Pseudomonas aeruginosa LysR PA4203 Regulator NmoR Acts as a Repressor of the PA4202nmoAGene, Encoding a Nitronate Monooxygenase

The PA4203 gene encodes a LysR regulator and lies between theppgLgene (PA4204), which encodes a periplasmic gluconolactonase, and, in the opposite orientation, the PA4202 (nmoA) gene, coding for a nitronate monooxygenase, andddlA(PA4201), encoding ad-alanine alanine ligase. The intergenic regions between PA4203 andppgLand between PA4203 andnmoAare very short (79 and 107 nucleotides, respectively). Here we show that PA4203 (nmoR) represses its own transcription and the expression ofnmoA. A chromatin immunoprecipitation analysis showed the presence of a single NmoR binding site betweennmoAandnmoR, which was confirmed by electrophoretic mobility shift assays (EMSAs) with the purified NmoR protein. Despite this observation, a transcriptome analysis revealed more genes to be affected in annmoRmutant, including genes known to be part of the MexT LysR activator regulon. The PA1225 gene, encoding a quinone oxidoreductase, was the most highly upregulated gene in thenmoRdeletion mutant, independently of MexT. Finally, deletion of thenmoAgene resulted in an increased sensitivity of the cells to 3-nitropropionic acid (3-NPA), confirming the role of the nitronate monooxygenase protein in the detoxification of nitronate.

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Distinct functional contributions of 2 GABP–NRF-2 recognition sites within the context of the human TOMM70 promoter

Biochemistry and Cell Biology ◽

10.1139/o06-064 ◽

2006 ◽

Vol 84 (5) ◽

pp. 813-822 ◽

Cited By ~ 10

Author(s):

José R. Blesa ◽

José Hernández-Yago

Keyword(s):

Chromatin Immunoprecipitation ◽

Binding Sites ◽

Site Directed Mutagenesis ◽

Outer Mitochondrial Membrane ◽

Mobility Shift ◽

A Subunit ◽

Expression Evidence ◽

Electrophoretic Mobility Shift Assays

TOMM70 is a subunit of the outer mitochondrial membrane translocase that plays a major role as a receptor of hydrophobic preproteins targeted to mitochondria. We have previously reported 2 binding sites for the transcription factor GABP–NRF-2 in the promoter region of the human TOMM70 gene that are important in activating transcription. To assess the functionality and actual role of these sites, chromatin immunoprecipitation, site-directed mutagenesis, and electrophoretic mobility shift assays were carried out. We conclude that GABP–NRF-2 binds in vivo to the TOMM70 promoter, and that the 2 GABP–NRF-2 binding sites of the promoter have different functional contributions in promoting TOMM70 expression. Evidence is provided that they work in an additive manner as single sites.

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Zygotic Vsx1 Plays a Key Role in Defining V2a Interneuron Sub-Lineage by Directly Repressing tal1 Transcription in Zebrafish

International Journal of Molecular Sciences ◽

10.3390/ijms21103600 ◽

2020 ◽

Vol 21 (10) ◽

pp. 3600

Author(s):

Qi Zhang ◽

Haomang Xu ◽

Wei Zhao ◽

Jianbo Zheng ◽

Lei Sun ◽

...

Keyword(s):

