scholarly journals Cloning and characterization of the 5′-flanking region of the oxalate decarboxylase gene from Flammulina velutipes

2002 ◽  
Vol 367 (1) ◽  
pp. 67-75 ◽  
Author(s):  
Mohammad AZAM ◽  
Meenu KESARWANI ◽  
Subhra CHAKRABORTY ◽  
Krishnamurthy NATARAJAN ◽  
Asis DATTA
Keyword(s):  
Transcription Factor ◽  
Oxalic Acid ◽  
Specific Binding ◽  
Low Ph ◽  
Flammulina Velutipes ◽  
Upstream Region ◽  
Oxalate Decarboxylase ◽  
Mobility Shift ◽  
Cell Extracts ◽  
Flanking Region

The oxalate-degrading enzyme, oxalate decarboxylase (OXDC), was purified and characterized from Flammulina velutipes, a basidiomycetous fungus [Mehta and Datta (1991) J. Biol. Chem. 266, 23548—23553]. The cDNA cloning and analyses revealed that OXDC transcription was induced by oxalic acid. However, in this report, we show that OXDC transcription is induced by low pH, not by oxalate. To understand the regulatory mechanism of OXDC expression, we have cloned and analysed a 580-bp genomic fragment from the 5′-flanking region of the OXDC gene. Sequence analysis showed the presence of several eukaryotic transcription factor binding motifs within the −580bp of the upstream region. Electrophoretic-mobility-shift assays with partially purified cell extracts revealed specific binding of a factor in acid-induced, but not in uninduced, extracts. Furthermore, DNase I protection assays using the partially purified fraction from oxalic acid-induced extract revealed a footprint of a 13-bp sequence 5′GCGGGGTCGCCGA3′, termed low pH responsive element (LPRE), corresponding to the −287 to −275bp region of the OXDC promoter. Our results suggest that in F. velutipes cells, activation of OXDC transcription in response to low pH is mediated by the binding of a novel transcription factor through the LPRE site in the OXDC promoter.

Glucocorticoids stimulate human sgk1 gene expression by activation of a GRE in its 5′-flanking region

2002 ◽  
Vol 283 (5) ◽  
pp. E971-E979 ◽  
Author(s):  
Omar A. Itani ◽  
Kang Z. Liu ◽  
Kristyn L. Cornish ◽  
Jason R. Campbell ◽  
Christie P. Thomas
Keyword(s):  
Transcription Initiation ◽  
Collecting Duct ◽  
A549 Cells ◽  
Initiation Site ◽  
Specific Protein ◽  
Upstream Region ◽  
Mobility Shift ◽  
Cell Extracts ◽  
Flanking Region ◽  
Gel Mobility Shift

In lung and collecting duct epithelia, glucocorticoid (GC)-stimulated Na+ transport is preceded by an increase in the protein kinase sgk1, which in turn regulates the activity of the epithelial Na+ channel (ENaC). We investigated the mechanism for GC-regulated human sgk1 expression in lung and renal epithelia. sgk1 mRNA was increased in these epithelia by GCs, and this was inhibited by actinomycin D and superinduced by cycloheximide, consistent with a transcriptional effect that did not require protein synthesis. To understand the basis for transcriptional regulation, the transcription initiation site was mapped and the 5′-flanking region cloned by PCR. A 3-kb fragment of the upstream region was coupled to luciferase and transfected into A549 cells. By deletion analysis, an imperfect GC response element (GRE) was identified that was necessary and sufficient for GC responsiveness. When tested with cell extracts, a specific protein recognized by an anti-GC receptor (GR) antibody bound the GRE in gel mobility shift assays. We conclude that GCs stimulate sgk1 expression in human epithelial cells via activation of a GRE in the 5′-flanking region of sgk1.

