scholarly journals Identification of a cAMP response element within the glucose- 6-phosphatase hydrolytic subunit gene promoter which is involved in the transcriptional regulation by cAMP and glucocorticoids in H4IIE hepatoma cells

1999 ◽  
Vol 338 (2) ◽  
pp. 457-463 ◽  
Author(s):  
Dieter SCHMOLL ◽  
Christina WASNER ◽  
Carolyn J. HINDS ◽  
Bernard B. ALLAN ◽  
Reinhard WALTHER ◽  
...  
Keyword(s):  
Reporter Gene ◽  
Consensus Sequence ◽  
Gene Promoter ◽  
Hepatoma Cells ◽  
Luciferase Reporter ◽  
Regulation Of Transcription ◽  
Dibutyryl Camp ◽  
Luciferase Reporter Gene ◽  
Camp Response Element ◽  
Response Element

The expression of a luciferase reporter gene under the control of the human glucose 6-phosphatase gene promoter was stimulated by both dexamethasone and dibutyryl cAMP in H4IIE hepatoma cells. A cis-active element located between nucleotides -161 and -152 in the glucose 6-phosphatase gene promoter was identified and found to be necessary for both basal reporter-gene expression and induction of expression by both dibutyryl cAMP and dexamethasone. Nucleotides -161 to -152 were functionally replaced by the consensus sequence for a cAMP response element. An antibody against the cAMP response element-binding protein caused a supershift in gel-electrophoretic-mobility-shift assays using an oligonucleotide probe representing the glucose 6-phosphatase gene promoter from nucleotides -161 to -152. These results strongly indicate that in H4IIE cells the glucose 6-phosphatase gene-promoter sequence from -161 to -152 is a cAMP response element which is important for the regulation of transcription of the glucose 6-phosphatase gene by both cAMP and glucocorticoids.

scholarly journals Polymorphisms in the osteopontin promoter affect its transcriptional activity

2004 ◽  
Vol 20 (1) ◽  
pp. 87-96 ◽  
Author(s):  
Francesca Giacopelli ◽  
Renato Marciano ◽  
Angela Pistorio ◽  
Paolo Catarsi ◽  
Silvana Canini ◽  
...  
Keyword(s):  
Cell Lines ◽  
Reporter Gene ◽  
Molecular Mechanisms ◽  
Association Studies ◽  
Susceptibility Gene ◽  
Organ Damage ◽  
Luciferase Reporter ◽  
Regulation Of Transcription ◽  
Luciferase Reporter Gene ◽  
Encoding Gene

Understanding the molecular mechanisms that underlie regulation of transcription of the human osteopontin encoding gene (OPN) may help to clarify several processes, such as fibrotic evolution of organ damage, tumorigenesis and metastasis, and immune response, in which OPN overexpression is observed. With the aim to evaluate variants with functional effect on transcription, we have analyzed the promoter region and focused our investigation on three common variants present in the first 500 bp upstream of the transcription start site. Transfection of constructs carrying the four most frequent haplotypes relative to variants at −66, −156, and −443 fused to the luciferase reporter gene in a panel of different cell lines showed that one haplotype conferred a significantly reduced level of reporter gene expression in all tested cell lines. We describe that the −66 polymorphism modifies the binding affinity for the SP1/SP3 transcription factors, the −156 polymorphism is included in a yet uncharacterized RUNX2 binding site, and the −443 polymorphism causes differential binding of an unknown factor. The finding of differential effects of various combination of variants in haplotypes may contribute to explain data of association studies reported in several already published articles. Future association studies using haplotypes instead of single OPN variants will allow to achieve more accurate results referable to differential expression of OPN in several common diseases, in which OPN is considered a candidate susceptibility gene.

scholarly journals Cloning and Functional Analysis of Rat Tweety-Homolog 1 Gene Promoter

2021 ◽  
Author(s):  
Malgorzata Gorniak-Walas ◽  
Karolina Nizinska ◽  
Katarzyna Lukasiuk
Keyword(s):  
Transcriptional Regulation ◽  
Reporter Gene ◽  
Hippocampal Neurons ◽  
Gene Promoter ◽  
Promoter Sequence ◽  
Regulatory Elements ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Gene Assay

