Showing papers on "Upstream activating sequence published in 2015"
TL;DR: Exposure to an ortholog of Arabidopsis CIPK25 and its high active form differentially increased salt and water-deficit tolerance demonstrated by improved growth and reduced leaf chlorosis suggesting that the kinase activity of CaCIPK 25 was required for these functions.
Abstract: Calcium signaling plays an important role in adaptation and developmental processes in plants and animals. A class of calcium sensors, known as Calcineurin B-like (CBL) proteins sense specific temporal changes in cytosolic Ca(2+) concentration and regulate activities of a group of ser/thr protein kinases called CBL-interacting protein kinases (CIPKs). Although a number of CIPKs have been shown to play crucial roles in the regulation of stress signaling, no study on the function of CIPK25 or its orthologs has been reported so far. In the present study, an ortholog of Arabidopsis CIPK25 was cloned from chickpea (Cicer arietinum). CaCIPK25 gene expression in chickpea increased upon salt, dehydration, and different hormonal treatments. CaCIPK25 gene showed differential tissue-specific expression. 5'-upstream activation sequence (5'-UAS) of the gene and its different truncated versions were fused to a reporter gene and studied in Arabidopsis to identify promoter regions directing its tissue-specific expression. Replacement of a conserved threonine residue with an aspartic acid at its catalytic site increased the kinase activity of CaCIPK25 by 2.5-fold. Transgenic tobacco plants overexpressing full-length and the high active versions of CaCIPK25 displayed a differential germination period and longer root length in comparison to the control plants. Expression of CaCIPK25 and its high active form differentially increased salt and water-deficit tolerance demonstrated by improved growth and reduced leaf chlorosis suggesting that the kinase activity of CaCIPK25 was required for these functions. Expressions of the abiotic stress marker genes were enhanced in the CaCIPK25-expressing tobacco plants. Our results suggested that CaCIPK25 functions in root development and abiotic stress tolerance.
36 citations
TL;DR: In this paper, small molecules were used to interfere with the interaction process between NM23-H2 and the guanine-rich promoter sequence of c-MYC, resulting in downregulation of the c-myC transcription and dramatically suppressed HeLa cell growth.
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.
36 citations
TL;DR: The Split KalTA4 system should be useful for expression of genes-of-interest in an intersectional manner, allowing for more refined manipulations of cell populations in zebrafish and in transient mosaic expression assays, when hemi-drivers are preceded by two distinct promoters.
Abstract: In this study, we describe the adaptation of the split Gal4 system for zebrafish. The Gal4-UAS system is widely used for expression of genes-of-interest by crossing driver lines expressing the transcription factor Gal4 (under the control of the promoter of interest) with reporter lines where upstream activating sequence (UAS) repeats (recognized by Gal4) drive expression of the genes-of-interest. In the Split Gal4 system, hemi-drivers separately encode the DNA-binding domain (DBD) and the activation domain (AD) of Gal4. When encoded under two different promoters, only those cells in the intersection of the promoters' expression pattern and in which both promoters are active reconstitute a functional Gal4 and activate expression from a UAS-driven transgene. We split the zebrafish-optimized version of Gal4, KalTA4, and generated a hemi-driver encoding the KalTA4 DBD and a hemi-driver encoding KalTA4's AD. We show that split KalTA4 domains can assemble in vivo and transactivate a UAS reporter transgene and that each hemi-driver alone cannot transactivate the reporter. Also, transactivation can happen in several cell types, with similar efficiency to intact KalTA4. Finally, in transient mosaic expression assays, we show that when hemi-drivers are preceded by two distinct promoters, they restrict the expression of an UAS-driven reporter from a broader pattern (sox10) to its constituent smaller neuronal pattern. The Split KalTA4 system should be useful for expression of genes-of-interest in an intersectional manner, allowing for more refined manipulations of cell populations in zebrafish.
29 citations
TL;DR: 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 for use in Saccharomyces cerevisiae is described.
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.
28 citations
TL;DR: An activating but not essential role of RUNX1 is found in CD69 gene transcription by site-directed mutagenesis and RNA silencing, probably through the interaction with this potent enhancer specifically in the hematopoietic lineage.
19 citations
TL;DR: The mechanism by which Sir2 is regulated under heat stress is demonstrated, which 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.
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.
16 citations
TL;DR: It is found that UAS-linked gene expression was transcriptionally amplified by Gal4/UAS during early developmental stages and that the amplification effects tended to weaken during later stages and even disappear in subsequent generations.
