scholarly journals Characterization of the human liver fructose-1,6-bisphosphatase gene promoter

2000 ◽  
Vol 351 (2) ◽  
pp. 385-392 ◽  
Author(s):  
Birger HERZOG ◽  
Mary WALTNER-LAW ◽  
Donald K. SCOTT ◽  
Klaus ESCHRICH ◽  
Daryl K. GRANNER
Keyword(s):  
Hormonal Regulation ◽  
Promoter Activity ◽  
Gene Promoter ◽  
Nuclear Factor Κb ◽  
Site Directed Mutagenesis ◽  
Upstream Stimulatory Factor ◽  
Mobility Shift ◽  
Fructose 1,6 Bisphosphatase

Fructose-1,6-bisphosphatase (FBPase; EC 3.1.3.11), an important gluconeogenic enzyme, catalyses the hydrolysis of fructose 1,6-bisphosphate to fructose 6-phosphate and Pi. Enzyme activity is mainly regulated by the allosteric inhibitors fructose 2,6-bisphosphate and AMP. Although some observations about hormonal regulation of the enzyme have been published, the FBPase promoter has not been studied in detail. Here we report an in vitro characterization of the FBPase promoter with respect to the elements that are required for basal promoter activity. Transient transfection of H4IIE rat hepatoma cells, combined with site-directed mutagenesis, demonstrated that an enhancer box, three GC-boxes and a nuclear factor κB (NF-κB)-binding element are important for hepatic FBPase promoter activity. These elements are found in the region located between -405 to +25bp relative to the transcription start site. Electrophoretic-mobility-shift assays and supershift analysis confirmed that upstream stimulatory factor 1 (USF1)/USF2, specificity protein 1 (Sp1)/Sp3 and NF-κB respectively bind to these sites. The present study provides the basis for a more comprehensive screening for mutations in FBPase-deficient patients and for further studies of the transcriptional regulation of this gene.

scholarly journals Upstream stimulatory factor activates the vasopressin promoter via multiple motifs, including a non-canonical E-box

2003 ◽  
Vol 369 (3) ◽  
pp. 549-561 ◽  
Author(s):  
Judy M. COULSON ◽  
Jodie L. EDGSON ◽  
Zoe V. MARSHALL-JONES ◽  
Robert MULGREW ◽  
John P. QUINN ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Dominant Negative ◽  
Site Directed Mutagenesis ◽  
Upstream Stimulatory Factor ◽  
Mobility Shift ◽  
Transcriptional Initiation ◽  
E Box ◽  
E Boxes ◽  
Stimulatory Factor

We have described previously a complex E-box enhancer (-147) of the vasopressin promoter in small-cell lung cancer (SCLC) extracts [Coulson, Fiskerstrand, Woll and Quinn, (1999) Biochem. J. 344, 961—970]. Upstream stimulatory factor (USF) heterodimers were one of the complexes binding to this site in vitro. We now report that USF overexpression in non-SCLC (NSCLC) cells can functionally activate vasopressin promoter-driven reporters that are otherwise inactive in this type of lung cancer cell. Site-directed mutagenesis and electrophoretic mobility-shift analysis demonstrate that although the −147 E-box contributes, none of the previously predicted E-boxes (-147, −135, −34) wholly account for this USF-mediated activation in NSCLC. 5′ Deletion showed the key promoter region as −52 to +42; however, USF-2 binding was not reliant on the −34 E-box, but on a novel adjacent CACGGG non-canonical E-box at −42 (motif E). This mediated USF binding in both SCLC and USF-2-transfected NSCLC cells. Mutation of motif E or the non-canonical TATA box abolished activity, implying both are required for transcriptional initiation on overexpression of USF-2. Co-transfected dominant negative USF confirmed that binding was required through motif E for function, but that the classical activation domain of USF was not essential. USF-2 bound motif E with 10-fold lower affinity than the −147 E-box. In NSCLC, endogenous USF-2 expression is low, and this basal level appears to be insufficient to activate transcription of arginine vasopressin (AVP). In summary, we have demonstrated a novel mechanism for USF activation, which contributes to differential vasopressin expression in lung cancer.

