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

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.

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Potency of isothiocyanates to induce luciferase reporter gene expression via the electrophile-responsive element from murine glutathione S-transferase Ya

Toxicology in Vitro ◽

10.1016/j.tiv.2009.02.005 ◽

2009 ◽

Vol 23 (4) ◽

pp. 617-621 ◽

Cited By ~ 8

Author(s):

Martijn Vermeulen ◽

Anne-Marie M.J.F. Boerboom ◽

Barry M.G. Blankvoort ◽

Jac M.M.J.G. Aarts ◽

Ivonne M.C.M. Rietjens ◽

...

Keyword(s):

Gene Expression ◽

Reporter Gene ◽

Reporter Gene Expression ◽

Luciferase Reporter ◽

Glutathione S Transferase ◽

Luciferase Reporter Gene ◽

Responsive Element

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Non-invasive somatotransgenic bioimaging in living animals

F1000Research ◽

10.12688/f1000research.25274.1 ◽

2020 ◽

Vol 9 ◽

pp. 1216 ◽

Cited By ~ 1

Author(s):

Juliette M. Delhove ◽

Rajvinder Karda ◽

Lorna M. FitzPatrick ◽

Suzanne M.K. Buckley ◽

Simon N. Waddington ◽

...

Keyword(s):

Gene Expression ◽

Reporter Gene ◽

Viral Vectors ◽

Es Cells ◽

Embryonic Stem ◽

Whole Body ◽

Luciferase Reporter ◽

Gene Promoters ◽

Luciferase Reporter Gene ◽

Endogenous Gene

Bioluminescence imaging enables noninvasive quantification of luciferase reporter gene expression in transgenic tissues of living rodents. Luciferase transgene expression can be regulated by endogenous gene promoters after targeted knock-in of the reporter gene, usually within the first intron of the gene. Even using CRISPR/Cas9 mediated genome editing this can be a time consuming and costly process. The generation of germline transgenic (GLT) rodents by targeted genomic integration of a gene expression cassette in embryonic stem (ES) cells is commonplace but results in the wastage of large numbers of animals during colony generation, back-crossing and maintenance. Using a synthetic/truncated promoter-driven luciferase gene to study promoter activity in a given tissue or organ of a GLT also often results in unwanted background luciferase activity during whole-body bioluminescent imaging as every cell contains the reporter. We have developed somatotransgenic bioimaging; a method to generate tissue-restricted transcription factor activated luciferase reporter (TFAR) cassettes in rodents that substantially reduces the number of animals required for experimentation. Bespoke designed TFARs are delivered to newborn pups using viral vectors targeted to specific organs by tissue-tropic pseudotypes. Retention and proliferation of TFARs is facilitated by stem/progenitor cell transduction and immune tolerance to luciferase due to the naïve neonatal immune system. We have successfully applied both lentiviral and adeno-associated virus (AAV) vectors in longitudinal rodent studies, targeting TFARs to the liver and brain during normal development and in well-established disease models. Development of somatotransgenic animals has broad applicability to non-invasively determine mechanistic insights into homeostatic and disease states and assess toxicology and efficacy testing. Somatotransgenic bioimaging technology is superior to current whole-body, light-emitting transgenic models as it reduces the numbers of animals used by generating only the required number of animals. It is also a refinement over current technologies given the ability to use conscious, unrestrained animals.

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In vivo regulation of β-MHC gene in rodent heart: role of T3 and evidence for an upstream enhancer

AJP Cell Physiology ◽

10.1152/ajpcell.1999.276.4.c883 ◽

1999 ◽

Vol 276 (4) ◽

pp. C883-C891 ◽

Cited By ~ 46

Author(s):

Carola E. Wright ◽

F. Haddad ◽

A. X. Qin ◽

P. W. Bodell ◽

K. M. Baldwin

Keyword(s):

