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PubMed2001

Transcription factor NF-kappaB as a potential biomarker for oxidative stress.

There is increasing interest in the involvement of transcription factors, such as of the transcription factor NF-kappaB (nuclear factor-kappaB), in the pathogenesis of various diseases. NF-kappaB is involved in the control of the transcription of a variety of cellular genes that regulate the inflammatory response by the production of cytokines, chemokines, cell adhesion molecules and acute phase proteins. The involvement of NF-kappaB is especially of interest as it is activated by oxidative stress and its activation can be modulated by antioxidant compounds. The activation of NF-kappaB can be determined by the electromobility shift assay (EMSA) with a NF-kappaB binding-site-specific probe. EMSA can also be used on human mononuclear cells isolated from peripheral blood, which could make the assay applicable for clinical trials. The critical steps of the EMSA are discussed, addressing some pitfalls of the assay. The procedure that can be used to express NF-kappaB activity in human subjects is evaluated. This offers the possibility to use NF-kappaB as a functional biomarker of oxidative stress as illustrated by several examples of in vitro and in vivo studies.

Biomarkers↗

PubMed1999

Transcriptional regulation of the CDK inhibitor p16INK4a gene by a novel pRb-associated repressor, RBAR1.

p16, also known as INK4a, CDKN2, or MTS1, plays an important role in the control of the cell cycle progression, and retinoblastoma protein (pRb) is suggested to be involved in the transcriptional regulation of p16. However, it is not fully understood how pRb regulates transcription of the p16 gene. Nuclear proteins prepared only from the pRb-nonfunctional human tumor cells were found to bind to the 5'-flanking sequence of the p16 gene in the presence of Zn2+ by electrophoretic mobility shift assay (EMSA). EMSA using mutagenized 5'-flanking sequences as competitors suggested that the sequence at position -97 to -87 relative to first ATG of the p16 gene was critical for protein binding. Transient reporter assay indicated that the sequence identified by EMSA acted as a silencer element in the pRb-nonfunctional tumor cells, showing the presence of a transcriptional repressor associated with functional pRb (RBAR1).

Cyclin-Dependent Kinase Inhibitor p16↗

PubMed2002

Effects of vomitoxin (deoxynivalenol) on the binding of transcription factors AP-1, NF-kappaB, and NF-IL6 in raw 264.7 macrophage cells.

The effects of vomitoxin (VT) on the binding activity of three transcription factors critical to pro-inflammatory cytokine regulation were assessed in the RAW 264.7 murine macrophage model by electrophoretic mobility shift assay (EMSA). When cells were treated with 100 to 250 ng/ml of VT, activator protein-1 (AP-1 binding) was increased after 2 and 8 h. This effect was potentiated when cells were coincubated with lipopolysaccharide (LPS) (synchronous model) but not when preincubated with LPS (delayed synchronous model). Supershift EMSA revealed that VT preferentially induced JunB, JunD, phosphorylated c-Jun, c-Fos, and Fra-2 binding activities of the AP-1 family. Nuclear factor kappaB (NF-kappaB) binding was increased at 2 and 8 h in cells subjected to synchronous and delayed synchronous VT exposure in the presence of LPS. Supershift EMSA indicated that the p-50 and c-Rel subunits of NF-kappaB/ Rel were specifically affected. Nuclear factor-IL6 (NF-IL6) binding was increased at 2 and 8 h with or without LPS in synchronous and delayed synchronous VT-exposure models. Here, the C/EBPbeta subunit was primarily involved in enhanced NF-IL6 binding. The capacity of VT to elevate binding of AP-1, NF-kappaB, and NF-IL6 may contribute to the VT-mediated cytokine up-regulation in vitro and in vivo. The observations that VT was active in synchronous and delayed synchronous models suggest that macrophages activated simultaneously or prior to toxin exposure were vulnerable to the effects of this trichothecene.

Animals↗

PubMed2005

Quantitative chemiluminescent immunoassay for NF-kappaB-DNA binding activity.

