Activation of NF-kappa B and elevation of MnSOD gene expression by thiol reducing agents in lung adenocarcinoma (A549) cells

1995 ◽  
Vol 269 (5) ◽  
pp. L588-L602 ◽  
Author(s):  
K. C. Das ◽  
Y. Lewis-Molock ◽  
C. W. White
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Transcription Factors ◽  
Steady State ◽  
A549 Cells ◽  
Reducing Agents ◽  
Mrna Levels ◽  
Nf Kappa B ◽  
Oxygen Intermediates ◽  
Mnsod Gene

The effect of reducing agents, including N-acetylcysteine (NAC), dithiothreitol (DTT), and 2-mercaptoethanol (2-ME) on nuclear transcription factor-kappa B (NF-kappa B) activation and manganese superoxide dismutase (MnSOD) expression was investigated in a pulmonary adenocarcinoma (A549) cell line. NAC, DTT, and 2-ME each activated the transcription factor NF-kappa B and increased steady-state levels of MnSOD mRNA and enzyme activity in these cells. In addition, NAC, DTT, and 2-ME increased chloramphenicol acetyltransferase (CAT) activity in cells transfected with a construct containing the CAT gene under the control of the rat MnSOD promoter. SOD and catalase (500 U/ml) plus ethanol (1 mM) did not inhibit activation of NF-kappa B or elevation of steady-state MnSOD mRNA levels by NAC, DTT, or 2-ME. Controls in which comparable amounts of O2-. to those produced by thiols were generated by hypoxanthine and xanthine oxidase, or in which H2O2 was added directly, had neither activated NF-kappa B nor elevated MnSOD mRNA. This shows that reactive oxygen intermediates, which may be formed during autooxidation, may not contribute to activation of NF-kappa B. Because the MnSOD promoter also contains potential binding sites for other transcription factors, such as promoter-selective transcription factor-1 (SP-1), activator protein-1 (AP-1), AP-2, adenosine 3',5'-cyclic monophosphate-regulator element binding factor (CREB), and transcription factor IID complex (TFIID), the effect of thiols on their activation also were evaluated. In contrast to findings with NF-kappa B, there was only minor activation of AP-1 by thiols, and none of the other transcription factors were activated by thiols. AP-1 activation was inhibited by catalase (500 U/ml) plus SOD plus ethanol (1 mM). Addition of 700 microM H2O2 also activated AP-1, and catalase at 500 U/ml prevented this activation. This indicates that H2O2 produced as a result of autooxidation of thiols can activate AP-1 but not NF-kappa B. Thus a close association between exposure to reducing agents, activation of NF-kappa B, and elevation of MnSOD gene expression is demonstrated.

Mechanism of RSV-induced IL-8 gene expression in A549 cells before viral replication

1996 ◽  
Vol 271 (6) ◽  
pp. L963-L971 ◽  
Author(s):  
M. A. Fiedler ◽  
K. Wernke-Dollries ◽  
J. M. Stark
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Viral Replication ◽  
Transcriptional Activation ◽  
Mutational Analysis ◽  
A549 Cells ◽  
Nf Kappa B ◽  
Mobility Shift ◽  
Syncytial Virus ◽  
Time Point

Previous studies demonstrated that respiratory syncytial virus (RSV) infection of A549 cells induced interleukin (IL)-8 gene expression and protein release from the cells as early as 2 h after treatment [M. A. Fiedler, K. Wernke-Dollries, and J. M. Stark. Am. J. Physiol. 269 (Lung Cell. Mol. Physiol. 13): L865-L872, 1995; J. G. Mastronarde, M. M. Monick, and G. W. Hunninghake. Am. J. Respir. Cell Mol. Biol. 13: 237-244, 1995]. Furthermore, the effects of RSV at the 2-h time point were not dependent on viral replication. The studies reported here were designed to test the hypothesis that active and inactive RSV induce IL-8 gene expression in A549 cells at the 2-h time point by a mechanism dependent on the activation of the nuclear transcription factor NF-kappa B Northern blot analysis indicated that IL-8 gene expression occurred independent of protein synthesis 2 h after A549 cells were treated with RSV. Analysis of nuclear extracts from RSV-treated A549 cells by electrophoretic mobility shift assays demonstrated that NF-kappa B was activated as early as 15 min after RSV was added to the cells and remained activated for at least 90 min. In contrast, baseline levels of NF-IL-6 and activator protein-1 (AP-1) did not change over this period of time. Deoxyribonuclease footprint analysis of a portion of the 5'-flanking region of the IL-8 gene demonstrated two potential regions for transcription factor binding, which corresponded to the potential AP-1 binding site, and potential NF-IL-6 and NF-kappa B binding sites. Mutational analysis of the 200-bp 5'-untranslated region of the IL-8 gene demonstrated that activation of NF-kappa B and NF-IL-6 were required for RSV-induced transcriptional activation of the IL-8 gene.