Gene Expression ◽

Thyroid Hormone ◽

Reporter Gene ◽

Normal Control ◽

Transcriptional Activity ◽

Luciferase Reporter ◽

Luciferase Reporter Gene ◽

Promoter Construct ◽

Hormone Responsiveness

Cardiac β-myosin heavy chain (β-MHC) gene expression is mainly regulated through transcriptional processes. Although these results are based primarily on in vitro cell culture models, relatively little information is available concerning the interaction of key regulatory factors thought to modulate MHC expression in the intact rodent heart. Using a direct gene transfer approach, we studied the in vivo transcriptional activity of different-length β-MHC promoter fragments in normal control and in altered thyroid states. The test β-MHC promoter was fused to a firefly luciferase reporter gene, whereas the control α-MHC promoter was fused to the Renilla luciferase reporter gene and was used to account for variations in transfection efficiency. Absolute reporter gene activities showed that β- and α-MHC genes were individually and reciprocally regulated by thyroid hormone. The β-to-α ratios of reporter gene expression demonstrated an almost threefold larger β-MHC gene expression in the longest than in the shorter promoter fragments in normal control animals, implying the existence of an upstream enhancer. A mutation in the putative thyroid response element of the −408-bp β-MHC promoter construct caused transcriptional activity to drop to null. When studied in the −3,500-bp β-MHC promoter, construct activity was reduced (∼100-fold) while thyroid hormone responsiveness was retained. These findings suggest that, even though the bulk of the thyroid hormone responsiveness of the gene is contained within the first 215 bp of the β-MHC promoter sequence, the exact mechanism of triiodothyronine (T3) action remains to be elucidated.

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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

Biochemical Journal ◽

10.1042/bj3380457 ◽

1999 ◽

Vol 338 (2) ◽

pp. 457-463 ◽

Cited By ~ 35

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.

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Haemoglobin interferes with the ex vivo luciferase luminescence assay: consequence for detection of luciferase reporter gene expression in vivo

Gene Therapy ◽

10.1038/sj.gt.3301248 ◽

2000 ◽

Vol 7 (15) ◽

pp. 1333-1336 ◽

Cited By ~ 47

Author(s):

M Colin ◽

S Moritz ◽

H Schneider ◽

J Capeau ◽

C Coutelle ◽

...

Keyword(s):

Gene Expression ◽

Reporter Gene ◽

Ex Vivo ◽

Reporter Gene Expression ◽

Luciferase Reporter ◽

Luciferase Reporter Gene ◽

Luminescence Assay

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Oxygen-evoked changes in transcriptional activity of the 5′-flanking region of the human amiloride-sensitive sodium channel (αENaC) gene: role of nuclear factor κB

Biochemical Journal ◽

10.1042/bj20011651 ◽

2002 ◽

Vol 364 (2) ◽

pp. 537-545 ◽

Cited By ~ 12

Author(s):

Deborah L. BAINES ◽

Mandy JANES ◽

David J. NEWMAN ◽

Oliver G. BEST

Keyword(s):

Sodium Channel ◽

Transcriptional Activation ◽

Transcriptional Activity ◽

Consensus Sequence ◽

A549 Cells ◽

Nuclear Factor Κb ◽

Luciferase Reporter ◽

Mrna Levels ◽

Nuclear Factor ◽

Α Subunit

Expression of the α-subunit of the amiloride-sensitive sodium channel (αENaC) is regulated by a number of factors in the lung, including oxygen partial pressure (Po2). As transcriptional activation is a mechanism for raising cellular mRNA levels, we investigated the effect of physiological changes in Po2 on the activity of the redox-sensitive transcription factor nuclear factor κB (NF-κB) and transcriptional activity of 5′-flanking regions of the human αENaC gene using luciferase reporter-gene vectors transiently transfected into human adult alveolar carcinoma A549 cells. By Western blotting we confirmed the presence of NF-κB p65 but not p50 in these cells. Transiently increasing Po2 from 23 to 42mmHg for 24h evoked a significant increase in NF-κB DNA-binding activity and transactivation of a NF-κB-driven luciferase construct (pGLNF-κBpro), which was blocked by the NF-κB activation inhibitor sulphasalazine (5mM). Transcriptional activity of αENaC-luciferase constructs containing 5′-flanking sequences (including the NF-κB consensus) were increased by raising Po2 from 23 to 142mm Hg if they contained transcriptional initiation sites (TIS) for exons 1A and 1B (pGL3E2.2) or the 3′ TIS of exon 1B alone (pGL3E0.8). Sulphasalazine had no significant effect on the activity of these constructs, suggesting that the Po2-evoked rise in activity was not a direct consequence of NF-κB activation. Conversely, the relative luciferase activity of a construct that lacked the 3′ TIS, a 3′ intron and splice site but still retained the 5′ TIS and NF-κB consensus sequence was suppressed significantly by raising Po2. This effect was reversed by sulphasalazine, suggesting that activation of NF-κB mediated Po2-evoked suppression of transcription from the exon 1A TIS of αENaC.

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Analyzing the whole-transcriptome profiles of ncRNAs and predicting the competing endogenous RNA networks in cervical cancer cell lines with cisplatin resistance

Cancer Cell International ◽

10.1186/s12935-021-02239-6 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Huimin Lv ◽

Shanshan Jin ◽

Binbin Zou ◽

Yuxiang Liang ◽

Jun Xie ◽

...