Nuclear factor-kappaB (NF-kappaB) is a ubiquitous redox-sensitive transcription factor involved in the pro-inflammatory response to several factors, including cytokines and oxidative stress. Upon activation, NF-kappaB translocates into the nucleus and binds to specific nucleotide sequences. The cellular responses to inflammatory and stress signals have been implicated in disease conditions, such as atherosclerosis, cancer, diabetes, and Alzheimer's disease. The conventional method for detection of NF-kappaB -DNA binding activity is the electrophoretic mobility shift assay (EMSA), which is time-consuming and non-quantitative. Here, we report (a) development of a rapid, sensitive and quantitative chemiluminescent immunoassay (QCI) for analysis of NF-kappaB DNA-binding activity, and (b) validation of the QCI with the EMSA using nuclear and cytosolic extracts from cultured prostate cancer cells (PC3), rat liver homogenates and human lymphocytes. The QCI for analysis of NF-kappaB DNA binding activity has advantages over the EMSA: (1) Higher speed: 3-5h post sample preparation, (2) Greater sensitivity: 10pg NF-kappaB/well, (3) Quantitative: linear range: 10-1000pg NF-kappaB; r2 = 0.999 (4) High throughput adaptability: 96-well plate format can analyze up to 40 samples in duplicate, (5) SAFETY: No radioactive isotopes, (6) Simplicity, and (7) Capability of measurement of both activated (free) NF-KB which is translocated into the nucleus and total (bound + unbound) NF-kappaB present in the cytosol/cell.

Animals↗

PubMed2003

Localization of IFN-gamma-activated Stat1 and IFN regulatory factors 1 and 2 in breast cancer cells.

The aim of the present work was to evaluate the induction and localization of Stat1, interferon (IFN) regulatory factor-1 (IRF-1), and IRF-2 after IFN-gamma exposure of human breast cancer cell lines, SKBR3, MDA468, MCF7, and BT20. Results from growth assays, Western staining, electrophoretic mobility shift assay (EMSA), and immunohistochemical staining were collated to test our hypothesis that immunohistochemical analysis of Stat1, IRF-1, and IRF-2 would provide additional information about the functionality of the IFN-gamma signaling pathway in human tumor lines. EMSA results showed that in each of four cell lines, Stat1 expression was increased and demonstrated functional activity after IFN-gamma stimulation. Western and EMSA analysis showed upregulation of IRF-1 but not IRF-2 in each cell line. Confocal microscopy of cells stained for Stat1, IRF-1, and IRF-2 confirmed the results and also provided novel information about the intracellular localization of proteins and intercellular variations in responses. The proportion of cells with IRF-1 stimulation and translocation was positively correlated with the IFN-gamma growth suppression in vitro. In conclusion, using four independent assays, we have demonstrated that heterogeneity in IFN-gamma-mediated upregulation of signal transduction proteins can be detected in vitro and that these differences can explain distinct cellular growth effects.

Antibodies, Monoclonal↗

PubMed1997

Regulation of the hepatic CYP 2E1 gene during chronic alcohol exposure: lack of an ethanol response element in the proximal 5'-flanking sequence.