Zinc-sensitive genes as potential new target genes of the metal transcription factor-1 (MTF-1)

2005 ◽  
Vol 83 (2) ◽  
pp. 221-229 ◽  
Author(s):  
Birgit Kindermann ◽  
Frank Döring ◽  
Jan Budczies ◽  
Hannelore Daniel
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Steady State ◽  
Cell Line ◽  
Target Genes ◽  
Complement Factor ◽  
Mrna Levels ◽  
Control Of Gene Expression ◽  
Conditional Expression ◽  
Ht 29

Zinc is an essential trace element that serves as a structural constituent of a large number of transcription factors, which explains its pivotal role in the control of gene expression. Previous studies investigating the effect of zinc deficiency and zinc supplementation on gene expression in the human adenocarcinoma cell line HT-29 led to the identification of a considerable number of genes responding to alterations in cellular zinc status with changes in steady state mRNA levels. For 9 of 20 genes from these previous screenings that were studied in more detail, mRNA steady state levels responded to both high and low media zinc concentrations. As they are primarily zinc-dependent, we assessed whether these genes are controlled by the zinc-finger metal transcription factor MTF-1. To test this hypothesis we generated a doxycyline-inducible Tet-On HT-29 cell line overexpressing MTF-1. Using this conditional expression system, we present evidence that Kruppel-like factor 4 (klf4), hepatitis A virus cellular receptor 1 (hhav), and complement factor B (cfbp) are 3 potential new target genes of MTF-1. To support this, we used in silico analysis to screen for metal-responsive elements (MREs) within promotors of zinc-sensitive genes. We conclude that zinc responsiveness of klf4, hhav, and cfbp in HT-29 cells is mediated at least in part by MTF-1.Key words: zinc-sensitive genes, target genes, MTF-1, HT-29 cells, metal-response element.

Subnuclear compartmentalization of sequence-specific transcription factors and regulation of eukaryotic gene expression

2005 ◽  
Vol 83 (4) ◽  
pp. 535-547 ◽  
Author(s):  
Gareth N Corry ◽  
D Alan Underhill
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Transcription Factors ◽  
Transcriptional Activation ◽  
Current Knowledge ◽  
Nuclear Architecture ◽  
Subnuclear Localization ◽  
Modular Structures

To date, the majority of the research regarding eukaryotic transcription factors has focused on characterizing their function primarily through in vitro methods. These studies have revealed that transcription factors are essentially modular structures, containing separate regions that participate in such activities as DNA binding, protein–protein interaction, and transcriptional activation or repression. To fully comprehend the behavior of a given transcription factor, however, these domains must be analyzed in the context of the entire protein, and in certain cases the context of a multiprotein complex. Furthermore, it must be appreciated that transcription factors function in the nucleus, where they must contend with a variety of factors, including the nuclear architecture, chromatin domains, chromosome territories, and cell-cycle-associated processes. Recent examinations of transcription factors in the nucleus have clarified the behavior of these proteins in vivo and have increased our understanding of how gene expression is regulated in eukaryotes. Here, we review the current knowledge regarding sequence-specific transcription factor compartmentalization within the nucleus and discuss its impact on the regulation of such processes as activation or repression of gene expression and interaction with coregulatory factors.Key words: transcription, subnuclear localization, chromatin, gene expression, nuclear architecture.

