77 results on '"Upstream activating sequence"'
Search Results
2. Synthetic Promoters from Strawberry Vein Banding Virus (SVBV) and Dahlia Mosaic Virus (DaMV)
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Jeky Chanwala, I. Sriram Sandeep, Badrinath Khadanga, and Nrisingha Dey
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0106 biological sciences ,Staphylococcus aureus ,Agrobacterium ,Nicotiana tabacum ,Molecular Farming ,Gene Expression ,Bioengineering ,Microbial Sensitivity Tests ,01 natural sciences ,Applied Microbiology and Biotechnology ,Biochemistry ,Defensins ,Viral Proteins ,03 medical and health sciences ,Upstream activating sequence ,Transformation, Genetic ,Ascomycota ,Plant Growth Regulators ,Caulimovirus ,010608 biotechnology ,Tobacco ,Exigua ,Humans ,Transgenes ,Promoter Regions, Genetic ,Molecular Biology ,Gene ,Plant Proteins ,030304 developmental biology ,0303 health sciences ,biology ,Chemistry ,Alternaria ,Promoter ,Phoma exigua ,biology.organism_classification ,Molecular biology ,Plant Leaves ,Agrobacterium tumefaciens ,Strawberry vein banding virus ,Pseudomonas aeruginosa ,Salicylic Acid ,Abscisic Acid ,Biotechnology - Abstract
We have constructed two intra-molecularly shuffled promoters, namely S100 and D100. The S100 recombinant promoter (621 bp) was generated by ligation of 250 bp long upstream activation sequence (UAS) of Strawberry vein banding virus (SV10UAS; − 352 to − 102 relative to TSS) with its 371 bp long TATA containing core promoter domain (SV10CP; − 352 to + 19). Likewise, 726 bp long D100 promoter was constructed by fusion of 170 bp long UAS of Dahlia mosaic virus (DaMV14UAS; − 203 to − 33) with its 556 bp long core promoter domain (DaMV4CP; − 474 to + 82). S100 and D100 promoters showed 1.8 and 2.2 times stronger activities than that of the CaMV35S promoter. The activity of the promoters is comparable to that of the CaMV35S2 promoter. Transcript analysis employing qRT-PCR and histochemical assays supported the above findings. Abscisic acid and salicylic acid induce the activity of the D100 promoter. Leaf protein obtained from Nicotiana tabacum plant expressing NSD2 gene (Nigella sativa L. defensin 2) driven by the D100 promoter showed antifungal activity against Alternaria alternata and Phoma exigua var. exigua and antibacterial activity against Pseudomonas aeruginosa and Staphylococcus aureus. Strong S100 and D100 promoters have potential to become efficient candidates for plant metabolic engineering and molecular pharming.
- Published
- 2021
3. Dynamic visualization of mRNA splicing variants with a transactivating reporter
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Haifeng Zheng, Si Chen, Wenjie Shu, Miao Li, Zhe Ma, and Fu Wang
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Reporter gene ,RNA Splicing ,Metals and Alloys ,Intron ,General Chemistry ,Biology ,Fusion protein ,Catalysis ,Surfaces, Coatings and Films ,Electronic, Optical and Magnetic Materials ,Cell biology ,Upstream activating sequence ,Dynamic visualization ,RNA splicing ,Trans-Activators ,Materials Chemistry ,Ceramics and Composites ,Humans ,Luciferase ,RNA, Messenger - Abstract
Dynamic changes in intron sequences, with their loss and gain, are poorly detected due to the limited methods for the non-invasive monitoring of the pre-mRNA splicing process. Here, we describe the design of a two-step transcriptional activation (TSTA) reporter for the real-time imaging of the splicing process in living subjects. By taking advantage of the strong transactivating properties of the GAL4-VP16 fusion protein, which can target upstream activation sequence (UAS) elements to boost subsequent firefly luciferase reporter gene expression, we successfully and consistently detected the dynamic pre-mRNA splicing activity in response to exogenous splicing modulators in living cells and animals. Our findings provide a valuable tool for the high-throughput screening of splicing modulators, which could speed up the development of new drugs for the treatment of disordered splicing diseases.
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- 2021
4. Construction and characterization of a zinc‐inducible gene expression vector in fission yeast
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Yota Murakami, Takahiro Asanuma, Shinya Takahata, and Miyuki Mori
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0106 biological sciences ,TATA box ,Genes, Fungal ,Genetic Vectors ,Bioengineering ,Biology ,01 natural sciences ,Applied Microbiology and Biotechnology ,Biochemistry ,03 medical and health sciences ,Upstream activating sequence ,Plasmid ,Gene Expression Regulation, Fungal ,010608 biotechnology ,Schizosaccharomyces ,Gene expression ,Genetics ,Gene ,030304 developmental biology ,0303 health sciences ,Expression vector ,Promoter ,Yeast ,Cell biology ,Zinc ,Genetic Engineering ,Plasmids ,Biotechnology - Abstract
Gene expression vectors are useful and important tools that are commonly used in a variety of experiments, including expression of foreign genes, functional analysis of genes of interest and complementation experiments. In this study, a hybrid promoter, combining the adh1+ upstream activating sequence (UAS) of fission yeast and the GAL10 core promoter of budding yeast, was constructed to enable high level expression depending on the presence of zinc in culture medium for fission yeast. When the hybrid promoter was cloned on the multicopy plasmid, it was fully induced and repressed within 10 h in the presence and absence of zinc, respectively. The kinetics of induction and reduction were similar to those of the endogenous adh1+ mRNA. In contrast, native adh1+ promoter lost its tight repression in zinc-depleted condition when it was cloned on the plasmid. Because adh1+ UAS-specific transcription factors have not yet been identified, we identified UAS elements involved in zinc sensing by characterizing this hybrid promoter. We also found that the expression level increased by the TATA box mutation, GATAA, in the presence of zinc.
- Published
- 2020
5. New inducible promoter for gene expression and synthetic biology in Yarrowia lipolytica.
- Author
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Trassaert, Marion, Vandermies, Marie, Carly, Fréderic, Denies, Olivia, Thomas, Stéphane, Fickers, Patrick, and Nicaud, Jean-Marc
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PROMOTERS (Genetics) , *GENE expression , *YEAST , *RECOMBINANT proteins , *PROTEIN expression , *OLEIC acid , *SYNTHETIC biology - Abstract
Background: The oleaginous yeast Yarrowia lipolytica is increasingly used as alternative cell factory for the production of recombinant proteins. At present, several promoters with different strengths have been developed based either on the constitutive pTEF promoter or on oleic acid inducible promoters such as pPOX2 and pLIP2. Although these promoters are highly efficient, there is still a lack of versatile inducible promoters for gene expression in Y. lipolytica. Results: We have isolated and characterized the promoter of the EYK1 gene coding for an erythrulose kinase. pEYK1 induction was found to be impaired in media supplemented with glucose and glycerol, while the presence of erythritol and erythrulose strongly increased the promoter induction level. Promoter characterization and mutagenesis allowed the identification of the upstream activating sequence UAS1EYK1. New hybrid promoters containing tandem repeats of either UAS1XPR2 or UAS1EYK1 were developed showing higher expression levels than the native pEYK1 promoter. Furthermore, promoter strength was improved in a strain carrying a deletion in the EYK1 gene, allowing thus the utilization of erythritol and erythrulose as free inducer. Conclusions: Novel tunable and regulated promoters with applications in the field of heterologous protein production, metabolic engineering, and synthetic biology have been developed, thus filling the gap of the absence of versatile inducible promoter in the yeast Y. lipolytica. [ABSTRACT FROM AUTHOR]
- Published
- 2017
- Full Text
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6. Expanding Toolbox for Genes Expression of Yarrowia lipolytica to Include Novel Inducible, Repressible, and Hybrid Promoters
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Xiaochao Xiong and Shulin Chen
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0106 biological sciences ,0303 health sciences ,biology ,Chemistry ,Biomedical Engineering ,Yarrowia ,Promoter ,General Medicine ,Computational biology ,biology.organism_classification ,01 natural sciences ,Biochemistry, Genetics and Molecular Biology (miscellaneous) ,Yeast ,Metabolic engineering ,03 medical and health sciences ,Upstream activating sequence ,Synthetic biology ,010608 biotechnology ,Gene expression ,Gene ,030304 developmental biology - Abstract
Promoters are critical tools to precisely control gene expression for both synthetic biology and metabolic engineering. Although Yarrowia lipolytica has demonstrated many industrially relevant advantages, promoter discovery efforts on this non-conventional yeast are limited due to the challenge in finding suitable inducible and repressible promoters. Six copper-inducible promoters and five repressible promoters were isolated in this work. Especially, Cu2+-repressible promoters showed relatively high activity under non-repressing conditions compared with a constitutive promoter, but the strength could be almost fully repressed by a supplement of a low content of Cu2+. The six Cu2+-inducible promoters were engineered to improve their dynamic regulation range with a tandem upstream activation sequence. An engineered promoter was successfully used to construct a more productive pathway for production of a novel bioproduct, wax ester, than that used for both Cu2+-inducible promoter and constitutive promoter. This study provides effective tools applicable to fine-tune the gene expression in this microbial host.
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- 2020
7. Synthetic control devices for gene regulation in Penicillium chrysogenum
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Zsófia Büttel, Yvonne Nygård, László Mózsik, Roel A. L. Bovenberg, Arnold J. M. Driessen, and Molecular Microbiology
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Models, Molecular ,STRAIN ,Genes, Fungal ,lcsh:QR1-502 ,PROTEIN ,Bioengineering ,ORGANIZATION ,Penicillium chrysogenum ,Applied Microbiology and Biotechnology ,lcsh:Microbiology ,Neurospora crassa ,Fungal Proteins ,03 medical and health sciences ,Upstream activating sequence ,CORE PROMOTERS ,TET-ON ,Gene Expression Regulation, Fungal ,Gene cluster ,Transgenes ,Secondary metabolite production ,Hybrid transcription factor ,Gene ,Transcription factor ,030304 developmental biology ,Regulation of gene expression ,0303 health sciences ,biology ,Organisms, Genetically Modified ,PARTS ,030306 microbiology ,Chemistry ,Research ,EXPRESSION SYSTEM ,CLUSTER ,DNA-binding domain ,biology.organism_classification ,Q-SYSTEM ,Biosynthetic Pathways ,Gene regulation ,Synthetic expression system ,Biochemistry ,Synthetic gene cluster ,Biotechnology ,Transcription Factors - Abstract
BackgroundOrthogonal, synthetic control devices were developed forPenicillium chrysogenum, a model filamentous fungus and industrially relevant cell factory. In the synthetic transcription factor, the QF DNA-binding domain of the transcription factor of the quinic acid gene cluster ofNeurospora crassais fused to the VP16 activation domain. This synthetic transcription factor controls the expression of genes under a synthetic promoter containing quinic acid upstream activating sequence (QUAS) elements, where it binds. A gene cluster may demand an expression tuned individually for each gene, which is a great advantage provided by this system.ResultsThe control devices were characterized with respect to three of their main components: expression of the synthetic transcription factors, upstream activating sequences, and the affinity of the DNA binding domain of the transcription factor to the upstream activating domain. This resulted in synthetic expression devices, with an expression ranging from hardly detectable to a level similar to that of highest expressed native genes. The versatility of the control device was demonstrated by fluorescent reporters and its application was confirmed by synthetically controlling the production of penicillin.ConclusionsThe characterization of the control devices in microbioreactors, proved to give excellent indications for how the devices function in production strains and conditions. We anticipate that these well-characterized and robustly performing control devices can be widely applied for the production of secondary metabolites and other compounds in filamentous fungi.
- Published
- 2019
8. Measuring the buffering capacity of gene silencing in Saccharomyces cerevisiae
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Rohinton T. Kamakaka, Kenneth Wu, Kelvin Du, and Namrita Dhillon
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Upstream activating sequence ,Multidisciplinary ,Histone ,biology ,Chemistry ,biology.protein ,Nucleosome ,Gene silencing ,Epigenetics ,Enhancer ,Gene dosage ,Cell biology ,Chromatin - Abstract
Gene silencing in budding yeast is mediated by Sir protein binding to unacetylated nucleosomes to form a chromatin structure that inhibits transcription. Transcriptional silencing is characterized by the high-fidelity transmission of the silent state. Despite its relative stability, the constituent parts of the silent state are in constant flux, giving rise to a model that silent loci can tolerate such fluctuations without functional consequences. However, the level of tolerance is unknown, and we developed methods to measure the threshold of histone acetylation that causes the silent chromatin state to switch to the active state as well as to measure the levels of the enzymes and structural proteins necessary for silencing. We show that loss of silencing required 50 to 75% acetyl-mimic histones, though the precise levels were influenced by silencer strength and upstream activating sequence (UAS) enhancer/promoter strength. Measurements of repressor protein levels necessary for silencing showed that reducing SIR4 gene dosage two- to threefold significantly weakened silencing, though reducing the gene copy numbers for Sir2 or Sir3 to the same extent did not significantly affect silencing suggesting that Sir4 was a limiting component in gene silencing. Calculations suggest that a mere twofold reduction in the ability of acetyltransferases to acetylate nucleosomes across a large array of nucleosomes may be sufficient to generate a transcriptionally silent domain.