Chronic exposure to ethanol is known to cause a dramatic increase in the level of CYP 2E1 apoprotein. More recently it has been demonstrated that under certain conditions the mRNA encoding cytochrome P450 2E1(CYP 2E1) is inducible; however, the mechanisms by which these increases occur are not well understood. In the current study, DNase I footprinting assays performed on the first kilobase of the CYP 2E1 5'-flanking sequences resulted in the identification of 13 sequence-specific protected regions using rat liver nuclear extracts isolated from either control or ethanol-treated animals. No differences were observed in the DNase I footprint patterns produced by the two different nuclear extracts. In addition, analysis by electrophoretic mobility shift assays (EMSA) revealed that with one exception, there were no differences in the level of binding complexes between the two extracts. However, EMSA analysis with an oligonucleotide to one footprint site (designated Site C) revealed that in nuclear extracts isolated from ethanol-treated animals there was a 2.9-fold increase in this binding complex when compared to control nuclear extracts. This site was previously shown to contain an HNF-1alpha binding site, and here we demonstrate that bacterially expressed HNF-1alpha in footprint assays bind Site C sequences and that HNF-1alpha transactivates the CYP 2E1 promoter in co-transfection experiments with HNF-1alpha expression plasmid and plasmids containing CYP 2E1 promoter sequences coupled to the chloramphenicol acetyl transferase gene. Furthermore, in contrast to the increase observed by EMSA in Site C binding, no increase was detected in the CYP 2E1 transcriptional rate supported by nuclear extracts from ethanol-treated animals over controls using in vitro transcription assays, suggesting that the increase by ethanol in CYP 2E1 transcription is not mediated through the HNF-1alpha site.

Animals↗

PubMed1994

Cis-acting DNA elements and cell type-specific nuclear proteins which may play a role in regulation of mouse CD8 alpha (Lyt-2) gene transcription.

Fusion of mouse CD8+ class I MHC-restricted T cells with the BW5147 thymoma invariably yields CD8- hybridomas in which RNA transcribed from the CD8 alpha (Lyt-2) gene is undetectable. To determine whether cis-acting DNA sequences may negatively regulate transcription of the Lyt-2 gene in BW5147 cells, one possible explanation for the above observation, BW5147 cells were stably transfected with the Lyt-2 gene containing 1 - 11,000 nucleotides of 5' flanking DNA and surface expression of Lyt-2 was monitored by flow microfluorometry. Initial results suggested the presence of a negative element between 1400 and 5000 nucleotides upstream of the site of transcription initiation. Further studies suggested the presence of two potential negative regulatory elements in this region, one of which includes a 269 nucleotide Accl - SstI fragment comprised of nucleotides -4700 to -4431 which bound nuclear proteins from CD8+ and CD8- cell lines in electrophoretic mobility shift assays (EMSA). EMSA studies performed using nuclear extracts from a variety of cell lines and tissues demonstrated that unique retarded complexes, called bands 1 and 2, correlated significantly with expression or non-expression of Lyt-2 respectively. EMSA analysis of proteins fractionated by SDS-PAGE from nuclear extract of the CD8+ VL3 T lymphoma cell line revealed proteins of approximately 110-130 kDa (called L2a-P1) and > 200 kDa (called L2a-P2) which bind within a 100 nucleotide region of this fragment (called L2a) to yield band 1 and 2 respectively, and which may play a role in regulation of Lyt-2 gene transcription.

Animals↗

PubMed1997

Four P-like elements are required for optimal transcription of the mouse IL-4 gene: involvement of a distinct set of nuclear factor of activated T cells and activator protein-1 family proteins.

We previously identified the P sequence as a critical regulatory element of the human IL-4 promoter. In the mouse IL-4 promoter, there are five elements homologous to the human P sequence designated conserved lymphokine element 0 (CLE0), P, P2, P3 and P4. To characterize the role of these P-like elements and their binding factors in the native promoter, we did transient transfection and electrophoretic mobility shift assays (EMSA). Transfection of EL-4 cells with the IL-4 promoter-reporter constructs carrying mutated P-like elements showed that four P-like elements, CLE0, P, P2 and P4, but not P3, were required for optimal activation of the IL-4 promoter. EMSA showed that both constitutive and inducible complexes bound to CLE0, P, P2 and P4, whereas only a constitutive complex bound to P3. In competition and antibody supershift assays in EMSA, complexes formed with P or P2 proved to contain nuclear factor of activated T cells (NFAT) family proteins as major components. Activator protein (AP)-1 family proteins interacted with CLE0, P, P2 and P4. NFAT/AP-1 complex formed only with P and P2. Cross-competition assays among the P-like elements revealed element-specific and common complexes. Six tandem repeats of the P element linked to the SV40 promoter responded to phorbol 12-myristate 13-acetate, while that of other elements did not. It would thus appear that components of each P-like element-binding complexes are not identical and may coordinately contribute to transcriptional activity.