scholarly journals The AML-associated K313 mutation enhances C/EBPα activity by leading to C/EBPα overexpression

2021 ◽  
Vol 12 (7) ◽  
Author(s):  
Ian Edward Gentle ◽  
Isabel Moelter ◽  
Mohamed Tarek Badr ◽  
Konstanze Döhner ◽  
Michael Lübbert ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Culture ◽  
Transcription Factor ◽  
Transcription Factors ◽  
Transcriptional Activity ◽  
Target Gene ◽  
Wild Type ◽  
Elevated Expression ◽  
Factor C ◽  
Acute Myeloid

AbstractMutations in the transcription factor C/EBPα are found in ~10% of all acute myeloid leukaemia (AML) cases but the contribution of these mutations to leukemogenesis is incompletely understood. We here use a mouse model of granulocyte progenitors expressing conditionally active HoxB8 to assess the cell biological and molecular activity of C/EBPα-mutations associated with human AML. Both N-terminal truncation and C-terminal AML-associated mutations of C/EBPα substantially altered differentiation of progenitors into mature neutrophils in cell culture. Closer analysis of the C/EBPα-K313-duplication showed expansion and prolonged survival of mutant C/EBPα-expressing granulocytes following adoptive transfer into mice. C/EBPα-protein containing the K313-mutation further showed strongly enhanced transcriptional activity compared with the wild-type protein at certain promoters. Analysis of differentially regulated genes in cells overexpressing C/EBPα-K313 indicates a strong correlation with genes regulated by C/EBPα. Analysis of transcription factor enrichment in the differentially regulated genes indicated a strong reliance of SPI1/PU.1, suggesting that despite reduced DNA binding, C/EBPα-K313 is active in regulating target gene expression and acts largely through a network of other transcription factors. Strikingly, the K313 mutation caused strongly elevated expression of C/EBPα-protein, which could also be seen in primary K313 mutated AML blasts, explaining the enhanced C/EBPα activity in K313-expressing cells.

scholarly journals Candida albicans Ferric Reductases Are Differentially Regulated in Response to Distinct Forms of Iron Limitation by the Rim101 and CBF Transcription Factors

2008 ◽  
Vol 7 (7) ◽  
pp. 1168-1179 ◽  
Author(s):  
Yong-Un Baek ◽  
Mingchun Li ◽  
Dana A. Davis
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Candida Albicans ◽  
Transcription Factors ◽  
Iron Acquisition ◽  
Mammalian Host ◽  
Regulatory Sequence ◽  
Ferric Reductase ◽  
Reductase Gene ◽  
Ferric Reductases

ABSTRACT Iron is an essential nutrient that is severely limited in the mammalian host. Candida albicans encodes a family of 15 putative ferric reductases, which are required for iron acquisition and utilization. Despite the central role of ferric reductases in iron acquisition and mobilization, relatively little is known about the regulatory networks that govern ferric reductase gene expression in C. albicans. Here we have demonstrated the differential regulation of two ferric reductases, FRE2 and FRP1, in response to distinct iron-limited environments. FRE2 and FRP1 are both induced in alkaline-pH environments directly by the Rim101 transcription factor. However, FRP1 but not FRE2 is also induced by iron chelation. We have identified a CCAAT motif as the critical regulatory sequence for chelator-mediated induction and have found that the CCAAT binding factor (CBF) is essential for FRP1 expression in iron-limited environments. We found that a hap5Δ/hap5Δ mutant, which disrupts the core DNA binding activity of CBF, is unable to grow under iron-limited conditions. C. albicans encodes three CBF-dependent transcription factors, and we identified the Hap43 protein as the CBF-dependent transcription factor required for iron-limited responses. These studies provide key insights into the regulation of ferric reductase gene expression in the fungal pathogen C. albicans.

