scholarly journals Impact of Promoter Polymorphisms on the Transcriptional Regulation of the Organic Cation Transporter OCT1 (SLC22A1)

2018 ◽  
Vol 8 (4) ◽  
pp. 42 ◽  
Author(s):  
Kristin Bokelmann ◽  
Jürgen Brockmöller ◽  
Mladen Tzvetkov
Keyword(s):  
Transcription Factor ◽  
Promoter Activity ◽  
Organic Cation ◽  
Specific Binding ◽  
Organic Cation Transporter ◽  
Luciferase Reporter ◽  
Potential Contribution ◽  
Luciferase Reporter Gene ◽  
Promoter Polymorphisms ◽  
Mobility Shift

The organic cation transporter 1 (OCT1, SLC22A1) is strongly expressed in the human liver and facilitates the hepatic uptake of drugs such as morphine, metformin, tropisetron, sumatriptan and fenoterol and of endogenous substances such as thiamine. OCT1 expression is inter-individually highly variable. Here, we analyzed SNPs in the OCT1 promoter concerning their potential contribution to the variability in OCT1 expression. Using electrophoretic mobility shift and luciferase reporter gene assays in HepG2, Hep3B, and Huh7 cell lines, we identified the SNPs −1795G>A (rs6935207) and −201C>G (rs58812592) as having effects on transcription factor binding and/or promoter activity. The A-allele of the −1795G>A SNP showed allele-specific binding of the transcription factor NF-Y leading to 2.5-fold increased enhancer activity of the artificial SV40 promoter. However, the −1795G>A SNP showed no significant effects on the native OCT1 promoter activity. Furthermore, the −1795G>A SNP was not associated with the pharmacokinetics of metformin, fenoterol, sumatriptan and proguanil in healthy individuals or tropisetron efficacy in patients undergoing chemotherapy. Allele-dependent differences in USF1/2 binding and nearly total loss in OCT1 promoter activity were detected for the G-allele of −201C>G, but the SNP is apparently very rare. In conclusion, common OCT1 promoter SNPs have only minor effects on OCT1 expression.

scholarly journals The myeloid-cell-specific c-fes promoter is regulated by Sp1, PU.1, and a novel transcription factor.

1996 ◽  
Vol 16 (4) ◽  
pp. 1676-1686 ◽  
Author(s):  
A Heydemann ◽  
G Juang ◽  
K Hennessy ◽  
M S Parmacek ◽  
M C Simon
Keyword(s):  
Transcription Factor ◽  
Vascular Endothelial Cells ◽  
Myeloid Cells ◽  
Myeloid Cell ◽  
Luciferase Reporter ◽  
Specific Gene ◽  
Luciferase Reporter Gene ◽  
Mobility Shift ◽  
Specific Expression ◽  
Flanking Sequences

The protein product of the c-fps/fes (c-fes) proto-oncogene has been implicated in the normal development of myeloid cells (macrophages and neutrophils). mRNA for c-fes has been detected exclusively in myeloid cells and vascular endothelial cells in adult mammals. Although a 13-kilobase-pair (kb) human c-fes transgene exhibits high levels of expression in mice, the sequences that confer myeloid-cell-specific expression of the human c-fes gene have not been defined. Transient-transfection experiments demonstrated that plasmids containing 446 bp of c-fes 5'-flanking sequences linked to a luciferase reporter gene were active exclusively in myeloid cells. No other DNA element within the 13-kb human c-fes locus contained positive cis-acting elements, with the exception of a weakly active region within the 3'-flanking sequences. DNase I footprinting assays revealed four distinct sites that bind myeloid nuclear proteins (-408 to -386, -293 to -254, -76 to -65, and -34 to +3). However, the first two footprints resided in sequences that were largely dispensable for transient activity. Plasmids containing 151 bp of 5'-flanking sequences confer myeloid-cell-specific gene expression. Electrophoretic mobility shift analyses demonstrated that the 151-bp region contains nuclear protein binding sites for Sp1, PU.1, and/or Elf-1, and a novel factor. This unidentified factor binds immediately 3' of the PU.1/Elf-1 sites and appears to be myeloid cell specific. Mutation of the PU.1/Elf-1 site or the 3' site (FP4-3') within the context of the c-fes promoter resulted in substantially reduced activity in transient transfections. Furthermore, transient-cotransfection assay demonstrated that PU.1 (and not Elf-1) can transactivate the c-fes promoter in nonmyeloid cell lines. We conclude that the human c-fes gene contains a strong myeloid-cell-specific promoter that is regulated by Sp1, PU.1, and a novel transcription factor.

