scholarly journals Identification of a functional hypoxia-responsive element that regulates the expression of the egl nine homologue 3 (egln3/phd3) gene

2005 ◽  
Vol 390 (1) ◽  
pp. 189-197 ◽  
Author(s):  
Nuria Pescador ◽  
Yolanda Cuevas ◽  
Salvador Naranjo ◽  
Marisa Alcaide ◽  
Diego Villar ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Initiation ◽  
Initiation Site ◽  
Egg Laying ◽  
Dependent Manner ◽  
Low Oxygen ◽  
Consensus Sequences ◽  
Homologous Protein ◽  
Responsive Element

Low oxygen levels induce an adaptive response in cells through the activation of HIFs (hypoxia-inducible factors). These transcription factors are mainly regulated by a group of proline hydroxylases that, in the presence of oxygen, target HIF for degradation. The expression of two such enzymes, EGLN1 [EGL nine homologous protein 1, where EGL stands for egg laying defective (Caenorhabditis elegans gene)] and EGLN3, is induced by hypoxia through a negative feedback loop, and we have demonstrated recently that hypoxic induction of EGLN expression is HIF-dependent. In the present study, we have identified an HRE (hypoxia response element) in the region of the EGLN3 gene using a combination of bioinformatics and biological approaches. Initially, we isolated a number of HRE consensus sequences in a region of 40 kb around the human EGLN3 gene and studied their evolutionary conservation. Subsequently, we examined the functionality of the conserved HRE sequences in reporter and chromatin precipitation assays. One of the HREs, located within a conserved region of the first intron of the EGLN3 gene 12 kb downstream of the transcription initiation site, bound HIF in vivo. Furthermore, this sequence was able to drive reporter gene expression under conditions of hypoxia in an HRE-dependent manner. Indeed, we were able to demonstrate that HIF was necessary and sufficient to induce gene expression from this enhancer sequence.

scholarly journals Domains within RbpA Serve Specific Functional Roles That Regulate the Expression of Distinct Mycobacterial Gene Subsets

2018 ◽  
Vol 200 (13) ◽  
Author(s):  
Jerome Prusa ◽  
Drake Jensen ◽  
Gustavo Santiago-Collazo ◽  
Steven S. Pope ◽  
Ashley L. Garner ◽  
...  
Keyword(s):  
Gene Expression ◽  
Mycobacterium Tuberculosis ◽  
Transcription Initiation ◽  
Dependent Manner ◽  
Interaction Domain ◽  
Structural Domains ◽  
Content Type ◽  
Group I

ABSTRACT The RNA polymerase (RNAP) binding protein A (RbpA) contributes to the formation of stable RNAP-promoter open complexes (RP o ) and is essential for viability in mycobacteria. Four domains have been identified in the RbpA protein, i.e., an N-terminal tail (NTT) that interacts with RNAP β′ and σ subunits, a core domain (CD) that contacts the RNAP β′ subunit, a basic linker (BL) that binds DNA, and a σ-interaction domain (SID) that binds group I and group II σ factors. Limited in vivo studies have been performed in mycobacteria, however, and how individual structural domains of RbpA contribute to RbpA function and mycobacterial gene expression remains mostly unknown. We investigated the roles of the RbpA structural domains in mycobacteria using a panel of rbpA mutants that target individual RbpA domains. The function of each RbpA domain was required for Mycobacterium tuberculosis viability and optimal growth in Mycobacterium smegmatis . We determined that the RbpA SID is both necessary and sufficient for RbpA interaction with the RNAP, indicating that the primary functions of the NTT and CD are not solely association with the RNAP. We show that the RbpA BL and SID are required for RP o stabilization in vitro , while the NTT and CD antagonize this activity. Finally, RNA-sequencing analyses suggest that the NTT and CD broadly activate gene expression, whereas the BL and SID activate or repress gene expression in a gene-dependent manner for a subset of mycobacterial genes. Our findings highlight specific outcomes for the activities of the individual functional domains in RbpA. IMPORTANCE Mycobacterium tuberculosis is the causative agent of tuberculosis and continues to be the most lethal infectious disease worldwide. Improved molecular understanding of the essential proteins involved in M. tuberculosis transcription, such as RbpA, could provide targets for much needed future therapeutic agents aimed at combatting this pathogen. In this study, we expand our understanding of RbpA by identifying the RbpA structural domains responsible for the interaction of RbpA with the RNAP and the effects of RbpA on transcription initiation and gene expression. These experiments expand our knowledge of RbpA while also broadening our understanding of bacterial transcription in general.