scholarly journals Identification and Characterization of Two Nuclear Factor-κB Sites in the Regulatory Region of the Dopamine D2 Receptor

Endocrinology ◽  
2007 ◽  
Vol 148 (5) ◽  
pp. 2563-2570 ◽  
Author(s):  
Sandra Bontempi ◽  
Chiara Fiorentini ◽  
Chiara Busi ◽  
Nicoletta Guerra ◽  
PierFranco Spano ◽  
...  
Keyword(s):  
Promoter Activity ◽  
Transcriptional Activity ◽  
Dopamine D2 Receptor ◽  
Regulatory Region ◽  
D2 Receptor ◽  
Nuclear Factor Κb ◽  
Site Directed Mutagenesis ◽  
Luciferase Reporter ◽  
Nuclear Factor

Regulation of D2 receptor (D2R) expression is crucial in the function of dopaminergic systems. Because alterations of D2R expression may contribute to the development of different disorders, it is important to elucidate the mechanisms regulating D2R gene transcription. We report the characterization of two putative nuclear factor-κB (NF-κB) motifs, referred to as D2-κB sites, in the human D2R promoter, and demonstrate that they bind NF-κB subunits and stimulate D2R promoter activity. D2-κB sites show different degrees of conservation and specificity, when compared with canonical kB sites. The D2-κB1 site (from −407 to −398) is highly conserved and binds p50/p65 and p50/c-Rel complexes, whereas D2-κB2 (from −513 to −504) is more degenerated and only binds p50/p65 heterodimers. Activation of D2-κB sites in COS-7 cells expressing a luciferase reporter vector containing the D2R promoter resulted in increased transcriptional activity. Site-directed mutagenesis of each D2-κB site differentially modified D2R promoter activity. In particular, mutation of the D2-κB1 motif did not affect D2R promoter response to p50/c-Rel complexes, whereas inactivation of the D2-κB2 site decreased it. Mutations of either D2-κB1 or D2-κB2 sites attenuated the D2R promoter transcriptional efficiency induced by p50/p65 complexes. Thus, D2R transcription mediated by p50/c-Rel is supported mainly by the D2-κB2 site, whereas both sites are necessary to support the full transcriptional activity mediated by p50/p65 complexes. A correlation was found between NF-κB activity and D2R expression in the pituitary and pituitary-derived cells but not in the striatum, suggesting that NF-κB regulation of D2R expression could be a pituitary-specific mechanism.

scholarly journals Expression of wnt4/5 during reproductive cycle of catfish and wnt5 promoter analysis

2017 ◽  
Vol 232 (1) ◽  
pp. 1-13 ◽  
Author(s):  
Yarikipati Prathibha ◽  
Balasubramanian Senthilkumaran
Keyword(s):  
Reproductive Cycle ◽  
Promoter Activity ◽  
Ovarian Development ◽  
Developmental Stages ◽  
Protein Localization ◽  
Site Directed Mutagenesis ◽  
Cloning And Expression ◽  
Mobility Shift ◽  
Mammalian Cell Lines