scholarly journals Transcription factor organic cation transporter 1 (OCT-1) affects the expression of porcine Klotho (KL) gene

2016 ◽  
Author(s):  
Yan Li ◽  
Lei Wang ◽  
Jiawei Zhou ◽  
Fenge Li
Keyword(s):  
Transcription Factor ◽  
Organic Cation ◽  
Core Promoter ◽  
Sequence Similarity ◽  
Organic Cation Transporter ◽  
Mobility Shift ◽  
Fragment Analysis ◽  
Intron 1 ◽  
Organic Cation Transporter 1 ◽  
Electrophoretic Mobility Shift Assays

Klotho (KL), originally discovered as an aging suppressor, was a membrane protein that shared sequence similarity with the β-glucosidase enzymes. Recent reports showed Klotho might have a role in adipocyte maturation and systemic glucose metabolism. However, little is known about the transcription factors involved in regulating the expression of porcine KL gene. Deletion fragment analysis identified KL-D2 (-418 bp to -3 bp) as the porcine KL core promoter. MARC0022311 in KL intron 1 appeared a polymorphism (A or G) in Landrace × DIV pigs, and relative luciferase activity of pGL3-D2-G was significantly higher than pGL3-D2-A. This was possibly the result of a change in KL binding ability with transcription factor organic cation transporter 1 (OCT-1), which was confirmed using electrophoretic mobility shift assays (EMSA) and chromatin immunoprecipitation (ChIP). Moreover, OCT-1 regulated endogenous KL expression by RNA interference. Our study indicates SNP MARC0022311 affects porcine KL expression by regulating its promoter activity via OCT-1.

Regulation of the human brain natriuretic peptide gene by GATA-4

2002 ◽  
Vol 283 (1) ◽  
pp. E50-E57 ◽  
Author(s):  
Quan He ◽  
Mariela Mendez ◽  
Margot C. LaPointe
Keyword(s):  
Brain Natriuretic Peptide ◽  
Natriuretic Peptide ◽  
Promoter Activity ◽  
Nuclear Protein ◽  
Ventricular Myocytes ◽  
Proximal Promoter ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Mobility Shift ◽  
Specific Expression

Brain natriuretic peptide (BNP) is a cardiac hormone constitutively expressed in the adult heart. We previously showed that the human BNP (hBNP) proximal promoter region from −127 to −40 confers myocyte-specific expression. The proximal hBNP promoter contains several putative cis elements. Here we tested whether the proximal GATA element plays a role in basal and inducible regulation of the hBNP promoter. The hBNP promoter was coupled to a luciferase reporter gene (1818hBNPLuc) and transferred into neonatal ventricular myocytes (NVM), and luciferase activity was measured as an index of hBNP promoter activity. Mutation of the putative GATA element at −85 of the hBNP promoter [1818(mGATA)hBNPLuc] reduced activity by 97%. To study transactivation of the hBNP promoter, we co-transfected 1818hBNPLuc with the GATA-4 expression vector. GATA-4 activated 1818hBNPLuc, and this effect was eliminated by mutation of the proximal GATA element. Electrophoretic mobility shift assay showed that an oligonucleotide containing the hBNP GATA motif bound to cardiomyocyte nuclear protein, which was competed for by a consensus GATA oligonucleotide but not a mutated hBNP GATA element. The β-adrenergic agonist isoproterenol and its second messenger cAMP stimulated hBNP promoter activity and binding of nuclear protein to the proximal GATA element. Thus the GATA element in the proximal hBNP promoter is involved in both basal and inducible transcriptional regulation in cardiac myocytes.

scholarly journals Ca2+ administration stimulates the binding of AP-1 factor to the 5′-flanking region of the rat gene for the Ca2+-binding protein regucalcin