Abstract 1384: Osteogenic Transcription Factor Runx2 Regulates Expression of RANKL during VSMC Calcification

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Chang Hyun Byon ◽  
Jay McDonald ◽  
Yabing Chen
Keyword(s):  
Oxidative Stress ◽  
Transcription Factor ◽  
Molecular Mechanism ◽  
Luciferase Reporter ◽  
Rankl Expression ◽  
Reporter System ◽  
Mobility Shift ◽  
Atherosclerotic Lesions ◽  
Flanking Region

The expression of receptor activator of nuclear factor κ B (RANKL) is up-regulated in calcified atherosclerotic lesions, whereas it is frequently undetectable in normal vessels. The underlying molecular mechanism of increased expression of RANKL in calcified vessels is not known. We have previously demonstrated that oxidative stress induces calcification of vascular smooth muscle cells (VSMC) in vitro . Therefore, we determined whether oxidative stress regulates RANKL expression in VSMC and the underlying molecular mechanism. Consistent with previous observations in vivo , we found that the expression of RANKL in VSMC isolated from mouse. However, hydrogen peroxide (H 2 O 2 ), which induces VSMC calcification, induced a 33-fold increase in the transcripts of RANKL as determined by real-time PCR. Increased expression of RANKL protein was further confirmed by ELISA. Using flow cytometry, we demonstrated that membrane-bound RANKL was increased by oxidative stress. To characterize the molecular mechanism underlying H 2 O 2 -induced RANKL expression, we employed the luciferase reporter system with a series of deletion mutants of the RANKL 5′-flanking region. The H 2 O 2 responsive region is located between −200 to −400 in the 5′-flanking region of RANKL gene. Analyses of the sequence of this region identified multiple binding sites for the key osteogenic transcription factor, Runx2, which we previously reported to be an essential regulator of VSMC calcification. Electrophoretic mobility shift analyses demonstrated increased binding of Runx2 on the RANKL promoter sequence in nuclear extracts from VSMC exposed to H 2 O 2 . To further determine the role of Runx2 in regulating RANKL expression, we generated stable Runx2 knockdown VSMC with the use of lentivirus-carrying shRNA for Runx2 gene. H 2 O 2 -induced RANKL expression was abrogated in VSMC with Runx2 knockdown. In addition, adenovirus-mediated overexpression of Runx2 in VSMC induced the expression of RANKL. In summary, we have demonstrated that H 2 O 2 induces the expression of RANKL in VSMC, which is regulated by the osteogenic transcription factor Runx2. These observations provide novel molecular insights into the regulation of RANKL and its role on the pathogenesis of calcified atherosclerotic lesions.

scholarly journals Identification of a novel factor that interacts with an immunoglobulin heavy-chain promoter and stimulates transcription in conjunction with the lymphoid cell-specific factor OTF2.

1990 ◽  
Vol 10 (5) ◽  
pp. 2145-2153 ◽  
Author(s):  
B K Yoza ◽  
R G Roeder
Keyword(s):  
B Cell ◽  
Heavy Chain ◽  
Regulatory Element ◽  
Immunoglobulin Heavy Chain ◽  
Upstream Region ◽  
Chain Gene ◽  
Specific Factor ◽  
Mobility Shift ◽  
Specific Expression ◽  
Cell Extracts

The tissue-specific expression of the MOPC 141 immunoglobulin heavy-chain gene was studied by using in vitro transcription. B-cell-specific transcription of this gene was dependent on the octamer element 5'-ATGCAAAG-3', located in the upstream region of this promoter and in the promoters of all other immunoglobulin heavy- and light-chain genes. The interaction of purified octamer transcription factors 1 and 2 (OTF1 and OTF2) with the MOPC 141 promoter was studied by using electrophoretic mobility shift assays and DNase I footprinting. Purified OTF1 from HeLa cells and OTF1 and OTF2 from B cells bound to identical sequences within the heavy-chain promoter. The OTF interactions we observed extended over the heptamer element 5'-CTCAGGA-3', and it seems likely that the binding of the purified factors involves cooperation between octamer and heptamer sites in this promoter. In addition to these elements, we identified a second regulatory element, the N element with the sequence 5'-GGAACCTCCCCC-3'. The N element could independently mediate low levels of transcription in both B-cell and HeLa-cell extracts, and, in conjunction with the octamer element, it can promote high levels of transcription in B-cell extracts. The N element bound a transcription factor, NTF, that is ubiquitous in cell-type distribution, and NTF was distinct from any of the previously described proteins that bind to similar sequences. Based on these results, we propose that NTF and OTF2 interactions (both with their cognate DNA elements and possibly at the protein-protein level) may be critical to B-cell-specific expression and that these interactions provide additional pathways for regulating gene expression.