Signaling molecules, Wnt4 and Wnt5, are essential for ovarian growth during developmental stages in mammals. Although these molecules were identified in several teleosts, their precise expression and role in reproductive processes have not yet been explored in any lower vertebrates. In view of this, using catfish, Clarias batrachus as an animal model, cloning and expression analysis of wnt4 and wnt5 were analyzed in different tissues, at various developmental stages, during ovarian reproductive cycle and after gonadotropin induction. These studies indicate a plausible influence of Wnts in ovarian development and recrudescence. Transcript and protein localization revealed their presence in peri-nucleolar, pre-vitellogenic, vitellogenic and follicular layer of post-vitellogenic oocytes. Synchronous expression of pax2 and wnt5 during the ovarian development and recrudescence of catfish led us to analyze the importance of putative binding element of Pax2 in the 5′-promoter motif of wnt5. Promoter activity of wnt5 was analyzed by luciferase assays after transfecting progressive deletion constructs in pGL3 basic vector into the mammalian cell lines (HEK 293 and CHO). The constructs having putative Pax2 motif showed high promoter activity compared with controls. Likewise, the constructs with site-directed mutagenesis showed increased activity after supplementing recombinant Pax2 indicating the prominence of this motif in wnt5 promoter, in vitro. Electrophoretic gel mobility shift, supershift and chromatin immunoprecipitation assays confirmed the binding of Pax2 to its corresponding cis-acting element in the upstream of wnt5. This study is the first of its kind to report the critical transcriptional interaction of Pax2 on wnt5 vis-à-vis ovarian development in teleosts.

Transcriptional regulation mechanisms of hypoxia-induced neuroglobin gene expression

2012 ◽  
Vol 443 (1) ◽  
pp. 153-164 ◽  
Author(s):  
Ning Liu ◽  
Zhanyang Yu ◽  
Shuanglin Xiang ◽  
Song Zhao ◽  
Anna Tjärnlund-Wolf ◽  
...  
Keyword(s):  
Gene Expression ◽  
Hypoxia Inducible Factor ◽  
Gene Promoter ◽  
Nuclear Factor Κb ◽  
Proximal Promoter ◽  
Mobility Shift ◽  
Hypoxic Conditions ◽  
Mobility Shift Assay

Ngb (neuroglobin) has been identified as a novel endogenous neuroprotectant. However, little is known about the regulatory mechanisms of Ngb expression, especially under conditions of hypoxia. In the present study, we located the core proximal promoter of the mouse Ngb gene to a 554 bp segment, which harbours putative conserved NF-κB (nuclear factor κB)- and Egr1 (early growth-response factor 1) -binding sites. Overexpression and knockdown of transcription factors p65, p50, Egr1 or Sp1 (specificity protein 1) increased and decreased Ngb expression respectively. Experimental assessments with transfections of mutational Ngb gene promoter constructs, as well as EMSA (electrophoretic mobility-shift assay) and ChIP (chromatin immunoprecipitation) assays, demonstrated that NF-κB family members (p65, p50 and cRel), Egr1 and Sp1 bound in vitro and in vivo to the proximal promoter region of the Ngb gene. Moreover, a κB3 site was found as a pivotal cis-element responsible for hypoxia-induced Ngb promoter activity. NF-κB (p65) and Sp1 were also responsible for hypoxia-induced up-regulation of Ngb expression. Although there are no conserved HREs (hypoxia-response elements) in the promoter of the mouse Ngb gene, the results of the present study suggest that HIF-1α (hypoxia-inducible factor-1α) is also involved in hypoxia-induced Ngb up-regulation. In conclusion, we have identified that NF-κB, Egr1 and Sp1 played important roles in the regulation of basal Ngb expression via specific interactions with the mouse Ngb promoter. NF-κB, Sp1 and HIF-1α contributed to the up-regulation of mouse Ngb gene expression under hypoxic conditions.

5-Aminolaevulinate synthase gene promoter contains two cAMP-response element (CRE)-like sites that confer positive and negative responsiveness to CRE-binding protein (CREB)

2001 ◽  
Vol 353 (2) ◽  
pp. 307-316 ◽  
Author(s):  
Luciana E. GIONO ◽  
Cecilia L. VARONE ◽  
Eduardo T. CÁNEPA
Keyword(s):  
Gene Expression ◽  
Promoter Activity ◽  
Molecular Mechanisms ◽  
Binding Protein ◽  
Phosphorylation Site ◽  
Gene Promoter ◽  
Site Directed Mutagenesis ◽  
Dependent Manner ◽  
Mobility Shift ◽  
Putative Transcription Factor