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- 2021
9. Cytokine exposure mediates transcriptional activation of the orphan nuclear receptor Nur77 in hematopoietic cells
- Author
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Orsola di Martino, Margaret A. Ferris, Haixia Niu, Gayla Hadwiger, and John S. Welch
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Transcriptional Activation ,Nerve growth factor IB ,MWCO, molecular weight cutoff ,medicine.medical_treatment ,NR4A1 ,Biochemistry ,Cell Line ,TFA, trifluoroacetic acid ,Transactivation ,Upstream activating sequence ,Mice ,MeCN, acetonitrile ,Granulocyte Colony-Stimulating Factor ,Transcriptional regulation ,medicine ,Nuclear Receptor Subfamily 4, Group A, Member 1 ,Animals ,Humans ,nuclear receptor ,mass spectrometry (MS) ,Molecular Biology ,Janus Kinases ,FA, formic acid ,Chemistry ,Kinase ,phosphorylation ,TOR Serine-Threonine Kinases ,IL-3 ,Nur77 mutants ,nano-LC-MS, capillary liquid chromatography interfaced to a mass spectrometer ,Cell Biology ,G-SCF ,Hematopoietic Stem Cells ,MS1, mass spectra of peptide precursors ,Cell biology ,TCEP, Tris (2-carboxyethyl) phosphine ,Cytokine ,Nuclear receptor ,DTT, dithiothreitol ,HCD, high-energy collision-induced dissociation ,Interleukin-3 ,Signal transduction ,transcription regulation ,MS2, fragmentation mass spectrum of peptide from precursor ion ,signal transduction ,Research Article ,proximity labeling - Abstract
The orphan nuclear receptor Nur77 is an immediate-early response gene that based on tissue and cell context is implicated in a plethora of cellular processes, including proliferation, differentiation, apoptosis, metabolism, and inflammation. Nur77 has a ligand-binding pocket that is obstructed by hydrophobic side groups. Naturally occurring, cell-endogenous ligands have not been identified, and Nur77 transcriptional activity is thought to be regulated through posttranslational modification and modulation of protein levels. To determine whether Nur77 is transcriptionally active in hematopoietic cells in vivo, we used an upstream activating sequence (UAS)-GFP transgenic reporter. We found that Nur77 is transcriptionally inactive in vivo in hematopoietic cells under basal conditions, but that activation occurs following cytokine exposure by G-CSF or IL-3. We also identified a series of serine residues required for cytokine-dependent transactivation of Nur77. Moreover, a kinase inhibitor library screen and proximity labeling-based mass spectrometry identified overlapping kinase pathways that physically interacted with Nur77 and whose inhibition abrogated cytokine-induced activation of Nur77. We determined that transcriptional activation of Nur77 by G-CSF or IL-3 requires functional JAK and mTor signaling since their inhibition leads to Nur77 transcriptional inactivation. Thus, intracellular cytokine signaling networks appear to regulate Nur77 transcriptional activity in mouse hematopoietic cells.
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- 2021
10. The Cdk8 kinase module regulates interaction of the mediator complex with RNA polymerase II
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Alexandra Stuetzer, Henning Urlaub, Fanni Laura Bazsó, Patrick Cramer, Eusra Mohammad, Sara Osman, Michael Lidschreiber, and Kerstin C. Maier
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0301 basic medicine ,Models, Molecular ,TF, transcription factor ,CTD, C-terminal repeat domain ,RNA polymerase II ,DMSO, dimethyl sulfoxide ,Biochemistry ,TBP, TATA box–binding protein ,Protein Interaction Maps ,transcription initiation ,4tU, 4-thiouracil ,Mediator Complex ,biology ,Chemistry ,Cell biology ,PI, protease inhibitor cocktail ,RNA Polymerase II ,Protein Binding ,Research Article ,CKM, Cdk8 kinase module ,Saccharomyces cerevisiae Proteins ,FDR, false discovery rate ,gene activation ,ITA, immobilized template assay ,Saccharomyces cerevisiae ,pol II, RNA polymerase II ,β-ME, β-mecaptoethanol ,PIC, preinitiation complex ,03 medical and health sciences ,Upstream activating sequence ,Mediator ,BS3, bis(sulfo)succinimidyl suberate ,LDS, lithium dodecyl sulfate ,cMed, core mediator ,Kinase activity ,Molecular Biology ,Transcription factor ,030102 biochemistry & molecular biology ,TATA-Box Binding Protein ,Cell Biology ,Cyclin-Dependent Kinase 8 ,1-NA-PP1, 1-Naphthyl-PP1 ,030104 developmental biology ,Cdk8 kinase module ,MBP, maltose binding protein ,Transcription preinitiation complex ,biology.protein ,Cyclin-dependent kinase 8 ,TSS, transcription start site ,UAS, upstream activation sequence ,mediator ,gene regulation - Abstract
The Cdk8 kinase module (CKM) is a dissociable part of the coactivator complex mediator, which regulates gene transcription by RNA polymerase II. The CKM has both negative and positive functions in gene transcription that remain poorly understood at the mechanistic level. In order to reconstitute the role of the CKM in transcription initiation, we prepared recombinant CKM from the yeast Saccharomyces cerevisiae. We showed that CKM bound to the core mediator (cMed) complex, sterically inhibiting cMed from binding to the polymerase II preinitiation complex (PIC) in vitro. We further showed that the Cdk8 kinase activity of the CKM weakened CKM–cMed interaction, thereby facilitating dissociation of the CKM and enabling mediator to bind the PIC in order to stimulate transcription initiation. Finally, we report that the kinase activity of Cdk8 is required for gene activation during the stressful condition of heat shock in vivo but not under steady-state growth conditions. Based on these results, we propose a model in which the CKM negatively regulates mediator function at upstream-activating sequences by preventing mediator binding to the PIC at the gene promoter. However, during gene activation in response to stress, the Cdk8 kinase activity of the CKM may release mediator and allow its binding to the PIC, thereby accounting for the positive function of CKM. This may impart improved adaptability to stress by allowing a rapid transcriptional response to environmental changes, and we speculate that a similar mechanism in metazoans may allow the precise timing of developmental transcription programs.
- Published
- 2021
11. Improved Tol2-mediated enhancer trap identifies weakly expressed genes during liver and β cell development and regeneration in zebrafish
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Wei Huang, Jiang Du, Yadong Zhong, Lingfei Luo, Zekun Wang, and Jianbo He
- Subjects
0301 basic medicine ,Cell type ,Biochemistry ,03 medical and health sciences ,Upstream activating sequence ,Animals ,Enhancer trap ,Enhancer ,Molecular Biology ,Zebrafish ,B-Lymphocytes ,030102 biochemistry & molecular biology ,biology ,Cell growth ,Promoter ,Cell Biology ,biology.organism_classification ,Cell biology ,Enhancer Elements, Genetic ,030104 developmental biology ,Gene Expression Regulation ,Liver ,DNA Transposable Elements ,Hepatocytes ,Cre-Lox recombination ,Developmental Biology - Abstract
The liver and pancreas are two major digestive organs, and among the different cell types in them, hepatocytes and the insulin-producing β cells have roles in both health and diseases. Accordingly, clinicians and researchers are very interested in the mechanisms underlying the development and regeneration of liver and pancreatic β cells. Gene and enhancer traps such as the Tol2 transposon-based system are useful for identifying genes potentially involved in developmental processes in the zebrafish model. Here, we developed a strategy that combines a Tol2-mediated enhancer trap and the Cre/loxP system by using loxP-flanked reporters driven by β cell– or hepatocyte-specific promoters and the upstream activating sequence (UAS)-driving Cre. Two double-transgenic reporter lines, Tg(ins:loxP-CFPNTR-loxP-DsRed; 10×UAS:Cre, cryaa:Venus) and Tg(fabp10:loxP-CFPNTR-loxP-DsRed; 10×UAS:Cre, cryaa:Venus), were established to label pancreatic β cells and hepatocytes, respectively. These two double-transgenic lines were each crossed with the Tol2-enhancer trap founder lines to screen for and identify genes expressed in the β cell and hepatocytes during development. This trap system coupled with application of nitroreductase (NTR)/metronidazole (Mtz)–mediated cell ablation could identify genes expressed during regeneration. Of note, pilot enhancer traps captured transiently and weakly expressed genes such as rab3da and ensab with higher efficiencies than traditional enhancer trap systems. In conclusion, through permanent genetic labeling by Cre/loxP, this improved Tol2-mediated enhancer trap system provides a promising method to identify transiently or weakly expressed, but potentially important, genes during development and regeneration.
- Published
- 2019
12. A Synthetic Oxygen Sensor for Plants Based on Animal Hypoxia Signaling
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Francesco Cardarelli, Lorenzo Cupellini, Luca Piccinini, Beatrice Giuntoli, Francesco Licausi, Pierdomenico Perata, Sandro Jurinovich, Benedetta Mennucci, Sergio Iacopino, Iacopino, Sergio, Jurinovich, Sandro, Cupellini, Lorenzo, Piccinini, Luca, Cardarelli, Francesco, Perata, Pierdomenico, Mennucci, Benedetta, Giuntoli, Beatrice, and Licausi, Francesco
- Subjects
0106 biological sciences ,hypoxia, prolyl hydroxylases, Gal4, plant synthetic biology, genetically encoded reporters ,plant synthetic biology ,Physiology ,Research Articles - Focus Issue ,Arabidopsis ,Biosensing Techniques ,Plant Science ,Hydroxylation ,01 natural sciences ,prolyl hydroxylases ,Synthetic biology ,Upstream activating sequence ,Gene Expression Regulation, Plant ,Genetics ,Transcriptional regulation ,Animals ,Gene ,genetically encoded reporters ,Regulation of gene expression ,biology ,hypoxia ,Chemistry ,Cell Hypoxia ,Genetic Engineering ,Oxygen ,Signal Transduction ,Synthetic Biology ,Transcription Factors ,Plant ,biology.organism_classification ,Ubiquitin ligase ,Cell biology ,Gene Expression Regulation ,biology.protein ,Gal4 ,Signal transduction ,010606 plant biology & botany - Abstract
Due to the involvement of oxygen in many essential metabolic reactions, all living organisms have developed molecular systems that allow adaptive physiological and metabolic transitions depending on oxygen availability. In mammals, the expression of hypoxia-response genes is controlled by the heterodimeric Hypoxia-Inducible Factor. The activity of this transcriptional regulator is linked mainly to the oxygen-dependent hydroxylation of conserved proline residues in its α-subunit, carried out by prolyl-hydroxylases, and subsequent ubiquitination via the E3 ligase von Hippel-Lindau tumor suppressor, which targets Hypoxia-Inducible Factor-α to the proteasome. By exploiting bioengineered versions of this mammalian oxygen sensor, we designed and optimized a synthetic device that drives gene expression in an oxygen-dependent fashion in plants. Transient assays in Arabidopsis (Arabidopsis thaliana) mesophyll protoplasts indicated that a combination of the yeast Gal4/upstream activating sequence system and the mammalian oxygen sensor machinery can be used effectively to engineer a modular, oxygen-inducible transcriptional regulator. This synthetic device also was shown to be selectively controlled by oxygen in whole plants when its components were expressed stably in Arabidopsis seedlings. We envision the exploitation of our genetically encoded controllers to generate plants able to switch gene expression selectively depending on oxygen availability, thereby providing a proof of concept for the potential of synthetic biology to assist agricultural practices in environments with variable oxygen provision.
- Published
- 2018
13. Improving the design of an oxidative stress sensing biosensor in yeast
- Author
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David R. McMillen and Louis C Dacquay
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0106 biological sciences ,Saccharomyces cerevisiae Proteins ,Saccharomyces cerevisiae ,Computational biology ,macromolecular substances ,Biosensing Techniques ,01 natural sciences ,Applied Microbiology and Biotechnology ,Microbiology ,cell-based biosensor ,03 medical and health sciences ,Upstream activating sequence ,Synthetic biology ,010608 biotechnology ,Biomanufacturing ,Promoter Regions, Genetic ,Transcription factor ,030304 developmental biology ,reactive oxygen species ,0303 health sciences ,AcademicSubjects/SCI01150 ,biology ,technology, industry, and agriculture ,Promoter ,General Medicine ,biology.organism_classification ,Yeast ,Saccharomyces boulardii ,Oxidative Stress ,Metabolic Engineering ,synthetic biology ,Biosensor ,promoter engineering ,Research Article ,Transcription Factors - Abstract
Transcription factor (TF)-based biosensors have proven useful for increasing biomanufacturing yields, large-scale functional screening, and in environmental monitoring. Most yeast TF-based biosensors are built from natural promoters, resulting in large DNA parts retaining considerable homology to the host genome, which can complicate biological engineering efforts. There is a need to explore smaller, synthetic biosensors to expand the options for regulating gene expression in yeast. Here, we present a systematic approach to improving the design of an existing oxidative stress sensing biosensor in Saccharomyces cerevisiae based on the Yap1 transcription factor. Starting from a synthetic core promoter, we optimized the activity of a Yap1-dependent promoter through rational modification of a minimalist Yap1 upstream activating sequence. Our novel promoter achieves dynamic ranges of activation surpassing those of the previously engineered Yap1-dependent promoter, while reducing it to only 171 base pairs. We demonstrate that coupling the promoter to a positive-feedback-regulated TF further improves the biosensor by increasing its dynamic range of activation and reducing its limit of detection. We have illustrated the robustness and transferability of the biosensor by reproducing its activity in an unconventional probiotic yeast strain, Saccharomyces boulardii. Our findings can provide guidance in the general process of TF-based biosensor design., The paper presents the design and optimization of a novel yeast biosensor using an engineered promoter to sense oxidative stress, and by extension, the presence of reactive oxygen species.
