Induced gene transcription: implications for biomarkers

1995 ◽  
Vol 41 (12) ◽  
pp. 1829-1834 ◽  
Author(s):  
C H Sewall ◽  
D A Bell ◽  
G C Clark ◽  
A M Tritscher ◽  
D B Tully ◽  
...  
Keyword(s):  
Gene Transcription ◽  
Target Genes ◽  
Northern Analysis ◽  
Critical Event ◽  
Gene Transcript ◽  
Cellular Functions ◽  
Vitro Assay ◽  
Pathological Alteration ◽  
Cytochrome P 450

Abstract Numerous xenobiotics regulate cellular functions by altering transcription of target genes. Use of sensitive and specific biomarkers based on gene transcript levels may help clarify the shape of the dose-response curve in the low-dose region associated with human exposures to environmental concentrations of chemicals. We have quantified gene transcription induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin in both animal models and humans with the use of Northern analysis and PCR-based methods. In addition, we describe a rapid and sensitive in vitro assay that we have used to screen chemicals and biological samples for their ability to alter gene transcription. Whereas some of the endpoints in our studies such as cytochrome P-450 1A1 are predictive indicators of exposure and dose, other gene responses such as growth factors are more complex and represent a critical event, progression, or adaptation to a pathological alteration. In conclusion, measurement of toxicant-induced gene transcription will contribute to the usefulness of biomarkers in addressing issues of human health and environmentally induced disease.

Development of rapid in vitro assay for oxidative liver injury and its application to 230 chemicals

2002 ◽  
Vol 46 (11-12) ◽  
pp. 337-341
Author(s):  
S.H. Kim ◽  
K. Ichikawa ◽  
I. Koshiishi ◽  
H. Utsumi
Keyword(s):  
Cell Injury ◽  
Liver Toxicity ◽  
Ecological Impacts ◽  
Hep G2 ◽  
Cellular Functions ◽  
Vitro Assay ◽  
Synthetic Chemicals ◽  
Liver Cell Injury ◽  
Oxidative Damage To Dna

Water environments are thought to be polluted with thousands of synthetic chemicals and biproducts involving persistent organic pollutants and endocrine disrupters, and their human and ecological impacts are causing serious anxiety. Many bioassays have been undertaken to evaluate the hazardous impacts of toxic chemicals dissolved in water. Reactive Oxygen Species (ROS) are well known to be involved in the toxicity of various chemicals. ROS are mostly generated in liver and cause oxidative damage to DNA, lipids and proteins, resulting in the failure of cellular functions. In order to develop an in vitro bioassay system to estimate ROS induced liver toxicity by chemicals, we investigated the lipid peroxidation, liver cell injury, and genotoxicity using the human hepatocellular carcinoma cell line, Hep-G2 (ATCC HB 8065), and applied it to 230 chemicals.

scholarly journals A Novel Assay for Viral MicroRNA Function Identifies a Single Nucleotide Polymorphism That Affects Drosha Processing

2006 ◽  
Vol 80 (11) ◽  
pp. 5321-5326 ◽  
Author(s):  
Eva Gottwein ◽  
Xuezhong Cai ◽  
Bryan R. Cullen
Keyword(s):  
Single Nucleotide Polymorphism ◽  
Target Genes ◽  
Human Herpesvirus ◽  
Northern Analysis ◽  
Luciferase Expression ◽  
Nucleotide Polymorphism ◽  
Single Nucleotide ◽  
Stem Loop