Codon replacement in the PGK1 gene of Saccharomyces cerevisiae: experimental approach to study the role of biased codon usage in gene expression

1987 ◽  
Vol 7 (8) ◽  
pp. 2914-2924
Author(s):  
A Hoekema ◽  
R A Kastelein ◽  
M Vasser ◽  
H A de Boer
Keyword(s):  
Gene Expression ◽  
Saccharomyces Cerevisiae ◽  
Steady State ◽  
Codon Usage ◽  
Experimental Approach ◽  
Mrna Translation ◽  
Yeast Cells ◽  
Expression Level ◽  
Start Codon ◽  
Mrna Levels

The coding sequences of genes in the yeast Saccharomyces cerevisiae show a preference for 25 of the 61 possible coding triplets. The degree of this biased codon usage in each gene is positively correlated to its expression level. Highly expressed genes use these 25 major codons almost exclusively. As an experimental approach to studying biased codon usage and its possible role in modulating gene expression, systematic codon replacements were carried out in the highly expressed PGK1 gene. The expression of phosphoglycerate kinase (PGK) was studied both on a high-copy-number plasmid and as a single copy gene integrated into the chromosome. Replacing an increasing number (up to 39% of all codons) of major codons with synonymous minor ones at the 5' end of the coding sequence caused a dramatic decline of the expression level. The PGK protein levels dropped 10-fold. The steady-state mRNA levels also declined, but to a lesser extent (threefold). Our data indicate that this reduction in mRNA levels was due to destabilization caused by impaired translation elongation at the minor codons. By preventing translation of the PGK mRNAs by the introduction of a stop codon 3' and adjacent to the start codon, the steady-state mRNA levels decreased dramatically. We conclude that efficient mRNA translation is required for maintaining mRNA stability in S. cerevisiae. These findings have important implications for the study of the expression of heterologous genes in yeast cells.

scholarly journals Genotype-dependent and non-gradient patterns of RSV gene expression

2019 ◽  
Author(s):  
Felipe-Andrés Piedra ◽  
Xueting Qiu ◽  
Michael N. Teng ◽  
Vasanthi Avadhanula ◽  
Annette A. Machado ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcriptional Regulation ◽  
Steady State ◽  
Transcription Initiation ◽  
Mrna Levels ◽  
Viral Pathogen ◽  
Negative Strand ◽  
Gene Start ◽  
Syncytial Virus ◽  
Conserved Gene

AbstractRespiratory syncytial virus (RSV) is a nonsegmented negative-strand (NNS) RNA virus and a leading cause of severe lower respiratory tract illness in infants and the elderly. Transcription of the ten RSV genes proceeds sequentially from the 3’ promoter and requires conserved gene start (GS) and gene end (GE) signals. Previous studies using the prototypical GA1 genotype Long and A2 strains have indicated a gradient of gene transcription. However, recent reports show data that appear inconsistent with a gradient. To better understand RSV transcriptional regulation, mRNA abundances from five RSV genes were measured by quantitative real-time PCR (qPCR) in three cell lines and cotton rats infected with virus isolates belonging to four different genotypes (GA1, ON, GB1, BA). Relative mRNA levels reached steady-state between four and 24 hours post-infection. Steady-state patterns were genotype-specific and non-gradient, where mRNA levels from the G (attachment) gene exceeded those from the more promoter-proximal N (nucleocapsid) gene across isolates. Transcript stabilities could not account for the non-gradient patterns observed, indicating that relative mRNA levels more strongly reflect transcription than decay. While the GS signal sequences were highly conserved, their alignment with N protein in the helical ribonucleocapsid, i.e., N-phase, was variable, suggesting polymerase recognition of GS signal conformation affects transcription initiation. The effect of GS N-phase on transcription efficiency was tested using dicistronic minigenomes. Ratios of minigenome gene expression showed a switch-like dependence on N-phase with a period of seven nucleotides. Our results indicate that RSV gene expression is in part sculpted by polymerases that initiate transcription with a probability dependent on GS signal N-phase.Author SummaryRSV is a major viral pathogen that causes significant morbidity and mortality, especially in young children. Shortly after RSV enters a host cell, transcription from its nonsegmented negative-strand (NNS) RNA genome starts at the 3’ promoter and proceeds sequentially. Transcriptional attenuation is thought to occur at each gene junction, resulting in a gradient of gene expression. However, recent studies showing non-gradient levels of RSV mRNA suggest that transcriptional regulation may have additional mechanisms. We show using RSV isolates belonging to four different genotypes that gene expression is genotype-dependent and one gene (the G or attachment gene) is consistently more highly expressed than an upstream neighbor. We hypothesize that variable alignment of highly conserved gene start (GS) signals with nucleoprotein (i.e., variable GS N-phase) can affect transcription and give rise to non-gradient patterns of gene expression. We show using dicistronic RSV minigenomes wherein the reporter genes differ only in the N-phase of one GS signal that GS N-phase affects gene expression. Our results suggest the existence of a novel mechanism of transcriptional regulation that might play a role in other NNS RNA viruses.