- Published
- 2021
14. Molecular and Phenotypic Characterization following RNAi Mediated Knockdown in Drosophila
- Author
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Saurabh Jayesh Kumar Mehta, Ram Kumar Mishra, and Pradyumna A. Joshi
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Regulation of gene expression ,Gene knockdown ,Strategy and Management ,Mechanical Engineering ,fungi ,Metals and Alloys ,Biology ,Industrial and Manufacturing Engineering ,Cell biology ,Small hairpin RNA ,Gene product ,Upstream activating sequence ,RNA interference ,Gene expression ,Gene - Abstract
Loss of function studies shed significant light on the involvement of a gene or gene product in different cellular processes. Short hairpin RNA (shRNA) mediated RNA interference (RNAi) is a classical yet straightforward technique frequently used to knock down a gene for assessing its function. Similar perturbations in gene expression can be achieved by siRNA, microRNA, or CRISPR-Cas9 methods also. In Drosophila genetics, the UAS-GAL4 system is utilized to express RNAi and make ubiquitous and tissue-specific knockdowns possible. The UAS-GAL4 system borrows genetic components of S. cerevisiae, hence rule out the possibility of accidental expression of the system. In particular, this technique uses a target-specific shRNA, and the expression of the same is governed by the upstream activating sequence (UAS). Controlled expression of GAL4, regulated by specific promoters, can drive the interfering RNA expression ubiquitously or in a tissue-specific manner. The knockdown efficiency is measured by RNA isolation and semiquantitative RT-PCR reaction followed by agarose gel electrophoresis. We have employed immunostaining procedure also to assess knockdown efficiency. RNAi provides researchers with an option to decrease the gene product levels (equivalent to hypomorph condition) and study the outcomes. UAS-GAL4 based RNAi method provides spatio-temporal regulation of gene expression and helps deduce the function of a gene required during early developmental stages also.
- Published
- 2021
15. Co-Expression of a Homologous Cytochrome P450 Reductase Is Required for In Vivo Validation of the Tetranychus urticae CYP392A16-Based Abamectin Resistance in Drosophila
- Author
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Aris Ilias, Maria Riga, John Vontas, and Vassilis Douris
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0106 biological sciences ,0301 basic medicine ,cytochrome P450 ,Tetranychus ,Biology ,01 natural sciences ,03 medical and health sciences ,Upstream activating sequence ,chemistry.chemical_compound ,transgenic Drosophila ,Tetranychus urticae ,detoxification ,lcsh:Science ,cytochrome P450 reductase ,fungi ,Cytochrome P450 ,Cytochrome P450 reductase ,abamectin ,Monooxygenase ,biology.organism_classification ,Cell biology ,010602 entomology ,030104 developmental biology ,chemistry ,Insect Science ,biology.protein ,Abamectin ,lcsh:Q ,Drosophila melanogaster - Abstract
Overexpression of the cytochrome P450 monooxygenase CYP392A16 has been previously associated with abamectin resistance using transcriptional analysis in the two-spotted spider mite Tetranychus urticae, an important pest species worldwide; however, this association has not been functionally validated in vivo despite the demonstrated ability of CYP392A16 to metabolize abamectin in vitro. We expressed CYP392A16 in vivo via a Gal4 transcription activator protein/Upstream Activating Sequence (GAL4/UAS) system in Drosophila melanogaster flies, driving expression with detoxification tissue-specific drivers. We demonstrated that CYP392A16 expression confers statistically significant abamectin resistance in toxicity bioassays in Drosophila only when its homologous redox partner, cytochrome P450 reductase (TuCPR), is co-expressed in transgenic flies. Our study shows that the Drosophila model can be further improved, to facilitate the functional analysis of insecticide resistance mechanisms acting alone or in combination.
- Published
- 2020
16. A Role for Mediator Core in Limiting Coactivator Recruitment in Saccharomyces cerevisiae
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Yaxin Yu, Chao Yan, Robert M. Yarrington, David J. Stillman, and Lu Bai
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Regulation of gene expression ,Genetics ,Transcriptional Activation ,0303 health sciences ,Mediator Complex ,Saccharomyces cerevisiae Proteins ,Transcription, Genetic ,Saccharomyces cerevisiae ,Biology ,Investigations ,03 medical and health sciences ,Upstream activating sequence ,Core mediator complex ,0302 clinical medicine ,Mediator ,Gene Expression Regulation, Fungal ,Transcription preinitiation complex ,Coactivator ,RNA Polymerase II ,Enhancer ,Promoter Regions, Genetic ,Transcription factor ,030217 neurology & neurosurgery ,030304 developmental biology - Abstract
Mediator is an essential, multisubunit complex that functions as a transcriptional coactivator in yeast and other eukaryotic organisms. Mediator has four conserved modules, Head, Middle, Tail, and Kinase, and has been implicated in nearly all aspects of gene regulation. The Tail module has been shown to recruit the Mediator complex to the enhancer or upstream activating sequence (UAS) regions of genes via interactions with transcription factors, and the Kinase module facilitates the transition of Mediator from the UAS/enhancer to the preinitiation complex via protein phosphorylation. Here, we analyze expression of the Saccharomyces cerevisiae HO gene using a sin4 Mediator Tail mutation that separates the Tail module from the rest of the complex; the sin4 mutation permits independent recruitment of the Tail module to promoters without the rest of Mediator. Significant increases in recruitment of the SWI/SNF and SAGA coactivators to the HO promoter UAS were observed in a sin4 mutant, along with increased gene activation. These results are consistent with recent studies that have suggested that the Kinase module functions negatively to inhibit activation by the Tail. However, we found that Kinase module mutations did not mimic the effect of a sin4 mutation on HO expression. This suggests that at HO the core Mediator complex (Middle and Head modules) must play a role in limiting Tail binding to the promoter UAS and gene activation. We propose that the core Mediator complex helps modulate Mediator binding to the UAS regions of genes to limit coactivator recruitment and ensure proper regulation of gene transcription.
- Published
- 2020
17. Facilitating fructose-driven metabolism exerts a protective effect on anoxic stress in Drosophila
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Myo-Jeong Kim, Mun-Cheol Kim, and H. Kim
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0106 biological sciences ,0301 basic medicine ,Fructose ,Biology ,01 natural sciences ,Fructokinases ,03 medical and health sciences ,Upstream activating sequence ,chemistry.chemical_compound ,Genetics ,Animals ,Humans ,Glycolysis ,Hypoxia ,Molecular Biology ,ATP synthase ,Glucose Transporter Type 5 ,fungi ,Transporter ,Metabolism ,Cell biology ,010602 entomology ,030104 developmental biology ,chemistry ,Insect Science ,biology.protein ,Alternative complement pathway ,Drosophila ,GLUT5 - Abstract
Hypoxic stress is linked to various cardiovascular disorders (e.g., stroke, myocardial infarction), mediated, at least in part, by a reduction in ATP synthesis. Fructose-driven glycolysis is proposed as an alternative pathway capable of sustaining ATP production even under anoxic conditions. Here, we tested the hypothesis that facilitating fructose-driven metabolism exerts a protective effect against anoxic stress in Drosophila. Genetically modified flies with the human fructose transporter (GluT5) and ketohexokinase (KHK) genes downstream of upstream activating sequence (UAS) were constructed. The GAL4-UAS system was confirmed to: (i) increase the expression of GluT5 and KHK in a tissue-specific and a time-dependent manner (i.e., whole flies [with Act5c-gene switch GAL4 driver], neurons [with elav-gene switch GAL4 driver]) and (ii) reduce mortality of flies when placed under anoxic stress. Taken together, these data suggest that increasing fructose metabolism may be a clinically relevant approach to minimize hypoxia-induced cellular damage.
- Published
- 2020
18. Transcriptome analysis reveals the effects of transgenic expression of the Gal4 protein on normal gene expression in silkworm tissues
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Yan Ma, Huang L, Hanfu Xu, Rongpeng Liu, Tingting Tan, and Chen T
- Subjects
Transcriptome ,Hippo signaling pathway ,Upstream activating sequence ,Transgene ,fungi ,Gene expression ,Rap1 ,Signal transduction ,Biology ,Gene ,Cell biology - Abstract
The Gal4/upstream activating sequence(UAS) system, a well-known genetic tool, has been widely used to analyze gene function in many organisms, including the silkworm (Bombyx mori), a model lepidopteran insect. Several studies have suggested that Gal4 protein activation in tissues can negatively affect transgenic individuals; however, whether and to what extent the Gal4 protein affects normal endogenous gene expression have rarely been studied. Here, we analyzed the transcriptomes of transgenic silkworms expressing the Gal4 protein at high levels in both the wing disc (WD) and epidermis (EP) and investigated gene expression changes in both tissues. Overall, 24,593 genes were identified in the WD and EP libraries, and 2,025 and 2,488 were identified as significant differentially expressed genes(DEGs) in the WD and EP between the transgenic and control groups, respectively. These DEGs were further annotated by gene function classification and pathway assessment using public databases. In addition, 506 DEGs were shared (common) between both tissues. Of these, 97 genes were commonly upregulated, and 234 were commonly downregulated; many of them were annotated to be involved in metabolic processes such as “fat digestion and absorption”, “glycine, serine and threonine metabolism” and “glutathione metabolism” and in signal transduction pathways such as the “Rap1 signaling pathway”, “MAPK signaling pathway” and “Hippo signaling pathway”. Overall, this work enhances understanding of the effects of transgenic Gal4 protein expression on normal gene expression in silkworm tissues and suggests that researchers should pay attention to unexpected effects when using the Gal4/UAS system to study gene function.
- Published
- 2020
19. Identification and Characterization of a Novel Basic Helix-Loop-Helix Transcription Factor of Phospholipid Synthesis Regulation in Aspergillus niger
- Author
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Hongzhi Dong, Dou Yu, Bin Wang, and Li Pan
- Subjects
Microbiology (medical) ,phosphatidylinositol ,yeast two-hybrid assay ,Two-hybrid screening ,lcsh:QR1-502 ,Protein degradation ,EMSA ,Microbiology ,lcsh:Microbiology ,03 medical and health sciences ,Upstream activating sequence ,chemistry.chemical_compound ,ino2 ,Transcription (biology) ,Electrophoretic mobility shift assay ,Phosphatidylinositol ,Gene ,Transcription factor ,transcription factor ,Original Research ,030304 developmental biology ,0303 health sciences ,030306 microbiology ,Chemistry ,Biochemistry ,Aspergillus niger ,comparative transcriptome - Abstract
The synthesis of phospholipids relies on a sort of genes, whose promoter regions contain inositol-sensitive upstream activation sequence (UASINO) and are regulated by the basic helix-loop-helix (bHLH)-type ino2/ino4 transcription factor (TF) pair. Ten putative bHLH TFs have been found through whole genome sequencing of Aspergillus niger, but none of these TFs have been characterized. In this study, we identified and characterized the bHLH-type TF ino2(An02g04350) in A. niger. Electrophoretic mobility shift assay (EMSA) and yeast two-hybrid assay demonstrated that ino2 functions as a homodimer in UASINO genes (e.g., ino1 and cho1) and binds to opi1(An1g02370) in vitro. Real-time quantitative PCR of ino1 and quantification of total phospholipid indicated that the ino2 disruptant downregulated the transcription of ino1 and the amount of total cellular phosphatidylinositol. In addition, phenotype analyses showed that a loss of ino2 led to resistance to cell wall interference and DNA damage. Comparative transcriptome analyses showed that more than 1000 genes and GO terms associated with UASINO, endoplasmic reticulum–associated protein degradation, phosphatidylinositol synthesis, chitin synthesis, and fatty acid synthesis were differentially expressed in Δino2 compared to the wild type (WT). Taken together, these observations indicate that the bHLH TF ino2 functions as a homodimer that regulates the synthesis of phosphatidylinositol, fatty acid, and chitin and influences the homeostasis of the endoplasmic reticulum membrane.