Chromatin Immunoprecipitation ◽

Consensus Sequence ◽

Mobility Shift ◽

Normal Expression ◽

The Common ◽

Necessary And Sufficient ◽

Electrophoretic Mobility Shift Assays ◽

Expression Program ◽

Lineage Diversification

In the spinal cord, excitatory V2a and inhibitory V2b interneurons are produced together by the final division of common P2 progenitors. During V2a and V2b diversification, Tal1 is necessary and sufficient to promote V2b differentiation and Vsx2 suppresses the expression of motor neuron genes to consolidate V2a interneuron identity. The expression program of Tal1 is triggered by a Foxn4-driven regulatory network in the common P2 progenitors. Why the expression of Tal1 is inhibited in V2a interneurons at the onset of V2a and V2b sub-lineage diversification remains unclear. Since transcription repressor Vsx1 is expressed in the P2 progenitors and newborn V2a cells in zebrafish, we investigated the role of Vsx1 in V2a fate specification during V2a and V2b interneuron diversification in this species by loss and gain-of-function experiments. In vsx1 knockdown embryos or knockout Go chimeric embryos, tal1 was ectopically expressed in the presumptive V2a cells, while the generation of V2a interneurons was significantly suppressed. By contrast, in vsx1 overexpression embryos, normal expression of tal1 in the presumptive V2b cells was suppressed, while the generation of V2a interneuron was expanded. Chromatin immunoprecipitation and electrophoretic mobility shift assays in combination with core consensus sequence mutation analysis further revealed that Vsx1 can directly bind to tal1 promoter and repress tal1 transcription. These results indicate that Vsx1 can directly repress tal1 transcription and plays an essential role in defining V2a interneuron sub-lineage during V2a and V2b sub-lineage diversification in zebrafish.

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ChIP-seq Analysis of the Global Regulator Vfr Reveals Novel Insights Into the Biocontrol Agent Pseudomonas protegens FD6

Frontiers in Microbiology ◽

10.3389/fmicb.2021.667637 ◽

2021 ◽

Vol 12 ◽

Author(s):

Qingxia Zhang ◽

Chenglin Xing ◽

Xiangwei Kong ◽

Cheng Wang ◽

Xijun Chen

Keyword(s):

Chromatin Immunoprecipitation ◽

Biocontrol Agent ◽

Antimicrobial Properties ◽

Nucleotide Sequencing ◽

Sequencing Analysis ◽

Mobility Shift ◽

Consensus Sequences ◽

Nucleotide Sequence Alignment ◽

Electrophoretic Mobility Shift Assays

Many Pseudomonas protegens strains produce the antibiotics pyoluteorin (PLT) and 2,4-diacetylphloroglucinol (2,4-DAPG), both of which have antimicrobial properties. The biosynthesis of these metabolites is typically controlled by multiple regulatory factors. Virulence factor regulator (Vfr) is a multifunctional DNA-binding regulator that modulates 2,4-DAPG biosynthesis in P. protegens FD6. However, the mechanism by which Vfr regulates this process remains unclear. In the present study, chromatin immunoprecipitation of FLAG-tagged Vfr and nucleotide sequencing analysis were used to identify 847 putative Vfr binding sites in P. protegens FD6. The consensus P. protegens Vfr binding site predicted from nucleotide sequence alignment is TCACA. The qPCR data showed that Vfr positively regulates the expression of phlF and phlG, and the expression of these genes was characterized in detail. The purified recombinant Vfr bound to an approximately 240-bp fragment within the phlF and phlG upstream regions that harbor putative Vfr consensus sequences. Using electrophoretic mobility shift assays, we localized Vfr binding to a 25-bp fragment that contains part of the Vfr binding region. Vfr binding was eliminated by mutating the TACG and CACA sequences in phlF and phlG, respectively. Taken together, our results show that Vfr directly regulates the expression of the 2,4-DAPG operon by binding to the upstream regions of both the phlF and phlG genes. However, unlike other Vfr-targeted genes, Vfr binding to P. protegens FD6 does not require an intact binding consensus motif. Furthermore, we demonstrated that vfr expression is autoregulated in this bacterium. These results provide novel insights into the regulatory role of Vfr in the biocontrol agent P. protegens.

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Matrix metalloproteinase-3 induction in rat brain astrocytes: focus on the role of two AP-1 elements

Biochemical Journal ◽

10.1042/bj20071207 ◽

2008 ◽

Vol 410 (3) ◽

pp. 605-611 ◽

Cited By ~ 15

Author(s):

Kwang Soo Kim ◽

Hee Young Kim ◽

Eun-hye Joe ◽

Ilo Jou

Keyword(s):