ABSTRACT MicroRNAs (miRNAs) are a class of ∼22-nucleotide noncoding RNAs that inhibit the expression of specific target genes at the posttranscriptional level. Recently, 11 miRNAs encoded by the pathogenic human herpesvirus Kaposi's sarcoma-associated herpesvirus (KSHV) were cloned from latently infected cells. While the expression of these miRNAs has been confirmed by Northern analysis, their ability to inhibit target gene expression has not been demonstrated. We have devised a novel assay for miRNA function that uses lentiviral indicator vectors carrying two perfectly complementary target sites for each given miRNA in the 3′ untranslated region of the Renilla luciferase gene. This assay allowed us to demonstrate the activity of each viral miRNA upon cotransduction of cells with the Renilla luciferase indicator vector together with a firefly luciferase control vector. In KSHV-infected BC-1 and BCBL-1 cells, but not uninfected control cells, Renilla luciferase expression was selectively reduced up to 10-fold. Interestingly, one of the viral miRNAs (miR-K5) exhibited much higher activity in BC-1 cells than in BCBL-1 cells. Sequence analysis of both viral genomes revealed a single nucleotide polymorphism in the miR-K5 precursor stem-loop, which inhibits the expression of mature miR-K5 in BCBL-1 cells. We show that the primary miR-K5 sequence present in BCBL-1 results in diminished processing by Drosha both in vivo and in vitro. This is the first report of a naturally occurring sequence polymorphism in an miRNA precursor that results in reduced processing and therefore lower levels of mature miRNA expression and function.

scholarly journals Semaphorin 3B Is a 1,25-Dihydroxyvitamin D3-Induced Gene in Osteoblasts that Promotes Osteoclastogenesis and Induces Osteopenia in Mice

2008 ◽  
Vol 22 (6) ◽  
pp. 1370-1381 ◽  
Author(s):  
Amelia L. M. Sutton ◽  
Xiaoxue Zhang ◽  
Diane R. Dowd ◽  
Yogendra P. Kharode ◽  
Barry S. Komm ◽  
...  
Keyword(s):  
Target Genes ◽  
Biological Effects ◽  
Osteoblastic Cells ◽  
Northern Analysis ◽  
Mineral Density ◽  
Dihydroxyvitamin D3 ◽  
1,25 Dihydroxyvitamin D3 ◽  
Skeletal Homeostasis

Abstract The vitamin D endocrine system is important for skeletal homeostasis. 1,25-Dihydroxyvitamin D3 [1,25(OH)2D3] impacts bone indirectly by promoting intestinal absorption of calcium and phosphate and directly by acting on osteoblasts and osteoclasts. Despite the direct actions of 1,25(OH)2D3 in bone, relatively little is known of the mechanisms or target genes that are regulated by 1,25(OH)2D3 in skeletal cells. Here, we identify semaphorin 3B (SEMA3B) as a 1,25(OH)2D3-stimulated gene in osteoblastic cells. Northern analysis revealed strong induction of SEMA3B mRNA by 1,25(OH)2D3 in MG-63, ST-2, MC3T3, and primary osteoblastic cells. Moreover, differentiation of these osteogenic cells enhanced SEMA3B gene expression. Biological effects of SEMA3B in the skeletal system have not been reported. Here, we show that osteoblast-derived SEMA3B alters global skeletal homeostasis in intact animals and osteoblast function in cell culture. Osteoblast-targeted expression of SEMA3B in mice resulted in reduced bone mineral density and aberrant trabecular structure compared with nontransgenic littermates. Histomorphometry studies indicated that this was likely due to increased osteoclast numbers and activity. Indeed, primary osteoblasts obtained from SEMA3B transgenic mice stimulated osteoclastogenesis to a greater extent than nontransgenic osteoblasts. This study establishes that SEMA3B is a 1,25(OH)2D3-induced gene in osteoblasts and that osteoblast-derived SEMA3B impacts skeletal biology in vitro and in vivo. Collectively, these studies support a putative role for SEMA3B as an osteoblast protein that regulates bone mass and skeletal homeostasis.

scholarly journals Selective silencing rather than targeted activation of gene expression underlies fate choice in human hematopoietic stem cells

2020 ◽  
Author(s):  
R Parmentier ◽  
A Moussy ◽  
S Chantalat ◽  
L Racine ◽  
R Sudharshan ◽  
...  
Keyword(s):  
Gene Expression ◽  
Gene Transcription ◽  
Target Genes ◽  
Expression Profiles ◽  
Second Phase ◽  
Gene Promoters ◽  
Human Cord Blood ◽  
Hematopoietic Stem ◽  
Cell Subpopulations

AbstractWhen human cord blood derived CD34+ cells are induced to differentiate in vitro, they undergo rapid and dynamic morphological and molecular transformation that are critical for the fate commitment. Using ATAC-seq and single-cell RNA sequencing, we detected two phases of this process. In the first phase, we observed that a rapid and widespread chromatin opening - that makes most of the gene promoters in the genome accessible - precedes a global upregulation of gene transcription and a concomitant increase in the cell-to-cell variability of gene expression. The second phase is marked by a slow chromatin closure that precedes an overall downregulation of gene transcription and the emergence of coherent expression profiles that characterize distinct cell subpopulations. We further showed that the accessibility of promoters has a crucial effect on whether transcription factor changes will lead to alterations in the expression of their target genes. Our observations are consistent with a model based on the spontaneous probabilistic organization of the cellular process of fate commitment.