- Published
- 2020
20. Knockdown transgenic Drosophila and Parkinson's disease
- Author
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Carlos Velez-Pardo and Marlene Jimenez-Del-Rio
- Subjects
Gene knockdown ,Upstream activating sequence ,animal structures ,biology ,RNA interference ,Transgene ,fungi ,Mutant ,Drosophila melanogaster ,biology.organism_classification ,Drosophila ,Parkin ,Cell biology - Abstract
In 2000, by using the binary galactose 4 (GAL4)/upstream activating sequence (UAS) system, Feany and Bender were the first to report a transgenic Drosophila melanogaster fly model expressing familial Parkinson's disease (PD)-linked mutants (Alanine53Treonine and Alanine30Proline) of human alpha-synuclein. Amazingly, these mutant Drosophila (as was not the case with normal Drosophila) replicated the essential features of human PD including adult-onset loss of dopaminergic neurons, filamentous intraneuronal inclusions containing alpha-synuclein, and locomotor dysfunction. We provide highlights in the history of Drosophila PD research with special attention paid to the knockdown transgenic Drosophila GAL4/UAS RNA interference system to characterize and understand the response of mutant parkin, dj-1β, and Lrrk flies to oxidative stress phenomena induced by PQ2+ intoxication, thereby exploring potential therapeutic treatments.
- Published
- 2020
21. Single-molecule studies reveal branched pathways for activator-dependent assembly of RNA polymerase II pre-initiation complexes
- Author
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Inwha Baek, Stephen Buratowski, Jeff Gelles, and Larry J. Friedman
- Subjects
General transcription factor ,biology ,TATA box ,RNA polymerase II ,Cell Biology ,Article ,Single Molecule Imaging ,Upstream activating sequence ,Transcription (biology) ,Transcription factor II H ,biology.protein ,Biophysics ,Transcription factor II F ,RNA Polymerase II ,Transcription factor II E ,Promoter Regions, Genetic ,Molecular Biology - Abstract
RNA polymerase II (RNA Pol II) transcription reconstituted from purified factors suggests pre-initiation complexes (PICs) can assemble by sequential incorporation of factors at the TATA box. However, these basal transcription reactions are generally independent of activators and co-activators. To study PIC assembly under more realistic conditions, we used single-molecule microscopy to visualize factor dynamics during activator-dependent reactions in nuclear extracts. Surprisingly, RNA Pol II, TFIIF, and TFIIE can pre-assemble on enhancer-bound activators before loading into PICs, and multiple RNA Pol II complexes can bind simultaneously to create a localized cluster. Unlike TFIIF and TFIIE, TFIIH binding is singular and dependent on the basal promoter. Activator-tethered factors exhibit dwell times on the order of seconds. In contrast, PICs can persist on the order of minutes in the absence of nucleotide triphosphates, although TFIIE remains unexpectedly dynamic even after TFIIH incorporation. Our kinetic measurements lead to a new branched model for activator-dependent PIC assembly.
- Published
- 2021
22. Zap1-dependent transcription from an alternative upstream promoter controls translation ofRTC4mRNA in zinc-deficientSaccharomyces cerevisiae
- Author
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David J. Eide, Spencer A. Haws, Janet Taggart, and Colin W. MacDiarmid
- Subjects
0301 basic medicine ,Sp1 transcription factor ,biology ,General transcription factor ,Response element ,RNA polymerase II ,Promoter ,E-box ,Microbiology ,Molecular biology ,03 medical and health sciences ,Upstream activating sequence ,030104 developmental biology ,biology.protein ,Enhancer ,Molecular Biology - Abstract
Maintaining zinc homeostasis is an important property of all organisms. In the yeast Saccharomyces cerevisiae, the Zap1 transcriptional activator is a central player in this process. In response to zinc deficiency, Zap1 activates transcription of many genes and consequently increases accumulation of their encoded proteins. In this report, we describe a new mechanism of Zap1-mediated regulation whereby increased transcription of certain target genes results in reduced protein expression. Transcription of the Zap1-responsive genes RTC4 and RAD27 increases markedly in zinc-deficient cells but, surprisingly, their protein levels decrease. We examined the underlying mechanism further for RTC4 and found that this unusual regulation results from altered transcription start site selection. In zinc-replete cells, RTC4 transcription begins near the protein-coding region and the resulting short transcript leader allows for efficient translation. In zinc-deficient cells, RTC4 RNA with longer transcript leaders are expressed that are not efficiently translated due to the presence of multiple small open reading frames upstream of the coding region. This regulation requires a potential Zap1 binding site located farther upstream of the promoter. Thus, we present evidence for a new mechanism of Zap1-mediated gene regulation and another way that this activator protein can repress protein expression.
- Published
- 2017
23. Synthetic regulatory RNAs selectively suppress the progression of bladder cancer
- Author
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Yaoting Gui, Changshui Zhuang, Chengle Zhuang, Xiaowei Zhang, Xiaomin Luo, Zhiming Cai, and Xinbo Huang
- Subjects
Male ,0301 basic medicine ,Human telomerase reverse transcriptase ,Cancer Research ,Cell Survival ,Biology ,lcsh:RC254-282 ,Proto-Oncogene Proteins c-myc ,Mice ,03 medical and health sciences ,Upstream activating sequence ,0302 clinical medicine ,Cell Movement ,Cell Line, Tumor ,microRNA ,medicine ,Animals ,Humans ,Telomerase reverse transcriptase ,Promoter Regions, Genetic ,Cell Proliferation ,Messenger RNA ,Cell growth ,Research ,Bladder cancer ,Cancer ,RNA ,medicine.disease ,lcsh:Neoplasms. Tumors. Oncology. Including cancer and carcinogens ,Xenograft Model Antitumor Assays ,Gene Expression Regulation, Neoplastic ,030104 developmental biology ,Artificial miRNA ,Urinary Bladder Neoplasms ,Oncology ,030220 oncology & carcinogenesis ,Cancer cell ,Disease Progression ,Cancer research ,GAL4 ,Female ,Protein Binding - Abstract
The traditional treatment for cancer is lack of specificity and efficacy. Modular synthetic regulatory RNAs, such as inhibitive RNA (iRNA) and active RNA (aRNA), may overcome these limitations. Here, we synthesize a new iRNA to bind the upstream activating sequence (UAS) of a minimal promoter that drives expression of artificial miRNAs (amiRNAs) targeting MYC, which represses the binding interaction between UAS and GAL4 fusion protein (GAL4-VP64) in GAL4/UAS system. The aRNA driven by a tumor-specific mutant human telomerase reverse transcriptase (hTERT) promoter is created to interact with iRNA to expose UAS again in bladder cancer. Without the aRNA, mRNA and protein levels of MYC, cell growth, cell apoptosis and cell migration were no significance in two bladder cancer cell lines, T24 and 5637, and human foreskin fibroblast (HFF) cells. The aRNA significantly inhibited the expression of MYC in mRNA and protein levels, as well as the proliferation and migration of the cancer cells, but not in HFF cells. These results indicated that regulatory RNAs selectively controlled the expression of amiRNAs and ultimately suppress the progression of bladder cancer cells without affecting normal cells. Synthetic regulatory RNAs might be a selective therapeutic approach for bladder cancer.
- Published
- 2017
24. Opi1p translocation to the nucleus is regulated by hydrogen peroxide inSaccharomyces cerevisiae
- Author
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Andreia P. Cepeda, Francisco R. Pinto, Filipe Vilas-Boas, Helena Soares, H. Susana Marinho, Carolina Camelo, Joana Barros‐Martins, Carla Real, and Luísa Cyrne
- Subjects
0301 basic medicine ,Regulation of gene expression ,030102 biochemistry & molecular biology ,Endoplasmic reticulum ,Saccharomyces cerevisiae ,Bioengineering ,Biology ,biology.organism_classification ,Applied Microbiology and Biotechnology ,Biochemistry ,Fusion protein ,Cell biology ,03 medical and health sciences ,Upstream activating sequence ,030104 developmental biology ,medicine.anatomical_structure ,Gene expression ,Genetics ,medicine ,Nuclear membrane ,Nucleus ,Biotechnology - Abstract
During exposure of yeast cells to low levels of hydrogen peroxide (H2 O2 ), the expression of several genes is regulated for cells to adapt to the surrounding oxidative environment. Such adaptation involves modification of plasma membrane lipid composition, reorganization of ergosterol-rich microdomains and altered gene expression of proteins involved in lipid and vesicle traffic, to decrease permeability to exogenous H2 O2 . Opi1p is a transcriptional repressor that is inactive when present at the nuclear membrane/endoplasmic reticulum, but represseses transcription of inositol upstream activating sequence (UASINO )-containing genes, many of which are involved in the synthesis of phospholipids and fatty acids, when it is translocated to the nucleus. We investigated whether H2 O2 in concentrations inducing adaptation regulates Opi1p function. We found that, in the presence of H2 O2 , GFP-Opi1p fusion protein translocates to the nucleus and, concomitantly, the expression of UASINO -containing genes is affected. We also investigated whether cysteine residues of Opi1p were implicated in the H2 O2 -mediated translocation of this protein to the nucleus and identified cysteine residue 159 as essential for this process. Our work shows that Opi1p is redox-regulated and establishes a new mechanism of gene regulation involving Opi1p, which is important for adaptation to H2 O2 in yeast cells. Copyright © 2017 John Wiley & Sons, Ltd.
- Published
- 2017
25. A synthetic hybrid promoter for D-xylonate production at low pH in the tolerant yeast Candida glycerinogenes
- Author
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Bin Zhuge, Hao Ji, Hong Zong, and Xinyao Lu
- Subjects
0301 basic medicine ,Bioengineering ,Dehydrogenase ,Biology ,Applied Microbiology and Biotechnology ,Green fluorescent protein ,law.invention ,03 medical and health sciences ,Upstream activating sequence ,law ,Promoter Regions, Genetic ,Gene ,Candida ,Xylose ,Promoter ,General Medicine ,Hydrogen-Ion Concentration ,Molecular biology ,Yeast ,Alcohol Oxidoreductases ,030104 developmental biology ,Biochemistry ,Recombinant DNA ,Fermentation ,Research Paper ,Biotechnology - Abstract
The tolerant yeast Candida glycerinogenes, with high D-xylonate and low-pH tolerances, was used as the host for D-xylonate production at low pH in this study. A low-pH inducible promoter, pGUKd, was engineered using the core promoter of the glyceraldehyde-3-phosphate dehydrogenase gene (pGAP) combined with the upstream activating sequence of the promoter of the guanylate kinase gene (pGUK1) that had substituted pH-responsive TF binding sites. The recombinant cells that expressed GFP from the hybrid promoter pGUKd displayed dramatically increased fluorescence intensity at pH 2.5, thus verifying that pGUKd is a low-pH inducible promoter. The promoter pGUKd was then used to express the D-xylose dehydrogenase gene xylB, resulting in increased expression levels of xylB at low pH. The recombinant protein exhibited higher specific activities under lower pH conditions and produced 38 g/l D-xylonate at pH 2.5. This rate is much higher than that produced by fermentation at pH 5.5. These results suggest that the novel hybrid promoter pGUKd functions to direct the production of D-xylonate at low pH, and we provide a candidate genetic tool for the stress tolerant yeast C. glycerinogenes.
- Published
- 2017
26. Phenotypic differences between Drosophila Alzheimer’s disease models expressing human Aβ42 in the developing eye and brain
- Author
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Yoon Seok Suh, Hye Sup Yun, Myoungchul Shin, Soojin Lee, Kyoung Sang Cho, Soojin Hwang, Youngjae Jeon, and Jang Ho Lee
- Subjects
0301 basic medicine ,Untranslated region ,Genetics ,Programmed cell death ,biology ,Transgene ,Articles ,biology.organism_classification ,Phenotype ,General Biochemistry, Genetics and Molecular Biology ,UAS-Aβ42 ,03 medical and health sciences ,Imaginal disc ,Upstream activating sequence ,030104 developmental biology ,Animal Science and Zoology ,Drosophila ,Drosophila melanogaster ,amyloid-β42 ,Alzheimer’s disease - Abstract
Drosophila melanogaster expressing amyloid-β42 (Aβ42) transgenes have been used as models to study Alzheimer's disease. Various Aβ42 transgenes with different structures induce different phenotypes, which make it difficult to compare data among studies which use different transgenic lines. In this study, we compared the phenotypes of four frequently used Aβ42 transgenic lines, UAS-Aβ422X , UAS-Aβ42BL33770 , UAS-Aβ4211C39 , and UAS-Aβ42H29.3 . Among the four transgenic lines, only UAS-Aβ422X has two copies of the upstream activation sequence-amyloid-β42 (UAS-Aβ42) transgene, while remaining three have one copy. UAS-Aβ42BL33770 has the 3' untranslated region of Drosophila α-tubulin, while the others have that of SV40. UAS-Aβ4211C39 and UAS-Aβ42H29.3 have the rat pre-proenkephalin signal peptide, while UAS-Aβ422X and UAS-Aβ42BL33770 have that of the fly argos protein. When the transgenes were expressed ectopically in the developing eyes of the flies, UAS-Aβ422X transgene resulted in a strongly reduced and rough eye phenotype, while UAS-Aβ42BL33770 only showed a strong rough eye phenotype; UAS-Aβ42H29.3 and UAS-Aβ4211C39 had mild rough eyes. The levels of cell death and reactive oxygen species (ROS) in the eye imaginal discs were consistently the highest in UAS-Aβ422X , followed by UAS-Aβ42BL33770 , UAS-Aβ4211C39 , and UAS-Aβ42H29.3 . Surprisingly, the reduction in survival during the development of these lines did not correlate with cell death or ROS levels. The flies which expressed UAS-Aβ4211C39 or UAS-Aβ42H29.3 experienced greatly reduced survival rates, although low levels of ROS or cell death were detected. Collectively, our results demonstrated that different Drosophila AD models show different phenotypic severity, and suggested that different transgenes may have different modes of cytotoxicity. Abbreviations: Aβ42: amyloid-β42; AD: Alzheimer's disease; UAS: upstream activation sequence.
