. The cAMP regulated pathway is ...

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Eukaryotic regulatory elements lurking in plasmid DNA: the activator protein-1 site in pUC.

Cited by 30 publications

References 6 publications

Genomic structure and transcriptional regulation of the human NAD+-dependent 15-hydroxyprostaglandin dehydrogenase gene

Genomic structure and transcriptional regulation of the human NAD+-dependent 15-hydroxyprostaglandin dehydrogenase gene

Transcriptional Activators Differ in Their Responses to Overexpression of TATA-Box-Binding Protein

Rat Angiotensin-Converting Enzyme Promoter Regulation by β-Adrenergics and cAMP in Endothelium

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