1998 ◽  
Vol 329 (1) ◽  
pp. 157-163 ◽  
Author(s):  
Tomiyasu MURATA ◽  
Masayoshi YAMAGUCHI
Keyword(s):  
Promoter Activity ◽  
Binding Protein ◽  
Luciferase Reporter ◽  
Rat Tissues ◽  
Luciferase Reporter Gene ◽  
Mobility Shift ◽  
Flanking Region ◽  
Gel Mobility Shift ◽  
Dna Complex ◽  
Regucalcin Gene

mRNA of the Ca2+-binding protein, regucalcin, is mainly expressed in the liver and only to a small extent in the kidney, and the expression of hepatic regucalcin mRNA is markedly stimulated by Ca2+ administration [Shimokawa and Yamaguchi (1992) FEBS Lett. 305, 151-154]. The existence of nuclear factors that bind to the 5ʹ-flanking region of the rat regucalcin gene was investigated. When nuclear proteins obtained from various rat tissues were used in gel mobility-shift assays, tissue-specific formation of a protein-DNA complex was found in the liver and kidney. An additional novel protein-DNA complex was formed when liver nuclear extracts obtained from Ca2+-administered rats (10 mg of Ca2+/100 g body weight) were used. Competition gel mobility-shift experiments using consensus and mutant oligonucleotides for AP-1 factor showed that the additional novel complex was formed from binding of the AP-1 factor to the regucalcin gene. Ca2+-induced binding of the AP-1 factor to the regucalcin gene was completely inhibited by simultaneous administration of trifluoperazine, an antagonist of calmodulin, suggesting that the activation of nuclear AP-1 protein is partly mediated through a Ca2+/calmodulin-dependent pathway. Moreover, the 5ʹ-flanking region of the rat regucalcin gene ligated to a luciferase reporter gene possessed the promoter activity in H4-II-E hepatoma cells. This promoter activity was enhanced by treatment with Bay K 8644, a Ca2+-channel agonist. The present study demonstrates that the Ca2+-response sequences are located within the 5ʹ-flanking region of the rat regucalcin gene.

Identification of the 2-tridecanone responsive region in the promoter of cytochrome P450 CYP6B6 of the cotton bollworm, Helicoverpa armigera (Lepidoptera: Noctuidae)

2014 ◽  
Vol 104 (6) ◽  
pp. 801-808 ◽  
Author(s):  
F. Li ◽  
X.N. Liu ◽  
Y. Zhu ◽  
J. Ma ◽  
N. Liu ◽  
...  
Keyword(s):  
Helicoverpa Armigera ◽  
Dna Sequences ◽  
Promoter Activity ◽  
Transcriptional Control ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Mobility Shift ◽  
Transient Transfection Assay ◽  
P450 Gene ◽  
Helicoverpa Armígera

AbstractEukaryote transcription is controlled by regulatory DNA sequences and transcription factors, so transcriptional control of gene plays a pivotal role in gene expression. In this study, we identified the region of the CYP6B6 gene promoter of Helicoverpa armigera which responds to the plant secondary toxicant 2-tridecanone. Transient transfection assay results from five of stepwise deletion fragments linked to the luciferase reporter gene revealed that the promoter activity of each CYP6B6 fragment was significantly higher than that of their basal activity after the Sf9 cells were treated with 2-tridecanone. Among all, the fragment spanning −373 to +405 bp of the CYP6B6 promoter showed an obviously 2-tridecanone inducibility (P<0.0001), which might have the 2-tridecanone responsive element based on promoter activity. Electrophoretic mobility shift assays revealed that the nuclear protein extracted from midgut of the 6th instar larva of H. armigera, reared on 10 mg 2-tridecanone per gram artificial diet for 48 h, could specifically bind to the active region from −373 to 21 bp of the CYP6B6 promoter. The combination feature also appeared when using a shorter fragment from −292 to −154 bp of the CYP6B6 promoter. Taken together, we found a 2-tridecanone core responsive region between −292 and −154 bp of the CYP6B6 promoter. This may lead us to a better understanding of transcriptional mechanism of P450 gene and provide very useful information for the pest control.

scholarly journals Transcription factor organic cation transporter 1 (OCT-1) affects the expression of porcine Klotho (KL) gene