scholarly journals Flow-dependent regulation of endothelial Tie2 by GATA3 in vivo

2021 ◽  
Vol 9 (1) ◽  
Author(s):  
Temitayo O. Idowu ◽  
Valerie Etzrodt ◽  
Thorben Pape ◽  
Joerg Heineke ◽  
Klaus Stahl ◽  
...  
Keyword(s):  
Gene Expression ◽  
Regulation Of Gene Expression ◽  
Experimental Models ◽  
Common Denominator ◽  
Dependent Manner ◽  
Pulmonary Endothelium ◽  
Delivery Platform ◽  
Transient Overexpression ◽  
Plasmid Delivery

Abstract Background Reduced endothelial Tie2 expression occurs in diverse experimental models of critical illness, and experimental Tie2 suppression is sufficient to increase spontaneous vascular permeability. Looking for a common denominator among different critical illnesses that could drive the same Tie2 suppressive (thereby leak inducing) phenotype, we identified “circulatory shock” as a shared feature and postulated a flow-dependency of Tie2 gene expression in a GATA3 dependent manner. Here, we analyzed if this mechanism of flow-regulation of gene expression exists in vivo in the absence of inflammation. Results To experimentally mimic a shock-like situation, we developed a murine model of clonidine-induced hypotension by targeting a reduced mean arterial pressure (MAP) of approximately 50% over 4 h. We found that hypotension-induced reduction of flow in the absence of confounding disease factors (i.e., inflammation, injury, among others) is sufficient to suppress GATA3 and Tie2 transcription. Conditional endothelial-specific GATA3 knockdown (B6-Gata3tm1-Jfz VE-Cadherin(PAC)-cerERT2) led to baseline Tie2 suppression inducing spontaneous vascular leak. On the contrary, the transient overexpression of GATA3 in the pulmonary endothelium (jet-PEI plasmid delivery platform) was sufficient to increase Tie2 at baseline and completely block its hypotension-induced acute drop. On the functional level, the Tie2 protection by GATA3 overexpression abrogated the development of pulmonary capillary leakage. Conclusions The data suggest that the GATA3–Tie2 signaling pathway might play a pivotal role in controlling vascular barrier function and that it is affected in diverse critical illnesses with shock as a consequence of a flow-regulated gene response. Targeting this novel mechanism might offer therapeutic opportunities to treat vascular leakage of diverse etiologies.

scholarly journals Modulatory Effects of Osthole on Lipopolysaccharides-Induced Inflammation in Caco-2 Cell Monolayer and Co-Cultures with THP-1 and THP-1-Derived Macrophages

Nutrients ◽  
2020 ◽  
Vol 13 (1) ◽  
pp. 123
Author(s):  
Natalia K. Kordulewska ◽  
Justyna Topa ◽  
Małgorzata Tańska ◽  
Anna Cieślińska ◽  
Ewa Fiedorowicz ◽  
...  
Keyword(s):  
Gene Expression ◽  
Inflammatory Reaction ◽  
Wide Spectrum ◽  
Cell Monolayer ◽  
In Vivo Studies ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Tnf Α

Lipopolysaccharydes (LPS) are responsible for the intestinal inflammatory reaction, as they may disrupt tight junctions and induce cytokines (CKs) secretion. Osthole has a wide spectrum of pharmacological effects, thus its anti-inflammatory potential in the LPS-treated Caco-2 cell line as well as in Caco-2/THP-1 and Caco-2/macrophages co-cultures was investigated. In brief, Caco-2 cells and co-cultures were incubated with LPS to induce an inflammatory reaction, after which osthole (150–450 ng/mL) was applied to reduce this effect. After 24 h, the level of secreted CKs and changes in gene expression were examined. LPS significantly increased the levels of IL-1β, -6, -8, and TNF-α, while osthole reduced this effect in a concentration-dependent manner, with the most significant decrease when a 450 ng/mL dose was applied (p < 0.0001). A similar trend was observed in changes in gene expression, with the significant osthole efficiency at a concentration of 450 ng/μL for IL1R1 and COX-2 (p < 0.01) and 300 ng/μL for NF-κB (p < 0.001). Osthole increased Caco-2 monolayer permeability, thus if it would ever be considered as a potential drug for minimizing intestinal inflammatory symptoms, its safety should be confirmed in extended in vitro and in vivo studies.