Animals↗

PubMed2002

Magnesium is required for specific DNA binding of the CREB B-ZIP domain.

We have examined binding of the CREB B-ZIP protein domain to double-stranded DNA containing a consensus CRE sequence (5'-TGACGTCA-3'), the related PAR, C/EBP and AP-1 sequences and the unrelated SP1 sequence. DNA binding was assayed in the presence or absence of MgCl2 and/or KCl using two methods: circular dichroism (CD) spectroscopy and electrophoretic mobility shift assay (EMSA). The CD assay allows us to measure equilibrium binding in solution. Thermal denaturation in 150 mM KCl indicates that the CREB B-ZIP domain binds all the DNA sequences, with highest affinity for the CRE site, followed by the PAR (5'-TAACGTTA-3'), C/EBP (5'-TTGCGCAA-3') and AP-1 (5'-TGAGTCA-3') sites. The addition of 10 mM MgCl2 diminished DNA binding to the CRE and PAR DNA sequences and abolished binding to the C/EBP and AP-1 DNA sequences, resulting in more sequence-specific DNA binding. Using 'standard' EMSA conditions (0.25x TBE), CREB bound all the DNA sequences examined. The CREB-CRE complex had an apparent Kd of approximately 300 pM, PAR of approximately 1 nM, C/EBP and AP-1 of approximately 3 nM and SP1 of approximately 30 nM. The addition of 10 mM MgCl2 to the polyacrylamide gel dramatically altered sequence-specific DNA binding. CREB binding affinity for CRE DNA decreased 3-fold, but binding to the other DNA sequences decreased >1000-fold. In the EMSA, addition of 150 mM KCl to the gels had an effect similar to MgCl2. The magnesium concentration needed to prevent non-specific electrostatic interactions between CREB and DNA in solution is in the physiological range and thus changes in magnesium concentration may be a cellular signal that regulates gene expression.

Animals↗

PubMed1999

Evaluation of methylated DNA binding protein-1 in mouse liver.

DNA methylation (DNA-5-methylcytosine [MeC]) plays a key role in regulation of gene expression. Highly methylated genes tend to be silenced whereas hypomethylated genes have an increased potential for expression. One way in which methylation may lead to inhibition of transcription is by permitting the binding of methylated DNA-binding proteins, which then block access of transcription factors to DNA. A particular methylated DNA-binding protein, MDBP-1, has been identified in nuclear extracts prepared from various human and rat tissues, and binds in a sequence-specific manner to DNA containing methylated cytosines (P. C. Supakar et al., 1988, Nucleic Acids Res. 16, 8029-8044). In the current study, an electrophoretic mobility shift assay (EMSA) was employed to assess the presence of MDBP-1 in protein isolated from mouse liver and to examine characteristics of its binding to DNA. The ligand used in our EMSA was a 32P-end-labeled, double-stranded, symmetrically methylated oligonucleotide containing the protein's binding site, 5'-CTAGATMGT-CAMGGMGAT-3' (M denotes 5-methyl-cytosine). Utilizing protein extracted from B6C3F1 mouse liver, we observed a complex that is competed by non-labeled, double-stranded, fully methylated ligand but not by an excess of a 26-mer, double-stranded oligonucleotide containing the binding site for AP-1. No complex was formed when a nonmethylated, double-stranded ligand, or our single-stranded ligands (methylated or unmethylated) were used as EMSA ligands. Complex formation was observed utilizing double-stranded, hemimethylated ligands; however, the affinity of MDBP-1 for these was one-tenth the affinity of MDBP-1 for fully methylated, double-stranded ligand. Additionally, protein isolated from C57BL/6 and C3H/He mouse liver was found to bind specifically to fully methylated ligand and hemimethylated ligands for MDBP-1, with similar characteristics as the binding of B6C3F1 mouse liver protein. These results indicate that MDBP-1 is present in mouse liver, and that the degree of methylation determines the strength of binding of MDBP-1 to DNA.