Inhibition of nitric oxide synthesis improves detoxication in inflammatory liver dysfunction in vivo

1997 ◽  
Vol 273 (2) ◽  
pp. G530-G536 ◽  
Author(s):  
A. Veihelmann ◽  
T. Brill ◽  
M. Blobner ◽  
I. Scheller ◽  
B. Mayer ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
In Vitro Assays ◽  
Serum Concentrations ◽  
Aminopyrine Breath Test ◽  
Vitro Assay ◽  
Synthase Inhibitor ◽  
Cytochrome P 450 ◽  
Stimulation Of

Inflammatory stimulation of the liver induces nitric oxide (NO) biosynthesis and suppression of detoxication. In this study the effect of NO biosynthesis on cytochrome P-450 (CYP) enzyme activity was investigated by comparing in vivo and in vitro assays. To establish liver inflammation, CD rats were injected with Corynebacterium parvum (C. parvum) suspension. After 5 days NO biosynthesis was highly induced as indicated by increased NO2- plus NO3- serum concentrations. At the same time the aminopyrine breath test (ABT), measuring CYP activity in vivo, was reduced to 42% and the in vitro assay of aminopyrine turnover was suppressed to 12% of NaCl- injected controls. When C. parvum-injected animals were treated with the NO synthase inhibitor NG-monomethyl-L-arginine (L-NMMA), CYP activities significantly improved with an ABT of 76% and an in vitro aminopyrine turnover of 47% of controls. Neither C. parvum injections nor L-NMMA treatment resulted in a significant change of CYP protein concentrations. These data indicate that suppression of xenobiotic metabolism can be attenuated by inhibition of NO biosynthesis during an ongoing process of inflammation.

scholarly journals Ehrlichia chaffeensis TRP32 Is a Nucleomodulin That Directly Regulates Expression of Host Genes Governing Differentiation and Proliferation

2016 ◽  
Vol 84 (11) ◽  
pp. 3182-3194 ◽  
Author(s):  
Tierra R. Farris ◽  
Paige S. Dunphy ◽  
Bing Zhu ◽  
Clayton E. Kibler ◽  
Jere W. McBride
Keyword(s):  
Dna Binding ◽  
Host Cell ◽  
Gene Transcription ◽  
Noncoding Rna ◽  
Target Genes ◽  
Immune Cell ◽  
Luciferase Reporter ◽  
Ehrlichia Chaffeensis ◽  
Content Type

Ehrlichia chaffeensis is an obligately intracellular bacterium that reprograms the mononuclear phagocyte through diverse effector-host interactions to modulate numerous host cell processes, including transcription. In a previous study, we reported that E. chaffeensis TRP32, a type 1 secreted effector, interacts with multiple host nucleus-associated proteins and also autoactivates reporter gene expression in yeast. In this study, we demonstrate that TRP32 is a nucleomodulin that binds host DNA and alters host gene transcription. TRP32 enters the host cell nucleus via a noncanonical translocation mechanism that involves phosphorylation of Y179 located in a C-terminal trityrosine motif. Both genistein and mutation of Y179 inhibited TRP32 nuclear entry. An electromobility shift assay (EMSA) demonstrated TRP32 host DNA binding via its tandem repeat domain. TRP32 DNA-binding and motif preference were further confirmed by supershift assays, as well as competition and mutant probe analyses. Using chromatin immunoprecipitation with next-generation sequencing (ChIP-seq), we determined that TRP32 binds a G-rich motif primarily within ±500 bp of the gene transcription start site. An ontology analysis identified genes involved in processes such as immune cell differentiation, chromatin remodeling, and RNA transcription and processing as primary TRP32 targets. TRP32-bound genes ( n = 1,223) were distributed on all chromosomes and included several global regulators of proliferation and inflammation such as those encoding FOS, JUN, AKT3, and NRAS and noncoding RNA genes microRNA 21 (miRNA 21) and miRNA 142. TRP32 target genes were differentially regulated during infection, the majority of which were repressed, and direct repression/activation of these genes by TRP32 was confirmed in vitro with a cellular luciferase reporter assay.