Analysis of Saccharomyces cerevisiae his3 transcription in vitro: biochemical support for multiple mechanisms of transcription

1990 ◽  
Vol 10 (6) ◽  
pp. 2832-2839
Author(s):  
A S Ponticelli ◽  
K Struhl
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Transcriptional Activation ◽  
Transcription Initiation ◽  
Molecular Mechanisms ◽  
Initiation Site ◽  
Activator Proteins ◽  
Nuclear Extracts ◽  
Transcription In Vitro

The promoter region of the Saccharomyces cerevisiae his3 gene contains two TATA elements, TC and TR, that direct transcription initiation to two sites designated +1 and +13. On the basis of differences between their nucleotide sequences and their responsiveness to upstream promoter elements, it has previously been proposed that TC and TR promote transcription by different molecular mechanisms. To begin a study of his3 transcription in vitro, we used S. cerevisiae nuclear extracts together with various DNA templates and transcriptional activator proteins that have been characterized in vivo. We demonstrated accurate transcription initiation in vitro at the sites used in vivo, transcriptional activation by GCN4, and activation by a GAL4 derivative on various gal-his3 hybrid promoters. In all cases, transcription stimulation was dependent on the presence of an acidic activation region in the activator protein. In addition, analysis of promoters containing a variety of TR derivatives indicated that the level of transcription in vitro was directly related to the level achieved in vivo. The results demonstrated that the in vitro system accurately reproduced all known aspects of in vivo his3 transcription that depend on the TR element. However, in striking contrast to his3 transcription in vivo, transcription in vitro yielded approximately 20 times more of the +13 transcript than the +1 transcript. This result was not due to inability of the +1 initiation site to be efficiently utilized in vitro, but rather it reflects the lack of TC function in vitro. The results support the idea that TC and TR mediate transcription from the wild-type promoter by distinct mechanisms.

scholarly journals Hypoxic-response elements in the oncolytic parvovirus Minute virus of mice do not allow for increased vector production at low oxygen concentration

2006 ◽  
Vol 87 (5) ◽  
pp. 1197-1201 ◽  
Author(s):  
Charlotte Servais ◽  
Perrine Caillet-Fauquet ◽  
Marie-Louise Draps ◽  
Thierry Velu ◽  
Yvan de Launoit ◽  
...  
Keyword(s):  
Cell Metabolism ◽  
Consensus Sequence ◽  
High Titre ◽  
Host Cells ◽  
Low Oxygen ◽  
Minute Virus Of Mice ◽  
Minute Virus ◽  
Responsive Element ◽  
Low Oxygen Concentration

Vectors derived from the autonomous parvovirus Minute virus of mice, MVM(p), are promising tools for the gene therapy of cancer. The validation of their in vivo anti-tumour effect is, however, hampered by the difficulty to produce high-titre stocks. In an attempt to increase vector titres, host cells were subjected to low oxygen tension (hypoxia). It has been shown that a number of viruses are produced at higher titres under these conditions. This is the case, among others, for another member of the family Parvoviridae, the erythrovirus B19 virus. Hypoxia stabilizes a hypoxia-inducible transcription factor (HIF-1α) that interacts with a ‘hypoxia-responsive element’ (HRE), the consensus sequence of which (A/GCGTG) is present in the B19 and MVM promoters. Whilst the native P4 promoter was induced weakly in hypoxia, vector production was reduced dramatically, and adding HRE elements to the P4 promoter of the vector did not alleviate this reduction. Hypoxia has many effects on cell metabolism. Therefore, even if the P4 promoter is activated, the cellular factors that are required for the completion of the parvoviral life cycle may not be expressed.

scholarly journals Enhancers Predominantly Regulate Gene Expression in vivo Via Transcription Initiation

2019 ◽  
Author(s):  
Martin Stephen Charles Larke ◽  
Takayuki Nojima ◽  
Jelena Telenius ◽  
Jacqueline A. Sharpe ◽  
Jacqueline A. Sloane-Stanley ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Initiation ◽  
Regulate Gene Expression ◽  
Regulate Gene

scholarly journals Expression of the Pseudorabies Virus Latency-Associated Transcript Gene during Productive Infection of Cultured Cells