Animals↗

PubMed2004

Polymorphic NF-Y dependent regulation of human nicotine C-oxidase (CYP2A6).

OBJECTIVES: In humans, cytochrome P450 2A6 (CYP2A6) constitutes the principal nicotine C-oxidase. Several different polymorphic CYP2A6 gene variants are known which contribute to the highly variable expression of this enzyme among individuals. In this study we report a novel polymorphism located in the 5' flanking region (-745A > G) of the CYP2A6 gene disrupting a CCAAT box. METHODS AND RESULTS: Electrophoretic mobility shift assays (EMSA) indicated that NF-YA is part of this nuclear protein complex. Chromatin immunoprecipitation revealed that NF-Y recognizes a region of the CYP2A6 5' flanking region located between -932 and -606. EMSA showed that out of the three CCAAT boxes in the CYP2A6 promoter, with CCAAT core sequences located between -839/-835, -748/-744, and -689/-685, only the one at -748/-744 was able to compete with the nuclear protein complex binding to the -748/-744 CCAAT box. Cotransfection experiments indicated that NF-Y acts as a positive regulatory element on CYP2A6 gene regulation. EMSA demonstrated that an NF-Y consensus oligonucleotide but not the -745A > G oligonucleotide competed efficiently with binding of the protein complex to the -748/-744 CCAAT box. Promoter activity of the -745A > G variant was significantly reduced to 78% relative to the wild-type allele in HepG2 cells transfected with luciferase reporter plasmids. Finally, haplotype analysis was carried out comprising the -745A > G variant in combination with all known CYP2A6 3' and 5' flanking single nucleotide polymorphisms: -1013A > G, -48T > G, and the CYP2A6/CYP2A7 3' flank conversion. CONCLUSION: A new haplotype, CYP2A6*1H was identified, with allele frequencies of 3.1% in Swedish and 5.2% in Turkish populations.

5' Flanking Region↗

PubMed2026

Azotobacter vinelandii AmrZ is a global regulator linking alginate production and c-di-GMP homeostasis.

Azotobacter vinelandii, a member of the Pseudomonadaceae, produces the exopolysaccharide alginate during vegetative growth; however, the circuitry linking alginate biosynthesis to lifestyle transitions remains poorly defined. Here, we show that the Ribbon-Helix-Helix transcription factor AmrZ coordinates alginate production, intracellular c-di-GMP levels and motility. Deletion of amrZ abolished alginate synthesis, whereas chromosomal complementation restored it. A PalgD-gusA fusion and RT-qPCR demonstrated that algD, the first gene in the alginate biosynthetic cluster, depends on AmrZ for expression. Motif analysis identified multiple AmrZ sites upstream of algD, and electrophoretic mobility-shift assays (EMSAs) confirmed specific binding to these regions. AmrZ also positively autoregulates: PamrZ-gusA activity decreased in ΔamrZ, and purified AmrZ bound the amrZ promoter in EMSA. Moreover, PamrZ activity required the sigma factor AlgU, consistent with the presence of an AlgU promoter; this positive, AlgU-dependent feedback may stabilize AmrZ under alginate-inducing conditions. To probe AmrZ control of c-di-GMP, we implemented a riboswitch-based biosensor in A. vinelandii. The ΔamrZ strain showed a markedly reduced signal, similar to a diguanylate cyclase (DGC) mutant, whereas a phosphodiesterase mutant displayed elevated output, validating the assay. RNA-seq and RT-qPCR identified two DGC genes, AVAEIV_RS11610 and AVAEIV_RS18795, as AmrZ-activated targets; EMSA verified direct binding at the RS11610 regulatory region. By contrast, transcription of the principal vegetative DGC AvGReg was not AmrZ-regulated. Lower c-di-GMP in ΔamrZ correlated with larger swimming halos. Collectively, these genetic, biochemical and transcriptomic data support a model in which AmrZ directly activates algD and elevates c-di-GMP via selected DGCs, thereby promoting alginate synthesis while reducing motility. RNA-seq data also indicate that AmrZ influences broader cellular programmes, including metabolism and iron homeostasis, positioning AmrZ as a central regulator that links c-di-GMP homeostasis to coordinated exopolysaccharide production in A. vinelandii. This work contributes to our understanding of the regulatory networks controlled by AmrZ outside the Pseudomonas genus and reveals important differences in its targets and regulatory mechanisms.