scholarly journals The ETS Transcription Factor GABPα Is Essential for Early Embryogenesis

2004 ◽  
Vol 24 (13) ◽  
pp. 5844-5849 ◽  
Author(s):  
Sika Ristevski ◽  
Debra A. O'Leary ◽  
Anders P. Thornell ◽  
Michael J. Owen ◽  
Ismail Kola ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Target Genes ◽  
Complex Function ◽  
Embryonic Stem ◽  
Transactivation Domain ◽  
Cellular Functions ◽  
Protein Levels ◽  
Ets Transcription Factor

ABSTRACT The ETS transcription factor complex GABP consists of the GABPα protein, containing an ETS DNA binding domain, and an unrelated GABPβ protein, containing a transactivation domain and nuclear localization signal. GABP has been shown in vitro to regulate the expression of nuclear genes involved in mitochondrial respiration and neuromuscular signaling. We investigated the in vivo function of GABP by generating a null mutation in the murine Gabpα gene. Embryos homozygous for the null Gabpα allele die prior to implantation, consistent with the broad expression of Gabpα throughout embryogenesis and in embryonic stem cells. Gabpα+/− mice demonstrated no detectable phenotype and unaltered protein levels in the panel of tissues examined. This indicates that Gabpα protein levels are tightly regulated to protect cells from the effects of loss of Gabp complex function. These results show that Gabpα function is essential and is not compensated for by other ETS transcription factors in the mouse, and they are consistent with a specific requirement for Gabp expression for the maintenance of target genes involved in essential mitochondrial cellular functions during early cleavage events of the embryo.

scholarly journals A Cell-Free Assay System for β-Catenin Signaling That Recapitulates Direct Inductive Events in the Early Xenopus laevis Embryo

1999 ◽  
Vol 147 (2) ◽  
pp. 367-374 ◽  
Author(s):  
Richard W. Nelson ◽  
Barry M. Gumbiner
Keyword(s):  
Protein Synthesis ◽  
Xenopus Laevis ◽  
Signaling Pathway ◽  
Target Genes ◽  
Degradation Pathway ◽  
Activation Mechanism ◽  
Independent Manner ◽  
Vitro Assay ◽  
A Cell

In vertebrate embryos, signaling via the β-catenin protein is known to play an essential role in the induction of the dorsal axis. In its signaling capacity, β-catenin acts directly to affect target gene transcription, in concert with transcription factors of the TCF/LEF family. We have developed a cell-free in vitro assay for β-catenin signaling activity that utilizes transcriptionally active nuclei and cytoplasm from cleavage-blocked Xenopus laevis embryos. Under these assay conditions, we demonstrate that either addition of β-catenin protein or upstream activation of the β-catenin signaling pathway can induce the expression of developmentally relevant target genes. Addition of exogenous β-catenin protein induced expression of Siamois, XTwin, Xnr3, and Cerberus mRNAs in a protein synthesis independent manner, whereas a panel of other Spemann organizer-specific genes did not respond to β-catenin. Lithium induction of the β-catenin signaling pathway, which is thought to cause β-catenin accumulation by inhibiting its proteasome-dependent degradation, caused increased expression of Siamois in a protein synthesis independent fashion. This result suggests that β-catenin derived from a preexisting pool can be activated to signal, and that accumulation of this activated form does not require ongoing synthesis. Furthermore, activation of the signaling pathway with lithium did not detectably alter cytoplasmic β-catenin levels and was insensitive to inhibition of the proteasome- dependent degradation pathway. Taken together, these results suggest that activation of β-catenin signaling by lithium in this system may occur through a distinct activation mechanism that does not require modulation of levels through regulation of proteasomal degradation.