The first and rate-controlling step of the haem biosynthetic pathway in mammals and fungi is catalysed by the mitochondrial-matrix enzyme 5-aminolaevulinate synthase (ALAS). The purpose of this work was to explore the molecular mechanisms involved in the cAMP regulation of rat housekeeping ALAS gene expression. Thus we have examined the ALAS promoter for putative transcription-factor-binding sites that may regulate transcription in a cAMP-dependent protein kinase (PKA)-induced context. Applying both transient transfection assays with a chloramphenicol acetyltransferase reporter gene driven by progressive ALAS promoter deletions in HepG2, and electrophoresis mobility-shift assays we have identified two putative cAMP-response elements (CREs) at positions -38 and -142. Functional analysis showed that both CRE-like sites were necessary for complete PKA induction, but only one for basal expression. Co-transfection with a CRE-binding protein (CREB) expression vector increased PKA-mediated induction of ALAS promoter transcriptional activity. However, in the absence of co-transfected PKA, CREB worked as a specific repressor for ALAS promoter activity. A CREB mutant deficient in a PKA phosphorylation site was unable to induce expression of the ALAS gene but could inhibit non-stimulated promoter activity. Furthermore, a DNA-binding mutant of CREB did not interfere with ALAS promoter basal activity. Site-directed-mutagenesis studies showed that only the nearest element to the transcription start site was able to inhibit the activity of the promoter. Therefore, we conclude that CREB, through its binding to CRE-like sites, mediates the effect of cAMP on ALAS gene expression. Moreover, we propose that CREB could also act as a repressor of ALAS transcription, but is able to reverse its role after PKA activation. Dephosphorylated CREB would interfere in a spatial-disposition-dependent manner with the transcriptional machinery driving inhibition of gene expression.

scholarly journals Functional Characterization of the mazEF Toxin-Antitoxin System in the Pathogenic Bacterium Agrobacterium tumefaciens

2021 ◽  
Vol 9 (5) ◽  
pp. 1107
Author(s):  
Wonho Choi ◽  
Yoshihiro Yamaguchi ◽  
Ji-Young Park ◽  
Sang-Hyun Park ◽  
Hyeok-Won Lee ◽  
...  
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Physiological Function ◽  
Functional Characterization ◽  
Site Directed Mutagenesis ◽  
In Vitro Assays ◽  
Cell Growth Inhibition ◽  
The Right

Agrobacterium tumefaciens is a pathogen of various plants which transfers its own DNA (T-DNA) to the host plants. It is used for producing genetically modified plants with this ability. To control T-DNA transfer to the right place, toxin-antitoxin (TA) systems of A. tumefaciens were used to control the target site of transfer without any unintentional targeting. Here, we describe a toxin-antitoxin system, Atu0939 (mazE-at) and Atu0940 (mazF-at), in the chromosome of Agrobacterium tumefaciens. The toxin in the TA system has 33.3% identity and 45.5% similarity with MazF in Escherichia coli. The expression of MazF-at caused cell growth inhibition, while cells with MazF-at co-expressed with MazE-at grew normally. In vivo and in vitro assays revealed that MazF-at inhibited protein synthesis by decreasing the cellular mRNA stability. Moreover, the catalytic residue of MazF-at was determined to be the 24th glutamic acid using site-directed mutagenesis. From the results, we concluded that MazF-at is a type II toxin-antitoxin system and a ribosome-independent endoribonuclease. Here, we characterized a TA system in A. tumefaciens whose understanding might help to find its physiological function and to develop further applications.

scholarly journals Silencing of the Gene for the α-Subunit of Human Chorionic Gonadotropin by the Embryonic Transcription Factor Oct-3/4

1997 ◽  
Vol 11 (11) ◽  
pp. 1651-1658 ◽  
Author(s):  
Limin Liu ◽  
Douglas Leaman ◽  
Michel Villalta ◽  
R. Michael Roberts
Keyword(s):  
Transcription Factor ◽  
Binding Site ◽  
Gene Promoter ◽  
Site Directed Mutagenesis ◽  
Mobility Shift ◽  
Α Subunit ◽  
Choriocarcinoma Cells ◽  
Human Choriocarcinoma Cells ◽  
Electrophoretic Mobility Shift Assays ◽  
Jar Cells