1999 ◽  
Vol 73 (12) ◽  
pp. 9781-9788 ◽  
Author(s):  
Ling Jin ◽  
Gail Scherba
Keyword(s):  
Gene Expression ◽  
Pseudorabies Virus ◽  
Cultured Cells ◽  
Initiation Site ◽  
Early Gene ◽  
Productive Infection ◽  
Virus Latency ◽  
Infected Cells ◽  
Transcript Gene

ABSTRACT Like other alphaherpesviruses, pseudorabies virus (PrV) exhibits restricted gene expression during latency. These latency-associated transcripts (LATs) are derived from the region located within 0.69 to 0.77 map units of the viral genome. However, the presence of such viral RNAs during a productive infection has not been described. Although several transcripts originating between 0.706 to 0.737 map units have been detected in PrV-infected cultured cells, their relationship to the LATs has not been examined. Therefore, to determine if any correlation exists between PrV LAT gene expression in the natural and laboratory systems, transcription from the LAT gene region during lytic infection of cultured neuronal and nonneuronal cells was evaluated. A Northern blot assay using single-stranded RNA probes complementary to the spliced in vivo 8.4-kb largest latency transcript (LLT) detected 1.0-, 2.0-, and 8.0-kb poly(A) RNAs in all PrV-infected cells lines. The 1.0- and 8.0-kb transcripts partially overlapped the first and second exons of the LLT, respectively. In contrast, portions of both LLT exons comprised the 2.0-kb RNA sequence, which lacked the same intron as the LLT. Generation of this transcript began about 243 bp downstream of the LLT initiation site and terminated near the junction of BamHI fragments 8′ and 8. Its synthesis was inhibited by cycloheximide but not by cytosine β-d-arabinofuranoside, which suggests that the 2.0-kb RNA is not an immediate-early gene product. Thus, although the PrV LAT gene is transcriptionally active during a productive infection of cultured cells, the resulting RNAs are distinctive from the LLT.

scholarly journals EnterotoxigenicE. colivirulence gene regulation in human infections

2018 ◽  
Vol 115 (38) ◽  
pp. E8968-E8976 ◽  
Author(s):  
Alexander A. Crofts ◽  
Simone M. Giovanetti ◽  
Erica J. Rubin ◽  
Frédéric M. Poly ◽  
Ramiro L. Gutiérrez ◽  
...  
Keyword(s):  
Gene Expression ◽  
Virulence Factor ◽  
Virulence Gene ◽  
Human Infection ◽  
Anaerobic Growth ◽  
Infection Model ◽  
Low Oxygen ◽  
Virulence Gene Expression ◽  
Factor Expression

EnterotoxigenicEscherichia coli(ETEC) is a global diarrheal pathogen that utilizes adhesins and secreted enterotoxins to cause disease in mammalian hosts. Decades of research on virulence factor regulation in ETEC has revealed a variety of environmental factors that influence gene expression, including bile, pH, bicarbonate, osmolarity, and glucose. However, other hallmarks of the intestinal tract, such as low oxygen availability, have not been examined. Further, determining how ETEC integrates these signals in the complex host environment is challenging. To address this, we characterized ETEC’s response to the human host using samples from a controlled human infection model. We found ETEC senses environmental oxygen to globally influence virulence factor expression via the oxygen-sensitive transcriptional regulator fumarate and nitrate reduction (FNR) regulator. In vitro anaerobic growth replicates the in vivo virulence factor expression profile, and deletion offnrin ETEC strain H10407 results in a significant increase in expression of all classical virulence factors, including the colonization factor antigen I (CFA/I) adhesin operon and both heat-stable and heat-labile enterotoxins. These data depict a model of ETEC infection where FNR activity can globally influence virulence gene expression, and therefore proximity to the oxygenated zone bordering intestinal epithelial cells likely influences ETEC virulence gene expression in vivo. Outside of the host, ETEC biofilms are associated with seasonal ETEC epidemics, and we find FNR is a regulator of biofilm production. Together these data suggest FNR-dependent oxygen sensing in ETEC has implications for human infection inside and outside of the host.

