New Transcriptional Reporters to Quantify and Monitor PPAR? Activity.
Ontology highlight
ABSTRACT:
SUBMITTER: Degrelle SA
PROVIDER: S-EPMC5684601 | biostudies-literature | 2017
REPOSITORIES: biostudies-literature
Publications
New Transcriptional Reporters to Quantify and Monitor PPAR<i>γ</i> Activity.
Degrelle Séverine A SA Shoaito Hussein H Fournier Thierry T
PPAR research 20171101
The peroxisome-proliferator-activated-receptor-<i>γ</i> (PPAR<i>γ</i>) is a member of the nuclear receptor superfamily that plays a critical role in diverse biological processes, including adipogenesis, lipid metabolism, and placental development. To study the activity of PPAR<i>γ</i>, we constructed two new reporter genes: a fluorescent GFP-tagged histone-2B (PPRE-H2B-eGFP) and a secreted nanoluciferase (PPRE-pNL1.3[secNluc]). This study demonstrates their usage to monitor PPAR<i>γ</i> activity ...[more]
Similar Datasets
Project description:PPAR? (peroxisome-proliferator-activated receptor ?) is a master transcription factor involved in adipogenesis through regulating adipocyte-specific gene expression. Recently, lipin1 was found to act as a key factor for adipocyte maturation and maintenance by modulating the C/EBP? (CCAAT/enhancer-binding protein ?) and PPAR? network; however, the precise mechanism by which lipin1 affects the transcriptional activity of PPAR? is largely unknown. The results of the present study show that lipin1 activates PPAR? by releasing co-repressors, NCoR1 (nuclear receptor co-repressor 1) and SMRT (silencing mediator of retinoid and thyroid hormone receptor), from PPAR? in the absence of the ligand rosiglitazone. We also identified a novel lipin1 TAD (transcriptional activation domain), between residues 217 and 399, which is critical for the activation of PPAR?, but not PPAR?. Furthermore, this TAD is unique to lipin1 since this region does not show any homology with the other lipin isoforms, lipin2 and lipin3. The activity of the lipin1 TAD is enhanced by p300 and SRC-1 (steroid receptor co-activator 1), but not by PCAF (p300/CBP-associated factor) and PGC-1? (PPAR co-activator 1?). The physical interaction between lipin1 and PPAR? occurs at the lipin1 C-terminal region from residues 825 to 926, and the VXXLL motif at residue 885 is critical for binding with and the activation of PPAR?. The action of lipin1 as a co-activator of PPAR? enhanced adipocyte differentiation; the TAD and VXXLL motif played critical roles, but the catalytic activity of lipin1 was not directly involved. Collectively, these data suggest that lipin1 functions as a key regulator of PPAR? activity through its ability to release co-repressors and recruit co-activators via a mechanism other than PPAR? activation.
| S-EPMC3690191 | biostudies-literature
Project description:We describe a ribonucleic acid (RNA) reporter system for live-cell imaging of gene expression to detect changes in polymerase II activity on individual promoters in individual cells. The reporters use strings of RNA aptamers that constitute IMAGEtags (Intracellular MultiAptamer GEnetic tags) that can be expressed from a promoter of choice. For imaging, the cells are incubated with their ligands that are separately conjugated with one of the FRET pair, Cy3 and Cy5. The IMAGEtags were expressed in yeast from the GAL1, ADH1 or ACT1 promoters. Transcription from all three promoters was imaged in live cells and transcriptional increases from the GAL1 promoter were observed with time after adding galactose. Expression of the IMAGEtags did not affect cell proliferation or endogenous gene expression. Advantages of this method are that no foreign proteins are produced in the cells that could be toxic or otherwise influence the cellular response as they accumulate, the IMAGEtags are short lived and oxygen is not required to generate their signals. The IMAGEtag RNA reporter system provides a means of tracking changes in transcriptional activity in live cells and in real time.