Keyword(s):

Gene Expression ◽

Cervical Cancer ◽

Reporter Gene ◽

Biological Function ◽

Malignant Tumors ◽

Luciferase Reporter ◽

Sequencing Analysis ◽

Luciferase Reporter Gene ◽

Network Analyses ◽

Whole Transcriptome

Abstract Background Cervical cancer (CC) is one of the most common malignant tumors in women. In order to identify the functional roles and the interaction between mRNA and non-coding RNA (ncRNA, including lncRNA, circRNA and miRNA) in CC cisplatin (DDP) resistance, the transcription profile analysis was performed and a RNA regulatory model of CC DDP resistance was proposed. Methods In this study, whole-transcriptome sequencing analysis was conducted to study the ncRNA and mRNA profiles of parental SiHa cells and DDP resistant SiHa/DDP cells. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were performed for pathway analysis based on the selected genes with significant differences in expression. Subsequently, ceRNA network analyses were conducted using the drug resistance-related genes and signal-transduction pathways by Cytoscape software. Furthermore, a ceRNA regulatory pathway, namely lncRNA-AC010198.2/hsa-miR-34b-3p/STC2, was selected by RT-qPCR validation and literature searching. Further validation was done by both dual-luciferase reporter gene assays and RNA pull-down assays. Besides that, the changes in gene expression and biological function were further studied by performing si-AC010198.2 transfection and DDP resistance analyses in the SiHa and SiHa/DDP cells, respectively. Results Using bioinformatics and dual-luciferase reporter gene analyses, we found that AC010198.2/miR-34b-3p/STC2 may be a key pathway for DDP resistance in CC cells. Significant differences in both downstream gene expression and the biological function assays including colony formation, migration efficiency and cell apoptosis were identified in AC010198.2 knockdown cells. Conclusions Our study will not only provide new markers and potential mechanism models for CC DDP resistance, but also discover novel targets for attenuating it.

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Screening for Discovery of Novel Peroxisome Proliferator-Activated Receptor-alpha and -gamma Agonists and Nuclear Factor-κB Inhibitors by Luciferase Reporter Gene Assays

Scientia Pharmaceutica ◽

10.3797/scipharm.oephg.21.po-41 ◽

2009 ◽

Vol 77 (1) ◽

pp. 240-240

Author(s):

Fakhrudin

Keyword(s):

Reporter Gene ◽

Nuclear Factor Κb ◽

Luciferase Reporter ◽

Peroxisome Proliferator ◽

Nuclear Factor ◽

Luciferase Reporter Gene ◽

Peroxisome Proliferator Activated Receptor ◽

Receptor Alpha ◽

Reporter Gene Assays

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Hepatocyte nuclear factor-4 mediates apolipoprotein A-IV transcriptional regulation by fatty acid in newborn swine enterocytes

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00072.2007 ◽

2007 ◽

Vol 293 (2) ◽

pp. G475-G483 ◽

Cited By ~ 18

Author(s):

Shuangying Leng ◽

Song Lu ◽

Ying Yao ◽

Zhisheng Kan ◽

Gabriel S. Morris ◽

...

Keyword(s):

Fatty Acid ◽

Large Subunit ◽

Dietary Lipid ◽

Lipid Absorption ◽

Luciferase Reporter ◽

Mrna Levels ◽

Nuclear Factor ◽

Hepatocyte Nuclear Factor ◽

Luciferase Reporter Gene

Hepatocyte nuclear factor-4α (HNF-4α) regulates transcription of several genes involved in lipid metabolism, including that of apolipoprotein (apo) A-IV, which is tightly regulated by lipid absorption and enhances enterocyte chylomicron secretion. Studies were performed to define the role of HNF-4α in the regulation of apo A-IV gene transcription by dietary fatty acid in neonatal swine small intestine. HNF-4α mRNA was expressed in liver > intestine > kidney in suckling, weanling, and weaned pigs. Jejunal HNF-4α mRNA and protein and apo A-IV and swine microsomal triglyceride transfer protein (MTP) large subunit mRNA expression were induced in parallel in 2-day-old swine by a 24-h high-fat intraduodenal infusion. In IPEC-1 cells, incubation with oleic acid (OA) resulted in coordinate induction of both HNF-4α, apo A-IV, and MTP mRNA, similar to that observed in vivo. When HNF-4α expression was driven by doxycycline by using the TET-On system in the absence of OA to observe the effect of HNF-4α directly on apo A-IV and MTP mRNA levels in the absence of other factors that might be concomitantly induced by fatty acid absorption, apo A-IV and MTP expression were increased. In luciferase reporter gene assays in IPEC-1 cells using apo A-IV/C-III intergenic region constructs, TET-On-regulated HNF-4α expression without OA increased luciferase activity, and incubation with OA did not further increase activity. These data suggest that acute induction of the apo A-IV and MTP genes by dietary lipid in newborn intestine occurs, at least in part, via ligand-independent transactivation by HNF-4α that is itself induced by a lipid-mediated mechanism.

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