scholarly journals Single Cell Transcriptomic Analysis of Spinal Dmrt3 Neurons in Zebrafish and Mouse Identifies Distinct Subtypes and Reveal Novel Subpopulations Within the dI6 Domain

2021 ◽  
Vol 15 ◽  
Author(s):  
Ana Belén Iglesias González ◽  
Jon E. T. Jakobsson ◽  
Jennifer Vieillard ◽  
Malin C. Lagerström ◽  
Klas Kullander ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Transcription Factors ◽  
Axon Guidance ◽  
Single Cell ◽  
Cellular Activity ◽  
Electrophysiological Properties ◽  
Related Transcription Factor ◽  
Unique Markers ◽  
Molecular Code

The spinal locomotor network is frequently used for studies into how neuronal circuits are formed and how cellular activity shape behavioral patterns. A population of dI6 interneurons, marked by the Doublesex and mab-3 related transcription factor 3 (Dmrt3), has been shown to participate in the coordination of locomotion and gaits in horses, mice and zebrafish. Analyses of Dmrt3 neurons based on morphology, functionality and the expression of transcription factors have identified different subtypes. Here we analyzed the transcriptomes of individual cells belonging to the Dmrt3 lineage from zebrafish and mice to unravel the molecular code that underlies their subfunctionalization. Indeed, clustering of Dmrt3 neurons based on their gene expression verified known subtypes and revealed novel populations expressing unique markers. Differences in birth order, differential expression of axon guidance genes, neurotransmitters, and their receptors, as well as genes affecting electrophysiological properties, were identified as factors likely underlying diversity. In addition, the comparison between fish and mice populations offers insights into the evolutionary driven subspecialization concomitant with the emergence of limbed locomotion.

Gene expression of human DNA polymerase alpha during cell proliferation and the cell cycle

1988 ◽  
Vol 8 (11) ◽  
pp. 5016-5025
Author(s):  
A F Wahl ◽  
A M Geis ◽  
B H Spain ◽  
S W Wong ◽  
D Korn ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Cell Proliferation ◽  
Steady State ◽  
Dna Polymerase ◽  
Transformed Cells ◽  
Mrna Levels ◽  
Dna Polymerase Alpha ◽  
Human Dna ◽  
Alpha Gene

We studied the expression of the human DNA polymerase alpha gene during cell proliferation, during cell progression through the cell cycle, and in transformed cells compared with normal cells. During the activation of quiescent cells (G0 phase) to proliferate (G1/S phases), the steady-state mRNA levels, rate of synthesis of nascent polymerase protein, and enzymatic activity in vitro exhibited a substantial and concordant increase prior to the peak of in vivo DNA synthesis. In transformed cells, the respective values were amplified greater than 10-fold. In actively growing cells separated into discrete stages of the cell cycle by counterflow elutriation or by mitotic shakeoff, levels of steady-state transcripts, translation rates, and enzymatic activities of polymerase alpha were constitutively and concordantly expressed at all stages of the cell cycle, with only a moderate elevation prior to the S phase and a slight decline in the G2 phase. These findings support the conclusion that the regulation of human DNA polymerase alpha gene expression is at the transcriptional level and strongly suggest that the regulatory mechanisms that are operative during the entrance of a cell into the mitotic cycle are fundamentally different from those that modulate polymerase alpha expression in continuously cycling cells.

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