| S-EPMC4066748 | biostudies-literature
Project description:Cell-to-cell fusion is involved in multiple fundamental biological processes. Prominent examples include osteoclast and giant cell formation, fertilization and skeletal myogenesis which involve macrophage, sperm-egg and myoblast fusion, respectively. Indeed, the importance of cell fusion is underscored by the wide range of homeostatic as well as pathologic processes in which it plays a key role. Therefore, rapid and sensitive systems to trace and measure cell fusion events in various experimental systems are in demand. Here, we introduce a bipartite cell fusion monitoring system based on a genetic switch responsive to the site-specific recombinase FLP. To allow flexible deployment in both dividing as well as non-dividing cell populations, inducer and reporter modules were incorporated in lentivirus vector particles. Moreover, the recombinase-inducible transcription units were designed in such a way as to minimize basal activity and chromosomal position effects in the "off" and "on" states, respectively. The lentivirus vector-based conditional gene expression assay was validated in primary human mesenchymal stem cells and in a differentiation model based on muscle progenitor cells from a Duchenne muscular dystrophy patient using reporter genes compatible with live- and single-cell imaging and with whole population measurements. Using the skeletal muscle cell differentiation model, we showed that the new assay displays low background activity, a 2-log dynamic range, high sensitivity and is amenable to the investigation of cell fusion kinetics. The utility of the bipartite cell fusion monitoring system was underscored by a study on the impact of drug- and RNAi-mediated p38 MAPK inhibition on human myocyte differentiation. Finally, building on the capacity of lentivirus vectors to readily generate transgenic animals the present FLP-inducible system should be adaptable, alone or together with Cre/loxP-based assays, to cell lineage tracing and conditional gene manipulation studies in vivo.
| S-EPMC2880594 | biostudies-literature
Project description:The impact of commensal bacteria in eukaryotic transcriptional regulation has increasingly been demonstrated over the last decades. A multitude of studies have shown direct effects of commensal bacteria from local transcriptional activity to systemic impact. The commensal bacterium Streptococcus salivarius is one of the early bacteria colonizing the oral and gut mucosal surfaces. It has been shown to down-regulate nuclear transcription factor (NF-?B) in human intestinal cells, a central regulator of the host mucosal immune system response to the microbiota. In order to evaluate its impact on a further important transcription factor shown to link metabolism and inflammation in the intestine, namely PPAR? (peroxisome proliferator-activated receptor), we used human intestinal epithelial cell-lines engineered to monitor PPAR? transcriptional activity in response to a wide range of S. salivarius strains. We demonstrated that different strains from this bacterial group share the property to inhibit PPAR? activation independently of the ligand used. First attempts to identify the nature of the active compounds showed that it is a low-molecular-weight, DNase-, proteases- and heat-resistant metabolite secreted by S. salivarius strains. Among PPAR?-targeted metabolic genes, I-FABP and Angptl4 expression levels were dramatically reduced in intestinal epithelial cells exposed to S. salivarius supernatant. Both gene products modulate lipid accumulation in cells and down-regulating their expression might consequently affect host health. Our study shows that species belonging to the salivarius group of streptococci impact both host inflammatory and metabolic regulation suggesting a possible role in the host homeostasis and health.
| S-EPMC4422599 | biostudies-literature
Project description:Sirtuin 3 (SIRT3) is a major regulator of oncometabolism. Indeed, the activity of SIRT3 significantly affects the response to oxidative stress, glycolytic proficiency, and tumorigenic potential of malignant cells. Thus, a system to accurately measure the transcriptional activity of the SIRT3 promoter could facilitate the identification of novel antineoplastic agents or have diagnostic applications. Here, we describe all the steps involved in the development of a luciferase-based reporter system to measure the activation of the human SIRT3 promoter, encompassing the design of appropriate primers, the cloning of the promoter fragment, and its site-directed mutagenesis. We validated this system in human embryonic kidney 293T cells, taking advantage of the renowned ability of the transcription factor estrogen-related receptor ? to transactivate SIRT3. Moreover, here we demonstrate that SIRT3 expression is responsive to rapamycin, a small inhibitor of mammalian target of rapamycin that has been extensively employed as a caloric restriction mimetic. Finally, we provide an overview of the complementary molecular biology techniques that might be employed to further verify the reliability of this system.