Rat Brain ◽

Interferon Γ ◽

Site Directed Mutagenesis ◽

Start Codon ◽

Activator Protein 1 ◽

Mobility Shift ◽

Matrix Metalloproteinase 3 ◽

Mitogen Activated Protein ◽

Electrophoretic Mobility Shift Assays

Many brain cells secrete MMPs (matrix metalloproteinases), and increased or misregulated MMP levels are found in neurodegenerative disorders. Here we report that MMP-3 transcription and protein secretion were increased in rat brain astrocytes stimulated with lipopolysaccharide, gangliosides or interferon-γ. Sequential deletion of the MMP-3 promoter revealed that sequences between −0.5 kb and the start codon were crucial for the transcriptional induction of MMP-3. In addition, experiments using pharmacological inhibitors of individual mitogen-activated protein kinases revealed that MMP-3 induction and promoter activity involved Jun N-terminal kinase, a representative upstream signal of AP-1 (activator protein-1). Sequence analyses of the region of the MMP-3 promoter 500 bp from the start codon indicated the presence of three AP-1 binding sequences. Among them, electrophoretic-mobility-shift assays as well as site-directed mutagenesis of individual AP-1 sequences revealed that distal and middle, but not proximal, sequences largely mediated its induction. Together, these results indicate that AP-1 could control MMP-3 induction in brain astrocytes and that its regulation through specific AP-1 elements could be exploited in the treatment of brain pathologies in which increased expression of MMP-3 plays crucial roles.

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Role of Cjun in Gγ-Globin Gene Trans-Activation.

Blood ◽

10.1182/blood.v112.11.1870.1870 ◽

2008 ◽

Vol 112 (11) ◽

pp. 1870-1870

Author(s):

Sirisha Kodeboyina ◽

Sima Zein ◽

Moosueng Lee ◽

Parimaladevi Balamurugan ◽

Xiao Yao ◽

...

Keyword(s):

Electrophoretic Mobility ◽

Globin Gene ◽

Fetal Hemoglobin ◽

K562 Cells ◽

Luciferase Reporter ◽

Complete Analysis ◽

Dependent Manner ◽

Mobility Shift ◽

Electrophoretic Mobility Shift Assays

Abstract Previous studies from our laboratory demonstrated the role of the G-CRE (Gγ-globin cAMP response element) in drug-mediated fetal hemoglobin induction. The G-CRE located at −1222 to −1229 in the promoter of Gγ-globin gene, contains binding site for trans-factors CREB1, ATF-2 and cJun. We previously demonstrated binding of phosphorylated CREB1 and ATF-2 to this element via p38 MAPK signaling triggered by sodium butyrate (NaB) and trichostatin A (TSA). Electrophoretic mobility shift assays with a probe containing the AC → TG mutation in the G-CRE (TGTGGTCA, m2) abolished trans-factor binding to the G-CRE. Furthermore, Gγ promoter activity was abolished in the PGL3 luciferase reporter vector driven by the Gγ promoter (−1500 to +36) carrying the m2 mutation. (Sangerman et al. Blood108:3590–9, 2006). Subsequent studies in our laboratory were aimed at understanding the role of trans-factor cJun, an AP-1 family member, as a regulator of Gγ-globin expression via the G-CRE site. In K562 cells treated with 2mM NaB or 0.3μM TSA for 48 hrs, cJun phosphorylation increased 2.8-fold and 6.4-fold respectively by western blot analysis. Chromatin immunoprecipitation studies showed 16-fold chromatin enrichment in the −1225 Gγ-globin region compared to IgG control studies indicative of significant cJun binding in vivo at steady state. Electrophoretic mobility shift assays using cJun monoclonal antibody demonstrated a supershifted DNA-protein complex confirming binding of cJun to the G-CRE probe. To gain evidence for a functional role of cJun, we performed enforced expression studies using the pLen-cJun vector. In a concentration dependent manner, over-expression of cJun increased luciferase activity up to 350-fold in the luciferase reporter plasmid controlled by the Gγ-promoter (−1500 to +36). As predicted from binding studies, the m2 mutation in this promoter abolished the cJunmediated trans-activation confirming that the G-CRE is required to mediate effects of cJun. We are currently investigating the ability of cJun to trans-activate the endogenous Gγ-globin gene in K562 cells. To achieve this goal, K562 stable lines were established with the expression vectors pLen-cJun and empty vector. A complete analysis of the stable lines is in progress. Future investigations to identify other components of the functional CREB1/ATF2/cJun enhanceosome complex bound to the G-CRE will be performed using affinity chromatography and mass spectrometry. This information will be used to develop strategies for fetal hemoglobin induction.