Azotobacter vinelandii↗

PubMed2005

Transcription and autoregulation of the Rv3134c-devR-devS operon of Mycobacterium tuberculosis.

DevR is a transcriptional regulator that mediates the genetic response of Mycobacterium tuberculosis to oxygen limitation and nitric oxide exposure. devR is co-transcribed along with devS, which encodes its cognate sensor kinase, and an upstream gene, Rv3134c. The transcriptional activity of this operon was characterized by primer extension, transcriptional fusion and electrophoretic mobility shift assays (EMSAs) under aerobic conditions. Transcription start points (Tsps) were detected upstream of both Rv3134c and devR, and the major transcript was derived from upstream of Rv3134c. Sequences with similarity to sigma factor consensus elements and to DevR-binding motifs were detected in the vicinity of the Tsps by in silico analysis. EMSAs with promoter regions and DevR protein showed that DevR binds to its own promoters in a sequence-specific manner with differing affinities. Consistent with the primer extension and EMSA data, Rv3134c promoters, and not devR promoters, were determined to be the principal promoters of this operon using reporter assays performed in Mycobacterium smegmatis and Escherichia coli. Furthermore, DevR modulated the activity of both devR and Rv3134c promoters. From these findings it is inferred that the Rv3134c-devR-devS operon is transcribed from multiple promoters and is autoregulated.

Bacterial Proteins↗

PubMed2004

Decreased expression of T-cell NF-kappaB p65 subunit in steroid-resistant nephrotic syndrome.

BACKGROUND: Although the etiology of childhood nephrotic syndrome is unclear, there is evidence to suggest an important role for T cells in the pathogenesis. Steroid resistance is considered a poor prognostic sign but the mechanism of the resistance is unknown. The study examined the potential role of T-cell nuclear transcription factors in the steroid resistance. METHODS: The expression of the nuclear transcription factors activating protein-1 (AP-1) and nuclear factor-kappaB (NF-kappaB) as well as that of lymphokines interleukin (IL)-2, IL-4, and interferon-gamma (IFN-gamma) were compared in T cells obtained from normal subjects, children with steroid-sensitive nephrotic syndrome (SSNS) and children with steroid-resistant nephrotic syndrome (SRNS) before any treatment was given. Changes in expression and binding of the nuclear transcription factors were studied with electrophoretic mobility shift assay (EMSA) and Western blot, whereas mRNA cytokine expression were evaluated by enzyme-linked immunosorbent assay (ELISA)-linked reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: A significant decrease of the p65 subunit protein of NF-kappaB but not in p50 was documented by both EMSA (N= 7) and Western blotting (N= 5) in five of five SRNS patients but not in control subjects or SSNS patients; there was a decrease in mRNA expression as shown by ELISA-linked RT-PCR. In contrast, there were no significant differences in AP-1 expression by EMSA. IL-2 mRNA level was higher in T cells from SRNS patients than in T cells from either SSNS or control subjects. IL-4 and IFN-gamma were equally decreased in both groups of patients. CONCLUSION: The results show differences in T cells between untreated SSNS and SRNS patients. The decrease of NF-kappaB p65 subunit and up-regulation of IL-2 are potential mechanism of glucocorticoid resistance in SRNS.