| S-EPMC5748892 | biostudies-literature
Project description:Maintenance of metabolic homeostasis is secured by metabolite-sensing systems, which can be overwhelmed by constant macronutrient surplus in obesity. Not only the uptake processes but also the consumption of energy substrates determine the cellular metabolic burden. We herein describe a novel transcriptional system in this context comprised of peroxisome proliferator-activated receptor alpha (PPARα), a master regulator for fatty acid oxidation, and C-terminal binding protein 2 (CtBP2), a metabolite-sensing transcriptional corepressor. CtBP2 interacts with PPARα to repress its activity, and the interaction is enhanced upon binding to malonyl-CoA, a metabolic intermediate increased in tissues in obesity and reported to suppress fatty acid oxidation through inhibition of carnitine palmitoyltransferase 1. In line with our preceding observations that CtBP2 adopts a monomeric configuration upon binding to acyl-CoAs, we determined that mutations in CtBP2 that shift the conformational equilibrium toward monomers increase the interaction between CtBP2 and PPARα. In contrast, metabolic manipulations that reduce malonyl-CoA decreased the formation of the CtBP2-PPARα complex. Consistent with these in vitro findings, we found that the CtBP2-PPARα interaction is accelerated in obese livers while genetic deletion of CtBP2 in the liver causes derepression of PPARα target genes. These findings support our model where CtBP2 exists primarily as a monomer in the metabolic milieu of obesity to repress PPARα, representing a liability in metabolic diseases that can be exploited to develop therapeutic approaches.
| S-EPMC10339064 | biostudies-literature
Project description:Thromboelastography is increasingly utilized in the management of bleeding and thrombotic complications where heparin management remains a cornerstone. This study assessed the feasibility of the cartridge-based TEG ® 6s system (Haemonetics Corp., Braintree, Massachusetts, United States) to monitor and quantify the effect of unfractionated and low-molecular-weight heparin (UFH and LMWH). Blood samples from healthy donors were spiked with UFH ( n = 23; 0-1.0 IU/mL) or LMWH (enoxaparin; n = 22; 0-1.5 IU/mL). Functional fibrinogen maximum amplitude (CFF.MA), RapidTEG activated clotting time (CRT.ACT), and kaolin and kaolin with heparinase reaction time (CK.R and CKH.R) were evaluated for their correlation with heparin concentrations, as well as the combination parameters ΔCK.R - CKH.R, ratio CK.R/CKH.R, and ratio CKH.R/CK.R. Nonlinear mixed-effect modelling was used to study the relationship between concentrations and parameters, and Bayesian classification modelling for the prediction of therapeutic ranges. CK.R and CRT.ACT strongly correlated with the activity of LMWH and UFH ( p < 0.001). Using combination parameters, heparin activity could be accurately quantified in the range of 0.05 to 0.8 IU/mL for UFH and 0.1 to 1.5 IU/mL for LMWH. CRT.ACT was able to quantify heparin activity at higher concentrations but was only different from the reference range ( p < 0.05) at >0.5 IU/mL for UFH and >1.5 IU/mL for LMWH. Combination parameters classified blood samples into subtherapeutic, therapeutic, and supratherapeutic heparin ranges, with an accuracy of >90% for UFH, and >78% for LMWH. This study suggests that TEG 6s can effectively monitor and quantify heparin activity for LMWH and UFH. Additionally, combination parameters can be used to classify blood samples into therapeutic ranges based on heparin activity.