AbstractTweety-homolog 1 protein (Ttyh1) is abundantly expressed in neurons in the healthy brain, and its expression is induced under pathological conditions. In hippocampal neurons in vitro, Ttyh1 was implicated in the regulation of primary neuron morphology. However, the mechanisms that underlie transcriptional regulation of the Ttyh1 gene in neurons remain elusive. The present study sought to identify the promoter of the Ttyh1 gene and functionally characterize cis-regulatory elements that are potentially involved in the transcriptional regulation of Ttyh1 expression in rat dissociated hippocampal neurons in vitro. We cloned a 592 bp rat Ttyh1 promoter sequence and designed deletion constructs of the transcription factors specificity protein 1 (Sp1), E2F transcription factor 3 (E2f3), and achaete-scute homolog 1 (Ascl1) that were fused upstream of a luciferase reporter gene in pGL4.10[luc2]. The luciferase reporter gene assay showed the possible involvement of Ascl1, Sp1, and responsive cis-regulatory elements in Ttyh1 expression. These findings provide novel information about Ttyh1 gene regulation in neurons.

scholarly journals Estradiol Reduces Nonclassical Transcription at Cyclic Adenosine 3′,5′-Monophosphate Response Elements in Glioma Cells Expressing Estrogen Receptor Alpha

Endocrinology ◽  
2006 ◽  
Vol 147 (4) ◽  
pp. 1796-1804 ◽  
Author(s):  
Andrew J. Mhyre ◽  
Robert A. Shapiro ◽  
Daniel M. Dorsa
Keyword(s):  
Gene Expression ◽  
Estrogen Receptor ◽  
Reporter Gene ◽  
Glioma Cells ◽  
Luciferase Reporter ◽  
Estradiol Treatment ◽  
Camp Response Element ◽  
Response Element ◽  
Functional Changes ◽  
Rapid Signaling

Estradiol can protect the brain from a variety of insults by activating membrane-initiated signaling pathways, and thereby modulate gene expression and lead to functional changes in neurons. These direct neuronal effects of the hormone have been well documented; however, it is less understood what effects estradiol may have on nonneuronal cells of the central nervous system. There is evidence that estradiol levels can induce the release of glial-derived growth factors and other cytokines, suggesting that estradiol may both directly and indirectly protect neurons. To determine whether 17β-estradiol (E2) can activate rapid signaling and modulate nonclassical transcription in astrocytes, we stably transfected the C6 rat glioblastoma cell line with human estrogen receptor (ER) α (C6ERα) or rat ERβ (C6ERβ). Introduction of a cAMP response element-luciferase reporter gene into C6, C6ERα, and C6ERβ cells leads to the observation that E2 treatment reduced isoproterenol-stimulated luciferase activity by 35% in C6ERα but had no effect on reporter gene expression in C6ERβ or untransfected C6 cells. A similar effect was seen with a membrane-impermeable estrogen (E2-BSA), suggesting the modulation of nonclassical transcription by estradiol treatment is mediated by the activation of a membrane-initiated signaling pathway. Furthermore, pretreatment with wortmannin (phosphatidylinsositol 3-kinase) or U73122 (phospholipase C) attenuated the E2-induced reduction in nonclassical transcription. We conclude that E2 treatment reduces cAMP response element-mediated transcription in glioma cells expressing ERα and that this reduction is dependent on the activation of membrane-initiated signaling. These findings suggest a novel model of estrogen rapid signaling in astrocytes that leads to modulation of nonclassical transcription.

Regulation of DHP receptor expression by elements in the 5′-flanking sequence

2000 ◽  
Vol 278 (4) ◽  
pp. H1153-H1162 ◽  
Author(s):  
Lei Liu ◽  
Q. Ivy Fan ◽  
Mohamad R. El-Zaru ◽  
Kathleen Vanderpool ◽  
Ronald N. Hines ◽  
...  
Keyword(s):  
Fusion Gene ◽  
Gene Promoter ◽  
Regulatory Elements ◽  
Receptor Expression ◽  
Neonatal Rat ◽  
Luciferase Reporter ◽  
Subunit Gene ◽  
Luciferase Reporter Gene ◽  
Response Element ◽  
Flanking Region

The α1-subunit of the cardiac/vascular Ca2+channel, which is the dihydropyridine (DHP)-binding site (the DHP receptor), provides the pore structure for Ca2+ entry. It contains the binding sites for multiple classes of drugs collectively known as Ca2+ antagonists. As an initial step toward understanding the mechanisms controlling transcription of the rat cardiac α1C-subunit gene, we have cloned a 2.3-kb fragment containing the 5′-flanking sequences and identified the α1C-subunit gene transcription start site. The rat α1C-subunit gene promoter belongs to the TATA-less class of such basal elements. Using deletion analysis of α1C-subunit promoter-luciferase reporter gene constructs, we have characterized the transcriptional modulating activity of the 5′-flanking region and conducted transient transfections in cultured neonatal rat cardiac ventricular myocytes and vascular smooth muscle cells. Sequence scanning identified several potential regulatory elements, including five consensus sequences for the cardiac-specific transcription factor Nkx2.5, an AP-1 site, a cAMP response element, and a hormone response element. Transient transfection experiments with the promoter-luciferase reporter fusion gene demonstrate that the 2-kb 5′-flanking region confers tissue specificity and hormone responsiveness to expression of the Ca2+ channel α1C-subunit gene. Electrophoretic mobility shift assays identified a region of the α1C-subunit gene promoter that can bind transcription factors and appears to be important for gene expression.