- Published
- 2017
27. An enhanced hTERT promoter-driven CRISPR/Cas9 system selectively inhibits the progression of bladder cancer cells
- Author
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Tiefu Xiong, Chengle Zhuang, Changshui Zhuang, Xinbo Huang, Yaoting Gui, and Yawen Li
- Subjects
0301 basic medicine ,Telomerase ,Genetic Vectors ,Gene Expression ,Apoptosis ,Biology ,Fusion gene ,03 medical and health sciences ,Upstream activating sequence ,Cell Movement ,Genes, Reporter ,Cell Line, Tumor ,Gene Order ,medicine ,Humans ,CRISPR ,Telomerase reverse transcriptase ,HRAS ,Promoter Regions, Genetic ,Molecular Biology ,Cell Proliferation ,Gene Editing ,Bladder cancer ,Cancer ,medicine.disease ,030104 developmental biology ,Urinary Bladder Neoplasms ,Disease Progression ,Cancer research ,CRISPR-Cas Systems ,RNA, Guide, Kinetoplastida ,Biotechnology - Abstract
The current therapies for treating tumors are lacking in efficacy and specificity. Synthetic biology principles may bring some new possible methods for curing cancer. Here we present a synthetic logic circuit based on the CRISPR/Cas9 system. The CRISPR/Cas9 technology has been applied in many biological fields, including cancer research. In this study, the expression of Cas9 nuclease was controlled indirectly by an enhanced hTERT promoter using the GAL4/upstream activating sequence (UAS) binding system. Cas9 was driven by 5XUAS, single guide RNA (sgRNA) was used to target mutant or wild-type HRAS, and the fusion gene GAL4-P65 was driven by the enhanced hTERT promoter. The system was tested in bladder cancer cells (T24 and 5637) and the results showed that the enhanced hTERT promoter could drive the expression of GAL4-P65 in these bladder cancer cell lines. Then all these devices were packed into lentivirus and the results of quantitative real-time PCR showed that the mRNA expression level of HRAS was selectively inhibited in the T24 and 5637 cells. The results of functional experiments suggested that the proliferation, cell migration and invasion were selectively suppressed, and that the apoptosis rate was increased in bladder cancer cells but not in human foreskin fibroblasts (HFF). In conclusion, we successfully constructed an enhanced hTERT promoter-driven CRISPR/Cas9 system and data showed that it could selectively suppress the progression of bladder cancer cells.
- Published
- 2017
28. Mediator Undergoes a Compositional Change during Transcriptional Activation
- Author
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Kevin Struhl, Yi Jin, Natalia Petrenko, and Koon Ho Wong
- Subjects
0301 basic medicine ,biology ,Enhancer RNAs ,RNA polymerase II ,Cell Biology ,Molecular biology ,Article ,Cell biology ,MED1 ,03 medical and health sciences ,Upstream activating sequence ,030104 developmental biology ,Mediator ,Transcription (biology) ,biology.protein ,Transcription factor II H ,Enhancer ,Molecular Biology - Abstract
Mediator is a transcriptional co-activator recruited to enhancers by DNA-binding activators, and it also interacts with RNA polymerase (Pol) II as part of the preinitiation complex (PIC). We demonstrate that a single Mediator complex associates with the enhancer and core promoter in vivo, indicating that it can physically bridge these transcriptional elements. However, the Mediator kinase module associates strongly with the enhancer, but not with the core promoter, and it dissociates from the enhancer upon depletion of the TFIIH kinase. Severing the kinase module from Mediator by removing the connecting subunit Med13 does not affect Mediator association at the core promoter, but increases occupancy at enhancers. Thus, Mediator undergoes a compositional change in which the kinase module, recruited via Mediator to the enhancer, dissociates from Mediator to permit association with Pol II and the PIC. As such, Mediator acts as a dynamic bridge between the enhancer and core promoter.
- Published
- 2016
29. Efficient expression vectors and host strain for the production of recombinant proteins by Yarrowia lipolytica in process conditions
- Author
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Paul Soudier, Patrick Fickers, Young-Kyoung Park, Stéphane Thomas, Jean-Marc Nicaud, Marie Vandermies, Samuel Telek, MICrobiologie de l'ALImentation au Service de la Santé (MICALIS), Institut National de la Recherche Agronomique (INRA)-AgroParisTech, Université Paris-Saclay, Microbial Processes and Interactions, ERRA Teaching and Research Centre, Université de Liège - Gembloux-Université de Liège - Gembloux, and INRA Biocatalysts Fonds de la Recherche Scientifique - FNRSKwanjeong Educational Foundation (KEF)
- Subjects
Yarrowia lipolytica ,[SDV.BIO]Life Sciences [q-bio]/Biotechnology ,lcsh:QR1-502 ,Gene Expression ,Yarrowia ,Bioengineering ,Synthetic promoter ,Erythritol ,Applied Microbiology and Biotechnology ,Upstream activating sequence ,lcsh:Microbiology ,Induction ,Fungal Proteins ,03 medical and health sciences ,chemistry.chemical_compound ,Lipase ,Promoter Regions, Genetic ,030304 developmental biology ,2. Zero hunger ,0303 health sciences ,Fungal protein ,Expression vector ,biology ,030306 microbiology ,Chemistry ,Research ,Promoter ,biology.organism_classification ,CalB ,Yeast ,Recombinant Proteins ,Biochemistry ,biology.protein ,Protein secretion ,Microorganisms, Genetically-Modified ,Biotechnology ,Regulation - Abstract
Background The oleaginous yeast Yarrowia lipolytica is increasingly used as an alternative cell factory for the production of recombinant proteins. Recently, regulated promoters from genes EYK1 and EYD1, encoding an erythrulose kinase and an erythritol dehydrogenase, respectively, have been identified and characterized in this yeast. Hybrid promoters up-regulated by polyols such as erythritol and erythrulose have been developed based on tandem copies of upstream activating sequences from EYK1 (UAS1EYK1) and XPR2 (encoding extracellular protease, UAS1XPR2) promoters. Results The strength of native (pEYD1) and engineered promoters (pEYK1-3AB and pHU8EYK) was compared using the extracellular lipase CalB from Candida antarctica as a model protein and a novel dedicated host strain. This latter is engineered in polyol metabolism and allows targeted chromosomal integration. In process conditions, engineered promoters pEYK1-3AB and pHU8EYK yielded 2.8 and 2.5-fold higher protein productivity, respectively, as compared to the reference pTEF promoter. We also demonstrated the possibility of multicopy integration in the newly developed host strain. In batch bioreactor, the CalB multi-copy strain RIY406 led to a 1.6 fold increased lipase productivity (45,125 U mL−1) within 24 h as compared to the mono-copy strain. Conclusions The expression system described herein appears promising for recombinant extracellular protein production in Y. lipolytica.
- Published
- 2019
30. Regulation of bistability in the std fimbrial operon of Salmonella enterica by DNA adenine methylation and transcription factors HdfR, StdE and StdF
- Author
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Lucía García-Pastor, Josep Casadesús, María Antonia Sánchez-Romero, Marcello Jakomin, Elena Puerta-Fernández, Ministerio de Ciencia, Innovación y Universidades (España), European Commission, García-Pastor, Lucía [0000-0001-7462-1860], Sánchez-Romero, María A. [0000-0003-3098-1033], Gutiérrez, Gabriel [0000-0002-1354-2883], Puerta Fernández, Elena [0000-0003-3559-6791], Casadesús, Josep [0000-0002-2308-293X], Universidad de Sevilla. Departamento de Genética, García-Pastor, Lucía, Sánchez-Romero, María A., Gutiérrez, Gabriel, Puerta Fernández, Elena, and Casadesús, Josep
- Subjects
Regulation of gene expression ,Transcriptional Activation ,Operon ,Adenine ,Gene regulation, Chromatin and Epigenetics ,Salmonella enterica ,Methylation ,Gene Expression Regulation, Bacterial ,Biology ,DNA Methylation ,Cell biology ,Upstream activating sequence ,Bacterial Proteins ,Transcription (biology) ,Fimbriae, Bacterial ,DNA methylation ,Mutation ,Genetics ,Epigenetics ,Promoter Regions, Genetic ,Transcription factor ,Protein Binding ,Transcription Factors - Abstract
13 páginas.- 10 figuras.- 44 referencias, Bistable expression of the Salmonella enterica std operon is controlled by an AND logic gate involving three transcriptional activators: the LysR-type factor HdfR and the StdE and StdF regulators encoded by the std operon itself. StdE activates transcription of the hdfR gene, and StdF activates std transcription together with HdfR. Binding of HdfR upstream of the std promoter is hindered by methylation of GATC sites located within the upstream activating sequence (UAS). Epigenetic control by Dam methylation thus antagonizes formation of the StdE-StdF-HdfR loop and tilts the std switch toward the StdOFF state. In turn, HdfR binding hinders methylation of the UAS, permitting activation of the StdE-StdF-HdfR loop and concomitant formation of StdON cells. Bistability is thus the outcome of competition between DNA adenine methylation and the StdE-StdF-HdfR activator loop., Ministerio de Ciencia, Innovación y Universidades, Spain [BIO2016–75235-P]; European Regional Fund (to J. C.). Funding for open access charge: Ministerio de Ciencia, Innovación y Universidades [BIO2016–75235-P]
- Published
- 2019
31. Synthetic Salicylic acid inducible recombinant promoter for translational research
- Author
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Debasish Deb and Nrisingha Dey
- Subjects
0106 biological sciences ,0301 basic medicine ,Agroinfiltration ,Antifungal Agents ,Transgene ,Bioengineering ,Microbial Sensitivity Tests ,01 natural sciences ,Applied Microbiology and Biotechnology ,law.invention ,Translational Research, Biomedical ,03 medical and health sciences ,Upstream activating sequence ,Ascomycota ,law ,Caulimovirus ,Gene Expression Regulation, Plant ,010608 biotechnology ,Gene expression ,Tobacco ,Promoter Regions, Genetic ,Gene ,Glucuronidase ,Recombination, Genetic ,biology ,Chemistry ,Phoma exigua ,General Medicine ,biology.organism_classification ,Plants, Genetically Modified ,Molecular biology ,030104 developmental biology ,Recombinant DNA ,Cassava vein mosaic virus ,Salicylic Acid ,Biotechnology - Abstract
In the present study, we have developed an inter-molecularly shuffled caulimoviral promoter for protein over-expression by placing the Upstream Activation Sequence (UAS) of Figwort Mosaic Virus (FMV; -249 to -54) at the 5'-end of the Cassava Vein Mosaic Virus (CsVMV) promoter fragment 8 (CsVMV8; -215 to +166) to design a hybrid promoter; FUASCsV8CP. The FUASCsV8CP promoter exhibited approximately 2.1 and 2.0 times higher GUS-activities than that obtained from the CaMV35S promoter, in tobacco (Xanthi Brad) protoplasts and in Agroinfiltration assays respectively. Hereto, when FUASCsV8CP was assayed using transgenic tobacco plants (T2- generation), it showed 2.0 times stronger activity than CaMV35S promoter and almost equivalent activity to that of CaMV35S2 promoter. The promoter displayed Salicylic acid (SA) inducibility and hence can also be used for ensuring effective gene expression in plants under constitutive as well as specific inducible conditions. Furthermore, FUASCsV8CP was used to drive the expression of victoviral Vin gene (encoding Victoriocin) transiently in tobacco. The recombinant Victoriocin could be successfully detected by western blotting three days post infiltration. Also, the in vitro Agar-based killing zone assays employing plant-derived Victoriocin-His (obtained from transient expression of Vin) revealed enhanced antifungal activity of Victoriocin against hemi-biotrophic pathogen Phoma exigua Desm. var. exigua.
- Published
- 2018
32. Single-molecule studies reveal branched pathways for activator-dependent assembly of RNA polymerase II pre-initiation complexes.