PeerJ ◽  
2016 ◽  
Vol 4 ◽  
pp. e2186 ◽  
Author(s):  
Yan Li ◽  
Lei Wang ◽  
Jiawei Zhou ◽  
Fenge Li
Keyword(s):  
Transcription Factor ◽  
Organic Cation ◽  
Core Promoter ◽  
Sequence Similarity ◽  
Organic Cation Transporter ◽  
Mobility Shift ◽  
Intron 1 ◽  
Organic Cation Transporter 1 ◽  
Immune Precipitation ◽  
Electrophoretic Mobility Shift Assays

Klotho (KL), originally discovered as an aging suppressor, is a membrane protein that shares sequence similarity with theβ-glucosidase enzymes. Recent reports showed Klotho might play a role in adipocyte maturation and systemic glucose metabolism. However, little is known about the transcription factors involved in regulating the expression of porcineKLgene. Deletion fragment analysis identified KL-D2 (−418 bp to −3 bp) as the porcineKLcore promoter. MARC0022311SNP (A or G) in KL intron 1 was detected in Landrace × DIV pigs using the Porcine SNP60 BeadChip. The pGL-D2-A and pGL-D2-G were constructed with KL-D2 and the intron fragment of different alleles and relative luciferase activity of pGL3-D2-G was significantly higher than that of pGL3-D2-A in the PK cells and ST cells. This was possibly the result of a change inKLbinding ability with transcription factor organic cation transporter 1 (OCT-1), which was confirmed using electrophoretic mobility shift assays (EMSA) and chromatin immune-precipitation (ChIP). Moreover, OCT-1 regulated endogenousKLexpression by RNA interference experiments. Our study indicates SNP MARC0022311 affects porcineKLexpression by regulating its promoter activity via OCT-1.

scholarly journals Transcriptional Regulation of Porcine Endogenous Retroviruses Released from Porcine and Infected Human Cells by Heterotrimeric Protein Complex NF-Y and Impact of Immunosuppressive Drugs

2002 ◽  
Vol 76 (24) ◽  
pp. 12553-12563 ◽  
Author(s):  
Gregor Scheef ◽  
Nicole Fischer ◽  
Egbert Flory ◽  
Isabel Schmitt ◽  
Ralf R. Tönjes
Keyword(s):  
Transcription Factor ◽  
Promoter Activity ◽  
Specific Binding ◽  
Immunosuppressive Drugs ◽  
Human Cells ◽  
Endogenous Retroviruses ◽  
Promoter Strength ◽  
Mobility Shift ◽  
Mammalian Cell Lines ◽  
Porcine Endogenous Retrovirus

ABSTRACT Recent studies revealed a significant promoter activity of porcine endogenous retrovirus (PERV) long terminal repeats (LTRs) in different human and mammalian cell lines, which is mediated by a 39-bp repeat located in the U3 region in different numbers, representing an enhancer (G. Scheef, N. Fischer, U. Krach, and R. R. Tönjes, J. Virol. 75:6933-6940, 2001). A statistical transcription factor analysis revealed putative binding sites for the CCAAT-binding transcription factor NF-Y inside the 39-bp repeat. Specific binding of NF-Y to the repeat sequence was demonstrated by electrophoretic mobility shift assays and supershift assays with specific antibodies directed against the three subunits of NF-Y. To identify further transcription-regulating elements, genetically modified LTRs lacking the repeat box, U3, R, or U5 were investigated. The results indicated a strong inhibitory element in the R region, as the deletion of R caused a significantly increased promoter activity. Since PERV might play a potential role in the application of xenogeneic cell therapy and xenotransplantation techniques, we have investigated whether immunosuppressive drugs that are routinely used in transplantation medicine have an impact on the promoter activity. Neither cyclosporine nor prednisolone had any influence on the promoter strength of the PERV LTRs. By performing a real-time PCR we were able to compare the proviral loads of porcine and infected human cells as well as the amount of released virions, which revealed a direct link between LTR activity and the number of released retroviruses.