Adolescent↗

PubMed2003

Enterohemorrhagic Escherichia coli O157:H7 disrupts Stat1-mediated gamma interferon signal transduction in epithelial cells.

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a clinically important bacterial enteropathogen that manipulates a variety of host cell signal transduction cascades to establish infection. However, the effect of EHEC O157:H7 on Jak/Stat signaling is unknown. To define the effect of EHEC infection on epithelial gamma interferon (IFN-gamma)-Stat1 signaling, human T84 and HEp-2 epithelial cells were infected with EHEC O157:H7 and then stimulated with recombinant human IFN-gamma. Cells were also infected with different EHEC strains, heat-killed EHEC, enteropathogenic E. coli (EPEC) O127:H6, and the commensal strain E. coli HB101. Nuclear and whole-cell protein extracts were prepared and were assayed by an electrophoretic mobility shift assay (EMSA) and by Western blotting, respectively. Cells were also processed for immunofluorescence to detect the subcellular localization of Stat1. The EMSA revealed inducible, but not constitutive, Stat1 activation upon IFN-gamma treatment of both cell lines. The EMSA also showed that 6 h of EHEC O157:H7 infection, but not 30 min of EHEC O157:H7 infection, prevented subsequent Stat1 DNA binding induced by IFN-gamma, whereas infection with EPEC did not. Immunoblotting showed that infection with EHEC, but not infection with EPEC, eliminated IFN-gamma-induced Stat1 tyrosine phosphorylation in both dose- and time-dependent fashions and disrupted inducible protein expression of the Stat1-dependent gene interferon regulatory factor 1. Immunofluorescence revealed that EHEC infection did not prevent nuclear accumulation of Stat1 after IFN-gamma treatment. Also, Stat1 tyrosine phosphorylation was suppressed by different EHEC isolates, including intimin-, type III secretion- and plasmid-deficient strains, but not by HB101 and heat-killed EHEC. These findings indicate the novel disruption of host cell signaling caused by EHEC infection but not by EPEC infection.

Cell Line↗

PubMed2006

Involvement of the HP0165-HP0166 two-component system in expression of some acidic-pH-upregulated genes of Helicobacter pylori.

About 200 genes of the gastric pathogen Helicobacter pylori increase expression at medium pHs of 6.2, 5.5, and 4.5, an increase that is abolished or much reduced by the buffering action of urease. Genes up-regulated by a low pH include the two-component system HP0165-HP0166, suggesting a role in the regulation of some of the pH-sensitive genes. To identify targets of HP0165-HP0166, the promoter regions of genes up-regulated by a low pH were grouped based on sequence similarity. Probes for promoter sequences representing each group were subjected to electrophoretic mobility shift assays (EMSA) with recombinant HP0166-His(6) or a mutated response regulator, HP0166-D52N-His(6), that can specifically determine the role of phosphorylation of HP0166 in binding (including a control EMSA with in-vitro-phosphorylated HP0166-His(6)). Nineteen of 45 promoter-regulatory regions were found to interact with HP0166-His(6). Seven promoters for genes encoding alpha-carbonic anhydrase, omp11, fecD, lpp20, hypA, and two with unknown function (pHP1397-1396 and pHP0654-0675) were clustered in gene group A, which may respond to changes in the periplasmic pH at a constant cytoplasmic pH and showed phosphorylation-dependent binding in EMSA with HP0166-D52N-His(6). Twelve promoters were clustered in groups B and C whose up-regulation likely also depends on a reduction of the cytoplasmic pH at a medium pH of 5.5 or 4.5. Most of the target promoters in groups B and C showed phosphorylation-dependent binding with HP0166-D52N-His(6), but promoters for ompR (pHP0166-0162), pHP0682-0681, and pHP1288-1289 showed phosphorylation-independent binding. These findings, combined with DNase I footprinting, suggest that HP0165-0166 is an acid-responsive signaling system affecting the expression of pH-sensitive genes. Regulation of these genes responds either to a decrease in the periplasmic pH alone (HP0165 dependent) or also to a decrease in the cytoplasmic pH (HP0165 independent).