scholarly journals Activation of a Delayed-Early Gene Encoding MHR3 by the Ecdysone Receptor Heterodimer EcR-B1–USP-1 but Not by EcR-B1–USP-2

1999 ◽  
Vol 19 (7) ◽  
pp. 4897-4906 ◽  
Author(s):  
Que Lan ◽  
Kiyoshi Hiruma ◽  
Xiao Hu ◽  
Marek Jindra ◽  
Lynn M. Riddiford
Keyword(s):  
Cell Extract ◽  
Nuclear Hormone Receptor ◽  
Orphan Receptor ◽  
Early Gene ◽  
Upstream Region ◽  
Ecdysone Receptor ◽  
Mobility Shift ◽  
Receptor Binding Site ◽  
Cell Extracts

ABSTRACT MHR3, a homolog of the retinoid orphan receptor (ROR), is a transcription factor in the nuclear hormone receptor family that is induced by 20-hydroxyecdysone (20E) in the epidermis of the tobacco hornworm, Manduca sexta. Its 2.7-kb 5′ flanking region was found to contain four putative ecdysone receptor response elements (EcREs) and a monomeric (GGGTCA) nuclear receptor binding site. Activation of this promoter fused to a chloramphenicol acetyltransferase (CAT) reporter by 2 μg of 20E per ml inManduca GV1 cells was similar to that of endogenous MHR3, with detectable CAT by 3 h. When the ecdysone receptor B1 (EcR-B1) and Ultraspiracle 1 (USP-1) were expressed at high levels under the control of a constitutive promoter, CAT levels after a 3-h exposure to 20E increased two- to sixfold. In contrast, high expression of EcR-B1 and USP-2 caused little increase in CAT levels in response to 20E. Moreover, expression of USP-2 prevented activation by EcR-B1–USP-1. Deletion experiments showed that the upstream region, including the three most proximal putative EcREs, was responsible for most of the 20E activation, with the EcRE3 at −671 and the adjacent GGGTCA being most critical. The EcRE1 at −342 was necessary but not sufficient for the activational response but was the only one of the three putative EcREs to bind the EcR-B1–USP-1 complex in gel mobility shift assays and was responsible for the silencing action of EcR-B1–USP-1 in the absence of hormone. EcRE2 and EcRE3 each specifically bound other protein(s) in the cell extract, but not EcR and USP, and so are not EcREs in this cellular context. When cell extracts were used, the EcR-B1–USP-2 heterodimer showed no binding to EcRE1, and the presence of excess USP-2 prevented the binding of EcR-B1–USP-1 to this element. In contrast, in vitro-transcribed-translated USP-1 and USP-2 both formed heterodimeric complexes with EcR-B1 that bound ponasterone A with the sameKd (7 × 10−10 M) and bound to both EcRE1 and heat shock protein 27 EcRE. Thus, factors present in the cell extract appear to modulate the differential actions of the two USP isoforms.

Platelet/Megakaryocyte PKC-Θ Is a Transcriptional Target of RUNX1/ CBFA2: Studies in Human RUNX1 Haplodeficiency.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1846-1846
Author(s):  
Gauthami S Jalagadugula ◽  
Gurpreet Kaur ◽  
Guangfen Mao ◽  
Danny Dhanasekaran ◽  
A. Koneti Rao
Keyword(s):  
Transcription Factor ◽  
Protein Binding ◽  
Platelet Function ◽  
Luciferase Reporter ◽  
Upstream Region ◽  
Mrna Levels ◽  
Consensus Binding Site ◽  
Mobility Shift ◽  
Hel Cells ◽  
Transcriptional Target