Inhibition of inducible TNF-α expression by oxaspirodion, a novel spiro-compound from the ascomycete Chaetomium subspirale

2004 ◽  
Vol 385 (9) ◽  
pp. 829-834 ◽  
Author(s):  
Jan Rether ◽  
Gerhard Erkel ◽  
Timm Anke ◽  
Olov Sterner
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
Transcription Factor ◽  
Mode Of Action ◽  
Promoter Activity ◽  
Spiro Compound ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Jurkat T Cells ◽  
Tnf Α

Abstract In a search for compounds inhibiting the inducible TNF-αa promoter activity in T cells, a new spiro-compound, designated oxaspirodion, was isolated from fermentations of the ascomycete Chaetomium subspirale. Oxaspirodion inhibited TNF-α promoter-driven luciferase reporter gene expression with an IC50 value of 2.5 µg/ml (10 µM) in TPA/ionomycin-stimulated Jurkat T cells. Studies on the mode of action of the compound revealed that the inhibition of the TNF-α promoter activity is caused by an inhibition of the phosphorylation of the ERK1/2 kinases. In addition, oxaspirodion inhibited the activation of the transcription factor NF-κB, which is involved in the inducible expression of many proinflammatory genes.

scholarly journals Transcription factor organic cation transporter 1 (OCT-1) affects the expression of porcine Klotho (KL) gene

2016 ◽  
Author(s):  
Yan Li ◽  
Lei Wang ◽  
Jiawei Zhou ◽  
Fenge Li
Keyword(s):  
Transcription Factor ◽  
Organic Cation ◽  
Core Promoter ◽  
Sequence Similarity ◽  
Organic Cation Transporter ◽  
Mobility Shift ◽  
Fragment Analysis ◽  
Intron 1 ◽  
Organic Cation Transporter 1 ◽  
Electrophoretic Mobility Shift Assays

Klotho (KL), originally discovered as an aging suppressor, was a membrane protein that shared sequence similarity with the β-glucosidase enzymes. Recent reports showed Klotho might have a role in adipocyte maturation and systemic glucose metabolism. However, little is known about the transcription factors involved in regulating the expression of porcine KL gene. Deletion fragment analysis identified KL-D2 (-418 bp to -3 bp) as the porcine KL core promoter. MARC0022311 in KL intron 1 appeared a polymorphism (A or G) in Landrace × DIV pigs, and relative luciferase activity of pGL3-D2-G was significantly higher than pGL3-D2-A. This was possibly the result of a change in KL binding ability with transcription factor organic cation transporter 1 (OCT-1), which was confirmed using electrophoretic mobility shift assays (EMSA) and chromatin immunoprecipitation (ChIP). Moreover, OCT-1 regulated endogenous KL expression by RNA interference. Our study indicates SNP MARC0022311 affects porcine KL expression by regulating its promoter activity via OCT-1.

Kv1.5 potassium channel gene regulation by Sp1 transcription factor and oxidative stress

2007 ◽  
Vol 293 (5) ◽  
pp. H2719-H2725 ◽  
Author(s):  
Samuel J. Fountain ◽  
Alex Cheong ◽  
Jing Li ◽  
Naciye Y. Dondas ◽  
Fanning Zeng ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Transcription Factor ◽  
Smooth Muscle ◽  
Potassium Channel ◽  
Promoter Activity ◽  
Dominant Negative ◽  
Luciferase Reporter ◽  
Therapeutic Drug ◽  
Luciferase Reporter Gene ◽  
Sp1 Transcription Factor

KV1.5, a voltage-gated potassium channel, has functional importance in regulating blood vessel tone and cardiac action potentials and is a target for numerous therapeutic drug development programs. Despite the importance of KV1.5, there is little knowledge of the mechanisms controlling expression of its underlying gene, Kcna5. We identified a 5′ flanking region of the murine Kcna5 gene that drives expression of a luciferase reporter gene in primary smooth muscle cells and a smooth muscle cell line. The promoter contained CACCC nucleotide motifs, which we have shown to bind the Sp1 transcription factor in the aorta under physiological conditions in vivo. Inhibition of Sp1- Kcna5 promoter interactions using mithramycin A, a dominant-negative Sp1 mutant, or disruption of the CACCC boxes by mutagenesis inhibited promoter activity. Conversely, expression of exogenous Sp1 augmented promoter activity. Sp1 has known sensitivity to oxidative stress and, consistent with this property, Kcna5 promoter activity was suppressed by hydrogen peroxide-induced oxidative stress. Our results show that Kcna5 promoter activity in vascular smooth muscle is critically dependent on Sp1 regulation via CACCC box motifs and identify mechanisms that potentially influence the expression of KV1.5 channel expression in physiological or pathological conditions.

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