scholarly journals A Master Regulator BrpR Coordinates the Expression of Multiple Loci for Robust Biofilm and Rugose Colony Development in Vibrio vulnificus

2021 ◽  
Vol 12 ◽  
Author(s):  
Seung-Ho Hwang ◽  
Hanhyeok Im ◽  
Sang Ho Choi
Keyword(s):  
Colony Formation ◽  
Vibrio Vulnificus ◽  
Colony Development ◽  
Mobility Shift ◽  
Master Regulator ◽  
Multiple Loci ◽  
Host Infection ◽  
Electrophoretic Mobility Shift Assays ◽  
Binding Sequence

Vibrio vulnificus, a fulminating human pathogen, forms biofilms to enhance its survival in nature and pathogenicity during host infection. BrpR is the transcriptional regulator governing robust biofilm and rugose colony formation in V. vulnificus, but little is known about both the direct regulon of BrpR and the role of BrpR in regulation of downstream genes. In this study, transcript analyses revealed that BrpR is highly expressed and thus strongly regulates the downstream gene in the stationary and elevated cyclic di-GMP conditions. Transcriptome analyses discovered the genes, whose expression is affected by BrpR but not by the downstream regulator BrpT. Two unnamed adjacent genes (VV2_1626-1627) were newly identified among the BrpR regulon and designated as brpL and brpG in this study. Genetic analyses showed that the deletion of brpL and brpG impairs the biofilm and rugose colony formation, indicating that brpLG plays a crucial role in the development of BrpR-regulated biofilm phenotypes. Comparison of the colony morphology and exopolysaccharide (EPS) production suggested that although the genetic location and regulation of brpLG are distinct from the brp locus, brpABCDFHIJK (VV2_1574-1582), brpLG is also responsible for the robust EPS production together with the brp locus genes. Electrophoretic mobility shift assays and DNase I protection assays demonstrated that BrpR regulates the expression of downstream genes in distinct loci by directly binding to their upstream regions, revealing a palindromic binding sequence. Altogether, this study suggests that BrpR is a master regulator coordinating the expression of multiple loci responsible for EPS production and thus, contributing to the robust biofilm and rugose colony formation of V. vulnificus.

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

2006 ◽  
Vol 84 (5) ◽  
pp. 813-822 ◽  
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.

scholarly journals Multi-Factor Regulation of the Master Modulator LeuO for the Cyclic-(Phe-Pro) Signaling Pathway in Vibrio vulnificus

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Na-Young Park ◽  
In Hwang Kim ◽  
Yancheng Wen ◽  
Keun-Woo Lee ◽  
Sora Lee ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Signaling Pathway ◽  
Vibrio Vulnificus ◽  
Dna Bending ◽  
Titration Calorimetry ◽  
Master Regulator ◽  
Bending Mechanism ◽  
Periplasmic Domain ◽  
Dependent Signaling Pathway

AbstractLeuO plays the role of a master regulator in the cyclic-L-phenylalanine-L-proline (cFP)-dependent signaling pathway in Vibrio vulnificus. cFP, as shown through isothermal titration calorimetry analysis, binds specifically to the periplasmic domain of ToxR. Binding of cFP triggers a change in the cytoplasmic domain of ToxR, which then activates transcription of leuO encoding a LysR-type regulator. LeuO binds to the region upstream of its own coding sequence, inhibiting its own transcription and maintaining a controlled level of expression. A five-bp deletion in this region abolished expression of LeuO, but a ten-bp deletion did not, suggesting that a DNA bending mechanism is involved in the regulation. Furthermore, binding of RNA polymerase was significantly lower both in the deletion of the ToxR binding site and in the five-bp deletion, but not in the ten-bp deletion, as shown in pull-down assays using an antibody against RNA polymerase subunit α. In summary, multiple factors are involved in control of the expression of LeuO, a master regulator that orchestrates downstream regulators to modulate factors required for survival and pathogenicity of the pathogen.

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

2008 ◽  
Vol 410 (3) ◽  
pp. 605-611 ◽  
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.

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

Blood ◽  
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.

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

Blood ◽  
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.

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

2010 ◽  
Vol 9 (12) ◽  
pp. 1835-1844 ◽  
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.

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

1996 ◽  
Vol 271 (4) ◽  
pp. G707-G713 ◽  
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.

scholarly journals Regulation ofperRExpression by Iron and PerR in Campylobacter jejuni

2011 ◽  
Vol 193 (22) ◽  
pp. 6171-6178 ◽  
Author(s):  
Minkyeong Kim ◽  
Sunyoung Hwang ◽  
Sangryeol Ryu ◽  
Byeonghwa Jeon
Keyword(s):  
Oxidative Stress ◽  
Campylobacter Jejuni ◽  
Regulatory Region ◽  
Transcriptional Level ◽  
Mobility Shift ◽  
Content Type ◽  
Dnase I Footprinting ◽  
Transcriptional Changes ◽  
Electrophoretic Mobility Shift Assays ◽  
Binding Sequence