Abstract Protein kinase C Θ (PKC-Θ) is an important signaling molecule and regulates platelet responses to activation including aggregation and secretion. In a patient with lifelong thrombocytopenia, impaired platelet aggregation and secretion, we have shown (Gabbeta et al 1996, Blood 87:1368–1376) that phosphorylation of pleckstrin (a PKC substrate) and myosin light chain (MLC) is impaired along with diminished GPIIb-IIIa activation. Platelet protein and mRNA levels of PKC-Θ were decreased with normal levels of other PKC isozymes. These findings were associated with a heterozygous nonsense mutation in transcription factor RUNX1 (also known as CBFA2 or AML1) (Sun et al 2004, Blood 103:948–54). RUNX1 is transcription factor that plays a major role in megakaryopoiesis, megakaryocytic maturation, and platelet production. Haplodeficiency of RUNX1 has been associated with familial thrombocytopenia, impaired megakaryopoiesis, impaired platelet function and predisposition to acute myeloid leukemia. Because of the important role of PKC-Θ in platelet activation and of RUNX1 in hematopoiesis, we addressed the hypothesis that PKC-Θ is a direct transcriptional target of RUNX1. Studies were performed using human erythroleukemia (HEL) cells treated with phorbol 12-myristate 13-acetate (PMA) for 24 h to induce megakaryocytic transformation. Chromatin immunoprecipitation (ChIP) assay using anti-RUNX1 antibody revealed RUNX1 binding to chromatin in the PKC-Θ 5’ upstream region −1225/−1056 bp from ATG codon. This region includes a RUNX1 consensus binding site ACCGCA at −1081/−1076 bp identified by TFSEARCH. We performed electrophoretic mobility shift assay (EMSA) using 20-mer probe −1088/−1069 containing the RUNX1 site and nuclear extracts from PMA-treated HEL cells. Protein binding to the probe was observed, which was competed by excess unlabelled probe, and anti-RUNX1 antibody inhibited this binding, indicating that RUNX1 was involved in the DNA binding. Moreover, protein binding to the wild type probe was not competed by an oligo with 4 nucleotides deleted from the RUNX1 consensus site. To determine the functional relevance of RUNX1 binding to PKC-Θ, transient transfections were performed in HEL cells with luciferase reporter constructs. The full length construct −1085/−206 showed ~14-fold activity compared to empty vector. A mutant construct with deletion of the RUNX1 site resulted in a ~50% decrease in activity indicating that the site was functional. siRNA-mediated knockdown of RUNX1 in HEL cells was associated with a decrease in both RUNX1 and PKC-Θ protein. Conclusion: These results and our findings in the patient provide the first evidence that PKC-Θ gene transcription in the megakaryocyte/platelet is regulated by RUNX1. They provide a cogent mechanism for the platelet PKC-Θ downregulation associated with RUNX1 haplodeficiency in our patient. RUNX1 dysregulation of PKC-Θ in megakaryocytic cells is an important aspect of the abnormal platelet function and production associated with human RUNX1 mutations.

scholarly journals The helix-loop-helix transcription factor USF (upstream stimulating factor) binds to a regulatory sequence of the human insulin gene enhancer

1993 ◽  
Vol 295 (1) ◽  
pp. 233-237 ◽  
Author(s):  
M L Read ◽  
A R Clark ◽  
K Docherty
Keyword(s):  
Transcription Factor ◽  
Human Insulin ◽  
Insulin Gene ◽  
Regulatory Sequence ◽  
Mobility Shift ◽  
Helix Loop Helix ◽  
Cell Extracts ◽  
Human Insulin Gene ◽  
Gene Enhancer ◽  
Stimulating Factor

Two important sequence elements, designated insulin enhancer binding site 1 (IEB1) or NIR and IEB2 or FAR, are involved in regulating expression of the rat insulin I gene. These elements bind a helix-loop-helix transcription factor, insulin enhancer factor 1 (IEF1). The IEB1 site is highly conserved among insulin genes but the IEB2 site is not conserved. To investigate the factors binding at the equivalent IEB1 and IEB2 sites in the human insulin gene enhancer, electrophoretic mobility shift assays were performed using a variety of cell extracts and probes specific for the homologous IEB1 and IEB2 sites. The results indicate that a factor with similar tissue distribution and binding characteristics to those of IEF1 binds to the IEB1 site in the human insulin gene, but that a separate factor, identified as the adenovirus major late transcription factor [MLTF, or upstream stimulating factor (USF)] binds to the IEB2 site.