- Author
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Baek, Inwha, Friedman, Larry J., Gelles, Jeff, and Buratowski, Stephen
- Subjects
- *
RNA polymerase II , *RNA polymerases , *NUCLEAR reactions , *TRANSCRIPTION factors , *POLYMERASES - Abstract
RNA polymerase II (RNA Pol II) transcription reconstituted from purified factors suggests pre-initiation complexes (PICs) can assemble by sequential incorporation of factors at the TATA box. However, these basal transcription reactions are generally independent of activators and co-activators. To study PIC assembly under more realistic conditions, we used single-molecule microscopy to visualize factor dynamics during activator-dependent reactions in nuclear extracts. Surprisingly, RNA Pol II, TFIIF, and TFIIE can pre-assemble on enhancer-bound activators before loading into PICs, and multiple RNA Pol II complexes can bind simultaneously to create a localized cluster. Unlike TFIIF and TFIIE, TFIIH binding is singular and dependent on the basal promoter. Activator-tethered factors exhibit dwell times on the order of seconds. In contrast, PICs can persist on the order of minutes in the absence of nucleotide triphosphates, although TFIIE remains unexpectedly dynamic even after TFIIH incorporation. Our kinetic measurements lead to a new branched model for activator-dependent PIC assembly. [Display omitted] • Single-molecule microscopy reveals unexpected dynamics of RNA Pol II and GTFs • Multiple RNA Pol IIs cluster on enhancer-bound activators before core promoter binding • RNA Pol II, TFIIF, and TFIIE, but not TFIIH, can pre-assemble at the UAS/enhancer • Activators increase the rates of RNA Pol II and GTF association with DNA Single-molecule microscopy experiments by Baek et al. show that RNA polymerase II and basal transcription factors TFIIF and TFIIE preassemble on UAS/enhancer-bound activators, poised for loading into initiation complexes, with TFIIH at the core promoter. Transcription activators kinetically enhance factor recruitment, creating a localized cluster of polymerases at the UAS/enhancer. [ABSTRACT FROM AUTHOR]
- Published
- 2021
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33. Transcriptional regulation of the IL-7Rα gene by dexamethasone and IL-7 in primary human CD8 T cells
- Author
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Parmvir Parmar, Scott M. Sugden, Feras M. Ghazawi, Juzer A. Kakal, Elliott M. Faller, and Paul MacPherson
- Subjects
0301 basic medicine ,Transcription, Genetic ,TATA box ,T cell ,Blotting, Western ,Immunology ,Anti-Inflammatory Agents ,CAAT box ,chemical and pharmacologic phenomena ,CD8-Positive T-Lymphocytes ,Regulatory Sequences, Nucleic Acid ,Biology ,Dexamethasone ,03 medical and health sciences ,Upstream activating sequence ,0302 clinical medicine ,Genetics ,medicine ,Humans ,Cytotoxic T cell ,IL-2 receptor ,Promoter Regions, Genetic ,Cells, Cultured ,Interleukin 3 ,Receptors, Interleukin-7 ,Interleukin-7 ,Promoter ,Flow Cytometry ,Molecular biology ,030104 developmental biology ,medicine.anatomical_structure ,Gene Expression Regulation ,Signal Transduction ,030215 immunology - Abstract
Interleukin-7 is essential for the development and maintenance of T cells, and the expression of the IL-7 receptor is tightly regulated at every stage of the T cell's lifespan. In mature CD8 T cells, IL-7 plays important roles in cell survival, peripheral homeostasis, and cytolytic function. The IL-7 receptor alpha-chain (CD127) is expressed at high levels on naïve and memory cells, but it is rapidly downregulated upon IL-7 stimulation. In this study, we illustrate the dynamicity of the CD127 promoter and show that it possesses positive as well as negative regulatory sites involved in upregulating and downregulating CD127 expression, respectively. We cloned the CD127 gene promoter and identified key cis-regulatory elements required for CD127 expression in mature resting primary CD8 T cells. The core promoter necessary for efficient basal transcription is contained within the first 262 bp upstream of the TATA box. Additional positive regulatory elements are located between -1200 and -2406 bp, conferring a further 2- to 4-fold enhancement in gene expression. While transcription of the CD127 gene is increased directly through a glucocorticoid response element located between -2255 and -2269 bp upstream of the TATA box, we identified a suppressive region that lies upstream of 1760 bp from the TATA box, which is likely involved in the IL-7-mediated suppression of CD127 transcription. Finally, we illustrated IL-7 does not bias alternative splicing of CD127 transcripts in primary human CD8 T cells.
- Published
- 2016
34. Fibroblast growth factor 21 induction by activating transcription factor 4 is regulated through three amino acid response elements in its promoter region
- Author
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Jun Inoue, Juan Li, Ryuichiro Sato, Makoto Shimizu, and Ryuto Maruyama
- Subjects
0301 basic medicine ,Genetic Vectors ,Response element ,Activating Transcription Factor 4 ,Biology ,Response Elements ,Transfection ,Applied Microbiology and Biotechnology ,Biochemistry ,Adenoviridae ,Analytical Chemistry ,03 medical and health sciences ,Upstream activating sequence ,0302 clinical medicine ,Genes, Reporter ,Cell Line, Tumor ,Serum response factor ,Humans ,Amino Acid Sequence ,Luciferases ,Promoter Regions, Genetic ,Molecular Biology ,Transcription factor ,Hormone response element ,Organic Chemistry ,Promoter ,General Medicine ,Endoplasmic Reticulum Stress ,Activating transcription factor 2 ,Cell biology ,Fibroblast Growth Factors ,030104 developmental biology ,Gene Expression Regulation ,Hepatocytes ,Cancer research ,biology.protein ,Sequence Alignment ,030217 neurology & neurosurgery ,Plasmids ,Signal Transduction ,Biotechnology - Abstract
Fibroblast growth factor 21 (FGF21) is an endocrine growth factor, a regulator of fatty acids and glucose metabolism. Recently, it has been reported that FGF21 expression is regulated by activating transcription factor 4 (ATF4), a transcription factor activated by various stimuli such as endoplasmic reticulum (ER) stress. ATF4 binds to the amino acid response element (AARE), a binding site for ATF4, in the promoter region of the target genes. The two response elements for ATF4 (AARE1 and AARE2) have been reported in the promoter region of FGF21 gene. In this study, we found a novel response element, located upstream of AARE1 and AARE2, essential for a promoter activation of FGF21. When this DNA sequence, named AARE3, was mutated, the promoter activation by ATF4 or ER stress was strongly decreased. Our results showed that the FGF21 promoter contains three response elements for ATF4, suggesting that FGF21 is a sensitive target of ATF4.
- Published
- 2016
35. Local potentiation of stress-responsive genes by upstream noncoding transcription
- Author
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Yutaka Suzuki, Takatomi Yamada, Charles S. Hoffman, Josephine Galipon, Tomoichiro Miyoshi, Naomichi Takemata, Kouji Hirota, Sumio Sugano, Kunihiro Ohta, and Arisa Oda
- Subjects
0301 basic medicine ,Chromatin Immunoprecipitation ,RNA, Untranslated ,Saccharomyces cerevisiae Proteins ,Transcription, Genetic ,Response element ,Saccharomyces cerevisiae ,Transcription coregulator ,Regulatory Sequences, Nucleic Acid ,Biology ,Histones ,03 medical and health sciences ,Upstream activating sequence ,0302 clinical medicine ,Stress, Physiological ,Gene Expression Regulation, Fungal ,Genetics ,General transcription factor ,Gene regulation, Chromatin and Epigenetics ,Pioneer factor ,High-Throughput Nucleotide Sequencing ,Acetylation ,Promoter ,Chromatin Assembly and Disassembly ,Molecular biology ,Glucose ,030104 developmental biology ,Gene Expression Regulation ,Transcription Coactivator ,Chromatin immunoprecipitation ,030217 neurology & neurosurgery ,Protein Binding - Abstract
It has been postulated that a myriad of long noncoding RNAs (lncRNAs) contribute to gene regulation. In fission yeast, glucose starvation triggers lncRNA transcription across promoter regions of stress-responsive genes including fbp1 (fructose-1,6-bisphosphatase1). At the fbp1 promoter, this transcription promotes chromatin remodeling and fbp1 mRNA expression. Here, we demonstrate that such upstream noncoding transcription facilitates promoter association of the stress-responsive transcriptional activator Atf1 at the sites of transcription, leading to activation of the downstream stress genes. Genome-wide analyses revealed that ∼50 Atf1-binding sites show marked decrease in Atf1 occupancy when cells are treated with a transcription inhibitor. Most of these transcription-enhanced Atf1-binding sites are associated with stress-dependent induction of the adjacent mRNAs or lncRNAs, as observed in fbp1. These Atf1-binding sites exhibit low Atf1 occupancy and high histone density in glucose-rich conditions, and undergo dramatic changes in chromatin status after glucose depletion: enhanced Atf1 binding, histone eviction, and histone H3 acetylation. We also found that upstream transcripts bind to the Groucho-Tup1 type transcriptional corepressors Tup11 and Tup12, and locally antagonize their repressive functions on Atf1 binding. These results reveal a new mechanism in which upstream noncoding transcription locally magnifies the specific activation of stress-inducible genes via counteraction of corepressors.
- Published
- 2016
36. Cloning and mutagenetic modification of the firefly luciferase gene and its use for bioluminescence microscopy of engrailed expression during Drosophila metamorphosis
- Author
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Katsunori Ogoh, Ryutaro Akiyoshi, and Hirobumi Suzuki
- Subjects
Firefly luciferase ,0301 basic medicine ,GAL4/UAS system ,Biophysics ,Biochemistry ,Drosophila pupa ,lcsh:Biochemistry ,03 medical and health sciences ,Upstream activating sequence ,0302 clinical medicine ,Fluorescence microscope ,Bioluminescence ,lcsh:QD415-436 ,Luciferase ,lcsh:QH301-705.5 ,Engrailed ,Cloning ,Gal4-UAS system ,Chemistry ,Bioluminescence microscopy ,engrailed ,Cell biology ,030104 developmental biology ,Segment polarity gene ,lcsh:Biology (General) ,030220 oncology & carcinogenesis ,sense organs - Abstract
Bioluminescence microscopy is an area attracting considerable interest in the field of cell biology because it offers several advantages over fluorescence microscopy, including no requirement for excitation light and being phototoxicity free. This method requires brighter luciferase for imaging; however, suitable genetic resource material for this purpose is not available at present. To achieve brighter bioluminescence microscopy, we developed a new firefly luciferase. Using the brighter luciferase, a reporter strain of Drosophila Gal4-UAS (Upstream Activating Sequence) system was constructed. This system demonstrated the expression pattern of engrailed, which is a segment polarity gene, during Drosophila metamorphosis by bioluminescence microscopy, and revealed drastic spatiotemporal change in the engrailed expression pattern during head eversion in the early stage of pupation.
- Published
- 2020
37. A zinc-finger fusion protein refines Gal4-defined neural circuits
- Author
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Claudia S. Barros, Adam Claridge-Chang, Sadhna Sahani, Mavis Loberas, Jia Yi Chua, Farhan Mohammad, Joanne Lam, and Shamprasad Varija Raghu
- Subjects
0301 basic medicine ,animal structures ,Computer science ,Transgene ,Recombinant Fusion Proteins ,Green Fluorescent Proteins ,Heterologous ,Gene Expression ,Computational biology ,lcsh:RC346-429 ,Domain (software engineering) ,Cellular and Molecular Neuroscience ,03 medical and health sciences ,Upstream activating sequence ,0302 clinical medicine ,Component (UML) ,Biological neural network ,Animals ,Drosophila Proteins ,Binding site ,Molecular Biology ,Transcription factor ,lcsh:Neurology. Diseases of the nervous system ,030304 developmental biology ,Zinc finger ,0303 health sciences ,fungi ,Methodology ,Zinc Fingers ,Fusion protein ,030104 developmental biology ,Drosophila melanogaster ,Nerve Net ,Protein Multimerization ,030217 neurology & neurosurgery ,Serotonergic Neurons ,Transcription Factors - Abstract
The analysis of behavior requires that the underlying neuronal circuits are identified and genetically isolated. In several major model species—most notably Drosophila—neurogeneticists identify and isolate neural circuits with a binary heterologous expression-control system: Gal4–UASG. One limitation of Gal4–UASG is that expression patterns are often too broad to map circuits precisely. To help refine the range of Gal4 lines, we developed an intersectional genetic AND operator. Interoperable with Gal4, the new system’s key component is a fusion protein in which the DNA-binding domain of Gal4 has been replaced with a zinc finger domain with a different DNA-binding specificity. In combination with its cognate binding site (UASZ) the zinc-finger-replaced Gal4 (‘Zal1’) was functional as a standalone transcription factor. Zal1 transgenes also refined Gal4 expression ranges when combined with UASGZ, a hybrid upstream activation sequence. In this way, combining Gal4 and Zal1 drivers captured restricted cell sets compared with single drivers and improved genetic fidelity. This intersectional genetic AND operation presumably derives from the action of a heterodimeric transcription factor: Gal4-Zal1. Configurations of Zal1–UASZ and Zal1-Gal4-UASGZ are versatile tools for defining, refining, and manipulating targeted neural expression patterns with precision. Electronic supplementary material The online version of this article (10.1186/s13041-018-0390-7) contains supplementary material, which is available to authorized users.