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Detection of a Complex That Associates With the Bβ Fibrinogen G−455-A Polymorphism

Blood ◽

10.1182/blood.v92.9.3286.421k20_3286_3293 ◽

1998 ◽

Vol 92 (9) ◽

pp. 3286-3293

Author(s):

Erika T. Brown ◽

Gerald M. Fuller

Keyword(s):

Luciferase Reporter ◽

Molecular Evidence ◽

Luciferase Reporter Gene ◽

Nucleotide Substitutions ◽

Mobility Shift ◽

Binding Characteristics ◽

Base Sequences ◽

American Society ◽

Electrophoretic Mobility Shift Assays

The promoter region of the Bβ fibrinogen gene containing the polymorphic site (G−455-A) shows an increase in fibrinogen levels for individuals containing an adenine rather than a guanine. Two methods were used to explore the possible functional role of this region. Electrophoretic mobility shift assays (EMSAs) were performed using specific DNA probes containing base sequences pertinent to the allelic site. Specific DNA binding proteins were detected and their binding characteristics were determined. Secondly, we placed DNA fragments containing different −455 nucleotide substitutions of the Bβ promoter upstream of a luciferase reporter gene and transfected them into HepG2 cells to determine their effect on transactivation. An adenine at position −455 resulted in greater luciferase activity than when a guanine was present. UV cross-linking bound protein to the DNA demonstrated a 47-kD protein binding preferentially to the site when a guanine rather than an adenine was present at −455. We hypothesize that a transactivation protein complex associates with the site, but its association is stronger when guanine is present, thereby slowing downstream Bβ gene transcription. These data provide the first molecular evidence that accounts for the increase in fibrinogen in individuals carrying this allele.© 1998 by The American Society of Hematology.

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Increased Rates of Genomic Deletions Generated by Mutations in the Yeast Gene Encoding DNA Polymerase δ or by Decreases in the Cellular Levels of DNA Polymerase δ

Molecular and Cellular Biology ◽

10.1128/mcb.20.20.7490-7504.2000 ◽

2000 ◽

Vol 20 (20) ◽

pp. 7490-7504 ◽

Cited By ~ 56

Author(s):

Robert J. Kokoska ◽

Lela Stefanovic ◽

Jeremy DeMai ◽

Thomas D. Petes

Keyword(s):

Dna Polymerase ◽

Genome Stability ◽

Gene Encoding ◽

Mutator Phenotype ◽

Genomic Deletions ◽

Dna Polymerase Δ ◽

Mutation Avoidance ◽

Increased Sensitivity

ABSTRACT In Saccharomyces cerevisiae, POL3 encodes the catalytic subunit of DNA polymerase δ. While yeastPOL3 mutant strains that lack the proofreading exonuclease activity of the polymerase have a strong mutator phenotype, little is known regarding the role of other Pol3p domains in mutation avoidance. We identified a number of pol3 mutations in regions outside of the exonuclease domain that have a mutator phenotype, substantially elevating the frequency of deletions. These deletions appear to reflect an increased frequency of DNA polymerase slippage. In addition, we demonstrate that reduction in the level of wild-type DNA polymerase results in a similar mutator phenotype. Lowered levels of DNA polymerase also result in increased sensitivity to the DNA-damaging agent methyl methane sulfonate. We conclude that both the quantity and the quality of DNA polymerase δ is important in ensuring genome stability.