Abstract CG is required for maintenance of the corpus luteum during pregnancy in higher primates. As CG is a heterodimeric molecule, some form of coordinated control must be maintained over the transcription of its two subunit genes. We recently found that expression of human CG β-subunit (hCGβ) in JAr human choriocarcinoma cells was almost completely silenced by the embryonic transcription factor Oct-3/4, which bound to a unique ACAATAATCA octameric sequence in the hCGβ gene promoter. Here we report that Oct-3/4 is also a potent inhibitor of hCG α-subunit (hCGα) expression in JAr cells. Oct-3/4 reduced human GH reporter expression from the −170 hCGα promoter in either the presence or absence of cAMP by about 70% in transient cotransfection assays, but had no effect on expression from either the −148 hCGα or the −99 hCGα promoter. Unexpectedly, no Oct-3/4-binding site was identified within the −170 to −148 region of the hCGα promoter, although one was found around position −115 by both methylation interference footprinting and electrophoretic mobility shift assays. Site-directed mutagenesis of this binding site destroyed the affinity of the promoter for Oct-3/4, but did not affect repression of the promoter. Therefore, inhibition of hCGα gene transcription by Oct-3/4 appears not to involve direct binding of this factor to the site responsible for silencing. When stably transfected into JAr cells, Oct-3/4 reduced the amounts of both endogenous hCGα mRNA and protein by 70–80%. Oct-3/4 is therefore capable of silencing both hCGα and hCGβ gene expression. We suggest that as the trophoblast begins to form, reduction of Oct-3/4 expression permits the coordinated onset of transcription from the hCGα and hCGβ genes.

The nuclear orphan receptors COUP-TFII and Ear-2 act as silencers of the human oxytocin gene promoter

1997 ◽  
Vol 19 (2) ◽  
pp. 163-172 ◽  
Author(s):  
K Chu ◽  
HH Zingg
Keyword(s):  
Receptor Binding ◽  
Promoter Activity ◽  
Transcriptional Activity ◽  
Gene Promoter ◽  
Fine Tuning ◽  
Orphan Receptor ◽  
Expression Vectors ◽  
Mobility Shift ◽  
Interaction Sites

We have previously shown that COUP-TFII and Ear-2, two members of the nuclear orphan receptor family, are able to repress oestrogen-stimulated transcriptional activity of the human oxytocin (OT) gene promoter by binding to a site that overlaps with the oestrogen response element (ERE) present in the 5' flanking region of the gene. Although most nuclear receptor-mediated transcriptional repression conforms with the paradigm of passive repression and involves competitive binding to an activator site, active repression, i.e. silencing of basal promoter activity, has been observed in a limited number of cases. Here we show by co-transfection experiments using COUP-TFII and Ear-2 expression vectors and reporter constructs containing OT gene promoter fragments linked to the chloramphenicol acetyltransferase gene that both COUP-TFII and Ear-2 are capable of silencing basal OT gene promoter activity by 54 and 75% respectively. 5' Deletion and footprint analyses revealed two areas of functionally important interaction sites: (1) a direct TGACC(T/C) repeat overlapping the ERE and (2) a more promoter-proximal area centred at - 90 containing three imperfect direct repeats (R1-R3) spaced by four nucleotides each. Mutagenesis of reporter constructs as well as electrophoretic mobility-shift assays demonstrated that each of the three proximal repeats R1-R3 contributed to orphan receptor binding and the silencing effect. Inasmuch as the orphan receptor-binding sites are not involved in mediating basal transcriptional activity of the OT gene promoter, the observed effects are best interpreted as active repression or promoter silencing. Moreover, since COUP-TFII and Ear-2 are both co-expressed in OT-expressing uterine epithelial cells, the novel transcriptional effects described here are likely to be of functional importance in the fine-tuning of uterine OT gene expression in vivo.