- Published
- 2018
38. Light-controllable Transcription System by Nucleocytoplasmic Shuttling of a Truncated Phytochrome B
- Author
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Takeaki Ozawa and Natsumi Noda
- Subjects
0301 basic medicine ,Cytoplasm ,Light ,Transcription, Genetic ,Active Transport, Cell Nucleus ,Arabidopsis ,Optogenetics ,Biochemistry ,03 medical and health sciences ,Upstream activating sequence ,0302 clinical medicine ,Transcription (biology) ,Phytochrome B ,Transcriptional regulation ,Basic Helix-Loop-Helix Transcription Factors ,Humans ,Physical and Theoretical Chemistry ,Cell Nucleus ,Expression vector ,Photoswitch ,Phytochrome ,Chemistry ,Arabidopsis Proteins ,General Medicine ,Cell biology ,030104 developmental biology ,HEK293 Cells ,Gene Expression Regulation ,030217 neurology & neurosurgery - Abstract
Transcriptional regulation is a useful strategy for gene therapy and for biomedical research. Unlike chemically regulated transcriptional approaches, spatiotemporal control of transcription using optogenetic tools is a powerful technology for the analysis of single cells. For light to penetrate into tissues, it is desired to use photoreceptors absorbing red/far-red light with a low-molecular mass applicable for the use of virus vectors, and a photoswitch using the photoreceptor needs to be constructed as a single expression vector. Herein, we describe an optogenetic tool based on Arabidopsis thaliana phytochrome (Phy) B and its binding partner, phytochrome-interacting factor (PIF) 6. We generated a truncated PhyB, which allowed for reversible association with PIF6 by red/far-red light illumination. The red light illumination only for 5 min induced PhyB translocation from the cytoplasm into the nucleus by the association with PIF6, resulting in transcriptional activation based on Gal4 DNA-binding domain and the upstream activating sequence of Gal system. The nucleocytoplasmic shuttling vector using PhyB and PIF6 might be applicable for transcriptional regulation in tissue experiments.
- Published
- 2018
39. Recombinant Promoter (MUASCsV8CP) Driven Totiviral Killer Protein 4 (KP4) Imparts Resistance Against Fungal Pathogens in Transgenic Tobacco
- Author
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Indu B. Maiti, Nrisingha Dey, Debasish Deb, and Ankita Shrestha
- Subjects
0106 biological sciences ,0301 basic medicine ,Phoma exigua ,recombinant promoter ,Transgene ,caulimovirus ,Plant Science ,Genetically modified crops ,lcsh:Plant culture ,01 natural sciences ,Microbiology ,law.invention ,03 medical and health sciences ,Upstream activating sequence ,law ,Exigua ,lcsh:SB1-1110 ,Original Research ,biology ,fungi ,food and beverages ,Promoter ,killer protein ,biology.organism_classification ,030104 developmental biology ,Recombinant DNA ,Alternaria alternata ,Cassava vein mosaic virus ,010606 plant biology & botany - Abstract
Development of disease-resistant plant varieties achieved by engineering anti-microbial transgenes under the control of strong promoters can suffice the inhibition of pathogen growth and simultaneously ensure enhanced crop production. For evaluating the prospect of such strong promoters, we comprehensively characterized the full-length transcript promoter of Cassava Vein Mosaic Virus (CsVMV; -565 to +166) and identified CsVMV8 (-215 to +166) as the highest expressing fragment in both transient and transgenic assays. Further, we designed a new chimeric promoter ‘MUASCsV8CP’ through inter-molecular hybridization among the upstream activation sequence (UAS) of Mirabilis Mosaic Virus (MMV; -297 to -38) and CsVMV8, as the core promoter (CP). The MUASCsV8CP was found to be ∼2.2 and ∼2.4 times stronger than the CsVMV8 and CaMV35S promoters, respectively, while its activity was found to be equivalent to that of the CaMV35S2 promoter. Furthermore, we generated transgenic tobacco plants expressing the totiviral ‘Killer protein KP4’ (KP4) under the control of the MUASCsV8CP promoter. Recombinant KP4 was found to accumulate both in the cytoplasm and apoplast of plant cells. The agar-based killing zone assays revealed enhanced resistance of plant-derived KP4 against two deuteromycetous foliar pathogenic fungi viz. Alternaria alternata and Phoma exigua var. exigua. Also, transgenic plants expressing KP4 inhibited the growth progression of these fungi and conferred significant fungal resistance in detached-leaf and whole plant assays. Taken together, we establish the potential of engineering “in-built” fungal stress-tolerance in plants by expressing KP4 under a novel chimeric caulimoviral promoter in a transgenic approach.
- Published
- 2018
40. Efficient expression of genes in the Drosophila germline using a UAS-promoter free of interference by Hsp70 piRNAs
- Author
-
Allan C. Spradling and Steven Z DeLuca
- Subjects
0301 basic medicine ,Genetics ,Somatic cell ,Endogeny ,Biology ,Oogenesis ,Germline ,Cell biology ,03 medical and health sciences ,Upstream activating sequence ,030104 developmental biology ,medicine.anatomical_structure ,medicine ,Transcription factor ,Gene ,Developmental biology ,Germ cell ,Function (biology) - Abstract
Using the yeast GAL4 transcription factor to control expression in Drosophila melanogaster has long been ineffective in female germ cells during oogenesis. Here, DeLuca and Spradling show that the expression problem of most Drosophila molecular tools... Controlling the expression of genes using a binary system involving the yeast GAL4 transcription factor has been a mainstay of Drosophila developmental genetics for nearly 30 years. However, most existing GAL4 expression constructs only function effectively in somatic cells, but not in germ cells during oogenesis, for unknown reasons. A special upstream activation sequence (UAS) promoter, UASp was created that does express during oogenesis, but the need to use different constructs for somatic and female germline cells has remained a significant technical limitation. Here, we show that the expression problem of UASt and many other Drosophila molecular tools in germline cells is caused by their core Hsp70 promoter sequences, which are targeted in female germ cells by Hsp70-directed Piwi-interacting RNAs (piRNAs) generated from endogenous Hsp70 gene sequences. In a genetic background lacking genomic Hsp70 genes and associated piRNAs, UASt-based constructs function effectively during oogenesis. By reducing Hsp70 sequences targeted by piRNAs, we created UASz, which functions better than UASp in the germline and like UASt in somatic cells.
- Published
- 2018
41. Utility of an appropriate reporter assay: Heliotrine interferes with GAL4/upstream activation sequence-driven reporter gene systems
- Author
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Alfonso Lampen, Claudia Luckert, Albert Braeuning, and Stefanie Hessel
- Subjects
Renilla ,Pregnane X receptor ,Reporter gene ,Fireflies ,Biophysics ,Cell Biology ,Biology ,Biochemistry ,Molecular biology ,Cell biology ,Upstream activating sequence ,Transactivation ,Nuclear receptor ,Genes, Reporter ,Luciferases, Firefly ,Animals ,Institut für Ernährungswissenschaft ,Luciferase ,RNA, Messenger ,Bioreporter ,Molecular Biology ,Transcription factor ,Pyrrolizidine Alkaloids ,Luciferases, Renilla - Abstract
Reporter gene assays are widely used for the assessment of transcription factor activation following xenobiotic exposure of cells. A critical issue with such assays is the possibility of interference of test compounds with the test system, for example, by direct inhibition of the reporter enzyme. Here we show that the pyrrolizidine alkaloid heliotrine interferes with reporter signals derived from GAL4-based nuclear receptor transactivation assays by a mechanism independent of luciferase enzyme inhibition. These data highlight the necessity to conduct proper control experiments in order to avoid perturbation of reporter assays by test chemicals. (C) 2015 Elsevier Inc. All rights reserved.
- Published
- 2015
42. Development of a Tightly Controlled Off Switch for Saccharomyces cerevisiae Regulated by Camphor, a Low-Cost Natural Product
- Author
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Jef D. Boeke, Shigehito Ikushima, and Yu Zhao
- Subjects
Operator (biology) ,Transcription, Genetic ,TetR homolog ,Saccharomyces cerevisiae ,Gene Expression ,Repressor ,Investigations ,Upstream activating sequence ,Genes, Reporter ,Gene Order ,Genetics ,Promoter Regions, Genetic ,Molecular Biology ,Genetics (clinical) ,Regulation of gene expression ,Biological Products ,Reporter gene ,biology ,Pseudomonas putida ,biology.organism_classification ,Fusion protein ,Camphor ,Repressor Proteins ,Gene Expression Regulation ,Biochemistry ,Tet system ,Genetic Engineering ,CamR ,Plasmids - Abstract
Here we describe the engineering of a distant homolog of the Tet repressor, CamR, isolated from Pseudomonas putida, that is regulated by camphor, a very inexpensive small molecule (at micromolar concentrations) for use in Saccharomyces cerevisiae. The repressor was engineered by expression from a constitutive yeast promoter, fusion to a viral activator protein cassette, and codon optimization. A suitable promoter responsive to the CamR fusion protein was engineered by embedding a P. putida operator binding sequence within an upstream activating sequence (UAS)-less CYC1 promoter from S. cerevisiae. The switch, named the Camphor-Off switch, activates expression of a reporter gene in camphor-free media and represses it with micromolar concentrations of camphor.
- Published
- 2015
43. Analysis of Polygenic Mutants Suggests a Role for Mediator in Regulating Transcriptional Activation Distance in Saccharomyces cerevisiae
- Author
-
Mark J. Hickman, Caitlin T. Reavey, Fred Winston, Krista C. Dobi, and David Botstein
- Subjects
Transcriptional Activation ,Genetics ,Mediator Complex ,Transcription, Genetic ,biology ,Mutant ,Eukaryotic transcription ,Saccharomyces cerevisiae ,Investigations ,Regulatory Sequences, Nucleic Acid ,biology.organism_classification ,Phenotype ,DNA-Binding Proteins ,Upstream activating sequence ,Mediator ,Gene Expression Regulation, Fungal ,Mutation ,Transcription Initiation Site ,Enhancer ,Gene - Abstract
Studies of natural populations of many organisms have shown that traits are often complex, caused by contributions of mutations in multiple genes. In contrast, genetic studies in the laboratory primarily focus on studying the phenotypes caused by mutations in a single gene. However, the single mutation approach may be limited with respect to the breadth and degree of new phenotypes that can be found. We have taken the approach of isolating complex, or polygenic mutants in the lab to study the regulation of transcriptional activation distance in yeast. While most aspects of eukaryotic transcription are conserved from yeast to human, transcriptional activation distance is not. In Saccharomyces cerevisiae, the upstream activating sequence (UAS) is generally found within 450 base pairs of the transcription start site (TSS) and when the UAS is moved too far away, activation no longer occurs. In contrast, metazoan enhancers can activate from as far as several hundred kilobases from the TSS. Previously, we identified single mutations that allow transcription activation to occur at a greater-than-normal distance from the GAL1 UAS. As the single mutant phenotypes were weak, we have now isolated polygenic mutants that possess strong long-distance phenotypes. By identification of the causative mutations we have accounted for most of the heritability of the phenotype in each strain and have provided evidence that the Mediator coactivator complex plays both positive and negative roles in the regulation of transcription activation distance.