Carbonic Anhydrases↗

PubMed2006

White spot syndrome virus annexes a shrimp STAT to enhance expression of the immediate-early gene ie1.

Although the Janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling pathway is part of the antiviral response in arthropods such as Drosophila, here we show that white spot syndrome virus (WSSV) uses a shrimp STAT as a transcription factor to enhance viral gene expression in host cells. In a series of deletion and mutation assays using the WSSV immediate-early gene ie1 promoter, which is active in shrimp cells and also in insect Sf9 cells, an element containing a STAT binding motif was shown to be important for the overall level of WSSV ie1 promoter activity. In the Sf9 insect cell line, a specific protein-DNA complex was detected by using electrophoresis mobility shift assays (EMSA) with the 32P-labeled STAT binding motif of the WSSV ie1 promoter as the probe. When recombinant Penaeus monodon STAT (rPmSTAT) was overexpressed in Sf9 cells, EMSA with specific antibodies confirmed that the STAT was responsible for the formation of the specific protein-DNA complex. Another EMSA showed that in WSSV-infected P. monodon, levels of activated PmSTAT were higher than in WSSV-free P. monodon. A transactivation assay of the WSSV ie1 promoter demonstrated that increasing the level of rPmSTAT led to dose-dependent increases in ie1 promoter activity. These results show that STAT directly transactivates WSSV ie1 gene expression and contributes to its high promoter activity. We conclude that WSSV successfully annexes a putative shrimp defense mechanism, which it uses to enhance the expression of viral immediate-early genes.

Animals↗

PubMed1992

The kappa B enhancer motifs in human immunodeficiency virus type 1 and simian virus 40 recognize different binding activities in human Jurkat and H9 T cells: evidence for NF-kappa B-independent activation of the kappa B motif.

The kappa B transcriptional enhancer motif, present in many viruses, is broadly active in many cell types. It is recognized by c-Rel/HIVEN86A in DNA affinity precipitation (DNAP) assays and by the Rel-related p50 and p65 subunits of the nuclear factor NF-kappa B in electrophoretic mobility shift assays (EMSA). We have analyzed activities that bind the human immunodeficiency virus type 1 and simian virus 40 kappa B motifs in two human leukemia cell lines, Jurkat and H9. In both DNAP and EMSA analyses of Jurkat cell extracts, we detected multiple kappa B motif-binding activities in addition to c-Rel/HIVEN86A and p50-p65 NF-kappa B. In Jurkat cell nuclear extracts, EMSA analysis revealed at least six specific DNA-protein complexes, of which one comigrated with the p50-p65 NF-kappa B complex. Formation of all six complexes was enhanced by stimulation of the cells with phorbol 12-myristate-13-acetate and phytohemagglutinin but was differentially affected by the salt concentration in the binding reaction and by the conditions of Jurkat cell growth. Nuclear extracts from both unstimulated and stimulated H9 cells revealed similar levels of five kappa B motif-specific complexes, all of which displayed mobilities distinct from those of the Jurkat cell complexes. Indeed, a complex corresponding to p50-p65 NF-kappa B was not detectable in nuclear extracts from unstimulated H9 cells although such a complex was apparent in nuclear extracts from stimulated H9 cells. In contrast to the inducibility of a p50-p65 NF-kappa B-like complex, transcriptional enhancers composed of multimerized kappa B motifs displayed similar high levels of activity in both the unstimulated and stimulated H9 cells. Thus, the activity of the kappa B motif in H9 cells corresponded to the abundance of the H9 cell-specific kappa B motif complexes and not to the levels of p50-p65 NF-kappa B complex. These results suggest that the broad activity of the kappa B enhancer element is not only due to the broadly distributed NF-kappa B activator but also to cell type-specific kappa B motif-binding activities.

Base Sequence↗