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The Sin3p PAH Domains Provide Separate Functions Repressing Meiotic Gene Transcription in Saccharomyces cerevisiae

Eukaryotic Cell ◽

10.1128/ec.00143-10 ◽

2010 ◽

Vol 9 (12) ◽

pp. 1835-1844 ◽

Cited By ~ 4

Author(s):

Michael J. Mallory ◽

Michael J. Law ◽

Lela E. Buckingham ◽

Randy Strich

Keyword(s):

Cell Division ◽

Vegetative Growth ◽

Mitotic Cell ◽

Amphipathic Helix ◽

Mobility Shift ◽

Mitotic Cell Division ◽

Meiotic Gene ◽

Electrophoretic Mobility Shift Assays ◽

Promoter Interaction

ABSTRACT Meiotic genes in budding yeast are repressed during vegetative growth but are transiently induced during specific stages of meiosis. Sin3p represses the early meiotic gene (EMG) by bridging the DNA binding protein Ume6p to the histone deacetylase Rpd3p. Sin3p contains four paired amphipathic helix (PAH) domains, one of which (PAH3) is required for repressing several genes expressed during mitotic cell division. This report examines the roles of the PAH domains in mediating EMG repression during mitotic cell division and following meiotic induction. PAH2 and PAH3 are required for mitotic EMG repression, while electrophoretic mobility shift assays indicate that only PAH2 is required for stable Ume6p-promoter interaction. Unlike mitotic repression, reestablishing EMG repression following transient meiotic induction requires PAH3 and PAH4. In addition, the role of Sin3p in reestablishing repression is expanded to include additional loci that it does not control during vegetative growth. These findings indicate that mitotic and postinduction EMG repressions are mediated by two separate systems that utilize different Sin3p domains.

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Construction and Characterization of aHelicobacter pylori clpB Mutant and Role of the Gene in the Stress Response

Journal of Bacteriology ◽

10.1128/jb.180.2.426-429.1998 ◽

1998 ◽

Vol 180 (2) ◽

pp. 426-429 ◽

Cited By ~ 24

Author(s):

Elaine Allan ◽

Peter Mullany ◽

Soad Tabaqchali

Keyword(s):

Helicobacter Pylori ◽

High Temperature ◽

Stress Response ◽

Structural Features ◽

High Temperature Stress ◽

Gene Encoding ◽

Increased Sensitivity ◽

H Pylori

ABSTRACT Antiserum raised against whole Helicobacter pyloricells identified a novel 94-kDa antigen. The nucleotide sequence of the gene encoding the 94-kDa antigen was determined, and analysis of the deduced amino acid sequence revealed structural features typical of the ClpB ATPase family of stress response proteins. An isogenic H. pylori clpB mutant showed increased sensitivity to high-temperature stress, indicating that the clpB gene product functions as a stress response protein in H. pylori.

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Regulation of sucrase and lactase in Caco-2 cells: relationship to nuclear factors SIF-1 and NF-LPH-1

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.1996.271.4.g707 ◽

1996 ◽

Vol 271 (4) ◽

pp. G707-G713 ◽

Cited By ~ 3

Author(s):

W. A. Olsen ◽

M. Lloyd ◽

H. Korsmo ◽

Y. Z. He

Keyword(s):

Gene Expression ◽

Cellular Differentiation ◽

Tissue Specificity ◽

Developmental Regulation ◽

Mobility Shift ◽

Nuclear Factors ◽

Relationship Of ◽

Electrophoretic Mobility Shift Assays ◽

The Relationship

Recent studies suggest the importance of two transcription factors, Cdx-2 and NF-LPH-1, in the regulation of sucrase-isomaltase (SI) and lactase-phlorizin hydrolase (LPH) gene expression, respectively. Cdx-2 accounts for the tissue specificity of sucrase expression (16), and NF-LPH-1 varies with postnatal changes in lactase activity, suggesting a role in its developmental regulation (22). We used electrophoretic mobility shift assays to study the relationship of Cdx-2 and NF-LPH-1 to SI and LPH gene expression in Caco-2 cells to provide evidence regarding the role of these factors in the development of sucrase and lactase with cellular differentiation. We found that Cdx-2 levels correlated with SI expression and that NF-LPH-1 did not correlate with LPH expression. These studies suggest a role for Cdx-2 but not for NF-LPH-1 in the development of carbohydrase expression in these cells.

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