- Published
- 2015
44. New insights on the transcriptional regulation of CD69 gene through a potent enhancer located in the conserved non-coding sequence 2
- Author
-
Teresa Laguna, Raffaella Pippa, Noemí Aguilera-Montilla, Miguel G. Fontela, Laura Notario, Miren Maicas, Pilar Lauzurica, Angel L. Corbí, Berta N. Vazquez, María D. Odero, Ministerio de Economía y Competitividad (España), Instituto de Salud Carlos III, and Ministerio de Educación y Ciencia (España)
- Subjects
Antigens, Differentiation, T-Lymphocyte ,Transcription, Genetic ,5' Flanking Region ,Molecular Sequence Data ,Immunology ,chemical and pharmacologic phenomena ,Enhancer RNAs ,Biology ,Transfection ,Conserved non-coding sequence ,Jurkat Cells ,Upstream activating sequence ,Antigens, CD ,Genes, Reporter ,Transcription (biology) ,Cell Line, Tumor ,Enhancers ,Transcriptional regulation ,Animals ,Humans ,Lectins, C-Type ,Amino Acid Sequence ,Transgenes ,CD69 ,Luciferases ,Enhancer ,Molecular Biology ,Conserved Sequence ,Genetics ,Regulation of gene expression ,Binding Sites ,Transcription regulation ,Gene regulation ,Enhancer Elements, Genetic ,Gene Expression Regulation ,Core Binding Factor Alpha 2 Subunit ,CNS ,K562 Cells ,Chromatin immunoprecipitation ,Protein Binding - Abstract
35 p.-8 fig., The CD69 type II C-type lectin is one ofthe earliestindicators of leukocyte activation acting in lymphocyte migration and cytokine secretion. CD69 expression in hematopoietic lineage undergoes rapid changes depending on the cell-lineage,the activation state or the localization ofthe cell where itis expressed, suggesting a complex and tightly controlled regulation. Here we provide new insights on the transcriptional regulation of CD69 gene in mammal species. Through in silico studies, we analyzed several regulatory features of the 4 upstream conserved non-coding sequences (CNS 1–4) previously described, confirming a major function of CNS2 in the transcriptional regulation of CD69. In addition, multiple transcription binding sites are identified in the CNS2 region by DNA cross-species conservation analysis. By functional approaches we defined a core region of 226 bp located within CNS2 as the main enhancer element of CD69 transcription in the hematopoietic cells analyzed. By chromatin immunoprecipitation, binding of RUNX1 to the core-CNS2 was shown in a T cell line. In addition, we found an activating but not essential role of RUNX1 in CD69 gene transcription by site-directed mutagenesis and RNA silencing, probably through the interaction with this potent enhancer specifically in the hematopoietic lineage. In summary, in this study we contribute with new evidences to the landscape of the transcriptional regulation of the CD69 gene., This work was supported by the Spanish Ministry of Economics (SAF2010-15649) and the Carlos III National Health Institute (ISCIII) – RTICC (RD12/0036/0063)
- Published
- 2015
45. Chemical intervention of the NM23-H2 transcriptional programme onc-MYCvia a novel small molecule
- Author
-
Hui-Yun Liu, Jia-Heng Tan, Zhi-Shu Huang, Ding Li, Chan Shan, Ai-Chun Chen, Shuo-Bin Chen, Lian-Quan Gu, Jin-Qiang Hou, Jing Lin, and Tian-Miao Ou
- Subjects
Transcription, Genetic ,Response element ,Down-Regulation ,Apoptosis ,Biology ,Proto-Oncogene Proteins c-myc ,Small Molecule Libraries ,Mice ,Upstream activating sequence ,Epigenetics of physical exercise ,Chemical Biology and Nucleic Acid Chemistry ,Transcription (biology) ,Genetics ,Transcriptional regulation ,Animals ,Humans ,Promoter Regions, Genetic ,Transcription factor ,Cells, Cultured ,Quinazolinones ,General transcription factor ,Promoter ,DNA ,NM23 Nucleoside Diphosphate Kinases ,Molecular biology ,Cell biology ,HeLa Cells ,Protein Binding - Abstract
c-MYC is an important oncogene that is considered as an effective target for anticancer therapy. Regulation of this gene's transcription is one avenue for c-MYC-targeting drug design. Direct binding to a transcription factor and generating the intervention of a transcriptional programme appears to be an effective way to modulate gene transcription. NM23-H2 is a transcription factor for c-MYC and is proven to be related to the secondary structures in the promoter. Here, we first screened our small-molecule library for NM23-H2 binders and then sifted through the inhibitors that could target and interfere with the interaction process between NM23-H2 and the guanine-rich promoter sequence of c-MYC. As a result, a quinazolone derivative, SYSU-ID-01: , showed a significant interference effect towards NM23-H2 binding to the guanine-rich promoter DNA sequence. Further analyses of the compound-protein interaction and the protein-DNA interaction provided insight into the mode of action for SYSU-ID-01: . Cellular evaluation results showed that SYSU-ID-01: could abrogate NM23-H2 binding to the c-MYC promoter, resulting in downregulation of c-MYC transcription and dramatically suppressed HeLa cell growth. These findings provide a new way of c-MYC transcriptional control through interfering with NM23-H2 binding to guanine-rich promoter sequences by small molecules.
- Published
- 2015
46. Characterization of the human α9 integrin subunit gene: Promoter analysis and transcriptional regulation in ocular cells
- Author
-
Céline Duval, Steeve Leclerc, Sylvain L. Guérin, Christian Salesse, and Karine Zaniolo
- Subjects
Sp1 Transcription Factor ,Response element ,E-box ,Biology ,Transfection ,03 medical and health sciences ,Cellular and Molecular Neuroscience ,Upstream activating sequence ,0302 clinical medicine ,Humans ,Promoter Regions, Genetic ,Enhancer ,Transcription factor ,Cells, Cultured ,030304 developmental biology ,0303 health sciences ,General transcription factor ,Gene Expression Profiling ,Epithelium, Corneal ,Epithelial Cells ,Promoter ,Fibroblasts ,Molecular biology ,Sensory Systems ,NFI Transcription Factors ,Ophthalmology ,Sp3 Transcription Factor ,Gene Expression Regulation ,030220 oncology & carcinogenesis ,TAF2 ,Integrin alpha Chains - Abstract
α9β1 is the most recent addition to the integrin family of membrane receptors and consequently remains the one that is the least characterized. To better understand how transcription of the human gene encoding the α9 subunit is regulated, we cloned the α9 promoter and characterized the regulatory elements that are required to ensure its transcription. Transfection of α9 promoter/CAT plasmids in primary cultured human corneal epithelial cells (HCECs) and uveal melanoma cell lines demonstrated the presence of both negative and positive regulatory elements along the α9 promoter and positioned the basal α9 promoter to within 118 bp from the α9 mRNA start site. In vitro DNaseI footprinting and in vivo ChIP analyses demonstrated the binding of the transcription factors Sp1, c-Myb and NFI to the most upstream α9 negative regulatory element. The transcription factors Sp1 and NFI were found to bind the basal α9 promoter individually but Sp1 binding clearly predominates when both transcription factors are present in the same extract. Suppression of Sp1 expression through RNAi also caused a dramatic reduction in the expression of the α9 gene. Most of all, addition of tenascin-C (TNC), the ligand of α9β1, to the tissue culture plates prior to seeding HCECs increased α9 transcription whereas it simultaneously decreased expression of the α5 integrin subunit gene. This dual regulatory action of TNC on the transcription of the α9 and α5 genes suggests that both these integrins must work together to appropriately regulate cell adhesion, migration and differentiation that are hallmarks of tissue wound healing.
- Published
- 2015
47. The GAL4 enhancer-trap line for analysis of definitive hematopoiesis in zebrafish
- Author
-
Jae-Ho Ryu, Hee Jeong Kong, Cheul Min An, Hyunju Ro, Kyung-Eun Lim, Sang-Yeob Yeo, Jehee Lee, and Woo-Jin Kim
- Subjects
GAL4/UAS system ,Reporter gene ,biology ,Transgene ,fungi ,biology.organism_classification ,Molecular biology ,General Biochemistry, Genetics and Molecular Biology ,Upstream activating sequence ,Enhancer trap ,Animal Science and Zoology ,Enhancer ,Developmental biology ,Zebrafish - Abstract
Zebrafish have been used as model vertebrates in several genetic studies. In developmental biology, gene function can be assessed through manipulation of the spatial expression or the temporal misexpression of target genes. In this study, we performed a developmental screen to identify a regulated enhancer of hematopoiesis capable of driving tissue-specific Gal4 expression in zebrafish. This Gal4/upstream activating sequence (UAS) system could be used to investigate regulatory interactions during embryonic development. We obtained a Tg[RBC:Gal4] transgenic line that showed red fluorescence protein (rfp) reporter gene expression during early embryonic hematopoiesis. Further analysis indicated the production of RFP-positive erythrocytes in a Tg[RBC:Gal4] transgenic line. By controlling the amount of metronidazole, which was used to induce production of an apoptosis-inducing reagent, we successfully created a conditional model of anemia using Tg[RBC:Gal4], Tg[UAS:RFP], and Tg[UAS:nfsB-mCherry] triple transge...
- Published
- 2015
48. MicroRNA-8 targets the Wingless signaling pathway in the female mosquito fat body to regulate reproductive processes
- Author
-
Alexander S. Raikhel, Jisu Ha, Keira J. Lucas, Vladimir A. Kokoza, Amanda L. Gervaise, and Sourav Roy
- Subjects
food.ingredient ,Wingless signaling ,1.1 Normal biological development and functioning ,Transgene ,Fat Body ,mosquito ,Vitellogenin ,Upstream activating sequence ,chemistry.chemical_compound ,food ,Underpinning research ,Yolk ,parasitic diseases ,microRNA ,Genetics ,Animals ,small RNA ,Antagomir ,Multidisciplinary ,biology ,Reproduction ,fungi ,Ovary ,Biological Sciences ,Blood meal ,Insect Vectors ,Wnt Proteins ,Vector-Borne Diseases ,MicroRNAs ,Culicidae ,Infectious Diseases ,Good Health and Well Being ,chemistry ,biology.protein ,Female ,Signal transduction ,Signal Transduction ,Biotechnology - Abstract
Female mosquitoes require a blood meal for reproduction, and this blood meal provides the underlying mechanism for the spread of many important vector-borne diseases in humans. A deeper understanding of the molecular mechanisms linked to mosquito blood meal processes and reproductive events is of particular importance for devising innovative vector control strategies. We found that the conserved microRNA miR-8 is an essential regulator of mosquito reproductive events. Two strategies to inhibit miR-8 function in vivo were used for functional characterization: systemic antagomir depletion and spatiotemporal inhibition using the miRNA sponge transgenic method in combination with the yeast transcriptional activator gal4 protein/upstream activating sequence system. Depletion of miR-8 in the female mosquito results in defects related to egg development and deposition. We used a multialgorithm approach for miRNA target prediction in mosquito 3' UTRs and experimentally verified secreted wingless-interacting molecule (swim) as an authentic target of miR-8. Our findings demonstrate that miR-8 controls the activity of the long-range Wingless (Wg) signaling by regulating Swim expression in the female fat body. We discovered that the miR-8/Wg axis is critical for the proper secretion of lipophorin and vitellogenin by the fat body and subsequent accumulation of these yolk protein precursors by developing oocytes.
- Published
- 2015
49. Heat Stress-Induced Cup9-Dependent Transcriptional Regulation of SIR2
- Author
-
Shyamasree Laskar, Sheeba K, Sunanda Bhattacharyya, Mrinal Kanti Bhattacharyya, Achuthsankar S. Nair, and Pawan K. Dhar
- Subjects
Saccharomyces cerevisiae Proteins ,Transcription, Genetic ,Heterochromatin ,Saccharomyces cerevisiae ,Biology ,Upstream activating sequence ,Sirtuin 2 ,Gene Expression Regulation, Fungal ,Heat shock protein ,Transcriptional regulation ,Animals ,Heat shock ,Molecular Biology ,Transcription factor ,Silent Information Regulator Proteins, Saccharomyces cerevisiae ,Homeodomain Proteins ,Regulation of gene expression ,Articles ,Cell Biology ,Telomere ,Molecular biology ,Chromatin ,enzymes and coenzymes (carbohydrates) ,Protein Processing, Post-Translational ,Heat-Shock Response ,Transcription Factors - Abstract
The epigenetic writer Sir2 maintains the heterochromatin state of chromosome in three chromosomal regions, namely, the silent mating type loci, telomeres, and the ribosomal DNA (rDNA). In this study, we demonstrated the mechanism by which Sir2 is regulated under heat stress. Our study reveals that a transient heat shock causes a drastic reduction in the SIR2 transcript which results in sustained failure to initiate silencing for as long as 90 generations. Hsp82 overexpression, which is the usual outcome of heat shock treatment, leads to a similar downregulation of SIR2 transcription. Using a series of genetic experiments, we have established that heat shock or Hsp82 overexpression causes upregulation of CUP9 that, in turn, represses SIR2 transcription by binding to its upstream activator sequence. We have mapped the cis regulatory element of SIR2. Our study shows that the deletion of cup9 causes reversal of the Hsp82 overexpression phenotype and upregulation of SIR2 expression in heat-induced Hsp82-overexpressing cells. On the other hand, we found that Cup9 overexpression represses SIR2 transcription and leads to a failure in the establishment of heterochromatin. The results of our study highlight the mechanism by which environmental factors amend the epigenetic configuration of chromatin.
- Published
- 2015
50. Encoding four gene expression programs in the activation dynamics of a single transcription factor
- Author
-
Anders S. Hansen and Erin K. O'Shea
- Subjects
Transcriptional Activation ,0301 basic medicine ,Genetics ,ATF3 ,Saccharomyces cerevisiae Proteins ,Response element ,TFE3 ,Biology ,General Biochemistry, Genetics and Molecular Biology ,Cell biology ,DNA-Binding Proteins ,03 medical and health sciences ,Upstream activating sequence ,SOX4 ,030104 developmental biology ,Sp3 transcription factor ,Gene Expression Regulation, Fungal ,Saccharomycetales ,PAX6 ,Promoter Regions, Genetic ,General Agricultural and Biological Sciences ,Transcription factor ,Fluorescent Dyes ,Signal Transduction ,Transcription Factors - Abstract
Summary Cellular signaling response pathways often exhibit a bow-tie topology [1,2]: multiple upstream stress signals converge on a single shared transcription factor, which is thought to induce different downstream gene expression programs (Figure 1A). However, if several different signals activate the same transcription factor, can each signal then induce a specific gene expression response? A growing body of literature supports a temporal coding theory where information about environmental signals can be encoded, at least partially, in the temporal dynamics of the shared transcription factor [1,2]. For example, in the case of the budding yeast transcription factor Msn2, different stresses induce distinct Msn2 activation dynamics: Msn2 shows pulsatile nuclear activation with dose-dependent frequency under glucose limitation, but sustained nuclear activation with dose-dependent amplitude under oxidative stress [3]. These dynamic patterns can then lead to differential gene expression responses [3–5], but it is not known how much specificity can be obtained. Thus, a major question of this temporal coding theory is how many gene response programs or cellular functions can be robustly encoded by dynamic control of a single transcription factor. Here we provide the first direct evidence that, simply by regulating the activation dynamics of a single transcription factor, it is possible to preferentially induce four distinct gene expression programs.
- Published
- 2016
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