Abstract O-1: Klf5 Regulates Cardiac Pparα and Med13 and affects Cardiac Fatty Acid Metabolism And Obesity

2014 ◽  
Vol 115 (suppl_1) ◽  
Author(s):  
Konstantinos Drosatos ◽  
Nina Pollak ◽  
Panagiotis Ntziachristos ◽  
Chad M Trent ◽  
Yunying Hu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Fatty Acid ◽  
Fatty Acid Metabolism ◽  
Binding Sites ◽  
Transcription Initiation ◽  
Acid Metabolism ◽  
Gene Promoter ◽  
Initiation Site ◽  
White Adipocytes ◽  
Metabolic Role

Krüppel-like factors (KLF) have been associated with metabolic phenotypes. Our study focused on the metabolic role of cardiac KLF5, as it showed the highest increase among all KLFs that were detected by whole genome microarrays of energy-starved hearts obtained from lipopolysaccharide (LPS)-treated mice. Analysis of ppara promoter indicated two potential binding sites for c-Jun (AP-1 sites), the transcriptional factor that is activated by LPS and reduces cardiac PPARα expression: −792/-772 bp and −719/-698 bp prior to the transcription initiation site. This analysis showed that both AP-1 sites overlap with potential KLF-binding sites. Adenovirus-mediated expression of constitutively active c-Jun in a mouse cardiomyocyte cell line (HL-1) reduced PPARα gene expression, while treatment with Ad-KLF5 had the opposite effect. Chromatin immunoprecipitation analysis (ChIP) showed that c-Jun binds both −792/-772 bp and −719/-698 sites of ppara promoter while KLF5 binds on −792/-772 bp. ChIP analysis also showed that LPS promotes c-Jun binding on −792/-772 bp, which prohibits occupation of this region by KLF5. A cardiomyocyte-specific KLF5 knockout mouse (αMHC-KLF5-/-) had normal cardiac function but reduced cardiac expression of PPARα (50%) and other fatty acid metabolism-associated genes such as CD36 (40%), LpL (20%), PGC1α (45%), AOX (28%) and Cpt1 (45%). High fat diet (HFD)-fed αMHC-KLF5-/- mice had a more profound body weight increase (35%) compared to HFD-fed WT mice (15%), as well as larger white adipocytes and brown adipocytes (H&E) and increased hepatic neutral lipid accumulation (Oil-Red-O). The obesogenic effect of cardiomyocyte-specific deletion of KLF5 resembles the phenotype of the αMHC-MED13-/- mice. We showed that KLF5 ablation reduced cardiac MED13 levels despite lack of changes in the expression levels of miR-208, a known regulator of MED13. Infection of HL-1 cells with Ad-KLF5 increased MED13 gene expression. ChIP identified a KLF5 binding site on med13 gene promoter region (-730/-714 bp). Thus, KLF5 regulates cardiac PPARα and MED13 and affects cardiac and systemic fatty acid metabolism and obesity, thus indicating KLF5 as a potential target for cardiac dysfunction associated with energetic complications, as well as for obesity

Comparison of gene expression and activation of transcription factors in organoid-derived monolayer intestinal epithelial cells and organoids

2021 ◽  
Author(s):  
Yu Takahashi ◽  
Yu Inoue ◽  
Keitaro Kuze ◽  
Shintaro Sato ◽  
Makoto Shimizu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Epithelial Cells ◽  
Gene Expression Regulation ◽  
Intestinal Epithelial Cells ◽  
Three Dimensional ◽  
Culture Conditions ◽  
Dependent Manner ◽  
Intestinal Epithelial

Abstract Intestinal organoids better represent in vivo intestinal properties than conventionally used established cell lines in vitro. However, they are maintained in three-dimensional culture conditions that may be accompanied by handling complexities. We characterized the properties of human organoid-derived two-dimensionally cultured intestinal epithelial cells (IECs) compared with those of their parental organoids. We found that the expression of several intestinal markers and functional genes were indistinguishable between monolayer IECs and organoids. We further confirmed that their specific ligands equally activate intestinal ligand-activated transcriptional regulators in a dose-dependent manner. The results suggest that culture conditions do not significantly influence the fundamental properties of monolayer IECs originating from organoids, at least from the perspective of gene expression regulation. This will enable their use as novel biological tools to investigate the physiological functions of the human intestine.

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