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.

Isolation of Lhcb3 sequences from Brassica napus: evidence for conserved genes encoding LHCII type III chlorophyll a/b binding proteins

Genome ◽  
1993 ◽  
Vol 36 (1) ◽  
pp. 139-146 ◽  
Author(s):  
Rodolphe Boivin ◽  
Diane Beauseigle ◽  
Chris L. Baszczynski ◽  
Guy Bellemare
Keyword(s):  
Brassica Napus ◽  
Chlorophyll A ◽  
Binding Proteins ◽  
Specific Binding ◽  
Evolutionary Relationship ◽  
Upstream Region ◽  
Type I ◽  
Mobility Shift ◽  
Type Iii ◽  
Genes Encoding

Three closely related sequences were isolated from Brassica napus genomic DNA and were identified as Lhcb3 (genes encoding type III chlorophyll a/b binding proteins of LHCII, the major light-harvesting complex of photosystem II). These genes, as was observed for a tomato Lhcb3, contain two introns and yield both divergent and conserved predicted amino acid segments as compared with type I and type II polypeptides. One of the B. napus genes, designated Lhcb3*1, is transcribed in vivo, since it is identical to corresponding sequences in a cDNA clone. The protein deduced from another sequence, Lhcb3*2, appears as the most divergent type III so far characterized. The partial sequence of a third gene, Lhcb3*3, was also recovered. The 5′ noncoding sequences of Lhcb3*1 and Lhcb3*2, in the far upstream region, are characterized by an extremely high AT content and extensive direct repeats. In the near upstream region, two long Lhcb3*2 segments are very similar to a segment proposed as containing regulatory signals in Lhcb3*1. Specific binding of nuclear proteins to Lhcb3*1 promoter fragments was detected by electrophoretic mobility-shift assays. The evolutionary relationship between genes for type III polypeptides and the other types present in LHCII is discussed.Key words: Brassica napus, chlorophyll a/b binding proteins, LHCII type III, promoter region, lhcb genes evolution.

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.

Identification of a novel factor that interacts with an immunoglobulin heavy-chain promoter and stimulates transcription in conjunction with the lymphoid cell-specific factor OTF2

1990 ◽  
Vol 10 (5) ◽  
pp. 2145-2153
Author(s):  
B K Yoza ◽  
R G Roeder
Keyword(s):  
B Cell ◽  
Heavy Chain ◽  
Regulatory Element ◽  
Immunoglobulin Heavy Chain ◽  
Upstream Region ◽  
Chain Gene ◽  
Specific Factor ◽  
Mobility Shift ◽  
Specific Expression ◽  
Cell Extracts

The tissue-specific expression of the MOPC 141 immunoglobulin heavy-chain gene was studied by using in vitro transcription. B-cell-specific transcription of this gene was dependent on the octamer element 5'-ATGCAAAG-3', located in the upstream region of this promoter and in the promoters of all other immunoglobulin heavy- and light-chain genes. The interaction of purified octamer transcription factors 1 and 2 (OTF1 and OTF2) with the MOPC 141 promoter was studied by using electrophoretic mobility shift assays and DNase I footprinting. Purified OTF1 from HeLa cells and OTF1 and OTF2 from B cells bound to identical sequences within the heavy-chain promoter. The OTF interactions we observed extended over the heptamer element 5'-CTCAGGA-3', and it seems likely that the binding of the purified factors involves cooperation between octamer and heptamer sites in this promoter. In addition to these elements, we identified a second regulatory element, the N element with the sequence 5'-GGAACCTCCCCC-3'. The N element could independently mediate low levels of transcription in both B-cell and HeLa-cell extracts, and, in conjunction with the octamer element, it can promote high levels of transcription in B-cell extracts. The N element bound a transcription factor, NTF, that is ubiquitous in cell-type distribution, and NTF was distinct from any of the previously described proteins that bind to similar sequences. Based on these results, we propose that NTF and OTF2 interactions (both with their cognate DNA elements and possibly at the protein-protein level) may be critical to B-cell-specific expression and that these interactions provide additional pathways for regulating gene expression.

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