scholarly journals Identification of sarV (SA2062), a New Transcriptional Regulator, Is Repressed by SarA and MgrA (SA0641) and Involved in the Regulation of Autolysis in Staphylococcus aureus

2004 ◽  
Vol 186 (16) ◽  
pp. 5267-5280 ◽  
Author(s):  
Adhar C. Manna ◽  
Susham S. Ingavale ◽  
MaryBeth Maloney ◽  
Willem van Wamel ◽  
Ambrose L. Cheung
Keyword(s):  
Staphylococcus Aureus ◽  
Target Genes ◽  
Transcriptional Regulator ◽  
Analysis Data ◽  
Hydrolase Activity ◽  
Northern Analysis ◽  
Wild Type ◽  
Expression Of Genes ◽  
Murein Hydrolase

ABSTRACT The expression of genes involved in the pathogenesis of Staphylococcus aureus is known to be controlled by global regulatory loci, including agr, sarA, sae, arlRS, lytSR, and sarA-like genes. Here we described a novel transcriptional regulator called sarV of the SarA protein family. The transcription of sarV is low or undetectable under in vitro conditions but is significantly augmented in sarA and mgrA (norR or rat) (SA0641) mutants. The sarA and mgrA genes act as repressors of sarV expression, as confirmed by transcriptional fusion and Northern analysis data. Purified SarA and MgrA proteins bound specifically to separate regions of the sarV promoter as determined by gel shift and DNase I footprinting assays. The expression of 19 potential target genes involved in autolysis and virulence, phenotypes affected by sarA and mgrA, was evaluated in an isogenic sarV mutant pair. Our data indicated that the sarV gene product played a role regulating some virulence genes and more genes involved in autolysis. The sarV mutant was more resistant to Triton X-100 and penicillin-induced lysis compared to the wild type and the sarA mutant, whereas hyperexpression of sarV in the parental strain or the sarV mutant rendered the resultant strain highly susceptible to lysis. Zymographic analysis of murein hydrolase activity revealed that inactivation of the sarV gene results in decreased extracellular murein hydrolase activity compared to that of wild-type S. aureus. We propose that sarV may be part of the common pathway by which mgrA and sarA gene products control autolysis in S. aureus.

scholarly journals MiR-193a-3p is an Important Tumour Suppressor in Lung Cancer and Directly Targets KRAS

2017 ◽  
Vol 44 (4) ◽  
pp. 1311-1324 ◽  
Author(s):  
Qian Fan ◽  
Xiuting Hu ◽  
Haiyang Zhang ◽  
Shengguang Wang ◽  
Huilai Zhang ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Cell Migration ◽  
Target Genes ◽  
Biological Effects ◽  
Tumour Suppressor ◽  
Luciferase Reporter ◽  
Cellular Functions ◽  
Reporter Assays

Background/Aims: MicroRNAs (miRNAs) have emerged as major regulators of tumour development and progression in non-small cell lung cancer (NSCLC). However, the role of miR-193a-3p in NSCLC is still unclear. Methods: Quantitative RT-PCR was used to detect miR-193a-3p expression levels in NSCLC tumour tissues. CCK8, EdU and cell migration assays were performed to analyse the biological functions of miR-193a-3p in NSCLC cells. Luciferase reporter assays were used to validate the bioinformatics-predicted target genes of miR-193a-3p. Western blotting and RNA/DNA interference carried out to evaluate the association between miR-193a-3p and KRAS. Results: miR-193a-3p expression was decreased in the NSCLC tumour tissues. We investigated the biological effects of miR-193a-3p both in vivo and in vitro and found that enforced expression of miR-193a-3p inhibited tumour formation and suppressed cell proliferation and cell migration. KRAS was found to be a potential target of miR-193a-3p, and dual luciferase reporter assays showed that miR-193a-3p directly binds to the 3’-untranslated region (3’-UTR) of KRAS mRNA. In addition, we found that changing the expression of KRAS had the opposite results to those induced by miR-193a-3p in the NSCLC cells. Importantly, simultaneous overexpression of miR-193a-3p and KRAS could counteract the effects of both on cellular functions. Conclusion: These findings highlight an important role for miR-193a-3p as a tumour suppressor in NSCLC pathogenesis via the regulation of KRAS expression.

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