Hepatocyte nuclear factor-1α regulates transcription of the guanylin gene

1997 ◽  
Vol 273 (4) ◽  
pp. G833-G841 ◽  
Author(s):  
J. A. Hochman ◽  
D. Sciaky ◽  
T. L. Whitaker ◽  
J. A. Hawkins ◽  
M. B. Cohen
Keyword(s):  
Gene Expression ◽  
Reporter Gene ◽  
Transcriptional Activation ◽  
Consensus Sequence ◽  
Luciferase Reporter ◽  
Intestinal Cell ◽  
Nuclear Factor ◽  
Hepatocyte Nuclear Factor ◽  
Luciferase Reporter Gene ◽  
Intestinal Cell Lines

To study the molecular mechanisms controlling guanylin expression, we have cloned the mouse guanylin gene, including 2.7 kb of upstream sequence. We show that the first 133 base pairs (bp) of the upstream guanylin promoter are sufficient to drive near maximal (6-fold over basal) luciferase reporter gene expression in Caco-2 intestinal cells; at least 300 bp of upstream promoter are required for reporter gene expression in HT-29 intestinal cell lines. Using electromobility shift assays, we demonstrate that nuclear proteins bind to the hepatocyte nuclear factor-1 (HNF-1) consensus sequence in the guanylin promoter. The HNF-1 consensus sequence, located in the immediate 5′ flanking region, is required for transcriptional activation of the guanylin gene in both intestinal cell lines. Mutagenesis of the HNF-1 consensus sequence abolishes transcriptional activation of guanylin promoter-luciferase reporter gene constructs. Cotransfection of these constructs with HNF-1α augments transcriptional initiation of the reporter gene. In contrast, HNF-1β has no significant effect on transcription of the reporter gene. These experiments demonstrate that HNF-1α is an important regulatory element in the transcriptional activation of guanylin.

Hypotonicity increases transcription, expression, and action ofEgr-1in murine renal medullary mIMCD3 cells

1997 ◽  
Vol 273 (5) ◽  
pp. F837-F842 ◽  
Author(s):  
Zheng Zhang ◽  
David M. Cohen
Keyword(s):  
Dna Binding ◽  
Reporter Gene ◽  
Collecting Duct ◽  
Consensus Sequence ◽  
Luciferase Reporter ◽  
Northern Analysis ◽  
Luciferase Reporter Gene ◽  
Hypotonic Stress ◽  
Gene Assay ◽  
In Cells

In cells of the murine renal inner medullary collecting duct (mIMCD3) cell line, acute hypotonic shock (50% dilution of medium with sterile water but not with sterile 150 mM NaCl) increased Egr-1 mRNA abundance 2.5-fold at 6 h, as determined by Northern analysis. This increase was accompanied by increased Egr-1 transcription, as quantitated by luciferase reporter gene assay. Increased transcription was dose dependent, additive with other Egr-1 transcriptional activators, and occurred in the absence of overt cytotoxicy, as quantitated via a fluorometric viability assay. In addition, hypotonic stress increased Egr-1 protein abundance, which was accompanied by augmented Egr-1-specific DNA binding ability, as measured via electrophoretic mobility shift assay. Increased DNA binding was further associated with increased transactivation by Egr-1, demonstrated through transient transfection of mIMCD3 cells with a luciferase reporter gene driven by tandem repeats of the Egr-1 DNA consensus sequence. Taken together, these data indicate that hypotonic stress activates Egr-1 transcription, translation, DNA binding, and transactivation in renal medullary cells. This phenomenon might play a role in the acquisition of the adaptive phenotype in response to hypotonic stress in cells of the renal medulla in vivo.

Influence of chlorophyll and tannins in plant extracts on cell-based luciferase reporter gene assays

Planta Medica ◽  
2009 ◽  
Vol 75 (09) ◽  
Author(s):  
S Vogl ◽  
P Picker ◽  
N Fakhrudin ◽  
A Atanasov ◽  
E Heiß ◽  
...  
Keyword(s):  
Reporter Gene ◽  
Plant Extracts ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Reporter Gene Assays
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