Campylobacter jejuniis a leading food-borne pathogen causing gastroenteritis in humans. Although OxyR is a widespread oxidative stress regulator in many Gram-negative bacteria,C. jejunilacks OxyR and instead possesses the metalloregulator PerR. Despite the important role played by PerR in oxidative stress defense, little is known about the factors influencingperRexpression inC. jejuni. In this study, aperRpromoter-lacZfusion assay demonstrated that iron significantly reduced the level ofperRtranscription, whereas other metal ions, such as copper, cobalt, manganese, and zinc, did not affectperRtranscription. Notably, aperRmutation substantially increased the level ofperRtranscription and intranscomplementation restored the transcriptional changes, suggestingperRis transcriptionally autoregulated inC. jejuni. In theperRmutant, iron did not repressperRtranscription, indicating the iron dependence ofperRexpression results fromperRautoregulation. Electrophoretic mobility shift assays showed that PerR binds to theperRpromoter, and DNase I footprinting assays identified a PerR binding site overlapping the −35 region of the twoperRpromoters, further supportingperRautoregulation at the transcriptional level. Alignment of the PerR binding sequence in theperRpromoter with the regulatory region of other PerR regulon genes ofC. jejunirevealed a 16-bp consensus PerR binding sequence, which shares high similarities to theBacillus subtilisPerR box. The results of this study demonstrated that PerR directly interacts with theperRpromoter and regulatesperRtranscription and thatperRautoregulation is responsible for the repression ofperRtranscription by iron inC. jejuni.

scholarly journals The transcriptional regulator IscR integrates host-derived nitrosative stress and iron starvation in activation of the vvhBA operon in Vibrio vulnificus

2020 ◽  
Vol 295 (16) ◽  
pp. 5350-5361 ◽  
Author(s):  
Garam Choi ◽  
Kyung Ku Jang ◽  
Jong Gyu Lim ◽  
Zee-Won Lee ◽  
Hanhyeok Im ◽  
...  
Keyword(s):  
Pathogenic Bacteria ◽  
Nitrosative Stress ◽  
Vibrio Vulnificus ◽  
Regulatory Region ◽  
Transcriptional Regulator ◽  
Iron Starvation ◽  
Mobility Shift ◽  
Food Borne ◽  
Successful Infection ◽  
Host Infection

For successful infection of their hosts, pathogenic bacteria recognize host-derived signals that induce the expression of virulence factors in a spatiotemporal manner. The fulminating food-borne pathogen Vibrio vulnificus produces a cytolysin/hemolysin protein encoded by the vvhBA operon, which is a virulence factor preferentially expressed upon exposure to murine blood and macrophages. The Fe-S cluster containing transcriptional regulator IscR activates the vvhBA operon in response to nitrosative stress and iron starvation, during which the cellular IscR protein level increases. Here, electrophoretic mobility shift and DNase I protection assays revealed that IscR directly binds downstream of the vvhBA promoter PvvhBA, which is unusual for a positive regulator. We found that in addition to IscR, the transcriptional regulator HlyU activates vvhBA transcription by directly binding upstream of PvvhBA, whereas the histone-like nucleoid-structuring protein (H-NS) represses vvhBA by extensively binding to both downstream and upstream regions of its promoter. Of note, the binding sites of IscR and HlyU overlapped with those of H-NS. We further substantiated that IscR and HlyU outcompete H-NS for binding to the PvvhBA regulatory region, resulting in the release of H-NS repression and vvhBA induction. We conclude that concurrent antirepression by IscR and HlyU at regions both downstream and upstream of PvvhBA provides V. vulnificus with the means of integrating host-derived signal(s) such as nitrosative stress and iron starvation for precise regulation of vvhBA transcription, thereby enabling successful host infection.

scholarly journals Role of folding kinetics of secondary structures in telomeric G-overhangs in the regulation of telomere maintenance in Saccharomyces cerevisiae

2020 ◽  
Vol 295 (27) ◽  
pp. 8958-8971 ◽  
Author(s):  
Katarina Jurikova ◽  
Martin Gajarsky ◽  
Mona Hajikazemi ◽  
Jozef Nosek ◽  
Katarina Prochazkova ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Secondary Structures ◽  
Telomere Maintenance ◽  
Folding Kinetics ◽  
Mobility Shift ◽  
Dna Structures ◽  
Electrophoretic Mobility Shift Assays ◽  
Kinetics Of

The ends of eukaryotic chromosomes typically contain a 3′ ssDNA G-rich protrusion (G-overhang). This overhang must be protected against detrimental activities of nucleases and of the DNA damage response machinery and participates in the regulation of telomerase, a ribonucleoprotein complex that maintains telomere integrity. These functions are mediated by DNA-binding proteins, such as Cdc13 in Saccharomyces cerevisiae, and the propensity of G-rich sequences to form various non-B DNA structures. Using CD and NMR spectroscopies, we show here that G-overhangs of S. cerevisiae form distinct Hoogsteen pairing–based secondary structures, depending on their length. Whereas short telomeric oligonucleotides form a G-hairpin, their longer counterparts form parallel and/or antiparallel G-quadruplexes (G4s). Regardless of their topologies, non-B DNA structures exhibited impaired binding to Cdc13 in vitro as demonstrated by electrophoretic mobility shift assays. Importantly, whereas G4 structures formed relatively quickly, G-hairpins folded extremely slowly, indicating that short G-overhangs, which are typical for most of the cell cycle, are present predominantly as single-stranded oligonucleotides and are suitable substrates for Cdc13. Using ChIP, we show that the occurrence of G4 structures peaks at the late S phase, thus correlating with the accumulation of long G-overhangs. We present a model of how time- and length-dependent formation of non-B DNA structures at chromosomal termini participates in telomere maintenance.

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