Abstract: The Gal4/upstream activating sequence (UAS) system is a powerful genetic tool for the temporal and spatial expression of target genes In this study, the dynamic activity of the Gal4/UAS system was monitored in zebrafish throughout the entire lifespan and during germline transmission, using an optimized Gal4/UAS, KalTA4/4xUAS, which is driven by two muscle-specific regulatory sequences We found that UAS-linked gene expression was transcriptionally amplified by Gal4/UAS during early developmental stages and that the amplification effects tended to weaken during later stages and even disappear in subsequent generations In the F2 generation, the transcription of a UAS-linked enhanced green fluorescent protein (EGFP) reporter was transcriptionally silent from 16 days post-fertilization (dpf) into adulthood, yet offspring of this generation showed reactivation of the EGFP reporter in some strains We further show that the transcriptional silencing and reactivation of UAS-driven EGFP correlated with the DNA methylation levels of the UAS regulatory sequences Notably, asymmetric DNA methylation of the 4xUAS occurred in oocytes and sperm Moreover, the paternal and maternal 4xUAS sequences underwent different DNA methylation dynamics after fertilization Our study suggests that the Gal4/UAS system may represent a powerful tool for tracing the DNA methylation dynamics of paternal and maternal loci during zebrafish development and that UAS-specific DNA methylation should be seriously considered when the Gal4/UAS system is applied in zebrafish
15 citations
TL;DR: It is shown 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.
10 citations
TL;DR: Adding 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, suggesting that both these integrins must work together to appropriately regulate cell adhesion, migration and differentiation that are hallmarks of tissue wound healing.
9 citations
TL;DR: The results suggested that the determinant for σ38-dependent promoter lies in the promoter upstream sequence, and it was found that the σ dependency was switched.
Abstract: σ38 in Escherichia coli is required for expression of a subset of stationary phase genes. However, the promoter elements for σ38-dependent genes are virtually indistinguishable from that for σ70-dependent house-keeping genes. hdeABp is a σ38-dependent promoter and LEE5p is a σ70-dependent promoter, but both are repressed by H-NS, a bacterial histone-like protein, which acts at promoter upstream sequence. We swapped the promoter upstream sequences of the two promoters and found that the σ dependency was switched. This was further verified using lacUV5 core promoter. The results suggested that the determinant for σ38-dependent promoter lies in the promoter upstream sequence.
3 citations
TL;DR: The results suggest that this transgenic line may be useful as model animal to study normal and disease-related hematopoiesis and this Gal4/upstream activating sequence (UAS) system could be used to investigate regulatory interactions during embryonic development.
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...
TL;DR: By identification of the causative mutations, this work has accounted for most of the heritability of the phenotype in each strain and provided evidence that the Mediator coactivator complex plays both positive and negative roles in the regulation of transcription activation distance.
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.
5 May 2015
TL;DR: The use of a GVG system developed and extensively used in plant research to synchronize Xa21 expression allows for a synchronized immune response across a large amount of rice tissue, well suited for studying XA21-mediated immunity by genome-wide approaches such as transcriptomics and proteomics.
Abstract: Inducible gene expression systems offer researchers the opportunity to synchronize target gene expression at particular developmental stages and in particular tissues. The glucocorticoid receptor (GR), a vertebrate steroid receptor, has been well adopted for this purpose in plants. To generate steroid-inducible plants, a construct of GAL4-binding domain-VP16 activation domain-GR fusion (GVG) with the target gene under the control of upstream activation sequence (UAS) has been developed and extensively used in plant research. Immune receptors perceive conserved molecular patterns secreted by pathogens and initiate robust immune responses. The rice immune receptor, XA21, recognizes a molecular pattern highly conserved in all sequenced genomes of Xanthomonas, and confers robust resistance to X. oryzae pv. oryzae (Xoo). However, identifying genes downstream of XA21 has been hindered because of the restrained lesion and thus limited defense response region in the plants expressing Xa21. Inducible expression allows for a synchronized immune response across a large amount of rice tissue, well suited for studying XA21-mediated immunity by genome-wide approaches such as transcriptomics and proteomics. In this protocol, we describe the use of this GVG system to synchronize Xa21 expression.
TL;DR: It is suggested that a specific DNA geometry of the nucleoprotein complex stabilized on concomitant binding of RNA polymerase molecules at the fis promoter and the upstream region acts as a topological device regulating the fIS transcription.