| S-EPMC6742498 | biostudies-literature
Project description:The bacterial Lux system is used as a gene expression reporter. It is fast, sensitive and non-destructive, enabling high frequency measurements. Originally developed for bacterial cells, it has also been adapted for eukaryotic cells, and can be used for whole cell biosensors, or in real time with live animals without the need for euthanasia. However, correct interpretation of bioluminescent data is limited: the bioluminescence is different from gene expression because of nonlinear molecular and enzyme dynamics of the Lux system. We have developed a computational approach that, for the first time, allows users of Lux assays to infer gene transcription levels from the light output. This approach is based upon a new mathematical model for Lux activity, that includes the actions of LuxAB, LuxEC and Fre, with improved mechanisms for all reactions, as well as synthesis and turn-over of Lux proteins. The model is calibrated with new experimental data for the LuxAB and Fre reactions from Photorhabdus luminescens-the source of modern Lux reporters-while literature data has been used for LuxEC. Importantly, the data show clear evidence for previously unreported product inhibition for the LuxAB reaction. Model simulations show that predicted bioluminescent profiles can be very different from changes in gene expression, with transient peaks of light output, very similar to light output seen in some experimental data sets. By incorporating the calibrated model into a Bayesian inference scheme, we can reverse engineer promoter activity from the bioluminescence. We show examples where a decrease in bioluminescence would be better interpreted as a switching off of the promoter, or where an increase in bioluminescence would be better interpreted as a longer period of gene expression. This approach could benefit all users of Lux technology.
| S-EPMC5619816 | biostudies-literature
Project description:The GFP folding reporter assay uses a C-terminal GFP fusion to report on the folding success of upstream fused polypeptides. The GFP folding assay is widely-used for screening protein variants with improved folding and solubility, but truncation artifacts may arise during evolution, i.e. from de novo internal ribosome entry sites. One way to reduce such artifacts would be to insert target genes within the scaffolding of GFP circular permuted variants. Circular permutants of fluorescent proteins often misfold and are non-fluorescent, and do not readily tolerate fused polypeptides within the fluorescent protein scaffolding. To overcome these limitations, and to increase the dynamic range for reporting on protein misfolding, we have created eight GFP insertion reporters with different sensitivities to protein misfolding using chimeras of two previously described GFP variants, the GFP folding reporter and the robustly-folding "superfolder" GFP. We applied this technology to engineer soluble variants of Rv0113, a protein from Mycobacterium tuberculosis initially expressed as inclusion bodies in Escherichia coli. Using GFP insertion reporters with increasing stringency for each cycle of mutagenesis and selection led to a variant that produced large amounts of soluble protein at 37 degrees C in Escherichia coli. The new reporter constructs discriminate against truncation artifacts previously isolated during directed evolution of Rv0113 using the original C-terminal GFP folding reporter. Using GFP insertion reporters with variable stringency should prove useful for engineering protein variants with improved folding and solubility, while reducing the number of artifacts arising from internal cryptic ribosome initiation sites.
| S-EPMC2408556 | biostudies-literature
Project description:CRISPR/Cas9 technologies have been employed for genome editing to achieve gene knockouts and knock-ins in somatic cells. Similarly, certain endogenous genes have been tagged with fluorescent proteins. Often, the detection of tagged proteins requires high expression and sophisticated tools such as confocal microscopy and flow cytometry. Therefore, a simple, sensitive and robust transcriptional reporter system driven by endogenous promoter for studies into transcriptional regulation is desirable. We report a CRISPR/Cas9-based methodology for rapidly integrating a firefly luciferase gene in somatic cells under the control of endogenous promoter, using the TGFβ-responsive gene PAI-1. Our strategy employed a polycistronic cassette containing a non-fused GFP protein to ensure the detection of transgene delivery and rapid isolation of positive clones. We demonstrate that firefly luciferase cDNA can be efficiently delivered downstream of the promoter of the TGFβ-responsive gene PAI-1. Using chemical and genetic regulators of TGFβ signalling, we show that it mimics the transcriptional regulation of endogenous PAI-1 expression. Our unique approach has the potential to expedite studies on transcription of any gene in the context of its native chromatin landscape in somatic cells, allowing for robust high-throughput chemical and genetic screens.
| S-EPMC4413877 | biostudies-literature