scholarly journals The nuclear factor κB inhibitor (E)-2-fluoro-4′-methoxystilbene inhibits firefly luciferase

2012 ◽  
Vol 32 (6) ◽  
pp. 531-537 ◽  
Author(s):  
Albert Braeuning ◽  
Silvia Vetter
Keyword(s):  
Photon Emission ◽  
Gene Promoter ◽  
Firefly Luciferase ◽  
Nuclear Factor Κb ◽  
Signalling Pathway ◽  
Luciferase Reporter ◽  
Nuclear Factor ◽  
Reporter System ◽  
Reporter Assays

Photinus pyralis (firefly) luciferase is widely used as a reporter system to monitor alterations in gene promoter and/or signalling pathway activities in vitro. The enzyme catalyses the formation of oxyluciferin from D-luciferin in an ATP-consuming reaction involving photon emission. The purpose of the present study was to characterize the luciferase-inhibiting potential of (E)-2-fluoro-4′-methoxystilbene, which is known as a potent inhibitor of the NF-κB (nuclear factor κB) signalling pathway that is used to modulate the NF-κB signalling pathway in vitro. Results show that (E)-2-fluoro-4′-methoxystilbene effectively inhibits firefly luciferase activity in cell lysates and living cells in a non-competitive manner with respect to the luciferase substrates D-luciferin and ATP. By contrast, the compound has no effect on Renilla and Gaussia luciferases. The mechanism of firefly luciferase inhibition by (E)-2-fluoro-4′-methoxystilbene, as well as its potency is comparable to its structure analogue resveratrol. The in vitro use of trans-stilbenes such as (E)-2-fluoro-4′-methoxystilbene or resveratrol compromises firefly luciferase reporter assays as well as ATP/luciferase-based cell viability assays.

Glucose induces expression of rat pyruvate carboxylase through a carbohydrate response element in the distal gene promoter

2010 ◽  
Vol 426 (2) ◽  
pp. 159-170 ◽  
Author(s):  
Kim B. Pedersen ◽  
Rebecca S. Buckley ◽  
Ray Scioneaux
Keyword(s):  
Pyruvate Carboxylase ◽  
Gene Promoter ◽  
Upstream Stimulatory Factor ◽  
Glucose Response ◽  
Response Element ◽  
Insulinoma Cell ◽  
Glucose Stimulated Insulin Secretion ◽  
Glucose Responsiveness ◽  
Distal Gene

Pyruvate carboxylase is an enzyme of the so-called pyruvate cycling pathways, which have been proposed to contribute to glucose-stimulated insulin secretion in pancreatic β-cells. In the rat insulinoma cell line 832/13, transcripts from both the distal and proximal gene promoter for pyruvate carboxylase are up-regulated by glucose, with pyruvate carboxylase being expressed mainly from the distal gene promoter. At position −408 to −392 relative to the transcription start site, the distal gene promoter was found to contain a ChoRE (carbohydrate response element). Its deletion abolishes glucose responsiveness of the promoter, and the sequence can mediate glucose responsiveness to a heterologous gene promoter. ChREBP (carbohydrate response element-binding protein) and its dimerization partner Mlx (Max-like protein X) bind to the ChoRE in vitro. ChREBP further binds to the distal promoter region at a high glucose concentration in situ. The E-box-binding transcription factors USF1/2 (upstream stimulatory factor 1/2) and E2A variant 2 [also known as E47 and TCF3 (transcription factor 3)] can also bind to the ChoRE. Overexpression of E2A diminishes the magnitude of the glucose response from the pyruvate carboxylase ChoRE. This illustrates that competition between ChREBP–Mlx and other factors binding to the ChoRE affects glucose responsiveness. We conclude that a ChoRE in the distal gene promoter contributes to the glucose-mediated expression of pyruvate carboxylase.

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